antibiotics

Review
Potential Therapeutic Targets for Combination Antibody
Therapy against Pseudomonas aeruginosa Infections
Luke L. Proctor 1 , Whitney L. Ward 1 , Conner S. Roggy 1 , Alexandra G. Koontz 1 , Katie M. Clark 1 ,
Alyssa P. Quinn 1 , Meredith Schroeder 2 , Amanda E. Brooks 1 , James M. Small 1 , Francina D. Towne 1, *
and Benjamin D. Brooks 1, *

1 Department of Biomedical Sciences, College of Osteopathic Medicine, Rocky Vista University,

Parker, CO 80134, USA; [email protected] (L.L.P.); [email protected] (W.L.W.);
[email protected] (C.S.R.); [email protected] (A.G.K.); [email protected] (K.M.C.);
[email protected] (A.P.Q.); [email protected] (A.E.B.); [email protected] (J.M.S.)
2 Inovan, Inc., Fargo, ND 58102, USA; [email protected]
* Correspondence: [email protected] (F.D.T.); [email protected] (B.D.B.)

Abstract: Despite advances in antimicrobial therapy and even the advent of some effective vaccines,
Pseudomonas aeruginosa (P. aeruginosa) remains a significant cause of infectious disease, primarily
due to antibiotic resistance. Although P. aeruginosa is commonly treatable with readily available



therapeutics, these therapies are not always efficacious, particularly for certain classes of patients


(e.g., cystic fibrosis (CF)) and for drug-resistant strains. Multi-drug resistant P. aeruginosa infections
Citation: Proctor, L.L.; Ward, W.L.;
are listed on both the CDC’s and WHO’s list of serious worldwide threats. This increasing emergence
Roggy, C.S.; Koontz, A.G.; Clark,
of drug resistance and prevalence of P. aeruginosa highlights the need to identify new therapeutic
K.M.; Quinn, A.P.; Schroeder, M.;
strategies. Combinations of monoclonal antibodies against different targets and epitopes have
Brooks, A.E.; Small, J.M.; Towne, F.D.;
demonstrated synergistic efficacy with each other as well as in combination with antimicrobial agents
et al. Potential Therapeutic Targets
for Combination Antibody Therapy
typically used to treat these infections. Such a strategy has reduced the ability of infectious agents
against Pseudomonas aeruginosa to develop resistance. This manuscript details the development of potential therapeutic targets for
Infections. Antibiotics 2021, 10, 1530. polyclonal antibody therapies to combat the emergence of multidrug-resistant P. aeruginosa infections.
https://doi.org/10.3390/ In particular, potential drug targets for combinational immunotherapy against P. aeruginosa are
antibiotics10121530 identified to combat current and future drug resistance.

Academic Editors: Jürgen Brem, Keywords: polyclonal antibodies; antibiotic resistance; antibiotics; combination therapies; Pseudomonas
Sónia Silva, Mark G. Moloney and aeruginosa; immunotherapies
Daniele Castagnolo

Received: 5 November 2021

Accepted: 9 December 2021
1. Introduction
Published: 14 December 2021
P. aeruginosa is a Gram-negative bacillus implicated in a wide variety of human infec-
Publisher’s Note: MDPI stays neutral
tions. In acute infections, individual P. aeruginosa organisms exhibit swarming motility via
with regard to jurisdictional claims in
a single flagellum and type 4 pili and express a wide variety of toxins, cell surface proteins,
published maps and institutional affil- and other molecules that contribute to its immunogenicity and pathogenicity [1]. In order
iations. to establish chronic infection, P. aeruginosa transitions to a sessile, non-motile state marked
by the formation of a mucoid biofilm, composed mainly of exo-polysaccharides, glycolipids,
and mucin, which often poses a barrier to successful clinical treatment [2]. Regardless
of if P. aeruginosa exists in an acute motile form or a chronic sessile biofilm, infection
Copyright: © 2021 by the authors.
with P. aeruginosa is particularly perilous for immunosuppressed patients [1], ventilator-
Licensee MDPI, Basel, Switzerland.
dependent patients, and cystic fibrosis patients. According to the CDC, P. aeruginosa
This article is an open access article
infections were responsible for 32,600 nosocomial infections and 2700 deaths in 2017.
distributed under the terms and Data collected from over 4500 hospitals in the United States’ National Healthcare Safety
conditions of the Creative Commons Network from 2011 to 2014 revealed the following rates of multidrug resistance among
Attribution (CC BY) license (https:// P. aeruginosa isolates [3]:
creativecommons.org/licenses/by/ Ventilator-associated pneumonia—20%
4.0/). Central line-associated bloodstream infection—18%

Antibiotics 2021, 10, 1530. https://doi.org/10.3390/antibiotics10121530 https://www.mdpi.com/journal/antibiotics

Antibiotics 2021, 10, x FOR PEER REVIEW 2 of 33

Antibiotics 2021, 10, 1530 2 of 31

Catheter-associated urinary tract infection—18%
Surgical site infection—4%
This culminates in an estimated cost to the healthcare system of USD 767 million [4].
Catheter-associated urinary tract infection—18%
In cystic fibrosis patients alone, mean healthcare costs per patient increase by 87% once a
Surgical site infection—4%
patient becomes colonized with P. aeruginosa, to nearly USD 67,000 annually [4]. Addi-
This culminates in an estimated cost to the healthcare system of USD 767 million [4].
tionally, P. aeruginosa has been recognized as the causative organism in catheter-associ-
In cystic fibrosis patients alone, mean healthcare costs per patient increase by 87% once
ated urinary tract infections, otitis externa, otitis media, contact lens keratitis, soft tissue
a patient becomes colonized with P. aeruginosa, to nearly USD 67,000 annually [4]. Addi-
infections in burn victims and AIDS patients, septic arthritis, folliculitis, meningitis, and
tionally, P. aeruginosa has been recognized as the causative organism in catheter-associated
sepsis. In fact, this broad array of associated disease states (Figure 1) has led P. aeruginosa
urinary tract infections, otitis externa, otitis media, contact lens keratitis, soft tissue infec-
to be recognized as the sixth leading cause of hospital-acquired infections, the second most
tions in burn victims and AIDS patients, septic arthritis, folliculitis, meningitis, and sepsis.
common cause of ventilator-associated pneumonia and the most common multidrug-re-
In fact, this broad array of associated disease states (Figure 1) has led P. aeruginosa to be
sistant Gram-negative cause of ventilator-associated pneumonia, the third most common
recognized as the sixth leading cause of hospital-acquired infections, the second most com-
cause of catheter-associated UTI, and the fifth most common cause of surgical site infec-
mon cause of ventilator-associated pneumonia and the most common multidrug-resistant
tions [1].
Gram-negative cause of ventilator-associated pneumonia, the third most common cause of
catheter-associated UTI, and the fifth most common cause of surgical site infections [1].

Figure 1.
Figure 1. Types
Types of
of Acute
Acute P.
P.Aeruginosa
AeruginosaInfections
Infections [5].
[5]. P.
P. aeruginosa
aeruginosa is
is prevalent
prevalent in
in skin
skin and
and soft
soft tissue
tissue infections (top right)
including trauma,
including trauma, burns,
burns, and
and dermatitis.
dermatitis. It
It also
also commonly
commonly causes causes swimmer’s’
swimmer’s’ earear (external
(external otitis),
otitis), hot
hot tub
tub folliculitis,
folliculitis, and
and
ocular infections,
ocular infections,bacteremia
bacteremiaandand septicemia,
septicemia, especially
especially in immunocompromised
in immunocompromised patients,
patients, and endocarditis
and endocarditis associated
associated with
with
IV IV users
drug drug and
users and prosthetic
prosthetic heart (bottom
heart valves valves (bottom
right). P.right). P. aeruginosa
aeruginosa can also
can also cause cause
central centralsystem
nervous nervous system
(CNS) (CNS)
infections
infections such as meningitis and brain abscess (top left), bone and joint infections, including osteomyelitis and osteochon-
such as meningitis and brain abscess (top left), bone and joint infections, including osteomyelitis and osteochondritis,
dritis, respiratory tract infections, and hospital-acquired urinary tract infections (UTIs; bottom left). P. aeruginosa is also
respiratory tract infections, and hospital-acquired urinary tract infections (UTIs; bottom left). P. aeruginosa is also resistant to
resistant to many common antibiotics [5].
many common antibiotics [5].

The vast array of infectious complications that can arise from normal commensal
and environmental strains of P. aeruginosa indicates that it is an opportunistic, adaptable,
Antibiotics 2021, 10, 1530 3 of 31

common environmental pathogen, making P. aeruginosa very robust and difficult to treat.
Several antimicrobial agents possess the ability to treat P. aeruginosa infections [3]; however,
successful clinical treatment regimens should include pre-treatment sensitivity testing, as
different strains possess widely different antimicrobial resistances. Importantly, treatment
is often dictated by the antibiogram of a specific hospital or region. P. aeruginosa is often sus-
ceptible to first-line agents, including beta-lactam antibiotics (e.g., piperacillin-tazobactam
and ticarcillin-clavulanate), cephalosporins (e.g., ceftazidime, cefoperazone, and cefepime),
and monobactams (e.g., Aztreonam). Carbapenems (e.g., meropenem and doripenem),
which were historically seen as the “big gun”, last-ditch antimicrobials, can be used to treat
highly resistant infections. However, as of 2019, the World Health Organization has listed
carbapenem-resistant P. aeruginosa as one of three bacterial diseases in critical need of new
treatment strategies, with up to 14% of P. aeruginosa isolates in the U.S. in 2019 expressing
carbapenem resistance (Figure 2) [6]. This highlights the need for expert guidance regard-
ing treating carbapenem-resistant infections. Interestingly, fluoroquinolones, especially
ciprofloxacin, are the only class of oral antibiotics with specifically antipseudomonal activ-
ity. Regardless, for those with risk for serious infections, combination therapy with agents
from different drug classes is generally suggested. Most commonly, a combination of a
beta-lactam along with an aminoglycoside is chosen [3,7]. However, resistance to these
standard antimicrobials is rapidly growing, particularly in hospital-acquired isolates [8]. In
fact, 2.8 million cases of multidrug-resistant bacterial infections were reported in the U.S.
alone in 2019 [9]. Unfortunately, although using two classes of drugs synergistically has
proven to be helpful, it has not solved the problem of multidrug-resistant, extensively drug-
resistant, or pandrug-resistant strains. Approaching pathogens with an “out of the box”
approach, such as that explored in this review, may be necessary to aid the antimicrobial
strategy of treatment in P. aeruginosa infections [3,10].
In the ongoing battle between humans and the pathogenic microbes that cause disease,
the CDC recognizes that the development of newer antimicrobial pharmacotherapeutics
continues to be a pressing need, despite several current pharmaceutical agents that are
reserved for the treatment of multidrug-resistant isolates [7]. In response to advancing
antimicrobial pharmacotherapies, particularly bactericidal therapies that impose selective
pressure, bacterial resistance mechanisms continue to evolve as opportunistic microbes
adapt to an ever-changing therapeutic landscape. The evolution of multidrug-resistant
P. aeruginosa can be considered as a case study based on its sophisticated quorum sensing
communication system and phenotypic plasticity that has allowed it to adapt, survive,
and thrive in a wide variety of environmental (e.g., aquatic and soil) and host condi-
tions [11]. Among clinical isolates, a wide range of phenotypic variation has been identified
including hyperpigmentation, small colony variant formation, autoaggregation, alginate
overproduction, and autolysis [12–16]. These phenotypes change and adapt as an infection
progresses, allowing for long-term survival in the differing conditions of the host [17].
The exact number of clinically significant strains of P. aeruginosa is unknown, but up to
40 individual strains were identified in the late 1970s, and as of 2019, there have been
at least 66 clinical strains and 19 environmental strains identified [18,19]. This versatil-
ity has led P. aeruginosa, which is prone to the development of antibiotic resistance that
renders current treatment options inadequate [2,8], to be categorized as part of the ESKAPE
(Enterococcus faecium Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii,
P. aeruginosa, and Enterobacter species) group of bacteria, deemed of particularly great
health concern by the Infectious Diseases Society of America (IDSA) [20]. Microbial evolu-
tion to circumvent and resist antimicrobial therapies necessitates distinctly new approaches
to targeting multi-resistant bacterial species, which cause human disease and place a large
burden on the healthcare system.
Antibiotics 2021,10,
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x FOR PEER REVIEW 44 of
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33

Figure 2.
Figure 2. Treatment
Treatment strategy
strategy for
for carbapenem-resistant
carbapenem-resistant P.
P.aeruginosa
aeruginosaisolates
isolates including
including future
future treatment
treatment options
options based
based on
on
combinatorial antibody therapies [21].
combinatorial antibody therapies [21].

Antibiotic
Antibiotic resistance
resistancecontinues
continues to increase
to increase through the development
through the development of beta-lactamase-
of beta-lac-
producing strains, shifts in porin conformations, efflux pumps, specific
tamase-producing strains, shifts in porin conformations, efflux pumps, specific antibiotic antibiotic inactivat-
ing enzymes,enzymes,
inactivating and non-specific porin modifications
and non-specific [8,22]. A deeper
porin modifications [8,22]. Aunderstanding of the
deeper understand-
mechanisms of resistance
ing of the mechanisms can informcan
of resistance drug discovery
inform and development
drug discovery efforts. A central
and development efforts.
mechanism in the development
A central mechanism of antibioticof
in the development resistance
antibiotic is the conversion
resistance to conversion
is the coordinatedto com-
co-
munal
ordinatedbiofilm growth.biofilm
communal growth. P.
As commensal Asaeruginosa
commensal changes to an opportunistic
P. aeruginosa changes to an pathogen
oppor-
or acquired
tunistic pathogenic
pathogen infection
or acquired is established,
pathogenic genotypic
infection and phenotypic
is established, genotypic changes occur.
and pheno-
Among the most important, P. aeruginosa switches from a more
typic changes occur. Among the most important, P. aeruginosa switches from a more anti- antibiotic susceptible, motile
planktonic phase tomotile
biotic susceptible, an antibiotic tolerant,
planktonic phase non-motile biofilm tolerant,
to an antibiotic phenotype. This switch
non-motile rep-
biofilm
resents a keyThis
phenotype. element
switchin the conversion
represents a keyto element
pathogenicity.
in the Importantly,
conversion tothis mucoid, biofilm
pathogenicity. Im-
phenotype is seen
portantly, this in overbiofilm
mucoid, 75% ofphenotype
CF-associated P. aeruginosa
is seen in over 75% infections [23,24]. Once
of CF-associated P. in its
aeru-
sessile phase, P. aeruginosa can attach to host epithelial cell layers to better
ginosa infections [23,24]. Once in its sessile phase, P. aeruginosa can attach to host epithelial avoid the host’s
immune
cell layers responses
to better[2]. avoid Although
the host’santi-P. aeruginosa
immune IgA antibodies
responses [2]. Althoughare also present
anti-P. lining
aeruginosa
the respiratory tract, limiting the spread of P. aeruginosa to the respiratory
IgA antibodies are also present lining the respiratory tract, limiting the spread of P. aeru- epithelium, they
have shown to be ineffective at eliminating infection [25]. Interestingly,
ginosa to the respiratory epithelium, they have shown to be ineffective at eliminating in- antibiotic use may
cause
fectionIgA deficiency
[25]. by inhibiting
Interestingly, antibiotic normal
use may microbiota
cause IgA anddeficiency
Toll-like receptor (TLR) signals
by inhibiting normal
that induce activating factors of IgA production in plasma cells.
microbiota and Toll-like receptor (TLR) signals that induce activating factors of IgA Therefore, excessive antibi-
pro-
otic use may further increase the risk of P. aeruginosa infection
duction in plasma cells. Therefore, excessive antibiotic use may further increase the or reinfection [25,26]. These
risk
problems underscore that passive immunity may provide the safest and most efficacious
of P. aeruginosa infection or reinfection [25,26]. These problems underscore that passive
therapeutic and even potentially prophylactic option. A thorough analysis of the varied
immunity may provide the safest and most efficacious therapeutic and even potentially
mechanisms of antibiotic resistance is beyond the scope of this review, but some of the
prophylactic option. A thorough analysis of the varied mechanisms of antibiotic resistance
types of drug resistance mechanisms in P. aeruginosa are shown in Figure 3 for reference.
Antibiotics 2021, 10, x FOR PEER REVIEW 5 of 33

Antibiotics 2021, 10, 1530 5 of 31

is beyond the scope of this review, but some of the types of drug resistance mechanisms
in P. aeruginosa are shown in Figure 3 for reference.

Figure
Figure3.3.Mechanisms
Mechanismsof
ofantibiotic
antibiotic resistance
resistance in P. aeruginosa. These
P. aeruginosa. These include
includeall
allof
ofthe
themechanisms
mechanismsin
in blue and biofilms.
blue and biofilms.

Chronic
Chronicbacterial
bacterialinfections
infectionsare
areoften
oftenheralded
heraldedby bythe
thepresence
presence ofof aasubpopulation
subpopulation of of
persister
persistercells,
cells,bacterial
bacterialcells
cellscapable
capableofofsurviving
survivingan anantibiotic
antibioticassault.
assault.This
Thissubpopulation
subpopulation
of
of bacteria
bacteria is typically
typically present
presentininhigh
highnumbers
numbers in in bacterial
bacterial biofilms
biofilms [26].
[26]. Although
Although ini-
initially
their their
tially abilityability
to persist in the in
to persist presence of a bactericidal
the presence agent may
of a bactericidal be may
agent indicative of some of
be indicative in-
nate antibiotic
some resistance,
innate antibiotic a deepera dive
resistance, deeperinto theinto
dive mechanism suggestssuggests
the mechanism that thisthat
population
this pop-is
able to decrease
ulation is able totheir metabolic
decrease theirand growth and
metabolic activity
growthto a state of senescence,
activity to a state ofcircumventing
senescence,
antibiotic action, which often requires active metabolism or proliferation. According
circumventing antibiotic action, which often requires active metabolism or proliferation. to this
hypothesis, as borne out by numerous studies, persistence may be a transient
According to this hypothesis, as borne out by numerous studies, persistence may be a state, which
may or may
transient notwhich
state, be passed
may toor subsequent
may not be generations based on the
passed to subsequent underlying
generations mechanism
based on the
of action. Alternatively, several genes have been identified (e.g., hip-high persistence [27]
and GlpD—sn-glycerol-3-phosphate dehydrogenase [28]) and have been suggested to
confer phenotypic persistence, a characteristic which may prove heritable [29–31]. The
persister cell’s phenotypic switch, which is likely the result of complex signaling cascade(s),
Antibiotics 2021, 10, 1530 6 of 31

between metabolically quiescent and metabolically active may prove to be an incredibly

important drug target; however, an in-depth discussion of the variability and complexity
is beyond the scope of the current paper and the reader is referred to several good recent
reviews of the topic, specifically that from Kaldalu et al., in 2020 [32] and Louwagie et al.,
in 2021 [30].
P. aeruginosa isolates taken from cystic fibrosis patients with chronic colonization have
shown high levels of persister cells, one of the leading hypotheses behind the inability
of antibiotics to effectively clear colonized bacteria [26,33,34]. While intrinsic antibiotic
resistance mechanisms lead to multidrug-resistant acute P. aeruginosa infections, chronic
infections may be more likely to display antibiotic-tolerant mechanisms such as these per-
sister cells and biofilms [33,34]. Many of the quorum sensing mechanisms to be described
later may underlie the development and proliferation of these persister cells, although
these processes remain poorly understood [26,35].

2. Host Immune Response

In addition to using mechanisms of P. aeruginosa antibiotic resistance to inform and
frame drug development efforts, knowledge of the host immune response to develop-
ing Pseudomonas infection can also inspire drug discovery and new clinical treatment
strategies. A brief discussion of the host/microbe interaction and immunity provides
necessary context.
P. aeruginosa infection commonly induces a robust humoral response including IgG an-
tibodies towards lipopolysaccharide (LPS), alginate, alkaline protease, elastase, exotoxin A,
and many other surface antigens and proteins of P. aeruginosa, which are often upregulated
as virulence factors during various stages of biofilm development [36,37]. Unfortunately,
the host antibodies produced typically have low affinity for their respective targets and
are not effective at eliminating the infection [25]. As an aside, host opsonizing antibodies
also cannot eliminate these mucoid microorganisms [38]. Nevertheless, anti-P. aeruginosa
IgG binds to its antigen and immune complexes are formed, activating complement and
recruiting macrophages. As macrophages and immune cells bind the anti-P. aeruginosa
IgG, they create reactive oxygen species (ROS), consuming oxygen, making the biofilm
environment more anaerobic and thus more favorable for the organism. The anaerobic
environment is unsuitable for host macrophages, neutrophils, and other immune cells.
Phagocytosis may occur, but without sufficient oxygen, ROS cannot be produced to elimi-
nate bacteria. This creates an inflammatory environment causing tissue damage without
efficient disruption of P. aeruginosa biofilms [25]. The ongoing inflammatory state in chronic
infections is thus not linked to immunogenicity of the bacterial organisms themselves, but
rather the secreted products that leave the biofilm and induce immune responses in the
airway epithelium [23,24]; hence, the humoral responses produced by many people in
response to P. aeruginosa infection are not effective in eliminating the infection.
The lack of a humoral immune response capable of eliminating P. aeruginosa has a
significant impact on vaccinologists’ ability to actively provoke the immune system with
a Pseudomonas vaccine. Vaccines against P. aeruginosa have been developed and tested
with the same unsatisfactory effect. While successful at producing antibodies, to date,
vaccines have been unsuccessful at preventing infection in many individuals [39]. For
example, a recent clinical trial studied the efficacy and safety of IC43 vaccination against
P. aeruginosa in ICU patients. IC43 contains the P. aeruginosa outer membrane proteins
OprI and OprF. Findings suggest that while the vaccination is highly immunogenic, the
candidate vaccine did not reduce overall mortality [40]. Further work here, specifically on
target identification, is warranted.

3. Description of Targets
Taking inspiration from the immune response and in the context of P. aeruginosa’s
life cycle and its antibiotic resistance mechanisms, several potential targets secreted by
P. aeruginosa were identified. These targets, outlined in Table 1, produce a wide variety of
Antibiotics 2021, 10, 1530 7 of 31

effects in hosts and the bacteria, contributing to the pathogenesis of the entire spectrum
of infections caused by this organism. Some of the most promising targets identified are
discussed in the following sections; however, a more complete list can be found in Table 1.
Identified targets are grouped into eight categories based on their function and location in
the cell.

Table 1. Potential Therapeutic Antibody Targets.

Location or
Examples Activity/Effects on Host
Class
Alginate Antiphagocytic, resists opsonic killing
Endotoxic, antiphagocytic, avoids preformed antibody to
Lipopolysaccharide
previously encountered O antigens
Twitching motility, biofilm formation, adherence to
Pili (produced by type IV secretion)
Cell surface host tissues
Motility, biofilm formation, adherence to host tissues and
Flagella
mucin components
PcrG, PcrV, PcrH, PopB, and PopD proteins form injection
Injection of type III secretion factors
bridge for type III effectors
Outer membrane Siderophore receptors Provides iron for microbial growth and survival
Efflux pumps Remove antibiotics
Secretion systems
Elastase, lipase, phospholipases, chitin-binding Variety of proteolytic, lipolytic, and toxic factors; degrade
• Type II protein, exotoxin A, and others host immune effectors
Intoxicates cells (ExoS, ExoT); cytotoxic (ExoU); disrupts
• Type III ExoS, ExoT, ExoU, ExoY
actin cytoskeleton
Poorly characterized but found in animal studies to be
Cytoplasmic and membrane-associated proteins,
• Type VI needed for optimal virulence, particularly in
ATPases, lipoproteins, Hcp1 protein
chronic infection
Iron acquisition Pyoverdin, pyochelin, HasAP Scavenge iron from the host for bacterial use
Kill leukocytes, hemolysis of red cells, degrade host cell
Secreted toxins Hemolysins, rhamnolipid phospholipases
surface glycolipids
Secreted Produce reactive oxygen species: H2 O2 , O2 −
Pyocyanin, ferric pyochelin, HCN
oxidative factors Inflammatory, disrupts epithelial cell function
Quorum sensing LasR/LasI, RhlR/RhlI, PQS Biofilm formation, regulation of virulence factor secretion
ATPases = adenosine triphosphatases; PQS = Pseudomonas quinolone signal.

3.1. Secreted Toxins and Invasins

Targeting extracellular-secreted toxins and invasins may have the greatest potential
to decrease virulence and improve clinical outcomes. “Secreted toxins,” here, is not ref-
erencing toxins injected into the cytoplasm of host cells (i.e., intracellular toxins) which
are not highly available to immunotherapeutics. Targeting secreted extracellular toxins
can be impactful not only to the host, but also to commensal flora. Invasins penetrate host
cells, allowing entry during the initial stage of infection. Commonly, these extracellular
toxins and invasins are available to interact with circulating and secreted (e.g., IgA and
IgM) antibodies.
Hence, this class has several very promising targets for antibody therapy development.
Additionally, they do not impose “life-or-death” selective pressure, allowing therapeutic
efficacy to be preserved beyond what is common for many antibiotics, which experience
emergence of resistance within a short time of their entrance to the market [31,37]. Several
common members of the toxin/invasin class, which are (1) ubiquitously and extracellularly
expressed or secreted, (2) key mediators of virulence and pathogenicity, (3) responsive to
Antibiotics 2021, 10, 1530 8 of 31

antibodies, and (4) specific to P. aeruginosa, are discussed below and should be strongly
considered as a part of any therapeutic antibody strategy.
Exotoxin A. Exotoxin A is the most potent virulence factor produced by most clinical
strains (88%) of P. aeruginosa [41]. Exotoxin A is a ribosylating enzyme that inhibits
protein synthesis and interferes with immune functions of the host and causes widespread
apoptosis [42]. The regulation of exotoxin A is not completely understood, although it is
thought to be upregulated for iron scavenging.43 However, it is known that exotoxin A is
secreted through the Type II Secretion System (T2SS, see Figure 3), making it a promising
target for therapeutic antibody development [43]. In several studies, exotoxin A antibodies
have provided protection to clinical P. aeruginosa infections, including in exotoxin A toxoid
vaccine trials [44].
Protease IV. Proteases are associated with the corneal damage seen with P. aeruginosa
keratitis and play an important role in the tissue damage seen with soft tissue P. aeruginosa
infections [45]. Protease IV, which acts to cleave or degrade host proteins such as im-
munoglobulins, complement, and fibrinogen, is a somewhat unique extracellular virulence
factor [46]; its expression and potency are induced by quorum sensing (see below) [47].
While host-derived antibodies to Protease IV fail to develop in acute infections, injection
of antibody–antigen complexes has been shown to develop strong, protective antibody
responses [48,49]. Alternatively, antibody inhibitors of Protease IV could be developed
as medications for P. aeruginosa infections. In one study, an antibody inhibitor showed
complete inhibition of the protease [46].
Lipase A (LipA). Lipase A is abundant and a major secreted protein of P. aeruginosa [50].
In fact, Lipase A is the main extracellular lipase of P. aeruginosa. LipA is transported across the
cell envelope by a type II secretion system, which is a two-step ATP-dependent process. It has
been shown that Lipase A binds to the extracellular polysaccharide, alginate, by electrostatic
interaction [50]. This interaction seems to localize the enzyme near the cell surface and is
thermo-stabilizing, which may be relevant for the growth and survival of biofilm resident
P. aeruginosa [50]. LipA is also an immunomodulator [51,52]. Therefore, therapies that include
Lipase A as a target would help decrease the establishment of infection and decrease virulence.
Phospholipase C. Phospholipase C (PLC), also known as lecithinase, is a common
class of enzymes that cleave exogenous phosphatidylcholine (PC) into fatty acids and
choline. P. aeruginosa uses the processed phospholipid products as an energy source.
P. aeruginosa has two types of PLC, hemolytic (PLCh) and nonhemolytic (PLCnh). The
hemolytic one seems to play a larger role in virulence [42]. It is hypothesized that PLCh
decreases oxidative burst activity in neutrophils, which are the primary cells responsible
for clearing P. aeruginosa from the lungs. In mouse models, PLCh has also been shown to
cause an increase in vascular permeability, end-organ damage, and death [53]. P. aeruginosa
strains with either disruptions or deletions in PLCh are less virulent than the wild-type
strain [53]. While commonly a membrane-bound protein, in P. aeruginosa infections, PLC is
a secreted toxin that plays a role in pathogenesis [54]. PLC’s ability to damage the host cell
membrane allows disseminated infection and wound colonization, indicating that PLC may
play an integral role in the establishment and maintenance of chronic wound infections [41].
Virulence factors expressed by P. aeruginosa isolates from chronic leg wounds include beta
hemolysins (92.3%), lipase (76.9%), and lecithinase (61.5%) [41]. Additionally, a study
of 123 environmental and clinical strains of P. aeruginosa also expressed beta hemolysins
(95.1%), lipase (100%), and lecithinase (100%), displaying high conservation of this class of
enzymes [55]. This high degree of conservation and known genetics (i.e., ExoT and AlgD,
coded for phospholipases and protease IV, respectively [41]), makes this a particularly
attractive target for therapeutic enzymes [53]. Antibodies to PLC have been detected early
and at high levels in many patients, but especially in CF patients with chronic P. aeruginosa
infections. The antibodies remain elevated throughout the course of infection and increase
with acute exacerbations [56]. The one particularly important caveat to targeting PLC for
therapeutic antibody development is that while PLCh is secreted, PLC may be sequestered
Antibiotics 2021, 10, 1530 9 of 31

in secreted outer membrane vesicles, providing only limited availability [57]. Nevertheless,
the potential of this class warrants further study.
LasA and LasB (Elastase A and B). LasA and LasB are two secreted proteases that
cleave immunoglobulins, inhibit cytokines, interfere with immune cell functions, and
increase the permeability of the tight junctions of the airway epithelium [58–65]. Both are
synthesized as proenzymes. LasA is secreted in its unprocessed form and then processed
extracellularly. LasA is known to enhance pseudomonal colonization and immune evasion
via enhanced syndecan-1, IL-6 receptor, CD14, and TNF-α shedding, and facilitates the
function of elastase [65]. LasA can also act on elastin but is limited to a few highly specific
amino acid sequences [66]. In contrast, LasB is a zinc metalloprotease that can cleave
proteins at multiple sites. LasB is a propeptide that is initially secreted and then partially
degraded extracellularly. LasB is able to degrade a wide range of host proteins, conferring
much more virulence when compared to LasA [45,66]. Part of this enhanced virulence is
also the ability of LasB to induce damage to host tissue and subvert immune responses. In
addition, LasB decreases the expression and activity of the CFTR ion channel in bronchial
epithelial cells and has been shown to have the ability to degrade IL-6 and trappin-2, which
are both important for antimicrobial defense [45]. LasB seems to exacerbate this weakened
system and cause increased morbidity and mortality for cystic fibrosis patients [45]. LasB
is prevalent and a major secreted protein in the secretome of the P. aeruginosa PA01 strain,
driving virulence in a large percent of CF patients [45]. LasB is secreted by the type II
secretion system (Figure 3) [45].
In addition to the central role of LasA and LasB in virulence, past studies of active
immunity indicate that antibody-based therapeutics might be particularly effective. Anti-
LasA and anti-elastase antibodies may decrease virulence by rendering P. aeruginosa more
vulnerable to host immune mechanisms or antibiotics. Vaccine candidates to LasB and LasA
were efficacious in preventing and decreasing the severity of pneumonia in minks, corneal
ulcers and lung infections in rats and burns in mice [67–70]. LasA and LasB may be secreted
in outer membrane vesicles and thus would have limited availability as drug targets [57].
Alkaline Protease (AprA). AprA, which is also a zinc metalloprotease [71] that has
activity and function similar to LasB, is a prevalent secreted protein of the P. aeruginosa
PA01 strain [71]. Its production is thought to be regulated not only by phosphatidylcholine,
but it appears that P. aeruginosa is also able to induce the production of alkaline protease
in conditions of limited inorganic phosphate [72], tagging it as potentially relevant target
to squelch virulence [73]. AprA is found to be active during an inflammatory phase of
infection and causes cellular necrosis and hemorrhage via increased vascular permeability
and proteolysis in infected wound sites. This activity is further expressed in septicemia,
in which proteolysis affects homeostatic mechanisms of plasma proteins [74]. Unlike
LasB, AprA is secreted as a part of the Type 1 secretion system which includes AprA, D,
E, and F. Unfortunately, there is some evidence to suggest that Alkaline Protease may
be sequestered in OMVs and thus may have limited availability [57]. Thus, AprX, a
caseinolytic extracellular protease, also secreted by T1SS, might serve as a good alternative
or supplementary target [75].
Caseinase. Caseinase is a major, soluble protease and virulence factor secreted by
P. aeruginosa that breaks down casein [41], although there is some evidence to suggest that
caseinase may be sequestered in OMVs after secretion, which may limit its utility as an
antibody drug target [57]. In isolated strains of P. aeruginosa, caseinase was expressed in
91.67% of chronic leg ulcers cultured [41]. Another study analyzed 123 clinical and environ-
mental P. aeruginosa strains and reported a 99.2% conservation of caseinase. Interestingly,
the clinical strains had considerably more proteolytic activity compared to environmental
strains [55]. In wounds, caseinase delays healing and contributes to chronic lesions by
limiting essential amino acids in the area [41]. Additionally, caseinase and other secreted
proteases aid in colonization, tissue damage, and immune evasion by P. aeruginosa [55].
Thus, therapies that include caseinase as a target would theoretically decrease the estab-
lishment of infection as well as the virulence of the strain.
Antibiotics 2021, 10, 1530 10 of 31

3.2. Secretion System Proteins

While the previous section focused on virulence factors and other key molecules
secreted, targeting key components of the secretion systems themselves may have the
same functional consequence. These secretion systems traverse bacterial membranes and
are classified into eight distinct types based on their specific structure, composition, and
activity. Types I, II, III, V, and VI are particularly relevant due to their significance in the
virulence of P. aeruginosa infections, although evidence suggests that P. aeruginosa possesses
all eight types [76].
Type 1 (T1SS) Secretion System. Two well-characterized, independent type I secre-
tion systems (T1SS) have been identified in P. aeruginosa. T1SS are associated with virulence
and found to be active during the inflammatory phase of P. aeruginosa infection, 77 making
Antibiotics 2021, 10, x FOR PEER REVIEW 11 of 33
them potential targets for pharmacotherapy. The Apr T1SS is an ABC transporter involved
in alkaline protease secretion (AprA) and (AprX) [76,77]. The HasF T1SS is a heme-binding
protein secretion system (HasF) [68,69]. AprF and HasF are the outer membrane proteins
inducing
of the HasFapoptosis. The T3SSinconsists
T1SS involved alkalineofprotease
a multimeric protein
secretion complex
and that is divided
heme uptake into
[78,79] (see
four major domains. Epitopes in each of the extracellular domains could serve
Figure 4), and as such would be accessible to antibody and other traditional therapeutics. as drug
targets
The mainto reduce
questionbacterial associatedof
is the availability morbidity. Other attempts
these membrane proteinstosince
actively
theirtarget the T3SS
immunogenic-
with antibody
ity has therapies
yet to be have yielded
characterized mixed
in detail [76]. results
Further[86], with enough promise
immunogenicity that anti-
and accessibility
body therapy against
characterization T3SS, which
is warranted. in conjunction
Two other with identified
T1SS have been T2SS is thought to significantly
using genomic analysis
contribute
but are notto bacterial virulence
well-characterized [76], should be pursued.
currently.

Figure
Figure 4.
4. Protein
Protein secretion
secretion systems
systems in
in P. aeruginosa described
P. aeruginosa described further
further in
in the
the text.
text.

PcrV:PcrG.
Type II (T2SS) PcrV and PcrG
Secretion are heterodimers
System. that aremajor
The T2SS secretes an integral part noted
toxins (e.g., of binding
abovethein
section complex
needle A, including LasA and
(NC)/T3SS LasB, type
apparatus IV protease,
(T3SA) of the T3SSandto exotoxin A) into
host cells. Theytheareextracellular
commonly
space during
found acutesecreted
in the main infections. Molecules
protein as partsecreted through of
of the secretome T2SS
thesecretions
P. aeruginosacause
PA01 a signifi-
strain
cant are
and number of the disease
anticipated pathologies
to be available associated with
to circulating P. aeruginosa
and secreted (IgAinfections
and IgM)[76]. XcpQ
antibody
and HxcQ are outer membrane proteins of the two major T2SSs
therapeutics. While PcrV’s role is not completely characterized, it (PcrV) seems to form a (see Figure 4), making
them accessible
scaffold at the tiptoofcirculating (e.g., IgGneedle
the T3SS injection and IgA) and secreted
[76,87,88]. PcrV and (IgA andare
PcrG IgM) therapeutic
found in 100%
antibodies.
of Unfortunately,
P. aeruginosa strains [89],as noted
while with
PcrV is the T1SS membrane
specifically found inproteins, these membrane
most P. aeruginosa strains
proteins have
associated withonly
poor limited characterization
clinical outcomes [90].of Intheir immunogenicity
addition, [80]. and structure
the PcrV sequence
Type III (T3SS)
are associated Secretionresistance
with antibiotic System. [90].The T3SS is the other
Vaccination major secretion
to produce mechanism
PcrV antibodies has
for toxins. More accurately, the T3SS injects molecules, commonly
been shown to be protective in mice infected with a lethal dose of virulent P. aeruginosa.DNA and toxins, into the
cytoplasm of the host cells [81]. These T3SS toxins play a role in
Passive immunization with anti-PcrV IgG has also been demonstrated to be protective in some localized infections,
including
animal the eye
models and lung
[91–99]. PcrV[82–84].
antibodies While the toxins to
are accessible arethenot secreted
humoral extracellularly
immune system, as in
many types of P. aeruginosa infections, antibodies
shown in clinical trials [92]. PcrG requires further study. to these toxins might be considered
for some
PcsF.indications.
PcsF is the needleThe hallmark
part of theof T3SS
needle is complex
the needle complex
(see Figure (NC)
4). Theormolecule
injectisome re-
(described below and in Figure 4). The T3SS is most often identified
mains poorly characterized, although the PcsF is highly conserved even between P. aeru- with Yersinia [76,81].
However,
ginosa andthe P. aeruginosa
Yersinia T3SSPcsF
[100]. Since injects numerous
is located exotoxins including
extracellularly, it should ExoU,S,T,Y,
represent MMP-12,
an avail-
and MMP-13 [85]. The injected
able target that should be pursued [100]. toxins from T3SS alter the host’s cell cycle, commonly
PoP B&D. PoPB and PoPD are part of the T3SS that forms a heterodimer on lipid
membranes, allowing for penetration of target cell membranes by assembling functional
translocons [101]. Their membrane location also makes them accessible to circulating and
secreted (IgA and IgM) antibodies [101]. PoP is active during an inflammatory phase of
Antibiotics 2021, 10, 1530 11 of 31

inducing apoptosis. The T3SS consists of a multimeric protein complex that is divided
into four major domains. Epitopes in each of the extracellular domains could serve as
drug targets to reduce bacterial associated morbidity. Other attempts to actively target the
T3SS with antibody therapies have yielded mixed results [86], with enough promise that
antibody therapy against T3SS, which in conjunction with T2SS is thought to significantly
contribute to bacterial virulence [76], should be pursued.
PcrV:PcrG. PcrV and PcrG are heterodimers that are an integral part of binding the
needle complex (NC)/T3SS apparatus (T3SA) of the T3SS to host cells. They are commonly
found in the main secreted protein as part of the secretome of the P. aeruginosa PA01 strain
and are anticipated to be available to circulating and secreted (IgA and IgM) antibody
therapeutics. While PcrV’s role is not completely characterized, it (PcrV) seems to form a
scaffold at the tip of the T3SS injection needle [76,87,88]. PcrV and PcrG are found in 100%
of P. aeruginosa strains [89], while PcrV is specifically found in most P. aeruginosa strains
associated with poor clinical outcomes [90]. In addition, the PcrV sequence and structure
are associated with antibiotic resistance [90]. Vaccination to produce PcrV antibodies has
been shown to be protective in mice infected with a lethal dose of virulent P. aeruginosa.
Passive immunization with anti-PcrV IgG has also been demonstrated to be protective in
animal models [91–99]. PcrV antibodies are accessible to the humoral immune system, as
shown in clinical trials [92]. PcrG requires further study.
PcsF. PcsF is the needle part of the needle complex (see Figure 4). The molecule remains
poorly characterized, although the PcsF is highly conserved even between P. aeruginosa
and Yersinia [100]. Since PcsF is located extracellularly, it should represent an available
target that should be pursued [100].
PoP B&D. PoPB and PoPD are part of the T3SS that forms a heterodimer on lipid
membranes, allowing for penetration of target cell membranes by assembling functional
translocons [101]. Their membrane location also makes them accessible to circulating and
secreted (IgA and IgM) antibodies [101]. PoP is active during an inflammatory phase of
infection. PopB and PopD are found in most P. aeruginosa strains associated with poor
clinical outcomes [102].
Type V (T5SS) secretion system. T5SS predominantly secretes virulence factors
and enzymes that support biofilm formation [103]. While the Type V secretion system
(T5SS) involves a two-step process: synthesis of a precursor molecule and a periplasmic
intermediate of the effector, it is considered the simplest secretion system [104].
Since they are autotransporters, the epitope of the antibody would need to target the
cleaved domain(s) of the transport protein. While EstA and LepA are potential targets
associated with T5SS, PlpD is currently more characterized and thus has more potential as
a drug target.
PlpD. PlpD is a phospholipase A1 enzyme in the patatin-like family that degrades
lipids, especially in membranes [104]. PlpD is composed of multiple domains including
a secreted domain that is fused to a transporter domain. Due to its high virulence and
availability, it makes a solid therapeutic target.
Type VI (T6SS) Secretion System. T6SSs are effector translocation systems that
resemble inverted bacteriophage-puncturing devices. Effectors of P. aeruginosa T6SS in-
clude Tse1-3, PldA, and PldB, well-known virulence factors [105]. Phospholipase D (PLD)
enzymes, including PldA and PldB, are enzymes that catalyze the hydrolysis of phosphodi-
ester bonds. In addition, T6SS machinery improves survival of P. aeruginosa by allowing
better delivery of toxins to neighboring organisms; suppressing nonpathogenic normal
flora may also help P. aeruginosa and translocate effector proteins directly into target host
cells [106]. Hence, T6SS, which also plays a role in biofilm formation, can be considered
a virulence factor of P. aeruginosa. T6SS consists of several protein components, namely
hexameric rings of hemolysin-coregulated protein (Hcp), Val-Gly repeat (Vgr) proteins,
and a PAAR protein. Hcp hexameric protein rings stack in vitro to form nanotubes that
resemble bacteriophage tail tubes while Vgr proteins are similar to the tail-spike puncturing
device of a phage. PAAR proteins are thought to aid in facilitating the puncture of target
Antibiotics 2021, 10, 1530 12 of 31

membranes [107]. Importantly, PAAR proteins as well as Hcp and Vgr complexes may
constitute a portion of the surface-exposed T6SS machine [106,107], making them potential
drug targets to reduce morbidity secondary to P. aeruginosa infection.

3.3. Quorum Sensing/Metabolites

In a process called quorum sensing, P. aeruginosa secretes small molecules to commu-
nicate local population density. As a result of these small quorum sensing (QS) molecules
bacteria will modulate gene expression to change their mode of growth (i.e., planktonic vs.
sessile), increase their virulence, improve their resilience in the face of antibiotics and other
therapeutics, and act in a coordinated way to “benefit the group” [108]. Since QS plays a
central role in virulence, the receptors and small molecules that coordinate these efforts
have earned a spot on the shortlist for drug targets. This section offers insight into potential
anti-virulence strategies using antibodies to small QS molecules and enzymes that produce
them. Antibody development to small molecules is technically difficult; however, these
molecules represent potentially highly efficacious therapeutic targets.
Pseudomonas Quinolone Signal (PQS; 2-heptyl-3-hydroxy-4-quinolone). PQS is a
ubiquitously expressed, small, intercellular signaling molecule that is actively expressed
during infection. Its intercellular nature means that it would be accessible to circulating and
secreted (IgA and IgM) antibody therapies. PQS regulates numerous virulence-related fac-
tors and functions [109] in P. aeruginosa and may participate in the biogenesis of OMVs [110].
The impact of PQS in surrounding tissues includes autolysis of damaged cells and de-
creased cellular metabolic activity. There is also evidence that PQS may act as a protective
stress response signal, iron scavenger, and host immune modulator [110]. Finally, and
perhaps most critically, PQS can both induce oxidative stress as well as provide an antiox-
idative response. It is these PQS functions that may prove most protective for P. aeruginosa
against damaging oxidative bursts from polymorphonuclear cells. These mechanisms kill
host tissue and provide selective pressure on bacteria in both chronic biofilm infections
and in acute infections [109]. The critical protective nature of PQS means that although
developing an antibody to PQS may be possible, it may lead to selective pressure and push
Pseudomonas to evolve [109]. However, delivered properly, any therapeutic strategy that
includes PQS could have a large impact on antibiotic-resistant P. aeruginosa.
Pyocyanin. Pyocyanin, or phenazine, which gives cultures their characteristic blue-
green coloration, is a secreted virulence factor found at the site of infection in the host.
Pyocyanin is a small-molecule toxin that is easily able to cross cell membranes usually
through a T2SS quorum-sensing dependent mechanism. Pyocyanin is a redox-active
tricyclic zwitterion shown to affect the respiratory, cardiovascular, urological, and central
nervous systems by inducing oxidative stress within cells [111,112]. Pyocyanin directly
oxidizes and depletes reduced glutathione, effectively neutralizing one of the major redox
pathways. Additionally, pyocyanin induces IL-8 and LTB4, which attracts neutrophils, and
although it seems counterintuitive, the compound then inhibits IL-2 and IL-2R expression,
causing neutrophil and lymphocyte apoptosis. Neutrophil apoptosis releases proteases,
leading to host tissue destruction and inflammation. Due to this cascade, pyocyanin
released by P. aeruginosa is especially toxic to cystic fibrosis patients, potentially due to
its ability to induce neutrophil extracellular traps (NETs), which are able to exacerbate
airway inflammation for their own benefit [113]. Thus, pyocyanin is a powerful mediator
of cell injury used by P. aeruginosa and is a prime target to halt disease [112]. In fact, in vitro
studies have demonstrated that anti-pyocyanin antibodies can provide protection [114].
However, just as with PQS, although antibody development to small molecules is often
feasible, it is difficult. Since pyocyanin is secreted, it is available for binding by both
circulating and secreted (IgA and IgM) antibodies. As with endotoxin A, pyocyanin is
ubiquitous, available, and pathologic, and should be explored as a target.
N-(3-oxododecanoyl)-l-HSL(3-oxo-C12-HSL/OdDHL). 3-oxo-C12-HSL, a specific type
of acyl homoserine lactone (AHL), is an auto-inducing, quorum-sensing associated viru-
lence factor that regulates swarming, toxin and protease production, and proper biofilm
Antibiotics 2021, 10, 1530 13 of 31

formation [115,116]. OdDHL inhibits naive T-Cell proliferation as well as subtype dif-
ferentiation. It acts as a quorum sensor signal molecule and inhibits IL-12 and TNF [117].
It also decreases antibody production at high concentrations and promotes IgE/IL-4 at
low concentrations. OdDHL also regulates Las gene expression, most notably LasR, and
upregulates IL-8 in corneal infections [115,116]. Additionally, it has been shown to inhibit
dendritic cell concentrations in a dose-dependent fashion. 3-oxo-C12-HSL inhibits PPAR
gamma functioning in host cells, leading to an active, expanding proinflammatory state
and bacterial swarming. Low-dose antibiotics can suppress the quorum sensing functions
of 3-oxo-C12-HSL; however, 3-oxo-C12-HSL can auto-induce Las systems, producing cy-
totoxic effects targeting macrophages and neutrophils via pro-apoptotic pathways. This
molecule also selectively regulates Nf-kB signaling, leading to decreased host TLR4 path-
way utilization to fight P. aeruginosa infections [118–121].
Evidence suggests that mice immunized with a carrier-conjugated 3-oxo-C12-HSL were
able to generate a protective humoral response. Thus, as with other QS molecules, antibody
generation, while challenging, is possible. Since 3-oxo-C12-HSL is secreted, it is available for
targeting and binding by circulating and secreted (IgA and IgM) antibodies [122].
Hydrogen cyanide (HCN). HCN production is an important mediator involved in
quorum sensing (QS). HCN-producing bacteria help to maintain cooperativity and eliminate
“cheating bacteria” [108]. Bacteria that participate in QS have increased resistance to HCN
intoxication and ROS damage than the mutant cheaters [123]. These cheaters threaten
cooperativity and thus stability of the P. aeruginosa bacterial population. HCN has been
detected in sputum cultures of P. aeruginosa-infected CF patients and P.aeruginosa-infected
burn cultures. Researchers postulate that HCN decreases pulmonary function, especially
in CF patients, by interfering with essential enzymes, such as superoxide dismutase, NO
synthase, cytochrome C oxidase, and others, disrupting aerobic respiration and cellular
immune functions [124]. In burn patients, HCN inhibits cytochrome c oxidase to decrease
metabolism at the site of infection [125]. Targeting HCN production may disrupt the
delicate nature of P. aeruginosa biofilms, helping to treat resistant infections [108,123].
To decrease HCN signaling, two targets are possible: (1) HCN, which is difficult
given the tiny size of the HCN molecule or (2) HCN synthase, a membrane-bound enzyme
that synthesizes HCN from glycine [125]. Targeting HCN synthase, which is a ubiquitous,
membrane-bound molecule, available to circulating and secreted (IgA and IgM) therapeutic
antibody delivery, may prove to be a viable strategy for combating QS.
Pyomelanin. Pyomelanin is a small molecule in the same family as melanin that is
secreted as a part of the P. aeruginosa QS system. Pyomelanin overproduction is a common
phenotype among patients with cystic fibrosis and urinary tract infections, giving colonies
a brown phenotype and heightened resistance to phagocytosis. Production of pyomelanin
is associated with an increased resistance to peroxide [126].
Since pyomelanin is a small molecule that is secreted, it is antibody accessible. How-
ever, synthesis of antibodies to pyomelanin may not be a viable strategy due to the difficulty
in production; nevertheless, if an antibody system could be developed against pyomelanin,
it may be effective based on the accessibility of the antigen [127].
Other candidate molecules. As more information is gathered, several other quorum-
sensing molecules may emerge as targets [128], including the Acyl Homoserine Lactones
(AHLs): OdDHL, ConA, and BHL which are produced by the majority of P. aeruginosa
strains. These molecules increase the organism’s virulence and have a wide array of
immunomodulatory effects. Generally, AHLs are modulated and expressed secondary to a
variety of quorum sensing signals. The variety of AHLs work in conjunction to divert the
immune response away from the P. aeruginosa organism [123,129]. AHLs suppress T-cell
growth and proliferation, particularly in CD4+ T-cells. They also promote Th2 over Th1
host immune responses. These molecules could serve as antibody-based drug targets.
Antibiotics 2021, 10, 1530 14 of 31

3.4. Antibiotic Resistance Determinants

Antibiotics remain the best tool to fight P. aeruginosa infections. Unfortunately, as
noted above, antibiotic resistance reduces antibiotic effectiveness. Targeting antibiotic
resistance mechanisms with antibody-based therapeutics may resurrect some antibiotics,
create synergistic new combination therapies, and expand the toolbox available to clinicians
in the arms race against opportunistic pathogens.
SecYEG and SecA. Antibiotic resistance may be in part due to the Sec proteins’ func-
tions as efflux pumps [76]. Hence, targeting the Sec proteins may be an effective coun-
termeasure in the fight against antibiotic resistance. Upregulated efflux pumps, which
can effectively eliminate intracellular acting antibiotics or other cytotoxic compounds
by pumping them out of the cell and into the periplasmic space, are a main driver of
antibiotic resistance [130].
The Sec proteins are all outer membrane accessible and thus potentially available
as drug targets. In addition, these proteins are conserved in most antibiotic-resistant
P. aeruginosa strains [76]. Sec are potentially accessible to circulating and secreted (IgA
and IgM) therapeutic antibodies. Since Sec proteins are ubiquitous and pathologic, any
therapeutic strategy could include these targets [76]. As noted with the T1SS membrane
proteins, these membrane proteins have only limited immunogenicity characterization.
OprM. OprM is a protein on the tip of an efflux pump that specifically confers
P. aeruginosa antibiotic resistance. The MexAB-OprM efflux system is in the resistance-
nodulation cell division (RND) family of exporters [131,132], which includes 12 total RND
pumps identified in the P. aeruginosa PAO1 genome [133]. MexA and MexB proteins
complex with the outer membrane porin-like OprM to form a one-step efflux pump respon-
sible for resistance to a large number of antibiotic classes, such as β-lactams, β-lactamase
inhibitors, fluoroquinolones, tetracyclines, novobiocin, thiolactomycin, sulfonamides,
macrolides, aminoglycosides, etc. [134].
As a ubiquitous outer membrane protein, OprM protein is accessible and thus poten-
tially available as a drug target [135], although the exposed part is a relatively small part
of the overall protein and prone to escape mutations. Nevertheless, OprM proteins are
conserved in most P. aeruginosa strains that are antibiotic resistant. As noted with other
membrane proteins, OprM has only limited immunogenic characteristics.

3.5. Other Membrane Biomolecules

Beyond conferring antibiotic resistance, P. aeruginosa has many other cell surface
molecules with a variety of functions. These functions include regulation of swarming
motility, host cell modification, biofilm composition, and adhesion, all of which are promis-
ing potential targets for antibody therapies. Unfortunately, generally speaking, these
biomolecules are difficult to target as they have evolved to evade immune systems and are
commonly poorly immunogenic; however, engineering antibodies to these targets could
prove a fruitful endeavor if successful. The following section explores some of the most
promising candidates.
LecA and LecB. The ability of P. aeruginosa to adhere to host cells is essential to
pathogenesis. Adhesion is mediated by pseudomonal lectin receptors to glycoproteins on
the surface of the host cell. LecA and LecB are located in the cytoplasm of P. aeruginosa
and bind to galactose and fructose. LecA has been shown to have a cytotoxic effect on
respiratory epithelial cells by decreasing their growth rate. It has also been shown to alter
the permeability of intestinal epithelium to allow for increased absorption of other secreted
toxins such as exotoxin A. LecB has been shown to play a role in pilus biogenesis and
protease IV activity increasing virulence [136].
In mouse studies, administration of carbohydrate inhibitors such as methyl-d-galactoside
(for LecA) led to the highest rates of survival, while administration of α-methyl-l-fucoside (for
LecB) did not seem to impact survival. The lectin inhibiting carbohydrates also reduced the
rates of lung injury and showed an increase in bacterial clearance from the lungs in the study
Antibiotics 2021, 10, 1530 15 of 31

population [136]. These results highlight the potential of LecA as a drug target; however, LecB
requires further study to assess its potential value as a drug target.
Phosphatidylcholine (PC) Metabolism and Transporting Enzymes. Phosphatidyl-
choline (PC), a highly prevalent lipid found in lung surfactant, has been identified as
a nutrient source for P. aeruginosa [54]. To serve as a nutrient source, PC is cleaved by
P. aeruginosa PLC into fatty acids, glycerol-3-phosphate and glycerol. Numerous enzymes
then transport these metabolic products into the bacterial cell. Glycerol-3-phosphate trans-
porter (GlpT) and glycerol uptake facilitator protein (GlpF) are transmembrane proteins
involved in the uptake of glycerol-3-phosphate and glycerol, respectively [137]. Block-
ing the metabolism of PC (as previously discussed for PLC) and/or transport of these
degraded components of PC should result in decreased replicative potential and biofilm
development of P. aeruginosa. Therefore, these metabolites and their transporters pose
great targets for pAbs [54]. Notably, there are other key transporters that may also provide
effective targets. Two choline symporters, BetT1 and BetT2, as well as an ABC transporter,
CbcXWV, are involved in choline transport into the bacterium [138], Fatty acid transporters,
such as long-chain fatty acid translocase (FadL), are utilized for free fatty acid uptake
by P. aeruginosa [54,137]. Blocking any of these transporters may have a similar effect as
blocking GlpT and GlpF.
Multivalent Adhesion Molecule 7. MAM7 is a protein that binds to the host cells
during the early stages of infection. MAM7 binds with fibronectin and phosphatidic acid
of the host’s cell membrane. Hence, MAM7 expression in P. aeruginosa is necessary for
virulence. In a recent study of burn patients, topical application of polymeric microbeads
functionalized (covalently attached) with the adhesin MAM7 led to positive clinical out-
comes, including reduction in P. aeruginosa bacterial loads by decreasing the available
receptors for the bacteria to attach in the wound and thus preventing bacterial spread [139].
The MAM7 protein is outer membrane accessible and conserved in most P. aeruginosa
strains [139]; therefore, it may prove an effective therapeutic target.
OprE and OprF. OprE and OprF are major outer membrane proteins in P. aeruginosa
that are associated with virulence, including cellular adhesion, virulence protein secretions
via T3SS (e.g., ExoT and ExoS), and secretion of the quorum sensing-dependent virulence
factors pyocyanin, elastase, lectin PA-1L, and exotoxin A. OprE and OmpA, are important in
biofilm development. A mutation in the ompA gene resulted in a thinner exopolysaccharide
layer of the biofilm [140]. Other surface membrane proteins are also involved in biofilm
development. OprE and, additionally, OmpH, interact with OmpA to aid in biofilm
development. OprF also binds to IFNγ, thereby increasing virulence [140]. OprE and
OprF are also found in secreted outer membrane vesicles (OMVs) [141] and may be good
therapeutic targets based on their accessibility [142]. However, as noted with the secretion
system membrane proteins, the availability of these membrane proteins has only limited
immunogenic characteristics, which may limit the development of therapeutic antibody-
based treatments using traditional antibody technology. Nevertheless, the variety of
proteins involved in biofilm development allows for the development of a multifaceted
approach to P.aeruginosa antibiotic resistance These different biofilm components contribute
to it being a suitable drug target for future pharmaceutical studies [143]. Further study
is warranted.
Lipopolysaccharide Lipopolysaccharide (LPS) is a major component of the outer
membrane of Gram-negative bacteria. For P. aeruginosa, LPS is a key virulence factor and
stimulates an inflammatory response in the host. It contains three primary structural
domains: lipid-A, variable O-antigen, and the core oligosaccharide, which can further be
divided into the inner and outer core. The core oligosaccharides link the lipid-A domain,
which is embedded into the outer membrane with the O-antigen, which in turn extends
from the cell to interact with the host environment. Lipid-A can have a variable structure
with differing levels of virulence and binds to the TLR-4 receptor, which is essential for LPS
recognition via lipid-A, to stimulate the host’s innate immune system. The inflammatory
response of the host increases the synthesis of LPS binding protein (LBP). The response
Antibiotics 2021, 10, 1530 16 of 31

that follows the binding of TLR-4 is dependent on the level of acetylation of lipid-A, which
can be penta- or hexa-acetylated. In general, the hexa-acetylated form of lipid-A results
in a much more robust inflammatory response.TLR-4 can also mediate host resistance
to infection [144,145].
There are two types of LPS, smooth and rough. The presence of the O-chain determines
whether the strain is considered rough. Rough LPS strains that are unable to produce the
O-antigen are present in the lungs of CF patients. The smooth LPS isolates are responsible
for systemic P. aeruginosa infections [144]. There have been attempts to develop a vaccine
against LPS for P. aeruginosa. A conjugate vaccine was trialed on CF patients and showed
initial promise. However, after moving on to phase III trials, the vaccine did not produce a
strong enough immunological response [146]. Newer vaccines being trialed are attempting
to elicit a stronger response. With incidence of P. aeruginosa antibiotic resistance becoming
more prevalent, an effective vaccine would be a game changer in the fight against the
bacteria [144,145]. Again, generating an antibody (or antibodies) to LPS is a difficult task
as LPS is both a non-protein target and has variable regions; however, the payoff for such a
development would be substantial as LPS is found in all P. aeruginosa strains and potentially
virtually all Gram-negative bacteria.
Rhamnolipids (A, B, C, G, R). In addition to LPS, which is a hallmark of Gram-
negative bacteria, P. aeruginosa also produces glycolipids called rhamnolipids with a rham-
nose head group (glycosyl) and fatty acid tails. These membrane-bound glycolipids
provide surfactant properties. The production of rhamnolipids is variable based on the
quorum sensing that regulates Rhl gene expression. Rhamnolipids are found on the cell
surface, in biofilms, and excreted into the environment. In fact, rhamnolipids are also found
in the sputa of people with cystic fibrosis with P. aeruginosa infection. The molecules have a
role in causing respiratory cell lysis and ciliary dysfunction in these patients by removing
ATP-ase dynein arms from cilia [147]. While the function of rhamnolipids is speculative, it
includes the uptake of hydrophobic substrates, improved motility, and mediating biofilm
growth. Rhamnolipids are particularly important in modulating the swarming motility
of colonies, which may serve to perpetuate bacterial survival and virulence. Reducing
P. aeruginosa cell tension and improving the organism’s adherence to host cells may be
inextricably connected to the rhamnolipid’s ability to modulate the bacteria’s swarming
behavior. As a multi-purpose molecule, targeting rhamnolipids may carry biological and
therapeutic benefits, but may also incur a host of side effects [148]. Nevertheless, in a drug
targeting context, the most relevant functional aspect of rhamnolipid may be its ability to
disrupt epithelial cell tight junctions, as evidenced by the presence of rhamnolipids in both
lung and corneal infections [149,150].
As noted above with LPS, the development of antibodies to rhamnolipids, as is the
case with many non-protein antibodies, is challenging for numerous reasons; however,
antibodies have been previously developed [151]. Rhamnose-binding lectins may also be
an option. Since rhamnolipids are membrane-bound, they have at least some epitopes
available for antibody or lectin therapy delivery. Since rhamnolipids are ubiquitous,
available, and potentially pathologic, any therapeutic strategy should include this target.
Psl EPS/Mucoid Exopolysaccharide (MEP). Psl exopolysaccharide (EPS/MEP) is a
major component of the biofilm produced by P. aeruginosa. It is produced by several
enzymes with increasing expression in sugar-rich environments. Psl EPS is composed
mainly of galactose and mannose and contributes to the matrix of the biofilm by initiating
and strengthening biofilm adhesions. The biofilm decreases antibiotic penetration into
P. aeruginosa allowing survival and propagation of antimicrobial-resistant strains [152]. As
with rhamnolipids and LPS, EPS is a valuable target, but it is difficult to generate antibodies
for numerous reasons. Nevertheless, antibodies have been previously developed. CF
patients often generate anti-EPS antibodies; however, these antibodies are non-opsonizing
and non-neutralizing [153]. Using a FAb (antigen-binding fragment of an antibody) and
recombinant opsonizing Fc (tail region of an antibody that interacts with cell surface
receptors) to engineer synthetic antibodies could improve efficacy in order to clear the
Antibiotics 2021, 10, 1530 17 of 31

infection [154]. Alternatively, as with other carbohydrate targets, lectins may also be an
option. Since EPSs are an integral membrane component, they are available to circulating
and secreted (IgA and IgM) antibody or lectin therapeutic delivery. Since EPS is ubiquitous,
available, and potentially pathologic, any therapeutic strategy could include this target.
Alginate. Alginate is another exopolysaccharide produced by P. aeruginosa. It spans
the organism’s inner and outer membrane. Alginate enhances cell to cell adhesions and
cell to host adhesions. These adhesions are protective for the organism; they increase
resistance to the host immune system [155]. P. aeruginosa evades the host immune system
during chronic disease by decreasing its virulence so as to not provoke an acute immune
reaction. Alginate is produced in large quantities during chronic infections. Inhibition of
macrophage clearance and efferocytosis have been observed, but the mechanism of alginate
in this process is still unknown [156]. Again, as with other lipids/carbohydrates discussed
in this section, alginate is a difficult, but potentially valuable, target.
Secreted outer membrane vesicles (OMVs). OMVs are ubiquitously generated and
secreted by P. aeruginosa during all stages of the bacteria’s life cycle [141,157], particularly
under conditions of stress. Growing experimental evidence suggests that OMVs are se-
creted with numerous virulence factors, antibiotic resistance molecules, and quorum sensing
molecules, including hemolysin, phospholipase C, elastase, and alkaline phosphatase,
antimicrobial quinolines, adhesions, CIF, and beta-lactamase into the host cells [158–161].
Additionally, these vesicles carry virulence factors that can cause cytotoxicity, increase
adherence, and carry factors such as cystic fibrosis transmembrane conductance regulator
inhibitory factor [141]. OMVs can then fuse with host cell membranes and alter their func-
tion, resulting in the breakdown of the host’s epithelial barrier cells, which can ultimately
facilitate the invasion of P. aeruginosa. Antibody therapies for these targets could include
several approaches. First, antibodies to CIF could be delivered inside the vesicle that fuses
to the secreted outer membrane vesicles. Second, antibodies to the OMV itself could be
developed with an opsonization effector function to eliminate the OMV entirely. More
study into OMVs as either a drug target or natural drug delivery system is warranted.
CbpD. CbpD is one of the conserved outer membrane vesicle proteins found in
the majority of studied strains of P. aeruginosa. Due to its conservative expression and
extracellular presence, CbpD may be a possible target of immunotherapy against the outer
membrane vesicles and the virulence factors, which they carry [162].

3.6. Motility Factors

Bacterial motility plays a key role in P. aeruginosa virulence and biofilm formation.
P. aeruginosa uses type IV pili and flagella for motility. Several studies have suggested that
targeting pili or flagella with drugs or vaccines would show strong efficacy [163].
Pili proteins. Type IV pili (TFP). TFP is a motorized fimbriae providing twitching
motility for P. aeruginosa [164,165]. TFP is essential to both biofilm formation and virulence.
Several adhesin proteins (the minor pilins) are at the tip of the pili including PilEXWV and
FimU (see Figure 3). The pilus is extended through major pilins and includes PilA [165].
Pili may also enable P. aeruginosa to perform transformation and conjugation, and exchange
virulence and antibiotic resistance genetic material with other bacteria and may at least
partially explain the organism’s ability to rapidly acquire antibiotic resistance.
The TFP proteins are all outer membrane accessible and are conserved in most
P. aeruginosa strains [166]. TFP proteins are potentially accessible to circulating and se-
creted (IgA and IgM) antibody therapy delivery. Since TFP proteins are ubiquitous and
pathologic, any therapeutic strategy could include these targets.
Flagellar Proteins. P. aeruginosa acquires its motility from a single glycosylated polar
flagellum. In addition to motility, the bacteria’s flagellum can play a role in adhesion to
host cells and stimulation of the immune system. The flagellum is an emerging drug target
due to the many roles it plays in the pathogenesis of P. aeruginosa [167]. There are two
common types of flagella proteins, PAK and PA01. Antibodies against PAK flagella and
PA01 flagella were used to assess their potential as a future drug target [168]. There have
Antibiotics 2021, 10, 1530 18 of 31

been varying results on the effectiveness of a vaccine targeting the P. aeruginosa flagellum.
Rabbits were passively immunized with antibodies against the monomeric flagella protein
and survival studies showed the antibodies provided little protection. However, antibodies
targeting polymeric flagella showed promising results with an 87% survival rate following
the P. aeruginosa challenge. Although anti-flagella antibodies against the PAK and PA01
strains showed only modest protection, flagella proteins appear to be a promising emerging
drug target [168].

3.7. Resource Scavenging Molecules

One of the key battlefronts between host and bacteria is molecular resource scavenging.
Iron is a prime example as it is an essential nutrient for the biological processes of P.
aeruginosa. Like host cells and other bacteria, P. aeruginosa requires iron as a cofactor for
many enzymes. It uses specialized iron scavenging molecules, siderophores, to steal iron
from its environment. Antibodies to these molecules commonly provide protection against
virulence and thus these molecules are excellent drug targets [169].
Pyoverdine. Pyoverdine is the major siderophore produced by P. aeruginosa that
extracts iron from host complexes [170]. Pyoverdine has such a high affinity for iron that it
can steal iron from transferrin and extract iron from host cells, leaving them metabolically
limited and damaging mitochondria [171]. Pyoverdine is an extracellular peptide found in
most P. aeruginosa infections, especially CF, and in most strains of P. aeruginosa [171]. As a
conserved, extracellular protein [172], its accessibility to antibody development makes it
an attractive candidate for therapeutic development.
Pyochelin (Pch). Pch is the second major siderophore secreted by P. aeruginosa to
scavenge iron. Pch chelates iron and other metals from the ECM (extracellular matrix). Pch
also provides a competitive advantage by inhibiting other bacteria, including S. aureus [173].
Pch is found extracellularly in most of the indications for P. aeruginosa infections and in most
strains of P. aeruginosa [174,175]. As with pyoverdine, Pch is an extracellular protein and is
conserved in most P. aeruginosa strains [175], making it a target worthy of consideration in
the development of new therapeutic antibody strategies. Notably, Pch is a small molecule
synthesized from cysteine and thus will require additional engineering to generate an
antibody to the molecule and so it may not be the best choice for the first generation of
antibodies developed.
HasAP. Aerobic metabolism of P. aeruginosa requires iron for many of the respiratory
enzymatic processes. HasAp is a secreted hemophore that, after being cleaved by a
Pseudomonas protease [176], captures iron as part of the heme acquisition system [176].
Its release is regulated via quorum sensing (QS) in response to cell growth and biofilm
establishment [177]. HasAp is not constitutively expressed, but of the QS-regulated proteins
studied, iron regulation proteins are the most abundant [178]. Targeting protease activity,
or the hemophore activity of HasAp, could help eliminate chronic infection by starving
P. aeruginosa of essential iron needed for aerobic metabolism, and thus may be a worthy
therapeutic antibody target.

3.8. Immunomodulators
Immunomodulators use regulatory molecules to adjust the immune response, includ-
ing inducing, attenuating, and amplifying the host immune response. P. aeruginosa uses
several mechanisms to modulate the immune system to reduce effective host immune
response, but P. aeruginosa also upregulates the immune response to fight competitors
such as S. aureus. For example, P. aeruginosa increases IL-8 production and in response,
S. aureus dampens Toll-like receptor (TLR1/TLR2)-mediated activation of the NF-κB path-
way [179]. As noted earlier, P. aeruginosa uses LasA to inhibit competing S. aureus. Here, a
few immunomodulators are highlighted as targets during a P. aeruginosa infection.
Lipoxygenase (LoxA). Lipoxygenases (LOXs) are extracellular, lipid-oxidizing enzymes
that have potent immunoregulatory properties in their hosts [180,181]. In P. aeruginosa, the
lipoxygenase LoxA oxidizes polyunsaturated fats, resulting in the expression of bioactive
Antibiotics 2021, 10, 1530 19 of 31

lipid mediators and suppression of chemotactic molecules [182]. With greater than 90%
conservation across P. aeruginosa strains, leukotrienes, a product of LoxA, are secreted
to balance lipoxins and resolvins. LoxA also activates host ferroptosis, resulting in host
cell death and inflammation. LoxA increases LXA4 along with other LOX expressions,
leading to an increase in neutrophil chemotaxis and pro-inflammatory signaling [183].
Evidence from preclinical animal studies shows that P. aeruginosa clinical isolates secrete
LoxA, indicating modulation of the host immune response during acute pneumonia [182].
However, LoxA decreases in expression after 24 h of lung infection and has decreased
expression in chronic infections. This indicates that LoxA is a significant pro-inflammatory
and bacterial invasion mediator in acute infections, while losing this effect when lung
infections become chronic. Thus, targeting LoxA during acute infection could help increase
the host immune response. Additionally, upregulation of CCR5, a receptor on white blood
cells for immune system chemokines, occurs after exposure of lung tissues to Lox through
unknown mechanisms. CCR5 acts as a chemokine receptor as well as a chemotactic agent,
resulting in increased bacterial invasion [182]. Since LoxA is accessible to both circulating
and secreted (IgA and IgM) antibodies, it may prove to be a good target for therapeutic
antibody development and delivery therapy.
Leukocidin. Leukocidin is a cytotoxin of P. aeruginosa that damages granulocytes
and lymphocytes by forming a beta-barrel pore in cell membranes, thereby inhibiting
essential bactericidal and phagocytic immune functions [184,185]. Leukocidin has long
been known as a common, secreted virulence factor in P. aeruginosa. Targeting Leukocidin,
which is accessible, ubiquitous, and pathogenic, could have direct influences on the course
of P. aeruginosa infection and could help increase the host immune response.

4. Antibodies as Therapeutics
As antibiotic resistance continues to increase throughout a wide spectrum of microbial
infections, new and innovative approaches to treating these infections must be sought.
The development of antibodies to the relevant bacterial targets outlined above and in
Table 1 may be the next step in combating antimicrobial resistance. Therapeutic antibodies
work by activating and modulating our own host effector mechanisms including direct
neutralization of toxins and pathogens, activation of the complement pathway, activation of
neutrophil and macrophage opsonophagocytosis, activation of natural killer cells, enhance-
ment of antigen presentation from dendritic cells to T cells and follicular dendritic cells to B
cells, and degranulation of mast cells, eosinophils, and basophils. Additionally, Fc receptors
that correspond to different classes of antibodies can activate the complement pathway,
induce innate immune responses, and enhance natural adaptive immunity, providing a
multi-faceted strategy to address disease [186].
Therapeutic monoclonal antibodies (mAbs) have emerged as a tour de force in the
drug market and are projected to capture a rising percentage of the worldwide drug
market—a trend that reflects increased prescription use of existing therapeutic mAbs and
a large number (~50) of new therapeutic mAb drugs approved for use [187]. Currently,
mAbs have been developed to treat a broad array of clinical conditions including cancer,
autoimmune diseases, transplants, infectious disease, and toxin neutralization [188].
In contrast to mAbs, polyclonal antibodies (pAbs) have multiple epitope binding sites,
thereby allowing greater coverage of neutralizing antigens. Much like our native immune
system, pAbs are produced as a collection of antibodies from multiple B cell lineages,
thus producing many different sites and affinities for the same antigen. Having multiple
epitope binding sites allows for greater neutralization options and less opportunities for
pathogens to develop escape mutants [189]. By harboring the ability to work at a wide
number of sites in microbial pathogenesis, polyclonal antibodies have the potential to curb
or slow the development of multidrug-resistant organisms and fill the gaps in treatment
left by antibiotics and vaccines. Passive polyclonal antibodies have been used to treat
diseases for over 100 years with the oldest being patient- and blood-derived products
(often “convalescent sera”) or intravenous immunoglobulins (IVIG) [190]. Additionally,
Antibiotics 2021, 10, 1530 20 of 31

more modern pAbs are currently used as antitoxins and antivenoms and are an effective
treatment post-infection or post-exposure for botulism, varicella, vaccinia, rabies, CMV,
hepatitis B, and tetanus [188]. This is not a new strategy, but merely a re-energized approach
to treating bacterial pathogenesis.
In an emerging appreciation of rational design for new therapeutic options, combi-
nations of mAbs with defined correlates of protection are driving an increasing number
of investigations into combination therapies, especially with a focus on broad effector
mechanisms of actions beyond just simple binding. This strategy has been useful in recent
viral outbreaks, where multiclonal cocktails, or convalescent sera (i.e., polyclonal) from
recovered patients, have been used to treat diseases [189]. Engineering combination ther-
apies with improved effector functions, specificity, affinity, and glycosylation generates
a stronger and more diverse immune response. Moreover, combination therapies also
generate a much higher barrier for pathogens to overcome; in other words, the pathogens
cannot easily evolve to circumvent the treatment [189].

Combinatorial Therapy
Recently, it has been shown that antibodies and antibiotics can act concomitantly
or even synergistically against pathogens. Several studies have shown that exposure to
antibiotic therapy alters the expression of secreted factors, cell surface proteins, and other
cellular products that may be viable immunotargets [190]. These altered levels of expres-
sion can change and potentially enhance the innate and humoral immune responses to
pathogens—namely through increased antigen exposure, increased protein secretion, and
potentially decreased virulence of pathogens [190]. Inspired by these same principles, com-
bination or conjugate antibiotic/antibody therapeutic strategies, particularly those based
on polyclonal antibodies, could be designed to capitalize on these same altered expression
levels to enhance therapeutic efficacy and combat resistance. Below is a non-exhaustive
look into the evidence demonstrating how combination antibiotic/antibody therapy can
have a synergistic effect and, in some cases, potentiate the antimicrobial response.
Several clinical isolates of Streptococcus pneumoniae have been shown to, when in the
presence of penicillins or cephalosporins, become more reactive to opsonization by the
innate immune response; acute phase reactants, such as c-reactive protein and serum
amyloid P, more efficiently bind cellular antigens and clear infection quicker than when
not in the presence of these antibiotics [190]. By examining the synergistic activity be-
tween antimicrobial drugs and innate immune responses in vivo, it can by deduced that
combinations between antimicrobial and antibody therapy will likely work in a similar
synergistic fashion.
Similar to making Streptococcus pneumoniae more readily opsonized, penicillins,
cephalosporins, and gentamicin have all been shown to alter gene expression and tran-
scription in Staphylococcus aureus, leading to a change in the molecular structure of the
cell walls of the organisms [191]. Surprisingly, changes in the expression of virulence
factors in S. aureus in response to antibiotic therapy may actually improve exposure to
cellular products targeted by polyclonal antibody therapies. Using this observation as a
foundation, further work is underway to characterize the response of organisms to spe-
cific antimicrobial therapies, allowing polyclonal antibodies to be tailored to the expected
upregulation of virulence factors. In 2015, Lehar et al. utilized rifamycin conjugated
to monoclonal antibodies against wall-teichoic acids (cell wall peptidoglycans) to elim-
inate residual methicillin-resistant S. aureus infections [191]. These antibiotic–antibody
conjugates effectively bound S. aureus cell walls allowing rifamycin to be taken up by
phagolysosomes and exert its antimicrobial effect intracellularly, showing improved eradi-
cation rates compared to Vancomycin treatment alone [192]. Such an effect was confirmed
in a recent in vivo study.
A monoclonal antibody to PcrV (in P. aeruginosa) was used in combination with either
ciprofloxacin, tobramycin, or ceftazidime to improve survival rates in mice, noting that the
effect was more pronounced in more virulent strains of P. aeruginosa [192]. Protecting the
Antibiotics 2021, 10, 1530 21 of 31

lung against the cytotoxic effects of exotoxin in addition to amplifying the antimicrobial
effects of the antibiotics highlights the value of antibiotic–antibody conjugate therapy and
punctuates the need for further research into the human applications of these treatments.

5. Discussion
Therapeutic antibodies are highly specific with long half-lives and minimal dosing
compared to other drug treatments [193]. Currently, there are 79 US FDA-approved mono-
clonal antibodies in use and well over 500 currently being studied in clinical trials [193].
Most therapeutic antibodies target cancer, autoimmune diseases, or prevent transplant
rejection, but their use in other disease realms is gaining momentum. Currently, there are
five approved infectious disease monoclonals: Bezlotoxumab for prevention of recurrent
Clostridiodes difficile, Ibalizumab for drug-resistant HIV, Palivizumab for prevention of
respiratory syncytial virus, and Raxibacumab and Obiltoxaximab for inhalational anthrax,
and casirivamab and imdevimab for COVID-19 [194].
New therapies and treatment options must be sought for P. aeruginosa, a highly prevalent
and exceedingly antibiotic-resistant microorganism. Patients with the highest risk of infection
include those with compromised immunity, seen with chronic burns, chronic wounds, and
HIV/AIDS patients. Those with deficient bacterial clearance mechanisms, as seen in cystic
fibrosis, COPD, and ventilator-dependent patients [195], are also particularly susceptible
to P. aeruginosa. P. aeruginosa’s widely varied environmental niches, from respiratory tract
infections, skin, and soft tissue infections, and even septicemia make treatment especially
challenging. Additionally, P. aeruginosa creates seemingly impenetrable biofilms, which are
especially prevalent on implanted and indwelling devices, such as catheters, endotracheal
tubes, and prostheses. These biofilms create a plethora of challenges for the host innate and
adaptive immunity as well as antimicrobials [92]. The most daunting challenge, however,
may be the ability of P. aeruginosa to modulate host immune responses.
As noted earlier, multidrug-resistant pathogens present an emergent, serious pub-
lic health threat at all levels. While we have detailed many of the potential targets of
P. aeruginosa, many other emerging infections are multipathogenic, multidrug-resistant,
and even more deadly. With poly- and oligoclonal mixtures of therapeutic antibodies,
increasing levels of efficacy can be reached by adjusting individual antibodies within
the mixture for specific or broad pathogenic threats. The oligomixture can also have
prophylactic applications with adjustable dosing. Further, oligomixtures and antibiotics
could be used concomitantly and potentially synergistically against individual or multiple
pathogens. While vaccination is a good option, oligomixtures have significant advantages
and offer a strong alternative to treat multidrug resistance pathogens. Most notable is the
ability to neutralize identified, virulent targets without the inherent variability in response
to vaccination.
The effector function of an antibody plays a critical role in pathogen neutralization.
With an oligomixture, the role of effector function can be engineered (see Figure 5). This is
especially true when compared to a vaccine in which effector function is merely a function
of genetic and/or environmental chance. With oligomixtures, the effector function options
could be optimized based on any number of factors, including genetics. Effector functions
could be standardized for the entire mixture or diversified to allow different parts of the
immune system to enter the fight. The isotypes and thus the effector functions of the
oligomixture could be highly dependent on the environment. For example, IgA could be
the most efficacious isotype for intestinal and endothelial pathogenic indications whereas
IgG isotypes would likely be most efficacious in sepsis indications.
Antibiotics 2021, 10, x FOR PEER REVIEW 23 of 33

Antibiotics 2021, 10, 1530 22 of 31

the human genome, the engineering of more efficacious antibody technologies for diverse
indications can also evolve, or even become personalized, just as P. aeruginosa evolves.

Effectorfunction
Figure5.5.Effector
Figure functionor
ormechanisms
mechanismsof
ofkilling
killingby
byantibodies.
antibodies.

Author Another major consideration

Contributions: moving
Conceptualization, forward
B.D.B., M.S. will
and be drugvalidation,
F.D.T.; delivery. As detailed
J.M.S. in the
and A.E.B.;
body of this review, the common indications that will benefit from polyclonal therapies
formal analysis, M.S., B.D.B., F.D.T. and J.M.S.; investigation, C.S.R., W.L.W., A.G.K., A.P.Q. and are
foundresources,
L.L.P.; in diverse environments.
C.S.R., In most
W.L.W., A.G.K., cases
A.P.Q. anddetailed in this
L.L.P.; data manuscript,
curation, the traditional
C.S.R., W.L.W., A.G.K.,
K.M.C. and L.L.P.;
therapeutic writing—original
concentrations draft preparation,
of antibiotics in serum C.S.R.,
and W.L.W., A.G.K.,
at the site A.P.Q., K.M.C.
of infection and
are below
L.L.P.; writing—review
MIC levels, and the
decreasing editing, C.S.R.,
efficacy ofW.L.W., A.G.K., L.L.P.,
the treatment J.M.S. and A.P.Q.;
and increasing supervision,
the probability of
J.M.S., B.D.B.resistance.
antibiotic and F.D.T.;Ideally,
project administration,
the route would M.S.be
and B.D.B. All authors
dependent haveofread
on the site and agreed
infection. For
tobacteremic
the published version of
infections, the manuscript.
intravenous would be the route of choice. Beyond that, it gets trickier.
A potential
Funding: This route would
research be topical
received administration
no external funding. of IgA/IgG via a nebulizer solution
for pneumonia or patients with cystic fibrosis. Studies have supported this possibility;
Institutional Review Board Statement: Not applicable.
IgG and IgA that have been nebulized in a variety of solutions and viscosities have been
Informed
shown toConsent Statement:
increase Not applicable.
immunoglobulin levels in bronchoalveolar lavage specimens up to
Data Availability Statement: Data sharing confer
48 h after administration as well as immunity to the specific immunoglobulin
not applicable.
targets [196]. Potential for topical preparations for keratitis, folliculitis, and other soft tissue
Conflicts ofexist
infections Interest: The expanding
as well, authors declare no conflict
the range of interest.
of these therapeutics [197]. Recently it has been
shown that antibodies and antibiotics can act concomitantly or even synergistically against
pathogens. In particular, bacterial cell surfaces can be altered by antibodies, resulting in
multiple mechanisms that improve drug efficacy [198,199].
Antibiotics 2021, 10, 1530 23 of 31

P. aeruginosa, like many other bacteria, continues to and will continue to find ways to
subvert the efficacy of antibiotics. The immune system has evolved to attack microbes on
multiple fronts, but P. aeruginosa likewise evolves counterattacks, allowing it to colonize
and infect hosts. Engineering combination antibody therapies with numerous targets as
presented here would be the best option currently available to improve therapeutic efficacy
and reduce the opportunity for resistance development. As the P. aeruginosa genome is
characterized or evolves, the combination of antibodies can be adapted and optimized.
Most importantly, with the improved engineering of antibodies and characterization of
the human genome, the engineering of more efficacious antibody technologies for diverse
indications can also evolve, or even become personalized, just as P. aeruginosa evolves.

Author Contributions: Conceptualization, B.D.B., M.S. and F.D.T.; validation, J.M.S. and A.E.B.;
formal analysis, M.S., B.D.B., F.D.T. and J.M.S.; investigation, C.S.R., W.L.W., A.G.K., A.P.Q. and
L.L.P.; resources, C.S.R., W.L.W., A.G.K., A.P.Q. and L.L.P.; data curation, C.S.R., W.L.W., A.G.K.,
K.M.C. and L.L.P.; writing—original draft preparation, C.S.R., W.L.W., A.G.K., A.P.Q., K.M.C. and
L.L.P.; writing—review and editing, C.S.R., W.L.W., A.G.K., L.L.P., J.M.S. and A.P.Q.; supervision,
J.M.S., B.D.B. and F.D.T.; project administration, M.S. and B.D.B. All authors have read and agreed to
the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

AHL Acyl Homoserine Lactone

AprA Alkaline Protease A
CF Cystic Fibrosis
CIF CFTR Inhibiting Factor
CNS Central Nervous System
CRPA Carbapenem Resistant Pseudomonas aeruginosa
ExoU Exotoxin U
ExoS Exotoxin S
ExoT Exotoxin T
ExoY Exotoxin Y
FAb antigen-binding region of an antibody
Fc cell receptor region of an antibody
GlpF Glycerol Uptake Facilitator Protein
GlpT Glycerol-2-Phosphate Transporter
HCN Hydrogen Cyanide
IDSA Infectious Disease Society of America
IgA Immunoglobulin A
IgE Immunoglobulin E
IgG Immunoglobulin G
IgM Immunoglobulin M
IVIG Intravenous Immunoglobulin
LOX Lipoxygenase
mABs Monoclonal Antibodies (is mABs in text)
MAM7 Multivalent Adhesion Molecule 7
MEP Mucoid Exopolysaccharide
MIC Minimum Inhibitory Concentration
MMP Matrix Metallopeptide
LPS Lipopolysaccharide
OMV Outer Membrane Vesicle
Antibiotics 2021, 10, 1530 24 of 31

NET Neutrophil Extracellular Trap

PAB Polyclonal Antibody
PC Phosphatidylcholine
PCH Pyochelin
PLC Phospholipase C
PQS Pseudomonas Quinolone Signal
QS Quorum Sensing
ROS Reactive Oxygen Species
TFP Type IV Pili
TNF Tumor Necrosis Factor
TLR Toll-Like Receptor
T1SS Type 1 Secretion System
T2SS Type 2 Secretion System
T3SS Type 3 Secretion System
UTI Urinary Tract Infection

References
1. Kanj, S.; Sexton, M.D.; Daniel, M.D. Epidemiology, Microbiology, and Pathogenesis of Pseudomonas aeruginosa Infection. Available
online: https://www.uptodate.com/contents/epidemiology-microbiology-and-pathogenesis-of-pseudomonas-aeruginosa-
infection?search=pseudomonas&source=search_result&selectedTitle=2~{}150&usage_type=default&display_rank=2 (accessed
on 21 September 2020).
2. Owlia, P.; Nosrati, R.; Alaghehbandan, R.; Lari, A.R. Antimicrobial susceptibility differences among mucoid and non-mucoid
Pseudomonas aeruginosa isolates. GMS Hyg. Infect. Control. 2014, 9, 13. [CrossRef]
3. Souha Kanj, M.D. Principles of Antimicrobial Therapy of Pseudomonas aeruginosa Infections. Available online: https:
//www-uptodate-com.proxy.rvu.edu/contents/principles-of-antimicrobial-therapy-of-pseudomonas-aeruginosa-infections#
H6675731 (accessed on 21 September 2020).
4. Centers for Disease Control and Prevention. Pseudomonas aeruginosa in Healthcare Settings. Available online: https://www.cdc.
gov/hai/organisms/pseudomonas.html (accessed on 21 September 2020).
5. Todar, K. Pseudomonas. Online Textbook of Bacteriology. Available online: http://textbookofbacteriology.net/pseudomonas_4.
html (accessed on 21 September 2020).
6. Nordmann, P.; Poirel, L. Epidemiology and Diagnostics of Carbapenem Resistance in Gram-negative Bacteria. Clin Infect Dis.
2019, 69 (Suppl. S7), S521–S528. [CrossRef] [PubMed]
7. Tamma, P.D.; Aitken, S.L.; Bonomo, R.A.; Mathers, A.J.; van Duin, D.; Clancy, C.J. Infectious Diseases Society of America
Guidance on the Treatment of Extended-Spectrum β-lactamase Producing Enterobacterales (ESBL-E), Carbapenem-Resistant
Enterobacterales (CRE), and Pseudomonas aeruginosa with Difficult-to-Treat Resistance (DTR-P. aeruginosa). Clin. Infect Dis. 2020,
72, e169–e183. [CrossRef]
8. Pang, Z.; Raudonis, R.; Glick, B.R.; Lin, T.-J.; Cheng, Z. Antibiotic resistance in Pseudomonas aeruginosa: Mechanisms and
alternative therapeutic strategies. Biotechnol. Adv. 2019, 37, 177–192. [CrossRef]
9. Centers for Disease Control and Prevention. Antibiotic-Resistant Germs: New Threats. Available online: https://www.cdc.gov/
drugresistance/biggest-threats.html (accessed on 6 May 2020).
10. Kmeid, J.G.; Youssef, M.M.; Kanafani, Z.A.; Kanj, S.S. Combination therapy for Gram-negative bacteria: What is the evidence?
Expert. Rev. Anti. Infect. Ther. 2013, 11, 1355–1362. [CrossRef]
11. Grekov, I.; Thöming, J.G.; Kordes, A.; Häussler, S. Evolution of Pseudomonas aeruginosa toward higher fitness under standard
laboratory conditions. ISME J. 2021, 15, 1165–1177. [CrossRef] [PubMed]
12. Fothergill, J.L.; Panagea, S.; Hart, C.A.; Walshaw, M.J.; Pitt, T.L.; Winstanley, C. Widespread pyocyanin over-production among
isolates of a cystic fibrosis epidemic strain. BMC Microbiol. 2007, 7, 45. [CrossRef] [PubMed]
13. Mathee, K.; Ciofu, O.; Sternberg, C.; Lindum, P.W.; Campbell, J.I.A.; Jensen, P.; Johnsen, A.H.; Givskov, M.; Ohman, D.E.; Søren,
M.; et al. Mucoid conversion of Pseudomonas aeruginos by hydrogen peroxide: A mechanism for virulence activation in the
cystic fibrosis lung. Microbiology 1999, 145, 1349–1357. [CrossRef] [PubMed]
14. Von Götz, F.; Häussler, S.; Jordan, D.; Saravanamuthu, S.S.; Wehmhöner, D.; Strüssmann, A.; Lauber, J.; Attree, I.; Buer, J.;
Tümmler, B.; et al. Expression Analysis of a Highly Adherent and Cytotoxic Small Colony Variant of Pseudomonas aeruginosa
Isolated from a Lung of a Patient with Cystic Fibrosis. J. Bacteriol. 2004, 186, 3837. [CrossRef] [PubMed]
15. Häußler, S.; Tümmler, B.; Weißbrodt, H.; Rohde, M.; Steinmetz, I. Small-Colony Variants of Pseudomonas aeruginosa in Cystic
Fibrosis. Clin. Infect. Dis. 1999, 29, 621–625. [CrossRef]
16. D’Argenio, D.A.; Calfee, M.W.; Rainey, P.B.; Pesci, E.C. Autolysis and Autoaggregation in Pseudomonas aeruginosa Colony
Morphology Mutants. J. Bacteriol. 2002, 184, 6481. [CrossRef] [PubMed]
17. Smith, E.E.; Buckley, D.G.; Wu, Z.; Saenphimmachak, C.; Hoffman, L.R.; D’Argenio, D.A.; Miller, S.I.; Ramsey, B.W.; Speert, D.P.;
Moskowitz, S.M.; et al. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc. Natl. Acad.
Sci. USA 2006, 103, 8487–8492. [CrossRef] [PubMed]
Antibiotics 2021, 10, 1530 25 of 31

18. Subedi, D.; Vijay, A.K.; Kohli, G.S.; Rice, S.A.; Willcox, M. Comparative genomics of clinical strains of Pseudomonas aeruginosa
strains isolated from different geographic sites. Sci. Rep. 2018, 8, 15668. [CrossRef] [PubMed]
19. Weiser, R.; Green, A.E.; Bull, M.J.; Cunningham-Oakes, E.; Jolley, K.A.; Maiden, M.C.J.; Hall, A.J.; Winstanley, C.; Weightman, A.J.;
Donoghue, D.; et al. Not all Pseudomonas aeruginosa are equal: Strains from industrial sources possess uniquely large multireplicon
genomes. Microb. Genom. 2019, 5, e000276. [CrossRef]
20. Boucher, H.W.; Talbot, G.H.; Bradley, J.S.; Edwards, J.E.; Gilbert, D.; Rice, L.B.; Scheld, M.; Spellberg, B.; Bartlett, J. Bad bugs, no
drugs: No ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am.
2009, 48, 1–12. [CrossRef]
21. Bassetti, M.; Peghin, M.; Vena, A.; Giacobbe, D.R. Treatment of Infections Due to MDR Gram-Negative Bacteria. Front. Med. 2019,
6, 74. [CrossRef]
22. Henrichfreise, B.; Wiegand, I.; Pfister, W.; Wiedemann, B. Resistance Mechanisms of Multiresistant Pseudomonas aeruginosa Strains
from Germany and Correlation with Hypermutation. Antimicrob. Agents Chemother. 2007, 51, 4062–4070. [CrossRef]
23. Song, Z.; Wu, H.; Ciofu, O.; Kong, K.-F.; Høiby, N.; Rygaard, J.; Kharazmi, A.; Mathee, K. Pseudomonas aeruginosa alginate is
refractory to Th1 immune response and impedes host immune clearance in a mouse model of acute lung infection. J. Med.
Microbiol. 2003, 52, 731–740. [CrossRef]
24. Worlitzsch, D.; Tarran, R.; Ulrich, M.; Schwab, U.; Cekici, A.; Meyer, K.C.; Birrer, P.; Bellon, G.; Berger, J.; Weiss, T.; et al. Effects
of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J. Clin. Investig. 2002, 109,
317–325. [CrossRef]
25. Mauch, R.M.; Jensen, P.Ø.; Moser, C.; Levy, C.E.; Høiby, N. Mechanisms of humoral immune response against Pseudomonas
aeruginosa biofilm infection in cystic fibrosis. J. Cyst. Fibros. 2018, 17, 143–152. [CrossRef]
26. Fauvart, M.; De Groote, V.N.; Michiels, J. Role of persister cells in chronic infections: Clinical relevance and perspectives on
anti-persister therapies. J. Med. Microbiol. 2011, 60, 699–709. [CrossRef]
27. Moyed, H.S.; Bertrand, K.P. hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after
inhibition of murein synthesis. J. Bacteriol. 1983, 155, 768–775. [CrossRef] [PubMed]
28. Spoering, A.L.; Vulic, M.; Lewis, K. GlpD and PlsB participate in persister cell formation in Escherichia coli. J. Bacteriol. 2006, 188,
5136–5144. [CrossRef] [PubMed]
29. Balaban, N.Q.; Merrin, J.; Chait, R.; Kowalik, L.; Leibler, S. Bacterial persistence as a phenotypic switch. Science 2004, 305,
1622–1625. [CrossRef] [PubMed]
30. Wainwright, J.; Hobbs, G.; Nakouti, I. Persister cells: Formation, resuscitation and combative therapies. Arch Microbiol. 2021, 203,
5899–5906. [CrossRef] [PubMed]
31. Louwagie, E.; Verstraete, L.; Michiels, J.; Verstraeten, N. Studying Bacterial Persistence: Established Methods and Current
Advances. Methods Mol. Biol. 2021, 2357, 3–20. [CrossRef] [PubMed]
32. Kaldalu, N.; Hauryliuk, V.; Turnbull, K.J.; La Mensa, A.; Putrinš, M.; Tenson, T. In Vitro Studies of Persister Cells. Microbiol. Mol.
Biol. Rev. 2020, 84, e00070-20. [CrossRef]
33. Mulcahy, L.R.; Burns, J.L.; Lory, S.; Lewis, K. Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells
in patients with cystic fibrosis. J. Bacteriol. 2010, 192, 6191–6199. [CrossRef]
34. Lewis, K. Multidrug tolerance of biofilms and persister cells. Curr. Top Microbiol. Immunol. 2008, 322, 107–131. [CrossRef]
[PubMed]
35. Möker, N.; Dean, C.R.; Tao, J. Pseudomonas aeruginosa increases formation of multidrug-tolerant persister cells in response to
quorum-sensing signaling molecules. J. Bacteriol. 2010, 192, 1946–1955. [CrossRef]
36. Robak, O.H.; Heimesaat, M.M.; Kruglov, A.A.; Prepens, S.; Ninnemann, J.; Gutbier, B.; Reppe, K.; Hochrein, H.; Suter, M.;
Kirschning, C.J.; et al. Antibiotic treatment-induced secondary IgA deficiency enhances susceptibility to Pseudomonas aeruginosa
pneumonia. J. Clin. Investig. 2018, 128, 3535–3545. [CrossRef] [PubMed]
37. Schroeder, M.; Brooks, B.D.; Brooks, A.E. The Complex Relationship between Virulence and Antibiotic Resistance. Genes 2017, 8,
39. [CrossRef] [PubMed]
38. Meluleni, G.J.; Grout, M.; Evans, D.J.; Pier, G.B. Mucoid Pseudomonas aeruginosa growing in a biofilm in vitro are killed by opsonic
antibodies to the mucoid exopolysaccharide capsule but not by antibodies produced during chronic lung infection in cystic
fibrosis patients. J. Immunol. 1995, 155, 2029. [PubMed]
39. Rello, J.; Krenn, C.-G.; Locker, G.; Pilger, E.; Madl, C.; Balica, L.; Dugernier, T.; Laterre, P.-F.; Spapen, H.; Depuydt, P.; et al. A
randomized placebo-controlled phase II study of a Pseudomonas vaccine in ventilated ICU patients. Crit. Care Lond. Engl. 2017, 21, 22.
[CrossRef]
40. Adlbrecht, C.; Wurm, R.; Depuydt, P.; Spapen, H.; Lorente, J.A.; Staudinger, T.; Creteur, J.; Zauner, C.; Meier-Hellmann, A.;
Eller, P.; et al. Efficacy, immunogenicity, and safety of IC43 recombinant Pseudomonas aeruginosa vaccine in mechanically ventilated
intensive care patients—A randomized clinical trial. Crit. Care 2020, 24, 74. [CrossRef]
41. Georgescu, M.; Gheorghe, I.; Curutiu, C.; Lazar, V.; Bleotu, C.; Chifiriuc, M.-C. Virulence and resistance features of Pseudomonas
aeruginosa strains isolated from chronic leg ulcers. BMC Infect. Dis. 2016, 16, 3–9. [CrossRef]
42. Michalska, M.; Wolf, P. Pseudomonas Exotoxin A: Optimized by evolution for effective killing. Front. Microbiol. 2015, 6, 963.
[CrossRef]
Antibiotics 2021, 10, 1530 26 of 31

43. Morlon-Guyot, J.; Méré, J.; Bonhoure, A.; Beaumelle, B. Processing of Pseudomonas aeruginosa Exotoxin A Is Dispensable for Cell
Intoxication. Infect. Immun. 2009, 77, 3090–3099. [CrossRef]
44. Hertle, R.; Mrsny, R.; Fitzgerald, D.J. Dual-Function Vaccine for Pseudomonas aeruginosa: Characterization of Chimeric Exotoxin
A-Pilin Protein. Infect. Immun. 2001, 69, 6962–6969. [CrossRef]
45. Saint-Criq, V.; Villeret, B.; Bastaert, F.; Kheir, S.; Hatton, A.; Cazes, A.; Xing, Z.; Sermet-Gaudelus, I.; Garcia-Verdugo, I.; Edelman,
A.; et al. Pseudomonas aeruginosa LasB protease impairs innate immunity in mice and humans by targeting a lung epithelial cystic
fibrosis transmembrane regulator-IL-6-antimicrobial-repair pathway. Thorax 2018, 73, 49–61. [CrossRef]
46. Engel, L.S.; Hill, J.M.; Caballero, A.R.; Green, L.C.; O’Callaghan, R.J. Protease IV, a Unique Extracellular Protease and Virulence
Factor from Pseudomonas aeruginosa. J. Biol. Chem. 1998, 273, 16792–16797. [CrossRef] [PubMed]
47. Oh, J.; Li, X.-H.; Kim, S.-K.; Lee, J.-H. Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa. Sci.
Rep. 2017, 7, 4416. [CrossRef]
48. Sharma, A.; Krause, A.; Worgall, S. Recent developments for Pseudomonas vaccines. Hum. Vaccin. 2011, 7, 999–1011. [CrossRef]
[PubMed]
49. Thibodeaux, B.A.; Caballero, A.R.; Dajcs, J.J.; Marquart, M.E.; Engel, L.S.; O’Callaghan, R.J. Pseudomonas aeruginosa Protease IV: A
Corneal Virulence Factor of Low Immunogenicity. Ocul. Immunol. Inflamm. 2005, 13, 169–182. [CrossRef] [PubMed]
50. Tielen, P.; Kuhn, H.; Rosenau, F.; Jaeger, K.-E.; Flemming, H.-C.; Wingender, J. Interaction between extracellular lipase LipA and
the polysaccharide alginate of Pseudomonas aeruginosa. BMC Microbiol. 2013, 13, 159. [CrossRef]
51. Funken, H.; Knapp, A.; Vasil, M.L.; Wilhelm, S.; Jaeger, K.-E.; Rosenau, F. The lipase LipA (PA2862) but not LipC (PA4813) from
Pseudomonas aeruginosa influences regulation of pyoverdine production and expression of the sigma factor PvdS. J. Bacteriol. 2011,
193, 5858–5860. [CrossRef]
52. Jaeger, K.-E.; Kharazmi, A.; Høiby, N. Extracellular lipase of Pseudomonas aeruginosa: Biochemical characterization and effect on
human neutrophil and monocyte function in vitro. Microb. Pathog. 1991, 10, 173–182. [CrossRef]
53. Terada, L.S.; Johansen, K.A.; Nowbar, S.; Vasil, A.I.; Vasil, M.L. Pseudomonas aeruginosa Hemolytic Phospholipase C Suppresses
Neutrophil Respiratory Burst Activity. Orndorff, P.E., Ed. Infect. Immun. 1999, 67, 2371–2376. [CrossRef] [PubMed]
54. Sun, Z.; Kang, Y.; Norris, M.; Troyer, R.M.; Son, M.S.; Schweizer, H.P.; Dow, S.W.; Hoang, T.T. Blocking phosphatidylcholine
utilization in Pseudomonas aeruginosa, via mutagenesis of fatty acid, glycerol and choline degradation pathways, confirms the
importance of this nutrient source in vivo. PLoS ONE 2014, 9, e103778. [CrossRef]
55. Maroui, I.; Aboulkacem, A.; Mohammed, T.; Belhaj, A. Virulence profiles of clinical and environmental Pseudomonas aeruginosa
isolates from Central Morocco. Afr. J. Microbiol. Res. 2016, 10, 473–480. [CrossRef]
56. Granström, M.; Ericsson, A.; Strandvik, B.; Wretlind, B.; Pavlovskis, O.R.; Berka, R.; Vasil, M.L. Relation between Antibody
Response to Pseudomonas aeruginosa Exoproteins and Colonization/Infection in Patients with Cystic Fibrosis. Acta. Paediatr. 1984,
73, 772–777. [CrossRef]
57. Yu, Y.-J.; Wang, X.-H.; Fan, G.-C. Versatile effects of bacterium-released membrane vesicles on mammalian cells and infec-
tious/inflammatory diseases. Acta Pharmacol. Sin. 2018, 39, 514–533. [CrossRef] [PubMed]
58. Döring, G.; Dalhoff, A.; Vogel, O.; Brunner, H.; Dröge, U.; Botzenhart, K. In Vivo Activity of Proteases of Pseudomonas aeruginosa
in a Rat Model. J. Infect. Dis. 1984, 149, 532–537. [CrossRef] [PubMed]
59. Fick, R.B., Jr.; Baltimore, R.S.; Squier, S.U.; Reynolds, H.Y. IgG Proteolytic Activity of Pseudomonas aeruginosa in Cystic Fibrosis. J.
Infect. Dis. 1985, 151, 589–598. [CrossRef] [PubMed]
60. Parmely, M.; Gale, A.; Clabaugh, M.; Horvat, R.; Zhou, W.W. Proteolytic inactivation of cytokines by Pseudomonas aeruginosa.
Infect. Immun. 1990, 58, 3009. [CrossRef]
61. Horvat, R.T.; Parmely, M.J. Pseudomonas aeruginosa alkaline protease degrades human gamma interferon and inhibits its bioactivity.
Infect. Immun. 1988, 56, 2925. [CrossRef]
62. Kharazmi, A.; Eriksen, H.O.; DÖRing, G.; Goldstein, W.; HØIby, N. Effect of pseudomonas aeruginosa proteases on human leukocyte
phagocytosis and bactericidal activity. Acta. Pathol. Microbiol. Scand. Ser. C Immunol. 1986, 94, 175–179. [CrossRef]
63. Theander, T.G.; Kharazmi, A.; Pedersen, B.K.; Christensen, L.D.; Tvede, N.; Poulsen, L.K.; Odum, N.; Svenson, M.; Bendtzen, K.
Inhibition of human lymphocyte proliferation and cleavage of interleukin-2 by Pseudomonas aeruginosa proteases. Infect. Immun.
1988, 56, 1673. [CrossRef]
64. Pedersen, B.K.; Kharazmi, A. Inhibition of human natural killer cell activity by Pseudomonas aeruginosa alkaline protease and
elastase. Infect. Immun. 1987, 55, 986. [CrossRef]
65. Nomura, K.; Obata, K.; Keira, T.; Miyata, R.; Hirakawa, S.; Takano, K.-I.; Kohno, T.; Sawada, N.; Himi, T.; Kojima, T. Pseudomonas
aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells.
Respir. Res. 2014, 15, 21. [CrossRef] [PubMed]
66. Kessler, E.; Safrin, M.; Gustin, J.K.; Ohman, D.E. Elastase and the LasA Protease of Pseudomonas aeruginosa Are Secreted with
Their Propeptides. J. Biol. Chem. 1998, 273, 30225–30231. [CrossRef] [PubMed]
67. Homma, J.Y.; Abe, C.; Tanamoto, K.; Hirao, Y.; Morihara, K.; Tsuzuki, H.; Yanagawa, R.; Honda, E.; Aoi, Y.; Fujimoto, Y.; et al.
Effectiveness of immunization with single and multi-component vaccines prepared from a common antigen (OEP), protease and
elastase toxoids of Pseudomonas aeruginosa on protection against hemorrhagic pneumonia in mink due to P. aeruginosa. Jpn. J. Exp.
Med. 1978, 48, 111–133. [PubMed]
Antibiotics 2021, 10, 1530 27 of 31

68. Hirao, Y.; Homma, J. Therapeutic effect of immunization with OEP, protease toxoid and elastase toxoid on corneal ulcers in mice
due to Pseudomonas aeruginosa infection. Jpn. J. Exp. Med. 1978, 48, 41–51.
69. Sokol, P.A.; Kooi, C.; Hodges, R.S.; Cachia, P.; Woods, D.E. Immunization with a Pseudomonas aeruginosa Elastase Peptide Reduces
Severity of Experimental Lung Infections Due to P. aeruginosa or Burkholderia cepacia. J. Infect. Dis. 2000, 181, 1682–1692.
[CrossRef]
70. Kawaharajo, K.; Homma, J. Effects of elastase, protease and common antigen (OEP) from Pseudomonas aeruginosa on protection
against burns in mice. Jpn. J. Exp. Med. 1977, 47, 495–500. [PubMed]
71. Casilag, F.; Lorenz, A.; Krueger, J.; Klawonn, F.; Weiss, S.; Häussler, S. The LasB Elastase of Pseudomonas aeruginosa Acts in Concert
with Alkaline Protease AprA To Prevent Flagellin-Mediated Immune Recognition. Infect. Immun. 2016, 84, 162. [CrossRef]
[PubMed]
72. Poole, K.; Hancock, R.E.W. Secretion of alkaline phosphatase and phsopholipase C in Pseudomonas aeruginosa is specific and does
not involve an increase in outer membrane permeability. FEMS Microbiol. Lett. 1983, 16, 25–29. [CrossRef]
73. Liu, X.; Long, D.; You, H.; Yang, D.; Zhou, S.; Zhang, S.; Li, M.; He, M.; Xiong, M.; Wang, X. Phosphatidylcholine affects the
secretion of the alkaline phosphatase PhoA in Pseudomonas strains. Microbiol. Res. 2016, 192, 21–29. [CrossRef]
74. Miyoshi, S.-I.; Shinoda, S. Bacterial Metalloprotease as the Toxic Factor in Infection. J. Toxicol. Toxin. Rev. 1997, 16, 177–194.
[CrossRef]
75. Zhang, C.; Bijl, E.; Svensson, B.; Hettinga, K. The Extracellular Protease AprX from Pseudomonas and its Spoilage Potential for
UHT Milk: A Review. Compr. Rev. Food Sci. Food Saf. 2019, 18, 834–852. [CrossRef]
76. Filloux, A. Protein Secretion Systems in Pseudomonas aeruginosa: An Essay on Diversity, Evolution, and Function. Front. Microbiol.
2011, 2, 155. [CrossRef]
77. Higgins, C.F. ABC transporters: Physiology, structure and mechanism—An overview. Res. Microbiol. 2001, 152, 205–210.
[CrossRef]
78. Duong, F.; Lazdunski, A.; Carni, B.; Murgier, M. Sequence of a cluster of genes controlling synthesis and secretion of alkaline
protease in Pseudomonas aeruginosa: Relationships to other secretory pathways. Gene 1992, 121, 47–54. [CrossRef] [PubMed]
79. Caza, M.; Kronstad, J.W. Shared and distinct mechanisms of iron acquisition by bacterial and fungal pathogens of humans. Front.
Cell Infect. Microbiol. 2013, 3, 80. [CrossRef] [PubMed]
80. Ball, G.; Durand, É.; Lazdunski, A.; Filloux, A. A novel type II secretion system in Pseudomonas aeruginosa. Mol. Microbiol. 2002,
43, 475–485. [CrossRef]
81. Cornelis, G.R. The type III secretion injectisome, a complex nanomachine for intracellular ‘toxin’ delivery. Biol. Chem. 2010, 391,
745–751. [CrossRef] [PubMed]
82. Fleiszig, S.M.; Zaidi, T.S.; Preston, M.J.; Grout, M.; Evans, D.J.; Pier, G.B. Relationship between cytotoxicity and corneal epithelial
cell invasion by clinical isolates of Pseudomonas aeruginosa. Infect. Immun. 1996, 64, 2288–2294. [CrossRef] [PubMed]
83. Finck-Barbançon, V.; Goranson, J.; Zhu, L.; Sawa, T.; Wiener-Kronish, J.P.; Fleiszig, S.M.J.; Wu, C.; Mende-Mueller, L.; Frank,
D.W. ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury. Mol. Microbiol. 1997, 25,
547–557. [CrossRef]
84. Roy-Burman, A.; Savel, R.H.; Racine, S.; Swanson, B.L.; Revadigar, N.S.; Fujimoto, J.; Sawa, T.; Frank, D.W.; Wiener-Kronish, J.P.
Type III Protein Secretion Is Associated with Death in Lower Respiratory and Systemic Pseudomonas aeruginosa Infections. J. Infect.
Dis. 2001, 183, 1767–1774. [CrossRef]
85. Hauser, A.R. The type III secretion system of Pseudomonas aeruginosa: Infection by injection. Nat. Rev. Microbiol. 2009, 7, 654–665.
[CrossRef] [PubMed]
86. Anantharajah, A.; Mingeot-Leclercq, M.-P.; Van Bambeke, F. Targeting the Type Three Secretion System in Pseudomonas aeruginosa.
Trends Pharmacol. Sci. 2016, 37, 734–749. [CrossRef]
87. Sato, H.; Frank, D.W. Multi-Functional Characteristics of the Pseudomonas aeruginosa Type III Needle-Tip Protein, PcrV Comparison
to Orthologs in other Gram-negative Bacteria. Front. Microbiol. 2011, 2, 142. [CrossRef] [PubMed]
88. Sawa, T.; Ito, E.; Nguyen, V.H.; Haight, M. Anti-PcrV antibody strategies against virulent Pseudomonas aeruginosa. Hum. Vaccines
Immunother. 2014, 10, 2843–2852. [CrossRef] [PubMed]
89. Lynch, S.V.; Flanagan, J.; Sawa, T.; Fang, A.; Baek, M.S.; Rubio-Mills, A.; Ajayi, T.; Yanagihara, K.; Hirakata, Y.; Kohno, S.; et al.
Polymorphisms in the Pseudomonas aeruginosa type III secretion protein, PcrV-implications for anti-PcrV immunotherapy. Microb.
Pathog. 2010, 48, 197–204. [CrossRef] [PubMed]
90. Moriyama, K.; Wiener-Kronish, J.P.; Sawa, T. Protective effects of affinity-purified antibody and truncated vaccines against
Pseudomonas aeruginosa V-antigen in neutropenic mice. Microbiol. Immunol. 2009, 53, 587–594. [CrossRef] [PubMed]
91. Sawa, T.; Yahr, T.; Ohara, M.; Kurahashi, K.; Gropper, M.A.; Wiener-Kronish, J.P.; Frank, D.W. Active and passive immunization
with the Pseudomonas V antigen protects against type III intoxication and lung injury. Nat. Med. 1999, 5, 392–398. [CrossRef]
92. Baer, M.; Sawa, T.; Flynn, P.; Luehrsen, K.; Martinez, D.; Wiener-Kronish, J.P.; Yarranton, G.; Bebbington, C. An engineered human
antibody fab fragment specific for Pseudomonas aeruginosa PcrV antigen has potent antibacterial activity. Infect. Immun. 2009, 77,
1083–1090. [CrossRef]
93. Faure, K.; Fujimoto, J.; Shimabukuro, D.W.; Ajayi, T.; Shime, N.; Moriyama, K.; Spack, E.G.; Wiener-Kronish, J.P.; Sawa, T. Effects
of monoclonal anti-PcrV antibody on Pseudomonas aeruginosa-induced acute lung injury in a rat model. J. Immun. Based Ther.
Vaccines. 2003, 1, 2. [CrossRef] [PubMed]
Antibiotics 2021, 10, 1530 28 of 31

94. Frank, D.W.; Vallis, A.; Wiener-Kronish, J.P.; Roy-Burman, A.; Spack, E.G.; Mullaney, B.P.; Megdoud, M.; Marks, J.D.; Fritz, R.;
Sawa, T. Generation and Characterization of a Protective Monoclonal Antibody to Pseudomonas aeruginosa PcrV. J. Infect. Dis. 2002,
186, 64–73. [CrossRef]
95. Imamura, Y.; Yanagihara, K.; Fukuda, Y.; Kaneko, Y.; Seki, M.; Izumikawa, K.; Miyazaki, Y.; Hirakata, Y.; Sawa, T.; Wiener-Kronish,
J.P.; et al. Effect of anti-PcrV antibody in a murine chronic airway Pseudomonas aeruginosa infection model. Eur. Respir. J. 2007, 29, 965.
[CrossRef]
96. Shime, N.; Sawa, T.; Fujimoto, J.; Faure, K.; Allmond, L.R.; Karaca, T.; Swanson, B.L.; Spack, E.G.; Wiener-Kronish, J.P. Therapeutic
Administration of Anti-PcrV F(ab’)2 in Sepsis Associated with Pseudomonas aeruginosa. J. Immunol. 2001, 167, 5880. [CrossRef]
97. Song, Y.; Baer, M.; Srinivasan, R.; Lima, J.; Yarranton, G.; Bebbington, C.; Lynch, S.V. PcrV antibody–antibiotic combination
improves survival in Pseudomonas aeruginosa-infected mice. Eur. J. Clin. Microbiol. Infect. Dis. 2012, 31, 1837–1845. [CrossRef]
[PubMed]
98. Wang, Q.; Li, H.; Zhou, J.; Zhong, M.; Zhu, D.; Feng, N.; Liu, F.; Bai, C.; Song, Y. PcrV antibody protects multi-drug resistant
Pseudomonas aeruginosa induced acute lung injury. Respir. Physiol. Neurobiol. 2014, 193, 21–28. [CrossRef]
99. Hosseinidoust, Z.; van de Ven, T.G.M.; Tufenkji, N. Evolution of Pseudomonas aeruginosa virulence as a result of phage predation.
Appl. Environ. Microbiol. 2013, 79, 6110–6116. [CrossRef]
100. Vance, R.E.; Rietsch, A.; Mekalanos, J.J. Role of the Type III Secreted Exoenzymes S, T, and Y in Systemic Spread of Pseudomonas
aeruginosa PAO1 In Vivo. Infect. Immun. 2005, 73, 1706–1713. [CrossRef] [PubMed]
101. Hauser, A.R.; Cobb, E.; Bodí, M.; Mariscal, D.; Vallés, J.; Engel, J.N.; Rello, J. Type III protein secretion is associated with poor
clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit. Care Med. 2002, 30,
521–528. [CrossRef]
102. Leo, J.C.; Grin, I.; Linke, D. Type V secretion: Mechanism(s) of autotransport through the bacterial outer membrane. Philos. Trans.
R. Soc. B Biol. Sci. 2012, 367, 1088–1101. [CrossRef]
103. Salacha, R.; Kovacic, F.; Brochier-Armanet, C.; Wilhelm, S.; Tommassen, J.; Filloux, A.; Voulhoux, R.; Bleves, S. The Pseudomonas
aeruginosa patatin-like protein PlpD is the archetype of a novel Type V secretion system. Environ. Microbiol. 2010, 12, 1498–1512.
[CrossRef]
104. Chen, L.; Zou, Y.; She, P.; Wu, Y. Composition, function, and regulation of T6SS in Pseudomonas aeruginosa. Microbiol. Res. 2015,
172, 19–25. [CrossRef] [PubMed]
105. The Type VI Secretion System: A Dynamic System for Bacterial Communication? Available online: https://www-ncbi-nlm-nih-
gov.proxy.rvu.edu/pmc/articles/PMC5532429/ (accessed on 20 August 2021).
106. Wang, M.; Schaefer, A.L.; Dandekar, A.A.; Greenberg, E.P. Quorum sensing and policing of Pseudomonas aeruginosa social cheaters.
Proc. Natl. Acad. Sci. USA 2015, 112, 2187. [CrossRef]
107. Häussler, S.; Becker, T. The Pseudomonas Quinolone Signal (PQS) Balances Life and Death in Pseudomonas aeruginosa Populations.
Ausubel, F.M., Ed. PLoS Pathog. 2008, 4, e1000166. [CrossRef] [PubMed]
108. Lin, J.; Cheng, J.; Wang, Y.; Shen, X. The Pseudomonas Quinolone Signal (PQS): Not Just for Quorum Sensing Anymore. Front. Cell
Infect. Microbiol. 2018, 8, 230. [CrossRef] [PubMed]
109. Castañeda-Tamez, P.; Ramírez-Peris, J.; Pérez-Velázquez, J.; Kuttler, C.; Jalalimanesh, A.; Saucedo-Mora, M.Á.; Jiménez-Cortés,
J.G.; Maeda, T.; González, Y.; Tomás, M.; et al. Pyocyanin Restricts Social Cheating in Pseudomonas aeruginosa. Front. Microbiol.
2018, 9, 1348. [CrossRef] [PubMed]
110. Hall, S.; McDermott, C.; Anoopkumar-Dukie, S.; McFarland, A.J.; Forbes, A.; Perkins, A.V.; Davey, A.K.; Chess-Williams, R.; Kiefel,
M.J.; Arora, D.; et al. Cellular Effects of Pyocyanin, a Secreted Virulence Factor of Pseudomonas aeruginosa. Toxins 2016, 8, 236.
[CrossRef]
111. Branzk, N.; Lubojemska, A.; Hardison, S.E.; Wang, Q.; Gutierrez, M.G.; Brown, G.D.; Papayannopoulos, V. Neutrophils sense
microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat. Immunol. 2014, 15,
1017–1025. [CrossRef] [PubMed]
112. Susilowati, H.; Artanto, S.; Yulianto, H.; Sosroseno, W.; Hutomo, S. The protective effects of antigen-specific IgY on pyocyanin-
treated human lymphoma Raji cells [version 2; peer review: 2 approved]. F1000Research 2019, 8, 1008. [CrossRef]
113. Telford, G.; Wheeler, D.; Williams, P.; Tomkins, P.T.; Appleby, P.; Sewell, H.; Stewart, G.S.A.B.; Bycroft, B.W.; Pritchard, D.I. The
Pseudomonas aeruginosa Quorum-Sensing Signal MoleculeN-(3-Oxododecanoyl)-l-Homoserine Lactone Has Immunomodulatory
Activity. Infect. Immun. 1998, 66, 36–42. [CrossRef]
114. Zhu, H.; Conibear, T.C.R.; Thuruthyil, S.J.; Willcox, M.D.P. Pseudomonas aeruginosa Quorum-Sensing Signal Molecules Induce IL-8
Production by Human Corneal Epithelial Cells. Eye Contact Lens. Sci. Clin. Pract. 2008, 34, 179–181. [CrossRef] [PubMed]
115. Gaisford, W.; Pritchard, D.I.; Cooke, A. OdDHL Inhibits T Cell Subset Differentiation and Delays Diabetes Onset in NOD Mice.
Clin. Vaccine Immunol. 2011, 18, 1213–1220. [CrossRef]
116. Borges, A.; Sousa, P.; Gaspar, A.; Vilar, S.; Borges, F.; Simões, M. Furvina inhibits the 3-oxo-C12-HSL-based quorum sensing
system of Pseudomonas aeruginosa and QS-dependent phenotypes. Biofouling 2017, 33, 156–168. [CrossRef]
117. Hooi, D.S.W.; Bycroft, B.W.; Chhabra, S.R.; Williams, P.; Pritchard, D.I. Differential Immune Modulatory Activity of Pseudomonas
aeruginosa Quorum-Sensing Signal Molecules. Infect. Immun. 2004, 72, 6463–6470. [CrossRef] [PubMed]
118. Cooley, M.A.; Whittall, C.; Rolph, M.S. Pseudomonas signal molecule 3-oxo-C12-homoserine lactone interferes with binding of
rosiglitazone to human PPARγ. Microbes. Infect. 2010, 12, 231–237. [CrossRef] [PubMed]
Antibiotics 2021, 10, 1530 29 of 31

119. Jarosz, L.M.; Ovchinnikova, E.S.; Meijler, M.M.; Krom, B.P. Microbial Spy Games and Host Response: Roles of a Pseudomonas
aeruginosa Small Molecule in Communication with Other Species. PLoS Pathog. 2011, 7, e1002312. [CrossRef] [PubMed]
120. Miyairi, S.; Tateda, K.; Fuse, E.T.; Ueda, C.; Saito, H.; Takabatake, T.; Ishii, Y.; Horikawa, M.; Ishiguro, M.; Standiford, T.J.; et al.
Immunization with 3-oxododecanoyl-l-homoserine lactone–protein conjugate protects mice from lethal Pseudomonas aeruginosa
lung infection. J. Med. Microbiol. 2006, 55, 1381–1387. [CrossRef] [PubMed]
121. Yan, H.; Asfahl, K.L.; Li, N.; Sun, F.; Xiao, J.; Shen, D.; Dandekar, A.A.; Wang, M. Conditional quorum-sensing induction of a
cyanide-insensitive terminal oxidase stabilizes cooperating populations of Pseudomonas aeruginosa. Nat. Commun. 2019, 10, 4999.
[CrossRef] [PubMed]
122. Ryall, B.; Davies, J.C.; Wilson, R.; Shoemark, A.; Williams, H.D. Pseudomonas aeruginosa, cyanide accumulation and lung function
in CF and non-CF bronchiectasis patients. Eur. Respir. J. 2008, 32, 740. [CrossRef]
123. Pessi, G.; Haas, D. Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR
and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa. J. Bacteriol. 2000, 182, 6940–6949. [CrossRef]
124. Rodríguez-Rojas, A.; Mena, A.; Martín, S.; Borrell, N.; Oliver, A.; Blázquez, J. Inactivation of the hmgA gene of Pseudomonas
aeruginosa leads to pyomelanin hyperproduction, stress resistance and increased persistence in chronic lung infection. Microbiology
2009, 155, 1050–1057. [CrossRef]
125. Hunter, R.C.; Newman, D.K. A Putative ABC Transporter, HatABCDE, Is among Molecular Determinants of Pyomelanin
Production in Pseudomonas aeruginosa. J. Bacteriol. 2010, 192, 5962. [CrossRef] [PubMed]
126. Defoirdt, T. Quorum-Sensing Systems as Targets for Antivirulence Therapy. Trends. Microbiol. 2018, 26, 313–328. [CrossRef]
127. Gupta, R.K.; Chhibber, S.; Harjai, K. Acyl Homoserine Lactones from Culture Supernatants of Pseudomonas aeruginosa Accelerate
Host Immunomodulation. PLoS ONE 2011, 6, e20860. [CrossRef]
128. Morita, Y.; Tomida, J.; Kawamura, Y. MexXY multidrug efflux system of Pseudomonas aeruginosa. Front. Microbiol. 2012, 3, 408.
[CrossRef] [PubMed]
129. Li, X.-Z.; Plésiat, P.; Nikaido, H. The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria. Clin.
Microbiol. Rev. 2015, 28, 337. [CrossRef] [PubMed]
130. Brooks, B.D.; Brooks, A.E. Therapeutic strategies to combat antibiotic resistance. Adv. Drug. Deliv. Rev. 2014, 78, 14–27. [CrossRef]
131. Yang, L.; Chen, L.; Shen, L.; Surette, M.; Duan, K. Inactivation of MuxABC-OpmB transporter system in Pseudomonas aeruginosa
leads to increased ampicillin and carbenicillin resistance and decreased virulence. J. Microbiol. 2011, 49, 107–114. [CrossRef]
132. Ma, D.; Cook, D.N.; Hearst, J.E.; Nikaido, H. Efflux pumps and drug resistance in Gram-negative bacteria. Trends. Microbiol. 1994,
2, 489–493. [CrossRef]
133. Bianco, N.; Neshat, S.; Poole, K. Conservation of the multidrug resistance efflux gene oprM in Pseudomonas aeruginosa. Antimicrob.
Agents Chemother. 1997, 41, 853–856. [CrossRef] [PubMed]
134. Chemani, C.; Imberty, A.; de Bentzmann, S.; Pierre, M.; Wimmerová, M.; Guery, B.P.; Faure, K. Role of LecA and LecB Lectins in
Pseudomonas aeruginosa-Induced Lung Injury and Effect of Carbohydrate Ligands. Infect. Immun. 2009, 77, 2065–2075. [CrossRef]
135. Son, M.S.; Matthews, W.J., Jr.; Kang, Y.; Nguyen, D.T.; Hoang, T.T. In vivo evidence of Pseudomonas aeruginosa nutrient acquisition
and pathogenesis in the lungs of cystic fibrosis patients. Infect. Immun. 2007, 75, 5313–5324. [CrossRef]
136. Malek, A.A.; Chen, C.; Wargo, M.J.; Beattie, G.A.; Hogan, D.A. Roles of three transporters, CbcXWV, BetT1, and BetT3, in
Pseudomonas aeruginosa choline uptake for catabolism. J. Bacteriol. 2011, 193, 3033–3041. [CrossRef]
137. Huebinger, R.M.; Stones, D.H.; Santos, M.D.S.; Carlson, D.L.; Song, J.; Vaz, D.P.; Keen, E.; Wolf, S.; Orth, K.; Krachler, A.M.
Targeting bacterial adherence inhibits multidrug-resistant Pseudomonas aeruginosa infection following burn injury. Sci. Rep. 2016,
6, 39341. [CrossRef] [PubMed]
138. Wu, L.; Estrada, O.; Zaborina, O.; Bains, M.; Shen, L.; Kohler, J.E.; Patel, N.; Musch, M.W.; Chang, E.B.; Fu, Y.-X.; et al. Recognition
of Host Immune Activation by Pseudomonas aeruginosa. Science 2005, 309, 774. [CrossRef] [PubMed]
139. Metruccio, M.M.E.; Evans, D.J.; Gabriel, M.M.; Kadurugamuwa, J.L.; Fleiszig, S.M.J. Pseudomonas aeruginosa Outer Membrane
Vesicles Triggered by Human Mucosal Fluid and Lysozyme Can Prime Host Tissue Surfaces for Bacterial Adhesion. Front.
Microbiol. 2016, 7, 871. [CrossRef]
140. Moghaddam, E.K.; Owlia, P.; Jahangiri, A.; Rasooli, I.; Rahbar, M.R.; Aghajani, M. Conserved OprF as a Selective Immunogen
against Pseudomonas aeruginosa. Iran J. Pathol. 2017, 12, 165–170. [CrossRef]
141. Ezhang, W.; Esun, J.; Eding, W.; Lin, J.; Tian, R.; Elu, L.; Eliu, X.; Eshen, X.; Eqian, P.-Y. Extracellular matrix-associated proteins
form an integral and dynamic system during Pseudomonas aeruginosa biofilm development. Front. Cell. Infect. Microbiol. 2015, 5,
40. [CrossRef]
142. Pier, G. Pseudomonas aeruginosa lipopolysaccharide: A major virulence factor, initiator of inflammation and target for effective
immunity. Int. J. Med. Microbiol. 2007, 297, 277–295. [CrossRef]
143. Kronborg, G.; Fomsgaard, A.; Galanos, C.; Freudenberg, M.A.; Høiby, N. Antibody responses to lipid A, core, and O sugars of the
Pseudomonas aeruginosa lipopolysaccharide in chronically infected cystic fibrosis patients. J. Clin. Microbiol. 1992, 30, 1848–1855.
[CrossRef]
144. Cryz, S.J.; Sadoff, J.C.; Fürer, E. Octavalent Pseudomonas aeruginosa O-polysaccharide-toxin A conjugate vaccine. Microb. Pathog.
1989, 6, 75–80. [CrossRef]
145. Hastie, A.T.; Hingley, S.T.; Higgins, M.L.; Kueppers, F.; Shryock, T. Rhamnolipid from Pseudomonas aeruginosa inactivates
mammalian tracheal ciliary axonemes. Cell. Motil. Cytoskeleton. 1986, 6, 502–509. [CrossRef]
Antibiotics 2021, 10, 1530 30 of 31

146. Caiazza, N.C.; Shanks, R.M.Q.; O’Toole, G.A. Rhamnolipids Modulate Swarming Motility Patterns of Pseudomonas aeruginosa. J.
Bacteriol. 2005, 187, 7351–7361. [CrossRef]
147. Zhu, H.; Bandara, R.; Conibear, T.C.R.; Thuruthyil, S.J.; Rice, S.A.; Kjelleberg, S.; Givskov, M.; Willcox, M.D.P. Pseudomonas
aeruginosa with LasI Quorum-Sensing Deficiency during Corneal Infection. Investig. Ophthalmol. Vis. Sci. 2004, 45, 1897–1903.
[CrossRef]
148. Alarcon, I.; Kwan, L.; Yu, C.; Evans, D.J.; Fleiszig, S.M.J. Role of the corneal epithelial basement membrane in ocular defense
against Pseudomonas aeruginosa. Infect. Immun. 2009, 77, 3264–3271. [CrossRef]
149. Giagkas, D.C.; Choli-Papadopoulou, T.; Pantazaki, A.A. Development of an Antibody for Detection of Rhamnolipids Character-
ized as a Major Bacterial Virulence Factor. Antibodies 2013, 2, 501–516. [CrossRef]
150. Ma, L.; Lu, H.; Sprinkle, A.; Parsek, M.R.; Wozniak, D.J. Pseudomonas aeruginosa Psl Is a Galactose- and Mannose-Rich Exopolysac-
charide. J. Bacteriol. 2007, 189, 8353–8356. [CrossRef] [PubMed]
151. Pier, G.B.; Matthews, W.J., Jr.; Eardley, D.D. Immunochemical Characterization of the Mucoid Exopolysaccharide of Pseudomonas
aeruginosa. J. Infect. Dis. 1983, 147, 494–503. [CrossRef] [PubMed]
152. Liu, X.; Pop, L.M.; Vitetta, E.S. Engineering therapeutic monoclonal antibodies. Immunol. Rev. 2008, 222, 9–27. [CrossRef]
[PubMed]
153. May, T.B.; Shinabarger, D.; Maharaj, R.; Kato, J.; Chu, L.; DeVault, J.D.; Roychoudhury, S.; Zielinski, N.A.; Berry, A.; Rothmel, R.K.
Alginate synthesis by Pseudomonas aeruginosa: A key pathogenic factor in chronic pulmonary infections of cystic fibrosis patients.
Clin. Microbiol. Rev. 1991, 4, 191–206. [CrossRef]
154. McCaslin, C.A.; Petrusca, D.N.; Poirier, C.; Serban, K.A.; Anderson, G.G.; Petrache, I. Impact of alginate-producing Pseudomonas
aeruginosa on alveolar macrophage apoptotic cell clearance. J. Cyst. Fibros. Off. J. Eur. Cyst. Fibros. Soc. 2015, 14, 70–77. [CrossRef]
155. Ellis, T.N.; Kuehn, M.J. Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles. Microbiol. Mol. Biol. Rev.
2010, 74, 81. [CrossRef] [PubMed]
156. Augustin, D.K.; Heimer, S.R.; Tam, C.; Li, W.Y.; Le Due, J.M.; Evans, D.J.; Fleiszig, S.M.J. Role of defensins in corneal epithelial
barrier function against Pseudomonas aeruginosa traversal. Infect. Immun. 2011, 79, 595–605. [CrossRef]
157. Baumgarten, T.; Sperling, S.; Seifert, J.; Von Bergen, M.; Steiniger, F.; Wick, L.Y.; Heipieper, H.J. Membrane vesicle formation as a
multiple-stress response mechanism enhances Pseudomonas putida DOT-T1E cell surface hydrophobicity and biofilm formation.
Appl. Environ. Microbiol. 2012, 78, 6217–6224. [CrossRef]
158. Bloemberg, G.V.; O’Toole, G.A.; Lugtenberg, B.J.; Kolter, R. Green fluorescent protein as a marker for Pseudomonas spp. Appl.
Environ. Microbiol. 1997, 63, 4543–4551. [CrossRef]
159. Bomberger, J.M.; Maceachran, D.P.; Coutermarsh, B.A.; Ye, S.; O’Toole, G.A.; Stanton, B.A. Long-distance delivery of bacterial
virulence factors by Pseudomonas aeruginosa outer membrane vesicles. PLoS Pathog. 2009, 5, e1000382. [CrossRef]
160. Koeppen, K.; Barnaby, R.; Jackson, A.A.; Gerber, S.A.; Hogan, D.A.; Stanton, B.A. Tobramycin reduces key virulence determinants
in the proteome of Pseudomonas aeruginosa outer membrane vesicles. PLoS ONE 2019, 14, e0211290. [CrossRef]
161. Potvin, E.; Lehoux, D.E.; Kukavica-Ibrulj, I.; Richard, K.L.; Lau, G.W.; Levesque, R.C. In vivo functional genomics of Pseudomonas
aeruginosa for high-throughput screening of new virulence factors and antibacterial targets. Environ. Microbiol. 2003, 5, 1294–1308.
[CrossRef]
162. Comolli, J.C.; Hauser, A.R.; Waite, L.; Whitchurch, C.B.; Mattick, J.S.; Engel, J.N. Pseudomonas aeruginosa Gene Products PilT and
PilU Are Required for Cytotoxicity In Vitro and Virulence in a Mouse Model of Acute Pneumonia. Infect. Immun. 1999, 67, 3625.
[CrossRef]
163. Burrows, L.L. Pseudomonas aeruginosa Twitching Motility: Type IV Pili in Action. Annu. Rev. Microbiol. 2012, 66, 493–520.
[CrossRef]
164. Leighton, T.L.; Mok, M.C.; Junop, M.S.; Howell, P.L.; Burrows, L.L. Conserved, unstructured regions in Pseudomonas aeruginosa
PilO are important for type IVa pilus function. Sci. Rep. 2018, 8, 2600. [CrossRef]
165. Curran, C.S.; Bolig, T.; Torabi-Parizi, P. Mechanisms and Targeted Therapies for Pseudomonas aeruginosa Lung Infection. Am. J.
Respir. Crit. Care Med. 2018, 197, 708–727. [CrossRef]
166. Campodónico, V.L.; Llosa, N.J.; Grout, M.; Döring, G.; Maira-Litrán, T.; Pier, G.B. Evaluation of Flagella and Flagellin of
Pseudomonas aeruginosa as Vaccines. Infect. Immun. 2010, 78, 746–755. [CrossRef]
167. Yeung, Y.A.; Foletti, D.; Deng, X.; Abdiche, Y.; Strop, P.; Glanville, J.; Pitts, S.; Lindquist, K.; Sundar, P.D.; Sirota, M.; et al.
Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire. Nat. Commun.
2016, 7, 13376. [CrossRef] [PubMed]
168. Peek, M.E.; Bhatnagar, A.; McCarty, N.A.; Zughaier, S.M. Pyoverdine, the Major Siderophore in Pseudomonas aeruginosa, Evades
NGAL Recognition. Interdiscip. Perspect. Infect. Dis. 2012, 2012, 843509. [CrossRef]
169. Kang, D.; Kirienko, D.R.; Webster, P.; Fisher, A.L.; Kirienko, N.V. Pyoverdine, a siderophore from Pseudomonas aeruginosa,
translocates into C. elegans, removes iron, and activates a distinct host response. Virulence 2018, 9, 804–817. [CrossRef]
170. Cezard, C.; Farvacques, N.; Sonnet, P. Sonnet Chemistry and Biology of Pyoverdines, Pseudomonas Primary Siderophores. Curr.
Med. Chem. 2015, 22, 165–186. [CrossRef]
171. Orazi, G.; O’Toole, G.A. Pseudomonas aeruginosa Alters Staphylococcus aureus Sensitivity to Vancomycin in a Biofilm Model of
Cystic Fibrosis Infection. mBio 2017, 8, e00873-17. [CrossRef]
Antibiotics 2021, 10, 1530 31 of 31

172. Braud, A.; Hannauer, M.; Mislin, G.L.A.; Schalk, I.J. The Pseudomonas aeruginosa Pyochelin-Iron Uptake Pathway and Its Metal
Specificity. J. Bacteriol. 2009, 191, 3517. [CrossRef]
173. Cox, M.J.; Allgaier, M.; Taylor, B.; Baek, M.S.; Huang, Y.J.; Daly, R.A.; Karaoz, U.; Andersen, G.L.; Brown, R.; Fujimura, K.E.; et al.
Airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients. PLoS ONE 2010, 5, e11044. [CrossRef]
174. Arevalo-Ferro, C.; Hentzer, M.; Reil, G.; Görg, A.; Kjelleberg, S.; Givskov, M.; Riedel, K.; Eberl, L. Identification of quorum-sensing
regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics. Environ. Microbiol. 2003, 5, 1350–1369.
[CrossRef]
175. Wandersman, C.; Delepelaire, P. Bacterial Iron Sources: From Siderophores to Hemophores. Annu. Rev. Microbiol. 2004, 58,
611–647. [CrossRef]
176. Létoffé, S.; Redeker, V.; Wandersman, C. Isolation and characterization of an extracellular haem-binding protein from Pseudomonas
aeruginosa that shares function and sequence similarities with the Serratia marcescens HasA haemophore. Mol. Microbiol. 1998, 28,
1223–1234. [CrossRef]
177. Bhagirath, A.Y.; Li, Y.; Somayajula, D.; Dadashi, M.; Badr, S.; Duan, K. Cystic fibrosis lung environment and Pseudomonas
aeruginosa infection. BMC Pulm. Med. 2016, 16, 174. [CrossRef]
178. Tam, V.C. Lipidomic profiling of bioactive lipids by mass spectrometry during microbial infections. Semin. Immunol. 2013, 25,
240–248. [CrossRef] [PubMed]
179. Dennis, E.A.; Norris, P.C. Eicosanoid storm in infection and inflammation. Nat. Rev. Immunol. 2015, 15, 511–523. [CrossRef]
180. Morello, E.; Pérez-Berezo, T.; Boisseau, C.; Baranek, T.; Guillon, A.; Bréa, D.; Lanotte, P.; Carpena, X.; Pietrancosta, N.; Hervé,
V.; et al. Pseudomonas aeruginosa Lipoxygenase LoxA Contributes to Lung Infection by Altering the Host Immune Lipid
Signaling. Front. Microbiol. 2019, 10, 1826. [CrossRef]
181. Serhan, C.N.; Chiang, N.; Van Dyke, T.E. Resolving inflammation: Dual anti-inflammatory and pro-resolution lipid mediators.
Nat. Rev. Immunol. 2008, 8, 349–361. [CrossRef] [PubMed]
182. Alonzo, F., 3rd; Torres, V.J. The bicomponent pore-forming leucocidins of Staphylococcus aureus. Microbiol. Mol. Biol. Rev. MMBR
2014, 78, 199–230. [CrossRef] [PubMed]
183. Scharmann, W. Cytotoxic effects of leukocidin from Pseudomonas aeruginosa on polymorphonuclear leukocytes from cattle.
Infect. Immun. 1976, 13, 836–843. [CrossRef]
184. Abbas, A.; Lichtman, A.; Pillai, S. Basic Immunology: Functions and Disorders of the Immune System, 5th ed.; Elsevier: Amsterdam,
The Netherlands, 2016.
185. Development of Antibody-Based Therapeutics—Translational Considerations. Available online: http://www.springer.com/
biomed/pharmacology+%26+toxicology/book/978-1-4419-5953-9 (accessed on 24 September 2014).
186. Hoffman, W.; Lakkis, F.G.; Chalasani, G. B Cells, Antibodies, and More. Clin. J. Am. Soc. Nephrol. CJASN 2016, 11, 137–154.
[CrossRef]
187. Ascoli, C.A.; Aggeler, B. Overlooked benefits of using polyclonal antibodies. BioTechniques 2018, 65, 127–136. [CrossRef]
188. Arnold, J.N.; Wormald, M.R.; Sim, R.B.; Rudd, P.M.; Dwek, R.A. The Impact of Glycosylation on the Biological Function and
Structure of Human Immunoglobulins. Annu. Rev. Immunol. 2007, 25, 21–50. [CrossRef]
189. Justiz Vaillant, A.A.; Ramphul, K. Immunoglobulin. In StatPearls; StatPearls Publishing: Treasure Island, FL, USA, 2020. Available
online: http://www.ncbi.nlm.nih.gov/books/NBK513460/ (accessed on 10 August 2020).
190. Domenech, M.; Sempere, J.; de Miguel, S.; Yuste, J. Combination of Antibodies and Antibiotics as a Promising Strategy Against
Multidrug-Resistant Pathogens of the Respiratory Tract. Front. Immunol. 2018, 9, 2700. [CrossRef]
191. Mariathasan, S.; Tan, M.-W. Antibody–Antibiotic Conjugates: A Novel Therapeutic Platform against Bacterial Infections. Trends
Mol. Med. 2017, 23, 135–149. [CrossRef] [PubMed]
192. Yuste, J.; Botto, M.; Bottoms, S.E.; Brown, J.S. Serum Amyloid P Aids Complement-Mediated Immunity to Streptococcus
pneumoniae. PLoS Pathog. 2007, 3, 1208–1219. [CrossRef] [PubMed]
193. Subrt, N.; Mesak, L.R.; Davies, J. Modulation of virulence gene expression by cell wall active antibiotics in Staphylococcus aureus.
J. Antimicrob. Chemother. 2011, 66, 979–984. [CrossRef]
194. Lehar, S.M.; Pillow, T.; Xu, M.; Staben, L.; Kajihara, K.K.; Vandlen, R.; DePalatis, L.; Raab, H.; Hazenbos, W.L.; Morisaki, J.H.; et al.
Novel antibody–antibiotic conjugate eliminates intracellular S. aureus. Nature 2015, 527, 323–328. [CrossRef]
195. Hansel, T.T.; Kropshofer, H.; Singer, T.; Mitchell, J.A.; George, A.J.T. The safety and side effects of monoclonal antibodies. Nat.
Rev. Drug. Discov. 2010, 9, 325–338. [CrossRef] [PubMed]
196. Lu, R.-M.; Hwang, Y.-C.; Liu, I.-J.; Lee, C.-C.; Tsai, H.-Z.; Li, H.-J.; Wu, H.-C. Development of therapeutic antibodies for the
treatment of diseases. J. Biomed. Sci. 2020, 27, 1–30. [CrossRef]
197. Mulcahy, L.R.; Isabella, V.M.; Lewis, K. Pseudomonas aeruginosa biofilms in disease. Microb. Ecol. 2014, 68, 1–12. [CrossRef]
198. Vonarburg, C.; Loetscher, M.; Spycher, M.O.; Kropf, A.; Illi, M.; Salmon, S.; Roberts, S.; Steinfuehrer, K.; Campbell, I.; Koernig,
S.; et al. Topical application of nebulized human IgG, IgA and IgAM in the lungs of rats and non-human primates. Respir. Res.
2019, 20, 99. [CrossRef]
199. Jones, R.G.A.; Martino, A. Targeted localized use of therapeutic antibodies: A review of non-systemic, topical and oral applications.
Crit. Rev. Biotechnol. 2015, 36, 506–520. [CrossRef]