Bacterial Ghost as Delivery System

Prepared by : Salma A. Elfaham

Supervised by : Prof. Ahmed Hussain
Dr. Hoda Eid
Questions that will be covered through out the presentation

1. How we reached the concept of using bacteria as a drug

delivery system
2. Properties of bacterial ghosts & is it considered as GMO?
3. Methods for preparing BGs : E-mediated lysis & chemical
induction
4. Why BG as carriers of Antigen, Nucleic acid and drugs should
find their clinical applications
5. How BG can represent a promising technology platform for
novel Probiotic .
6. Advantages, drawbacks and challenges
The idea of using bacteria as a vector for drug delivery … !!

1819
Cancer patient
+ Regression
Clostridium in cancer our enemy's enemy could
perfringens cells be our friend
infection = patient is
(gas gangrene ) cured

Bacteria offer an Why ???

oncolytic activity
The idea of using bacteria as a vector for drug delivery … !!

1868

patient with The patient's

sarcoma cancer tumor shrunk
+ by half in a
accidentally week, but she
placed onto the died from the
bed of another Group A infection after
patient with an streptococcus (GAS), nine days
erysipelas is a facultative Gram-
infection. positive.
The idea of using bacteria as a vector for drug delivery … !!

William B. Coley cured a patient with malignant

In early
sarcoma of the neck cancer by introducing a severe
1900s erysipelas infection twice .

Dr. Coley found the patient still alive seven

years later, after which he repeatedly
applied the bacterial infection to treat cancers
The idea of using bacteria as a vector for drug delivery … !!

Strict anaerobes & faculitative

1955 anaerobes selectively colonize
In hypoxic & necrotic regions of solid tumor
Despite this
anecdotal clinical exclusive localization of
evidence, research bacteria inside the tumorous
into bacteria- tissues Infection-mediated oncolysis
mediated (destruction of tumor cells) was
cancer therapy did often accompanied by acute
not begin until the toxic effects , septic shock , and
mid-1900s death .
The idea of using bacteria as a vector for drug delivery … !!

It did not take long to realize

Therefore the tumor targeting strains that the same tool, namely
Salmonella, Bifidobacteria and genetic manipulation, could be
Clostridia had to be manipulated used to morph tumor targeting
genetically to reduce their bacteria into vectors for
pathogenicity
delivering therapeutic proteins
to tumor
 What is meant by bacterial ghost ?

 Empty non denatured cell envelopes of Gram –ve bacteria :

- devoid of any cytoplasmic content
- devoid of chromosomal and plasmid DNA
 What is meant by bacterial ghost ?

Transmission electron micrograph of an E. coli BG, im, inner membrane; om, outer
membrane. On the left edge and below the BG parts of full viable bacteria are
visible with contrasted cytoplasmic content in contrast to the empty inner
cytoplasmic lumen of the BG.
 Properties of bacterial ghost ?

 An important property for BGs that their

outer surface retain all their appendices
• pili PAMPs
• flagella Pathogen-associated molecular
• Lipopolysaccharide of the parent bacteria patterns

They give the BGs its natural outer surface make-

up , which provide them with the original targeting
functions of the bacteria they are derived from .
Thus , they are able to bind to or taken up by
specific tissues or cells.
 Properties of bacterial ghost ?

 BGs are devoid of any cytoplasmic content so the carrier capacity of

the inner cytoplasmic lumen provides an intracellular space of
approximately 250 femtoliter per BG.
 Properties of bacterial ghost ?

 The final BG preparations are freeze dried (lyophilized)

and are stable for many years at room temperature.
 Methods to prepare BGs ?

E-mediated-lysis Chemically
Method induced-lysis
Method
Most
Recently
common
used
2013
 How to prepare BGs --E-mediated-lysis Method

The concept of BGs has been emerged from the lysis mechanism of
bacteriophage PhiX174 after infection of Escherichia coli in 1966.

1. transformation of the host bacterium with a plasmid which carries the gene E
under an inducible promoter
2. Gene E  protein E
3. protein E is able to fuse the inner and outer membranes of Gram-negative
bacteria, thereby forming a transmembrane lysis tunnel in the bacterial
envelope through which the cytoplasmic content is released.
4. E-specific Transmembrane lysis tunnel is not randomly distributed all over the cell
envelope ,but is restricted to areas of potential division sites.

E-mediated lysis
 How to prepare BGs ?

Since Gene E product is very lethal for the recipient , the transforming plasmoid must contain Repressor system
How ?
Repressor systems can be : Temperature regulated or chemically inducible

• Using phage lamda promotor operator system for gene E works at 42°-44°C
• Controlled by thermosenstive c1857 repressor works at lower temp.
• So, by shifting temp. to 39°-44°C , gene E will be expressed
Left: Schematic representation of the ghost formation in Escherichia coli (Hjelm et al, 2015). Right: Structured
Illumination Microscopy time-lapse imaging of the generation of bacterial ghosts. Two consecutive images
capturing the generation of a ghost (red arrow). Time resolution ~ 500 ms.
 E- mediated lysis under micrograph

High resolution field emission scanning electron micrograph of protein E-lysed Gram-
negative bacteria. An arrow indicates the efflux of bacterial cytoplasm at the time
point of lysis onset through the E-specific lysis tunnel.
 E-Iysis system is restricted to Gram-negative bacteria

Gram-negative bacteria has an inner and an outer membrane. In

contrast, the large group of Gram-positive bacteria are killed but not
lysed by gene E expression
 Methods to prepare BGs ?

E-mediated-lysis Chemically
Method induced-lysis
Method
Most Recently
common used
 How to prepare BGs –Chemically induced Method

 The protocol depends on :

1- growing the bacterial cells in condition allowing a correct cell wall formation
2- subject the cells (in stationary growth phase) to chemicals
 We choose chemicals according to their permeabilization effect on call wall
 based on the MIC/MGC of each chemical

(Minimum Inhibition/growth Concentration)

MIC will give us the minimum quantity of the used
chemicals responsible for preventing bacterial growth ---
complete cells lysis should be avoided
 How to prepare BGs –Chemically induced Method

In a previous protocols  these chemicals were used at their MIC/MGC

• Hydrogen peroxide
(produce hydroxyl free radical , but since gram +ve lack catalase, this limit the its Effect
on gram +ve )
2013 • Sodium Hydroxide
• Acetic acid , Boric acid , Hydrochloric acid , Sulfuric acid
2018 • Tween 80
(non ionic surfactant that solubilize hydrophobic component )
 E-mediated lysis VS Chemically induced lysis

Limitation to gram negative bacteria Applied on both gram negative, positive, yeast.
Formation of one pore Not certain about number of pores &
With known pore size Unknown pore size
( diameter 40-80 nm ) Further investigation is needed to study their utilization
as a drug delivery systems, biological carriers or vaccines

Multi step process that is cost expensive More simple , economic & feasible.
and time consuming
Need experience & stringent process control
Some surface structures in the LPS may be lost or
Maintain LPS on their cell envelops in same modified .
way as untreated wild-type bacterial cell.
Using some chemicals as NaOH that displays
Alkaline Hydrolysis leads to breaking the link
between Lipid A and the core polysaccharide.
 BGs are not considered as GMOs !

 Although the use of plasmid encoded genetic information is essential for the final make up of BG,
BG are not to be considered as generically manipulated organisms (GMO)

Why ?
as they are nonliving and
devoid of genetic information

This aspect is of great importance for safety

as no pathogenic islands or antibiotic resistant genes
can be transferred to other bacteria by horizontal gene
transfer
 BGs are derived from :

BGs have been produced from various non-pathogenic, pathogenic

and probiotic strains :
PROBIOTIC STRAIN : play role in protection against GIT
 E.coli K12 infections
 E.coli Nissle 1917
 Salmonella enterica
 Vibrio Cholerae
 Helicobacter Pylori
 Mannheimia haemolytica colonic commensal
 Bacterial ghost

Principle
Mimic living
Have all
Maintain bacteria , so
structural,antigens
Native cell can be
and bioadhesive
wall attached to
properties of
components specific target
original bacterium
tissues or cells

 Envelopes made from

pathogenic enterobacteria, for instance, could be
used to target the gastrointestinal tract
 Applications of BGs as Delivery Systems

Candidate
Vaccine
itself

Vaccine ANTIGEN
NUCLEIC DNA
ACID vaccine
Tumor therapy DRUG
 Bacterial ghost as drug,DNA,antigen delivery system

Depending on the bacterial ghost species , it can be targeted to

( BGs can be phagocytosed by : .. )

1- Antigen presenting cells : Macrophages & dendritic cells to deliver Ag as Vaccine

2 Tumor cells : Melanoma , colon cancer & leukemia to deliver drug

3- Micro vascular endothelial cells ( The walls of capillaries are composed of a single
layer of micro vascular endothelial cells)
HOW BGs can be recognized by Antigen Presenting Cells ?

The Key reason is the PAMPs present on the BGs ( LPS )

they can be easily recognized by APC

thus phagocytized + stimulate immune response against the
carried Ag .
 HOW BGs can be recognized by Tumor Cells ?

TLR 4
Normally , All antigen presenting cells recognize pathogens ( PAMPS )
APC by the help of toll like receptors present on their surface .

TOLL like receptor 4 is expressed in certain types of tumor

( melanoma )
Mechanism 1

TLR 4 This may explain why melanoma cells have the

Tumor capacity to behave as non-professional APCs and can phagocyte
cell both apoptotic and live cells
stimulates a potent immune response in the
tumor area
 Damage-associated molecular patterns

Mechanism 2

Initiate
inflammatory
response

Cytotoxic T cells
that kills Cancer cell
 Why would we use BGs as Delivery
System , when we already have other
biological & Synthetic carriers ?
They have to be pathogenic in order to be immunogenic
Biological
carriers
Synthetic
carriers

Anti neoplastic drug currently used.

Liposome offers non-sufficient targeting
ability to Doxorubicin

Cardio toxic side effect still persist

 Why would we use BGs as Delivery System , when we
already have other biological & Synthetic carriers ?

1. They can be used as vaccine candidates themselves without the need of other adjuvant .
2. They can be employed as a delivery system for proteins/antigens, nucleic acids, drugs and
soluble compounds for various medical and technical applications.
3. Excellent carrier capacity
4. Are taken up very effectively by antigen-presenting cells, particularly suited as vaccines
for mucosal administration by oral, intranasal.
5. Specific Targeting properties for different tissues ( unlike liposomes ) in drug delivery.
6. Non-Living envelopes ,No Horizontal gene transfer, safe for immunocompromised
patients
7. Studies have shown that they completely preserve the enzymatic activity
of enzymes , and thus BG can be introduced as novel probiotics by carrying specific
enzymes with a certain preference for the gut system.
BGs offer several compartment for
Delivery of foreign antigen , DNA ,
Drug compared to other carriers
 Schematic line drawings of bacterial ghosts and their potential
Applications & possible ways to overcome leakage !

Empty BG BG with inner

membrane anchored
antigen

BG as carrier for antigen

in periplasmic space BG as carrier for immobilized
antigen

Biotinylated polymer
BG as carrier for nucleic acid
(dextran) carrying
drug is attached to Streptavidin anchored
streptavidin BG as carrier of water-
soluble active substances
BG as carrier of inner
membrane attached
active substances: vesicle attached by
drug, e.g., specific streptavidin-
doxorubicin. biotin interaction
 Depending on the high affinity of Streptavidin to Biotin
molecules with organic rings
binds unspecifically to
membrane component
attachment to the Foreign protein
outer localization within
membrane as bacterial
fusion proteins ghosts is performed by
with OmpA or pili fusion with specific anchor
sequences for attachment
on inner membrane

target Ag
exported into the
periplasmic space
by fusion
to the MalE signal
sequence
Nowadays , Where do we
stand with BGs as delivery
system ?
Applications of BGs as Delivery Systems
1
Doxorubicin-loaded bacterial ghosts have been used to
target colon carcinoma cells.
DOX bounded Mannheimia haemolytica
BG reduced cell proliferation with lower concentration of drug up to 300
times more effectively than using free drug . Therefore ,minimizing the
negative effects of drug .
Doxorubicin-loaded bacterial ghosts have been used to target colon
carcinoma cells.

To overcome these undesired effects of DOX without reducing drug potency

DOX is encapsulated in drug delivery vehicle that should allow

a site-specific targeting and prolong drug circulation time within the body

Reduce negative Allow the use of

impact on patients decreased drug
dosages
 Advantage of using BGs as Drug Delivery Vehicle

Selective Dramatic Decrease

increase in Reduction of
delivery of the required the side
drugs to efficiency of
particular medical dose of effect of
treatment
tissue
treatment drug
2
BGs as Enzyme reactors for Novel Probiotics

Here, the proteins of interest have enzymatic activity and the whole
construct is designed for carrying out metabolic activity .

 BG as carrier of beta-galactosidase would be able to cleave the milk sugar lactose

in glucose and galactose to avoid lactose intolerance

 BG as carrier of polyamine hydrolase to avoid unpleasant allergic or sickness

effects from food

There is a whole package of other metabolic enzymes that could support

digestion or make novel Substrates available as nutritive source
To sum up :

1. Non-living carriers  no risk of reversal to pathogenic form

2. long room temperature storage life after lyophilization
3. Strong adjuvant properties
4. Good recognition and uptake by antigen-presenting cells
5. High loading capacity for DNA ( 4000-6000 ) plasmid copy number .
6. Targeting properties for different tissues
7. Ability to express recombinant proteins in a variety of locations on the
Ghost