Capitulo 1 Mandell

1
Basic Principles in the

Diagnosis and Management
of Infectious Diseases
I
A Microbial Pathogenesis
A Molecular Perspective of Microbial

1 Pathogenicity
David A. Relman* and Stanley Falkow
DIVERSITY OF HUMAN-MICROBE exposure to more than 100 bacterial phyla in the surrounding environ-
RELATIONSHIPS ment, members of the phyla Firmicutes, Bacteroidetes, Actinobacteria,
Beginning immediately at birth, humans are colonized by a myriad of Proteobacteria, and Fusobacteria dominate human body sites, suggest-
microorganisms that assemble into complex stereotypic communities, ing a role for strong selective forces and microbial diversification over
creating a beneficial indigenous microbiota. The result is a “supra- hundreds of thousands of years of coevolution with their host. Within
organism” in which microbial symbionts outnumber human cells by the domain Archaea, diversity in the human body is apparently limited
10-fold. Most currently available information about the human indig- to a handful of methanogen species: Methanobrevibacter smithii is
enous microbiota concerns the bacterial component, although they are commonly found in the healthy distal gut, and Methanobrevibacter-
by no means the only important members. Bacteria are the focus of related species are found in the inflamed subgingival crevice in some
the following discussion. patients with moderate or severe chronic periodontitis. Of interest,
In contrast to the relatively rare harmful encounters with patho- patterns of bacterial diversity in humans display individual-specific
gens, indigenous human-microbe relationships in which either features. The distinctness of an individual’s microbiota is less evident
microbe or host benefits without causing harm (commensal relation- when viewed in terms of the overall functional capabilities of the
ships) and relationships in which both benefit (mutualistic relation- community, rather than in terms of the names and relatedness of the
ships) are the dominant forms of interaction and are fundamentally strains and species6; this difference probably reflects the functional
important to human biology. Coevolution, co-adaptation, and co- redundancy of strains and species within the human microbiota,
dependency are features of our relationships with our indigenous which, in turn, may contribute to stability of this ecologic system. Yet,
microbiota.1 The human microbiota facilitates nutrient acquisition and differences in the capability of strains may explain variation among
energy extraction from food, promotes terminal (postnatal) differen- individuals in the metabolism of drugs such as digoxin and other
tiation of mucosal structure and function, and stimulates both the exogenous chemicals.7 Differences in the capability of strains to toler-
innate and adaptive immune systems. By so doing, it helps to maintain ate normal inflammation may also influence the composition of the
epithelial boundary function and integrity, as well as to “educate” our microbiota. Although there is evidence for shared functional capabili-
innate immune defenses. It also provides “colonization resistance” ties among the intestinal microbial communities of different healthy
against pathogen invasion, regulates intermediary metabolism, pro- humans, host genetics is a source of variation in the makeup of the
cesses ingested chemicals, and provides small amounts of human human indigenous microbiota.8
accessory growth factors.2,3 The rules and features of microbial com- Infection (or colonization) is simply the establishment of a micro-
munity assembly are fundamentally important but, so far, are poorly organism on or within a host; it may be short lived, as in our encoun-
understood.4 In the neonatal period, the community assembly process ters with “transients” (Table 1-1), or be persistent and may result in
is especially dynamic and is influenced by early environmental (in only low gain or harm to either participant. The term infectious disease
particular, maternal) exposures and stochastic effects. The composition applies when an interaction with a microbe causes damage to the host
and functional capabilities of the indigenous microbiota evolve in a and the associated damage or altered physiology results in clinical signs
generally orderly fashion, as diet, hormonal environment, other envi- and symptoms of disease. A pathogen is usually defined as any micro-
ronmental factors, and occasional ecologic disturbances play out their organism that has the capacity to cause disease. It is a medical defini-
effects on a distinct, albeit diverse, human genetic background.5 tion; it is not a biologic definition, and certainly, not all pathogens have
Bacterial diversity in the indigenous communities of the human an equal probability of causing clinically apparent disease. Virulence
body is striking in its richness of distinct species and strains but also provides a quantitative measure of pathogenicity or the likelihood of
noteworthy for the limited number of phyla commonly found. Despite causing disease. For example, encapsulated pneumococci are more
virulent than nonencapsulated pneumococci, and Escherichia coli
*All material in this chapter is in the public domain, with the exception of any borrowed strains that express Shiga-like toxins are more virulent than those that
figures or tables. do not express these toxins. Virulence factors refer to the properties
1
Downloaded for Anonymous User (n/a) at Universidad Nacional Autonoma de Mexico from ClinicalKey.com by Elsevier on June 05, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
1.e1
KEYWORDS
bacteriophage; clonality; commensal; commensalism; diagnostics;
ecology; evolution; genomics; horizontal gene transfer; infectious
Chapter 1 A Molecular Perspective of Microbial Pathogenicity

diseases; intracellular parasites; metagenomics; microbiota;
opportunistic infection; pathogen; pathogenicity island; plasmid;
population biology; regulation of virulence; virulence
2
TABLE 1-1 Microbe-Human Host Interactions of the community and live in a perilous location—associated with
respiratory tract lymphatic tissue, where they regularly come into
Transient A microorganism that we encounter in our food contact with elements of an immune system that hold them at bay most
or elsewhere in our environment. In general,
of the time but occasionally fail to do so, resulting in disease?
Part I Basic Principles in the Diagnosis and Management of Infectious Diseases
it is just “passing through” and of little

consequence; however, regular encounters An understanding of the definition of a pathogen is not required
over extended periods of time might lead to when a clinician is faced with an infected patient who needs treatment.
host adaptation or even dependence. However, if we are to understand disease-associated microbes and
Commensal (literally, those A microorganism that is a normal inhabitant of discover effective therapies, we will also need to appreciate their fun-
that “eat at the same the human body. In commensal relationships,
table”) either the microbe or host derives benefit; in
damental biology and ecologic niche. And it is important to be
mutualistic relationships, both derive benefit. reminded that antibiotics do not always, and are increasingly less likely
Pathogen (derived from the A microbe that may or may not be a member to, work against many pathogens; that antibiotics incur a cost, in terms
Greek, pathos, meaning of the indigenous microbiota, but it regularly of resistance and collateral damage to commensals9; and that we still
the “birth of suffering”) causes disease in apparently normal lack effective vaccines against a multitude of infectious agents that are
individuals. encountered in everyday medical practice.
Opportunistic pathogen A microbe that causes disease only in humans Thus, the capacity of certain microorganisms to cause disease in
who are in some way compromised in their
normal defense mechanisms.
healthy, uncompromised human hosts on a regular basis should reflect
fundamental biologic differences in their virulence capabilities from
Accidental pathogen A microorganism that is encountered by
accidental contact with animals, insects, or those of opportunists and commensal species that rarely, if ever, cause
the environment. These microorganisms are disease. In the following sections, we address this issue and discuss how
often deadly in humans and sometimes the insight into pathogenesis has been applied to the practice of contem-
causative agent of disease in other animals. porary infectious disease medicine.
These microbes are often distinguished from
human-specific pathogens because they are
not directly or readily transmissible from ATTRIBUTES OF MICROBIAL
human to human. PATHOGENS
What are the distinguishing characteristics of microbes that live in
humans? A successful pathogen or commensal must do the following:
(e.g., gene products) that enable a microorganism to establish itself (1) enter the human host; (2) become established, which includes suc-
and replicate on or within a specific host species and that enhance the cessful competition with indigenous microbes; (3) acquire nutrients;
microbe’s potential to cause overt pathology. In many ways, what we (4) avoid or circumvent the host’s innate defenses and a powerful
refer to as virulence factors are in the biologic sense colonization immune system; (5) above all, replicate; (6) disseminate if necessary
factors that permit replication in the host and subsequent transmission to a preferred site; and (7) eventually be transmitted to a new suscep-
to a new susceptible host. tible host.
Thus, it is useful to distinguish pathogens that regularly cause Whether a pathogen or a commensal, a microorganism must also
disease in some proportion of susceptible individuals with apparently possess an interactive group of complementary genetic properties,
intact defense systems from other potentially pathogenic microorgan- sometimes coregulated, that promote its interaction with the human
isms, such as Pseudomonas aeruginosa. This microorganism does not host. For a given microorganism, the genetic traits define unique attri-
usually cause disease in individuals with intact host defense systems, butes that enable it to follow a common sequence of steps used in
yet it causes devastating disease in many immunocompromised establishing infection or, in some cases, subsequent disease.10,11
patients. Many microorganisms with a capacity for sustained multipli- Elegant molecular and genetic techniques now permit the identifi-
cation in humans, including members of the indigenous microbiota, cation, isolation, and characterization of many of these genes and their
cause disease more readily in individuals with underlying chronic products. We now also possess the complete genome sequences of
disease or in those who are otherwise compromised. The common virtually every major pathogenic bacterial species. This information
term opportunist suits this category of pathogen well (see Table 1-1). provides important clues and insight into the potential of a microor-
An emerging concept of microbial disease causation, with origins ganism for causing disease and facilitates new experimental strategies
in the field of ecology, is the notion of “community as pathogen,” in for understanding pathogens and commensals alike.12,13 The availabil-
which a conserved broad feature of the microbial community contrib- ity of the host (e.g., human) genome sequence also enables multiple
utes to pathology, rather than any one specific member or component. synergistic approaches for understanding virulence, including the
This concept may be relevant to a wide variety of chronic inflammatory identification of host susceptibility traits, genome-wide assessments of
processes of skin and mucosa, including inflammatory bowel disease host response, and clues about the mechanisms of host defense and
and chronic periodontitis. It suggests that studies of pathogenesis con- pathogen counterdefense.14 It is important to recognize that pathoge-
sider general properties of microbial communities, such as resilience, nicity can only be understood in the context of a specific host.
or conserved functional interactions, such as syntrophy (cross-feeding), These genomic analyses have lent credence to the working hypoth-
rather than the role of single microbes in isolation, especially for devel- esis of almost a half-century of research—that the distinguishing char-
opment of novel approaches for maintaining or restoring health. acteristic of microorganisms that regularly cause disease is a set of
The difficulty, therefore, is that the distinctions between a com- special genetic traits that provide them with the capacity to breach
mensal, an opportunist, and a pathogen can be blurred at times, in part intact host anatomic, cellular, or biochemical barriers that ordinarily
because some commensals cause disease, albeit usually in immuno- prevent entry by other microorganisms into sterile tissue sites. Thus,
compromised hosts, and some of the most feared pathogens can persist pathogens “go where other microbes dare not.” In addition, many
in humans for a lifetime without causing disease symptoms. In addi- pathogens, such as Mycobacterium tuberculosis, Treponema pallidum,
tion, microbial pathogenesis involves synergies between organisms, as Chlamydia trachomatis, and Salmonella typhi, have the capacity to
well as between gene products, each of which may be insufficient alone establish persistent (usually asymptomatic) infection in the human
in causing disease. For example, several members of the human health- host and have evolved the extraordinary capacity to live in the inner
associated nasopharyngeal microbiota, including Streptococcus pneu- sanctums of our innate and adaptive immune defenses or, in general,
moniae, Neisseria meningitidis, and Streptococcus pyogenes, regularly to compete well in the face of otherwise hostile host conditions. For
cause well-defined, well-known human diseases. Immunization against example, Salmonella profits from the inflammatory response that it
the first two of these microbes not only protects against disease but provokes in the gut by using the oxidized form of a locally produced
also prevents, in an antigen-specific fashion, their ability to colonize host factor for a selective growth advantage against commensals.15 A
the host. Such commensal pathogens persist in a significant proportion distinction, then, between a primary pathogen and opportunist is that
of the human population, the vast majority being asymptomatic carri- the pathogen has an inherent ability to breach the host barriers that
ers. Are they pathogens, or are these organisms members of the indig- ordinarily restrict other microbes, whereas the opportunist requires
enous microbiota that have evolved to compete with other members some underlying defect or alteration in the host’s defenses, whether it
3
be genetic, ecologic (altered microbiota), or caused by underlying (transient or long-term infection) and to ensure its successful trans-
disease, to establish itself in a usually privileged host niche. Clearly, the mission to a new susceptible host, while at the same time, no more
nature of the host plays as important a role as the pathogen in deter- than is tolerated by the host as it gains immunity from further incur-
mining outcome.16 sion by the same and even related pathogens.

An initial step required of a pathogen is to gain access to the host Why do some pathogens cause disease more readily than others?
in sufficient numbers. Such access requires that the microorganism not The strategy used for multiplication on or within the host (i.e., its
only make contact with an appropriate surface but also then reach its ability to overcome host barriers) often defines fundamental differ-
unique niche or microenvironment on or within the host. This require- ences between pathogens that commonly cause acute disease symp-
ment is not trivial. Some pathogens must survive for varying periods toms and those that do not. An organism that can reach and multiply
in the external environment. Others have evolved an effective and in privileged anatomic sites away from the competitive environment
efficient means of transmission. To accomplish this goal, the infecting of skin and mucosal surfaces is more likely to disrupt homeostasis in
microbe may make use of motility, chemotactic properties, and adhe- the host and cause disease than one that chooses a different strategy.
sive structures (or adhesins) that mediate binding to specific eukaryotic If a microorganism has evolved a means to nullify or destroy phago-
cell receptors or to other microorganisms.17,18 Pathogens that persist at cytic cells to multiply successfully, it is more likely to be found in
the surface of skin or mucosa usually rely upon multiple redundant deeper compartments and associated with acute disease. Commensal
adhesins and adherence mechanisms. If the adhesin is immunogenic, or mutualistic organisms are restrained so that they multiply just
expression is usually regulated; in addition, antigenic variants may enough, in the midst of competing microbiota, to persist but not
arise (see “Regulation of Bacterial Pathogenicity”). Preexisting micro- damage the host’s self-preserving homeostatic and innate immunity
organisms, the indigenous microbiota, provide competition against mechanisms. It is important to emphasize that a microorganism
establishment of the newcomer; furthermore, the latter must adapt, at equipped to multiply efficiently in a human may be exceptional in the
least temporarily, to the particular nutrient environment in which it biologic sense but unexceptional as a pathogen in the medical sense,
now finds itself. and only infrequently, if ever, a cause of clinically manifested disease.
Normal inherent host defense mechanisms pose the most difficult Some organisms, such as P. aeruginosa, are “only” opportunists in
set of obstacles for pathogens and commensals in establishing them- humans despite their impressive array of virulence factors. These viru-
selves in a host. For any set of specific host defenses, an individual lence factors work well in some plant hosts and in predators it encoun-
pathogen will have a unique and distinctive counterstrategy. Some of ters in the environment. However, for Pseudomonas, these same
the best-known mechanisms that pathogenic microbes use for coun- pathogenic determinants usually fail to overcome the average human’s
tering host defenses include the use of an antiphagocytic capsule and defenses. For opportunistic pathogens, the state of the host is the major
the elaboration of toxins and microbial enzymes that act on host determinant of whether disease is the outcome of their interaction with
immune cells and/or destroy anatomic barriers. Microorganisms also the host. Commensals and mutualists, for example, which are the usual
use subtle biochemical mechanisms to avoid, subvert, or, as we now cause of opportunistic infections, may be very adept at colonization,
increasingly understand, manipulate host defenses. These strategies but because of their preferred growth locale (e.g., at the mucosal
include the elaboration of immunoglobulin-specific proteases, iron surface) and preferred growth conditions (e.g., a microaerobic envi-
sequestration mechanisms, coating themselves with host proteins to ronment), they may have limited growth opportunities outside their
confuse the immune surveillance system, or causing host cells to signal restricted niche in an unimpaired individual. Innate immune factors
inappropriately, leading to dysregulation of host defenses or even host are difficult to overcome for the vast majority of commensals. Little
cell death. Examples of these mechanisms include the production of more than 50 years ago, there was a prevalent view that pathogens had
immunoglobulin A1 protease by the meningococci, the use of recep- undergone retrograde evolution and caused disease because they were
tors for iron-saturated human transferrin and lactoferrin by N. gonor- little more than parasites. Pathogens were then viewed as organisms
rhoeae, and the coating of T. pallidum with human soluble fibronectin. often unadapted to their hosts and that elaborated potent toxins or
Yersinia, Mycobacterium, and Bordetella induce host cell production of other powerful aggressive factors causing the signs and symptoms of
interleukin-10, which is a potent immunosuppressive cytokine, thereby disease. However, bacterial pathogenicity has been redefined over the
downregulating important elements of the innate immune defense. past quarter century by using the tools of molecular genetics, genom-
Antigenic variation and intracellular invasion are other common strat- ics, and cell biology. We can now directly address the question: why
egies used by successful pathogens to avoid immune detection.19,20 The are some bacteria pathogenic for humans, whereas other (closely
intimacy of the relationships between viral pathogens and host is related) bacteria are not?
reflected in the frequency with which these pathogens co-opt host We understand now that pathogenic bacteria are indeed often
molecules and pathways for subverting host defenses (see “Subversion exquisitely adapted microbes that use sophisticated biochemical strate-
of Host Cellular Processes and Immune Defenses”).19,21-23 gies to interfere with, or manipulate for their own benefit, the normal
The ability to multiply is a characteristic of all living organisms. function(s) of the host cell. They are impressive human cell biologists!
Whether the pathogen’s habitat in the relevant host is intracellular or It is now also quite clear that these sophisticated biochemical proper-
extracellular, mucosal or submucosal, within the bloodstream or ties that distinguish pathogens from their nonpathogenic brethren
within another privileged anatomic site, pathogens have evolved a derive from specialized genes possessed by pathogens but absent from
distinct set of biochemical tactics to achieve this goal. The ultimate nonpathogens. The driving force for the inheritance of pathogenic
success of a pathogen, indeed, of any microorganism, is measured by traits is not slow adaptation to the host but, rather, a more dynamic
the degree to which it can multiply. The pathogen must not only rep- process of horizontal (lateral) gene transfer via mobile genetic ele-
licate sufficiently to establish itself in a host on reaching its specific ments. Hence, the genes for many specialized “bacterial” products,
niche, but it also must replicate sufficiently at some point in its life cycle such as toxins and adhesins, actually reside on transposons (“jumping
to ensure its potential transmission to a new susceptible host. The rate genes”) and bacterial viruses (bacteriophages) (Table 1-2). Larger
of pathogen multiplication is appreciated by a clinician in terms of a packets of information have also been shared among bacteria by
characteristic incubation period spanning the time of exposure to the genetic transfer. The lateral inheritance of large blocks of genes, called
appearance of signs and symptoms of disease. pathogenicity islands, is often the key to the expression of pathogenicity
Infectious disease, in one sense, is simply a byproduct of the method in bacteria. Many of these virulence determinants acquired by lateral
and site chosen by pathogens for replication and persistence; disease gene transfer have several features that are apparent by simple inspec-
per se is not a measure of microbial success. Disease, in part, reflects tion of genome sequences, including distinct chromosomal nucleotide
the status of the host as much as it does the virulence characteristics composition and association with plasmids or phages, suggesting that
of the offending microorganism. Death of the host is fortunately a rare their ancestry derives from an unrelated microbe. One surprising
event and one that must be viewed with the dispassion of biology as finding is that the amount of acquired DNA associated with virulence
being detrimental to both parties involved! The usual rules of host- and adaptation to a host habitat in many bacteria can be substantial.
pathogen engagement most often produce a tie: sufficient multiplica- For example, uropathogenic, enterohemorrhagic, and extraintestinal
tion of the pathogen to ensure its establishment within the host types of E. coli all display mosaic genome structure, with hundreds of
4
TABLE 1-2 Examples of Plasmid- and Phage- instead, a general strategy for microbial specialization and success in
Encoded Virulence Determinants some environmental niches that are highly competitive. Why bacteria
have adapted this tactic to maximize their diversity and to increase
VIRULENCE their opportunity for continuing evolution is most likely a reflection
ORGANISM FACTOR BIOLOGIC FUNCTION of their haploid state and their need to conserve fundamental charac-
Plasmid Encoded teristics, such as the ability to live on a mucosal surface, while still
Enterotoxigenic Heat-labile, Activation of adenylate/ being able to try new combinations of genes. The sharing of genes
Escherichia coli heat-stable guanylate cyclase in the among seemingly disparate microorganisms occupying the same niche
enterotoxins small bowel, which leads
to diarrhea in a sense provides these microbes with an endless number of combi-
CFA/I and CFA/II Adherence/colonization
nations of genes for evolutionary experimentation, as it were, within a
factors habitat such as the human intestinal tract.26 Overall, across the bacte-
Extraintestinal E. coli Hemolysin Cytotoxin rial world, the number of such successful experiments resulting in the
Shigella spp. and Gene products Induces internalization by
emergence of a pathogen appears to have been quite rare (see “Clonal
enteroinvasive E. coli involved in invasion intestinal epithelial cells Nature of Bacterial Pathogens”). Yet, when successful, these experi-
Yersinia spp. Adherence factors Attachment/invasion ments are surprisingly efficient, at least from the perspective of the
and gene products microbe, yet manageable, from the standpoint of the host.
involved in invasion Some infectious diseases occur predominantly in dramatic epi-
Bacillus anthracis Edema factor, lethal Edema factor has adenylate demic form, which argues against the establishment of a balanced
factor, and cyclase activity; lethal host-parasite relationship; however, in many such epidemics, mitigat-
protective antigen factor is a metalloprotease
that acts on host signaling
ing circumstances involving herd immunity and other underlying
molecules social, economic, and political issues impinge on this relationship.
Staphylococcus aureus Exfoliative toxin Causes toxic epidermal So-called emerging infectious diseases reflect various aspects of imbal-
necrolysis ance in the relationships between host, pathogen, and environment.27
Clostridium tetani Tetanus neurotoxin Blocks the release Many of the most serious and feared infectious diseases occur when
of inhibitory humans are infected by microorganisms (accidental pathogens) that
neurotransmitter, which prefer and are better adapted to another mammalian host. In fact, most
leads to muscle spasms
emerging infectious diseases in humans are of zoonotic origin.28 As
Phage Encoded seen in many zoonotic diseases, the rules of engagement between the
Corynebacterium Diphtheria toxin Inhibition of eukaryotic host and the pathogen are blurred, often to the detriment of both the
diphtheriae protein synthesis host and the microbe. It is often an evolutionary dead end for both
Streptococcus pyogenes Erythrogenic toxin Rash of scarlet fever parties.
Clostridium botulinum Botulism neurotoxin Blocks synaptic acetylcholine Given the increasingly frequent and unexpected emergence of pre-
release, which leads to viously unrecognized pathogens, it is appropriate to question how well
flaccid paralysis
we appreciate the true diversity and distribution of extant microorgan-
Enterohemorrhagic E. Shiga-like toxin Inhibition of eukaryotic
coli protein synthesis
isms capable of causing human disease. Although most emerging
pathogens are zoonotic agents and already adapted to a different host,
Vibrio cholerae Cholera toxin Stimulates adenylate cyclase
in host cells the question also concerns a more basic uncertainty about how often,
in what phylogenetic backgrounds, and through what mechanisms
CFA, colonization factor antigen.
Data from Elwell LP, Shipley PL. Plasmid-mediated factors associated with virulence for humans among microbes can arise. Pathogenicity appears
virulence of bacteria to animals. Annu Rev Microbiol. 1980;34:465-496; Cheetham to have arisen on multiple occasions throughout the domain Bacteria
BR, Katz ME. A role for bacteriophages in the evolution and transfer of bacterial but only in a small fraction of the overall phyla, that is, those whose
virulence determinants. Mol Microbiol. 1995;18:201-208. members typically colonize humans (see “Diversity of Human-Microbe
Relationships”). Although there are currently no known traditional
pathogens within the domain Archaea, the methanogens, through a
gene islands distinct to each type, comprising as much as 40% of the synergistic interaction with other microbes, known as syntrophy, may
overall gene content in each of these strains.24 Each pathotype is as contribute to pathology in certain clinical settings.29-31 For example, in
distinct from others as each is from a nonpathogenic laboratory strain chronic periodontitis, methanogens in the subgingival crevice may
of E. coli. Conversely, no more than half of the combined gene set is enhance the growth of fermentative, would-be pathogenic bacteria by
common to all E. coli strains. From this and other similar findings consuming the hydrogen produced by the latter.
arises the concept of the “pan-genome” or the complete set of genes Finally, before considering several facets of pathogenicity in more
for a species. E. coli has a relatively “open” pan-genome, in that with detail, three further points should be considered: (1) pathogen detec-
every new genome sequence, a new set of approximately 300 unique tion and identification remain suboptimal, in part because of continu-
genes is discovered, suggesting ongoing evolution of this species by ing dependence on cultivation methods, and therefore a number of
gene acquisition.25 Bacillus anthracis and other accidental human novel pathogens may have gone undetected32; (2) some potential
pathogens with a restricted environmental habitat or a different host pathogens may not have had adequate contact with humans to have
preference instead display a relatively “closed” pan-genome and a much made themselves known (yet)33; and (3) dominant ideas of microbial
greater fraction of shared genes. disease causation (e.g., a single pathogenic agent in a susceptible host)
Hence, we can conclude that, in most cases, human-adapted patho- may be too restrictive. As mentioned earlier, some microbial diseases
gens have virulence genes not present in nonpathogenic relatives, and may require a consortium of agents (e.g., intra-abdominal abscess),
the distribution of these genes suggests that bacteria evolve to become thereby posing challenges for pathogen identification. If we define
pathogens by acquiring virulence determinants and not by the gradual success for a microbial pathogen as disease without a requirement for
loss of genes. This is not to say that, over time, some pathogens do not long-term survival, a much larger number of organisms may qualify,
dispense with some genes that are no longer useful for a pathogenic in being able to cause devastating human misery but only over a
lifestyle. Indeed, the study of host adaptation suggests that gene loss limited number of generations. These matters have obvious relevance
or gene inactivation is often associated with the adaptation of a par- to the troubling issue of bioterrorism and the potential malevolent use
ticular pathogen to a particular host. For example, S. typhi, compared and genetic manipulation of microorganisms.
with Salmonella typhimurium, has lost or inactivated a large number
of genes. Yet, it also has acquired by lateral transfer a unique surface CLONAL NATURE OF BACTERIAL
determinant, Vi, and a unique toxin. Thus, the fundamental evolution- PATHOGENS
ary push to pathogenicity results from gene acquisition. This is not As noted previously, pathogenicity is not a microbial trait that has
simply a mechanism that microbes use to become pathogenic but, become fixed by chance. Instead, particular microbial strains and
5
species adapted to a particular host have evolved to carry very specific numerous unrelated cell lines appear and disappear. E. coli urinary
37
arrays of virulence-associated genes. By examining the genetic organi- tract pathogens that cause symptomatic disease in humans may be
zation of pathogens, opportunists, and nonpathogenic bacteria, one even less genetically diverse than E. coli strains found in the intestinal
can begin to understand the origins of pathogenicity and why some microbiota or those that cause asymptomatic urinary tract coloniza-

pathogens are more pathogenic or more successful than their peers. tion.41 Perhaps the evolution of these E. coli strains to live in a more
Techniques used in the study of genetic relatedness include primary specialized epithelial niche results in constraints on recombination that
protein or nucleic acid sequence comparisons and DNA hybridization preserve their added degree of specialization. At the same time, spe-
methods, including DNA microarray-based approaches.12 Some cialization for one body site may not preclude fitness for another site:
genetic sequences, such as those of the small- and large-subunit ribo- in some individuals with recurrent urinary tract infections, there can
somal RNAs, have been used as reliable evolutionary clocks.34,35 Com- be a simultaneous and identical shift in the dominant E. coli population
parative analysis of these sequences allows one to infer phylogenetic of the bladder and distal gut between one episode and the next.42
relationships among all known cellular life, but these sequences provide Pathogens have even taught us about human prehistory: sequence-
only limited resolution between strains and limited insight into organ- based population analysis of human-restricted and human-adapted
ismal biology and function. The increasing ease with which primary bacterial pathogens, such as H. pylori, has clarified important aspects
genomic sequence information can be acquired, differences quantified, of human migration and human population structure.43
and these data shared has led to more precise methods of strain char-
acterization, such as multilocus sequence typing36 and whole-genome GENOMICS AND THE EVOLUTION
sequencing. Today, full-genome sequencing and genome-wide single OF PATHOGENICITY
nucleotide polymorphism analysis are feasible on a large-scale basis The first complete genome sequence for a free-living organism, H.
and offer the greatest insight into evolutionary relationships and popu- influenzae, was described in 1995.44 Since then, more than 2900 bac
lation biology of microbes.13 All of these sequence-based approaches terial and archaeal complete genome sequences have been released
avoid the pitfalls of classic comparisons of phenotypes (i.e., gross to public databases (see www.ncbi.nlm.nih.gov/genome/browse/).
observable characteristics of a microbe), which can be unreliable. Despite the obvious value of a primary genomic blueprint, it is increas-
When these sequence-based techniques are used, a consistent finding ingly clear that genetic, genomic, biochemical, and epidemiologic
emerges concerning the population structure of microorganisms: most approaches provide complementary advantages. Each contributes to
natural populations of microorganisms consist of a number of discrete the search for new determinants of virulence.
clonal lineages.37 As noted earlier, comparisons of pathogenic and nonpathogenic
A clonal population structure implies that the rates of recombina- representatives of a single genus or species usually demonstrate the
tion of chromosomal genes between different strains of the same nonpathogens to be relatively devoid of functional genetic sequences
species and between different bacterial species are very low. Clonal encoding the pathogenic trait or traits. Inactive mutational variants or
organization has been substantiated by the concordance between evo- portions of virulence-associated genes are sometimes found in non-
lutionary trees derived from unrelated chromosomal sequences.38 Even pathogenic strains of the same species. In general, as bacteria evolve
though bacteria possess well-established, naturally occurring genetic from free-living organisms with multiple habitats to obligate patho-
exchange mechanisms, they retain their individuality, just as the gens, host-restricted organisms, endosymbionts, or obligate intracel-
human- and other animal-associated bacterial communities demon- lular organisms, their genomes appear to become reduced in size, or
strate stable membership over long periods of time in an individual. they accumulate inactive or defective genes (pseudogenes), or both.13,45
We might have thought that in light of unmistakable gene shuffling For example, the evolution of B. pertussis as a host-specific, human-
among and between bacteria, we might see homogenization of bacte- adapted pathogen from a Bordetella bronchiseptica–like ancestor has
rial species and little specialization. In fact, the opposite is true. Bacte- been accompanied by extensive gene loss and gene inactivation (3816
rial species have remained discrete and distinct taxonomic entities39 coding sequences vs. 5007 for B. bronchiseptica; 9.4% of coding
because the bacterial chromosome is a highly integrated and co-adapted sequences are pseudogenes vs. 0.4% for B. bronchiseptica).46 In this
entity that has, in general, resisted rearrangement. The same may be case, a highly restricted host range (for B. pertussis, humans only) has
true of the overall structure of the indigenous bacterial communities meant loss of genetic diversity. In contrast to B. bronchiseptica, which
of humans. infects multiple animal hosts and can survive in the environment, B.
It is intriguing that analysis of natural populations of microorgan- pertussis varies little in gene content among different strains isolated
isms with pathogenic potential has revealed the prominent representa- over the past 50 years and across several continents.47 B. anthracis,
tion of a relatively few clones. In fact, most cases of serious disease may which exists predominantly as an inactive spore, and Mycobacterium
be caused by only a few of the many extant clones that constitute a tuberculosis, which exists primarily in a latent phase in human granu-
pathogenic bacterial species. For example, one sees this in meningo- lomas, also exhibit limited genomic diversity. Mycobacterium leprae
coccal disease, where there is a clear predominance of a particular displays an extreme degree of gene decay.48
clone in large areas worldwide. In contrast, in the case of the typhoid Not uncommonly, virulence-specific sequences are bounded by
bacillus, there is only one major clone worldwide, although antibiotic repeated DNA segments, some of which represent known insertion
resistance may be forcing diversity.40 Indeed, in some extreme cases, elements, which suggests that these virulence genes were once associ-
all members of a species, such as Shigella sonnei or Bordetella pertussis, ated with a mobile genetic element or that these genes formerly occu-
belong to the same clonal type or small group of closely related types. pied another chromosomal locale in either the same species or another
Not all pathogenic bacterial species reveal this pattern of clonal orga- microorganism altogether. Acquisition of an adhesin, toxin, or serum-
nization. Two notable exceptions are N. gonorrhoeae and Helicobacter resistance factor might enable a previously nonpathogenic organism to
pylori, which appear to use chromosomal recombination to increase cause disease in a host that had previously been nonsusceptible.
their genetic diversity. It may be that organisms such as N. gonorrhoeae Pathogenicity islands provide support for this concept.49 These
and H. pylori, which are quite specialized in their preference for dis- islands comprise clusters of virulence-associated genes that encode
crete human anatomic sites where they rarely encounter related species, specialized secretion systems, secreted effector molecules, adhesins,
must resort to constant recombination and genetic reassortment by and regulatory proteins. S. typhimurium is believed to have begun
using DNA transformation as a means of sharing the well-adapted evolving as a pathogen from a common ancestor that it shares with E.
alleles that accumulate within their populations. Thus, genetic vari- coli, from approximately 130 million years ago, through the sequential
ability among gonococcal and H. pylori isolates from discrete geo- acquisition of at least two pathogenicity islands, one of which mediates
graphic locations suggests that these organisms are essentially and internalization within host cells, and the other, survival and replication
appropriately sexual. within an intracellular vacuole. Yersinia pestis provides a dramatic
Clonal analysis has generated other important conclusions con- example of evolution through both acquisition and loss of genes. It is
cerning the evolution of bacterial species and pathogenic strains in estimated that Y. pestis evolved from the enteropathogenic Yersinia
particular. Study of E. coli populations in the human intestinal tract pseudotuberculosis only 2000 to 20,000 years ago. All pathogenic yer-
indicates that only a small number of clonal lineages persist, whereas siniae species harbor a 70-kb virulence plasmid (pYV) needed for
6
toxicity and to overcome host immune system; but there are two Y. RNA conformation, and protein conformation and stability mediate
pestis–specific plasmids that were recently acquired by horizontal gene thermal regulation of these diverse virulence determinants.55
transfer. One encodes a plasminogen activator, a surface molecule that The number of well-characterized virulence regulatory systems is
provides proteolytic, adhesive, and invasive functions and facilitates impressive. One common mechanism for bacterial transduction of
dissemination from an intradermal site of infection. The other plasmid environmental signals involves two-component regulatory systems
encodes a capsular antigen that blocks phagocytosis and a toxin needed that act on gene expression, usually at the transcriptional level.56,57 Such
for survival in the flea. Thus, this organism evolved to establish a dis- systems make use of similar pairs of proteins; one protein of the pair
tinct mammalian reservoir, to ensure its transmission by a flea, and spans the cytoplasmic membrane, contains a transmitter domain, and
gained attributes that permitted it to spread to systemic sites in its may act as a sensor of environmental stimuli, whereas the other is
preferred murine host and with obvious devastating effect in an acci- a cytoplasmic protein (response regulator) with a receiver domain
dental human host. In the process, it rearranged its chromosome and that regulates responsive genes or proteins. Sensor proteins are often
inactivated genes that were relics of its previous gastrointestinal life. kinases that phosphorylate themselves at a conserved histidine residue.
That a microorganism can accomplish this remarkable feat of evolution These high-energy intermediates then transfer their phosphate groups
in what is a relative blink of the eye, in evolutionary terms, should be to a conserved aspartate residue within the receiver domain of the
a cautionary lesson for what the future may hold in store for any living response regulator proteins. Competing dephosphorylases determine
entity that is host to microbes. an overall phosphorylation state of these response regulators and hence
Although genomic analyses provide us with fascinating stories of their level of activity. Many of these regulators are DNA-binding pro-
how pathogens evolved by genetic acquisition of specialized secretion teins that regulate transcription of multiple gene targets. Systems of
systems and the role of these systems in exporting a variety of genes this type control, for example, the permeability properties of the E. coli
that provide the microbe with extraordinary properties to survive in a cell envelope in response to osmotic stimuli (EnvZ/OmpR), motor
specific host, we still remain ignorant of the precise origins of these activity involved in E. coli chemotaxis (CheA/CheY, CheB), expression
and other virulence-associated systems. It seems likely, however, that of numerous virulence factors in Streptococcus pyogenes (CovR/CovS),
pathogenicity is an old and honorable bacterial trait that can be traced the switch from vegetative growth to sporulation by Bacillus subtilis
to a pathogen’s need for avoiding predation as more sophisticated (KinA/SpoOF, SpoOA), and even the ability of the soil bacterium Agro-
organisms evolved, such as free-living amebae, nematodes, and a host bacterium tumefaciens to induce tumors in susceptible plant cells in
of other equally invisible creatures that exploit microbes for food. response to phenols found within plant wound exudates (VirA/VirG).
The coordinated control of pathogenicity incorporates the impor-
REGULATION OF BACTERIAL tant concept of a regulon. A regulon is a group of operons or individual
PATHOGENICITY genes controlled by a common regulator, usually a protein activator or
If an organism possesses specialized gene products for its virulence, it repressor. This regulator may, in some cases, be the second component
must be able to use them when needed but not squander its metabolic of a two-component system. A regulon provides a means by which
energy producing them aimlessly or risk having them detected by host many genes can respond in concert to a particular stimulus. At other
defenses and prematurely neutralized. Consequently, regulating the times, the same genes may respond independently to other signals.
expression of virulence factors is an additional, yet essential complica- Global regulatory networks are a common feature of microbial viru-
tion of a pathogenic microbe’s life.50 The host presents an array of lence as well as basic microbial physiology (Table 1-3). The complexity
conditions strikingly distinct from those of the outside environment, of virulence regulation in a single microbial pathogen is magnified by
conditions that are not easily reproduced in the laboratory. In fact, the coexistence of multiple interacting (cross-talking) systems and by
laboratory culture conditions bias our understanding of microbial regulons within regulons. P. aeruginosa, for example, contains genes
adaptation to natural environments. This bias is reflected in the concept for 55 sensors and 89 response regulators, whereas H. pylori contains
of a “viable but nonculturable state” for bacteria in their natural exter- genes for 4 and 7, respectively. Perhaps the more restricted numbers
nal environment.51 Vibrio cholerae, for example, is thought to persist and types of microenvironments occupied by the latter organism limit
in this state in brackish estuaries and other saline aquatic environ- the number, while increasing the relative importance, of cues that it
ments, sometimes associated with the chitinous exoskeleton of various must recognize. It appears that some, but not all, regulatory systems
marine organisms.52 Transition from this milieu to the contrasting are essential for virulence.
environment of the human small intestinal lumen must be accompa- Regulation of the expression of virulence determinants by V. chol-
nied by substantial genetic regulatory events. erae involves a global regulatory protein that, in this case, serves a dual
The microbial cell is relatively simple, yet it possesses the means function. The toxR gene product is a transmembrane, DNA-binding
to detect, often simultaneously, changes in temperature; ionic condi- protein that regulates expression of cholera toxin, toxin-coregulated
tions; oxygen concentration; pH; and calcium, iron, and other metal pili, and specific outer membrane proteins. The ToxR protein is thought
concentrations that might appear to be subtle signals, but which are to sense a variety of environmental regulatory signals, including osmo-
essential for the precise mobilization of virulence determinants. Simi- larity, amino-acid concentration, temperature, and pH. ToxR directs
larly, environmental regulatory signals prepare the microbe for its tran- expression of these genes indirectly by activating transcription of toxT,
sition from an extracellular to an intracellular state. For example, iron whose product is a member of the AraC family of transcriptional regu-
is a critical component of many cell metabolic processes; therefore, it lators. At the level of amino-acid sequence, the ToxR protein contains
is not surprising that animals rely on high-affinity iron-binding and features of both sensor and regulator proteins from the two-component
storage proteins to deprive microorganisms of access to this nutrient, sensory transduction system. The combination of these features into
especially at the mucosal surface. In turn, most pathogens sense iron one protein may lead to increased specificity of action. The massive
availability and induce or repress various iron acquisition systems increase in the number of vibrios in cholera stools may presage a
accordingly.53 Indeed, many microorganisms possess toxins that are hyperinfectious state and enhanced transmissibility.58 The transcrip-
regulated by iron such that low iron concentrations trigger toxin bio- tional profile of these organisms as they exit patients suggests recent
synthesis. For the gastric pathogen H. pylori, pH may be a critical nutrient deprivation, iron limitation, downmodulated toxin expres-
signal. The H. pylori response to low pH involves changes in transcript sion, and reduced chemotactic activity.58,59
abundance for 7% of its genes and is associated with increased motility, Quorum sensing is an approach by which bacteria keep track of
perhaps as a means for penetrating the gastric mucous layer.54 their cell density and regulate their behavior accordingly.60 It is inex-
Reversible regulation of the expression of virulence genes by tem- tricably involved in the formation of complex community structures,
perature is a feature common to many pathogens, including entero- called biofilms, by bacteria on environmental surfaces for long-term
pathogenic and uropathogenic E. coli (K-88 and K-99 fimbriae, persistence and resistance to host defenses. Gram-negative organisms
pyelonephritis-associated pilus fimbriae, and K-1 capsular antigen), secrete and respond to acylated homoserine lactones as a means of
Shigella spp. (invasiveness and Shiga toxin), and Yersinia spp. quorum sensing and cell-cell communication. Production of light by
(virulence-associated determinants, including a low-calcium response marine vibrios and tissue-degrading enzymes by P. aeruginosa is acti-
and outer membrane proteins). Changes in DNA topology, messenger vated by these autoinducing compounds when they reach sufficient
7
TABLE 1-3 Examples of Bacterial Virulence Regulatory Systems

REGULATORY
ORGANISM GENE(S) ENVIRONMENTAL STIMULI REGULATED FUNCTIONS

Escherichia coli drdX Temperature Pyelonephritis-associated pili
fur Iron concentration Shiga-like toxin, siderophores
Bordetella pertussis bvgAS Temperature, ionic conditions, nicotinic acid Pertussis toxin, filamentous hemagglutinin, adenylate cyclase, others
Vibrio cholerae toxR Temperature, osmolarity, pH, amino acids Cholera toxin, pili, outer membrane proteins
Yersinia spp. lcr loci Temperature, calcium Secretion of effector proteins
virF Temperature Adherence, invasiveness
Shigella spp. virR Temperature Invasiveness
Salmonella typhimurium pag genes pH Virulence, macrophage survival
Staphylococcus aureus agr genes Cell density α-, β-Hemolysins; toxic shock syndrome toxin 1, protein A
Data from Miller JF, Mekalanos JJ, Falkow S. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science. 1989;243:916-922; Mekalanos JJ.
Environmental signals controlling the expression of virulence determinants in bacteria. J Bacteriol. 1992;174:1-7.
concentration.61 Gram-positive organisms such as Staphylococcus intoxication, or entry.10 A precise configuration of microbial surface
aureus use peptide autoinducers and repressors to sense cell density molecules might be viewed as an “attack complex” with properties not
and regulate toxin expression. The ability of bacterial pathogens to found in any of the individual components.
take their own census enables precise choreography of virulence factor
production during the course of growth in a vigilant host. For example, MICROBIAL PATHOGENS AS
in the early stages of a developing soft tissue abscess, S. aureus turns INTRACELLULAR PARASITES
on antiphagocytic toxins just as the bacteria reach numbers sufficient Despite their capacity for an extracellular existence, a wide variety of
to draw the attention of neutrophils.62 V. cholerae relies on quorum bacterial and protozoal pathogens have evolved the means to enter,
sensing to regulate biofilm formation on marine plankton and medi- survive, multiply, and even persist within host eukaryotic cells. By so
ates release from these biofilms upon entry into a human host.63 doing, a microorganism avoids host immune defenses and gains access
Quorum factors sometimes exhibit strain specificity and might serve to what are otherwise restricted nutrients. These advantages impose a
as targets for novel therapeutic approaches.64 strong selective evolutionary pressure that is dramatically reflected in
Some microbial pathogens (e.g., N. gonorrhoeae, Borrelia recurren- the refined strategies developed by microbial pathogens for life within
tis, and Trypanosoma brucei) periodically vary prominent antigenic a host cell. These strategies include molecular mimicry, coercion, and
components of their surface and, by so doing, avoid the host immune intimate adaptation to eukaryotic cellular processes, and they are
response. Antigenic variation in S. typhimurium and N. gonorrhoeae accompanied by genome reduction.
provides examples of alternative molecular mechanisms (i.e., DNA To a large degree, the mechanisms used by a microorganism to
rearrangements) that mediate regulation of the expression of virulence adhere to a eukaryotic cell dictate whether and how it enters the cell
factors. S. typhimurium varies an immunodominant antigen by alter- and its subsequent intracellular fate.17 Most, if not all, intracellular
nating between the expression of two different flagellin genes, H1 and pathogens have multiple means for attachment to a eukaryotic cell
H2. The mechanism for this form of variation involves inversion of a surface; the particular combination of microbial attachment factors
995-bp chromosomal DNA sequence containing a promoter.65 By alter- and cognate host receptors favors selection of one of several entry
ing expression of flagellin, S. typhimurium may avoid a host antibody pathways and predetermines basic features of the intracellular vacuole.
response directed against it. However, in a general sense, it is unclear to what extent microbial
Pili are essential for virulence of gonococci in the human host, pathogens accept preprogrammed pathways dictated by phagocytic
probably as a result of their role in adherence to the mucosal target (e.g., complement and Fc receptors) and nonphagocytic receptors and
surface.66 They also elicit a specific local and systemic host antibody to what extent they may be able to modify or exploit these pathways.
response. Intermittent production of pili, as well as variation in the Toxoplasma gondii invades and replicates within all types of nucleated
antigenic type of a pilus, may be strategies used by the gonococci to mammalian cells. After entry and through unidentified receptors, T.
avoid the host immune response. The molecular mechanisms behind gondii resides within a parasitophorous vacuole that is permanently
these strategies are complex. In general terms, phase and antigenic incapable of fusion with other intracellular organelles, including lyso-
variation result from DNA rearrangements that move pilin-related somes. Parasite survival within this vacuole depends on the accompa-
sequences scattered around the gonococcal chromosome (in silent pilS nying lack of acidification, exclusion of lysosomal contents, and specific
loci) to the expression site (pilE locus). Numerous different pilus types mechanisms for nutrient acquisition and environmental sensing.
may be expressed by derivatives of a single N. gonorrhoeae strain. However, when this organism is directed to enter eukaryotic cells by
Among other microbial pathogens, DNA rearrangements account for means of an alternative pathway (e.g., mediated by receptors for the
the antigenic variation of variant surface glycoproteins of T. brucei67 constant region of immunoglobulin G, Fc), this vacuole fusion block
and the antigenic variation of variable major proteins in Borrelia spp.68 is overcome. Presumably, parasite-directed modifications of the sur-
Proper presentation of certain virulence-associated gene products rounding vacuolar membrane and exclusion of certain host proteins
on the microbial surface is now recognized to be as important to during the earliest stages of entry help create conditions necessary for
pathogenicity as the initial expression of these genes. Presentation growth and development of the pathogen.70
entails export pathways, association with other periplasmic or surface Some pathogenic microorganisms seem to regulate when and
factors, and sometimes macromolecular assembly at the surface and is where they interact with host cells by using preexistent host signaling
also subject to regulation.69 Among bacterial pathogens, shared homol- pathways.17 Among the receptors that recognize pathogens and, in
ogy is apparent among families of proteins involved in these processes. some cases, mediate entry are integrins (Yersinia spp.), tight junction
One family consists of proteins that are known as chaperones and apparatus cadherins (Listeria monocytogenes)71 and scaffolding protein
ushers, concepts first proposed in a model for the assembly of ZO-1 (H. pylori),72 dystroglycans (arenaviruses),73 and growth factor
uroepithelium-adherent E. coli P pili. Periplasmic chaperones, such as receptors (S. typhimurium). In most of these cases, the pathogens do
PapD, escort protein subunits from the cytoplasmic to the outer mem- not depend on only one receptor family for cellular entry. In addition,
brane and assist in their proper folding. Outer membrane ushers, such cell or organ tropism may be determined by recognition of different
as PapC, target these complexes to a surface assembly site. Folding, members of the same family.
transport, and assembly enable a microorganism to present a specific Signaling events at the surface of the host cell, between pathogens
array of surface molecules necessary for eukaryotic cell tropism, of the same type, and between pathogen and host cell indicate a
8
Early escape from the vacuole is essential for the growth and viru-
lence of some intracellular pathogens. Listeria monocytogenes relies
on several molecules for lysis of the early phagosome, including a
pore-forming hemolysin (listeriolysin O) and two forms of phospho-
lipase C. Once in the cytoplasm, Listeria replicates and induces its

own movement through a remarkable process of host cell actin polym-
erization and formation of microfilaments within a comet-like tail.
Shigella also lyses the phagosomal vacuole and induces the formation
of similar structures for the purpose of intracytoplasmic movement
and cell-cell spread. In both cases, bacterial and host factors involved
in actin polymerization have been identified.76 In the same way that
microbial pathogens fare differently in their interactions with phago-
cytic cells, the outcome of intracellular parasitism for the host cell also
varies considerably, depending on the specific host cell and pathogen
involved.
SUBVERSION OF HOST CELLULAR

PROCESSES AND IMMUNE
FIGURE 1-1 Scanning electron micrograph depicting pseudopod, DEFENSES
or “pedestal,” formation by enteropathogenic Escherichia coli Pathogens can be distinguished from typical commensal microorgan-
(EPEC) as it interacts with the surface of an epithelial cell. This form isms by the degree to which they subvert host cellular processes to their
of intimate adherence requires a bacterial adhesin, intimin; a receptor of own advantage.17,19 Enhanced adherence or internalization of the
bacterial origin, Tir, that is injected into the host cell; and a series of EPEC- pathogen, inhibition of host cell antimicrobial activity, altered inflam-
initiated signaling events. Disruption of normal absorptive function results
in diarrhea. Other bacterial pathogens are also capable of inducing ped-
matory responses, enhanced pathogen multiplication, and host cell
estal formation on intestinal epithelial cells. (From Rosenshine I, Rusch- death are potential outcomes. As mentioned earlier, one common
kowski S, Stein M, et al. A pathogenic bacterium triggers epithelial signals mechanism by which bacterial pathogens alter or subvert the host cell
to form a functional bacterial receptor that mediates actin pseudopod involves specialized secretion systems, such as the type III and type VI
formation. EMBO J. 1996;15:2613-2624. Courtesy B. B. Finlay.) or contact-dependent secretion pathways and the type IV secretion
pathway.77-79 Type III secretion systems from diverse bacterial patho-
gens share structural and functional features that suggest an evolution-
ary relationship with the bacterial flagellar apparatus.77 These systems
complex, highly evolved process of co-adaptation and co-optation.10,19,60 are encoded by blocks of genes that are usually located within patho-
Many of these signals induce rearrangement of host cell cytoskeleton genicity islands. Using a supramolecular structure that spans the entire
to the advantage of the pathogen. In a particularly dramatic example, cell wall and resembles a hypodermic syringe,80 pathogens secrete
enteropathogenic E. coli induces the effacement of normal epithelial effector molecules directly across host cell membranes. Whereas Sal-
cell surface architecture and the formation of a specialized structure monella and Shigella use type III secretion systems (Salmonella patho-
containing reorganized actin that protrudes from the host cell surface genicity island 1 [SPI-1] and invasion plasmid systems, respectively)
and is called a “pedestal,” or pseudopod (Fig. 1-1). Pedestals facilitate to mediate entry into host cells, Salmonella relies on a second type III
intimate attachment, but not entry, of enteropathogenic E. coli to the system (SPI-2) for successful replication within an intracellular vacuole;
host cell; attachment is mediated by the bacterial adhesin intimin and this second system is expressed only when the organism occupies this
a receptor, Tir, that is secreted by enteropathogenic E. coli into the host privileged niche.
cell and then localized to the host cell membrane at the apical surface Type III and type VI secreted effector molecules mediate diverse
of the pedestal.74 These events require a specialized secretion system tasks. Salmonella SopE is secreted by the SPI-1 system and binds
(see “Subversion of Host Cellular Processes and Immune Defenses”) directly to members of the Rho small-molecular-weight guanosine
that delivers not only Tir but also effector proteins that direct host cell triphosphatase protein family in the host cell cytoplasm; this action
phosphorylation of Tir and stimulate other signaling pathways. All activates membrane ruffling.81 The Yersinia YopH effector protein is a
these factors are encoded by genes found within a pathogenicity island potent protein tyrosine phosphatase, virulence factor, and antiphago-
known as the locus for enterocyte effacement (LEE); this chromosomal cytic factor. YopJ induces apoptosis in activated macrophages, sup-
island is also found in some strains of Shiga toxin–producing E. coli. presses tumor necrosis factor-α production, and is critical for Yersinia
Tir from Shiga toxin–producing E. coli is immunogenic in humans and translocation from Peyer’s patches to lymphoid tissue and replication
exhibits sequence and antigenic diversity among different isolates.75 in the spleen. A number of pathogens, including Shigella and Salmo-
Other forms of cytoskeletal rearrangements are essential to the nella, are capable of inducing cell death in macrophages and dendritic
process by which Salmonella, Shigella, and other intracellular patho- cells but not epithelial cells. Although the induction of cell death and
gens enter host cells. Salmonella induces “ruffling” of the host cell frank apoptosis is a common shared strategy of many pathogens, each
membrane, which then engulfs the bacterium and leads to internaliza- accomplishes this outcome through different mechanisms and with a
tion through macropinocytosis. This response by nonphagocytic cells different precise temporal program.19,22 Contact-dependent type VI
is similar to that provoked by growth factors. Salmonella Sip proteins secretion has been associated with bactericidal activity in a variety of
and the related Shigella Ipa proteins are secreted into host cells after gram-negative bacteria and a form of “dueling” behavior between het-
surface contact and are each necessary for the cytoskeletal responses erologous bacterial species.82
of the host cell and for pathogen entry. Manipulation of host cell fate and orchestrated choreography of
Microbial pathogens that have adapted to an intracellular environ- inflammatory responses are recurrent themes in the strategies of
ment possess diverse and specific strategies for survival and replica- microbial pathogens. For example, mycobacteria shut down host-
tion. Some pathogens remain within a vacuole (e.g., Toxoplasma, beneficial myeloid differentiation primary response protein 88
Salmonella), and some lyse the initial phagosomal membrane and (MyD88)-dependent recruitment of macrophages and activate an
replicate within the host cell cytoplasm (Shigella, Listeria, Trypano- alternate recruitment program to gain residence in more permissive
soma, some Rickettsia spp.). Maintenance of specific and favorable macrophages. Hence, the granuloma, for 100 years assigned a central
vacuolar conditions may entail inhibition of phagolysosomal fusion role in “walling off ” M. tuberculosis infection, is as much a structure
and acidification (Toxoplasma), association of eukaryotic organelles built by mycobacteria to promote their expansion and dissemination
with bacteria-containing vacuoles (Legionella, Salmonella), regulation during early infection.83 Recent studies likewise suggest that long-term
of pH (Salmonella), and elevated intracellular levels of glucose in alter- Salmonella carriage is a reflection of the “selection” of a particular class
natively activated macrophages (Salmonella, Brucella). of phagocytic cells to achieve long-term survival.84
9
Because they establish dependent relationships with host cells, external and internal host and cellular environments. Two approaches
viruses often manipulate host cells in dramatic fashion. Human papil- allow investigators to select for genes and promoters that are preferen-
lomaviruses and other animal viruses induce expansion of their pre- tially expressed by a microbial pathogen within a host cell or within a
ferred host niche by interfering with critical cell-cycle controls.85 In an host organ. These approaches rely on specially designed vectors into

interesting analogy, Rickettsia rickettsii blocks a host cell apoptosis which a complete library of chromosomal genes are cloned such that
defensive strategy to prolong the life of the infected cell, facilitate when the promoters for these genes are activated, they turn on the
rickettsial replication, and then spread to other host cells.86 Molluscum expression of factors that can be easily selected, either by expression of
contagiosum virus protects its host cell from oxidative or ultraviolet- antibiotics, by complementation of an engineered growth-attenuating
induced damage by expressing a glutathione peroxidase–like seleno- mutation in the pathogen, or by expression of a fluorescent protein. In
protein that acts as a scavenger of toxic oxygen metabolites.87 Other the first approach, the application of in vivo expression technology to V.
opportunistic strategies of viral pathogens include suppression of viral cholerae and S. typhimurium has clarified the conditions encountered
antigen presentation by host cells and interference with host cytokine, by pathogens in vivo as well as their regulatory responses.91 By using
complement, and interferon activities.21-23 a second approach, termed differential fluorescence induction, research-
A wide variety of intracellular pathogens and their patterned ers identify promoters that are selectively induced within host cells or
molecular components are recognized by members of the NOD (nucle- tissues by fusing random fragments of a pathogen’s genome to the gene
otide oligomerization domain)-like family of receptors, which then encoding green fluorescent protein and then applying fluorescence-
leads to activation of a cytosolic protein complex known as the “inflam- activated flow cytometry to a pool of recombinant organisms bearing
masome” and activation of caspase-1, a protease that, in turn, promotes these reporter gene fusions.92
activation of proinflammatory cytokines and host cell death.88 The Broad-based, nonselective approaches for screening an entire
inflammasome can be viewed as a kind of ancient record of the long- genome and its complement of expressed genes are now quite feasible
standing interplay between phagocytes and microbes. The scope and with DNA microarray and DNA sequencing technologies. DNA
sophistication of the means by which pathogens overcome the host microarrays are high-density grids of probes displayed on a solid
barrier to their establishment, replication, persistence, and subsequent surface; tens or hundreds of thousands of probes can be arrayed in an
exit and transmission to a new host must be seen as one of the most area of 1 cm2. By displaying probes for every gene and intergenic
impressive examples of evolutionary diversity and adaptation. We feature of a given pathogen on a microarray and hybridizing labeled
cannot hope to do justice to the topic here, but we do hope to inspire genomic DNA or complementary DNA (cDNA), one can obtain a
appreciation and respect among clinicians for their microscopic complete gene content or expression profile, respectively, for the patho-
adversaries. gen under any desired condition. Comparative genomic hybridization
analysis of multiple strains of pathogens reveals localized regions of
IDENTIFICATION AND frequent gene gain and loss (“regions of difference,” equivalent in many
CHARACTERIZATION OF cases to pathogenicity islands) and can provide insight into pathogen
VIRULENCE GENES evolution as well as novel mechanisms of virulence.47 More recently,
Characterization of microbial pathogenicity at the molecular level has the ease with which complex pools of DNA or RNA molecules can be
traditionally begun with the identification of a virulence-associated sequenced simultaneously in parallel has facilitated other approaches
phenotype. Such identification may come from clinical observation, for screening large numbers of transposon mutant strains and identify-
epidemiologic investigation, or the use of a model system that reliably ing genes associated with microbial fitness.93 Today, quantitative mea-
reproduces the microbial phenotype in a manner similar to that seen surements of gene transcripts and comparisons of transcript abundance
in natural infection. Traditionally, a virulent strain was compared with are greatly facilitated by high-throughput random sequencing of cDNA
a naturally occurring avirulent variant. Such variants, however, may and the generation of millions of expressed sequence tags that are then
have complex genotypic alterations involving multiple genetic loci. mapped back to genes and genomes with a method called “RNAseq.”
Today, increasingly sophisticated computational tools and ever more- With these approaches, genes and their products are incriminated
easily acquired complete genome sequences have led to predictions of by their relationship with a disease-associated process. Final proof,
phenotype and identification of candidate virulence genes from the however, that a gene is associated with pathogenicity requires that
sequence. The inspection of genome sequences from multiple strains, certain criteria be met. In a manner analogous to Koch’s original pos-
each with variant virulence phenotypes, provides a powerful starting tulates, these criteria must include return of the putative causal agent
point for subsequent hypothesis testing by using specific engineered (the cloned virulence-associated gene mutated or intact) to the host of
mutant strains, when the microbial species is genetically tractable. origin. Unless one can demonstrate an effect on pathogenicity by this
Some pathogens and many commensals, however, remain genetically kind of controlled genetic manipulation, causality with respect to viru-
intractable or resistant to laboratory cultivation. For these organisms, lence has not been proved. Just as the original Henle-Koch postulates
emerging methods offer the possibility of examining their behavior have provided a reference point for later revised criteria of microbial
and putative properties directly from clinical specimens without the causality,94 a molecular form of Koch’s postulates95 can serve as a guide-
need for laboratory propagation (see “Molecular Microbiology at line for an experimental approach to the molecular genetic basis of
the Bedside: Pathogen Detection, Pathogen Discovery, and Genomic pathogenicity. These postulates continue to coevolve in conjunction
Profiling”). with emerging insights into microbial virulence and rapidly improving
Some of the now-standard approaches for identifying virulence- experimental approaches and technologies. For example, alternative
associated genes include the use of insertional elements (e.g., transpo- approaches for proof of causation are necessary for pathogens that
sons) as mutational agents for generating isogenic mutant strains. cannot be isolated and for disease in which a “pathogenic community”
Transposons have the advantage of marking the mutagenized genetic is believed to be the cause.96
locus with a new selectable phenotype, typically antibiotic resistance,
but the disadvantage of possible pleiotropic effects on cotranscribed MOLECULAR MICROBIOLOGY AT THE
genes or on overall microbial fitness. The development of broad-host- BEDSIDE: PATHOGEN DETECTION,
range plasmid vectors carrying well-defined transposons has extended PATHOGEN DISCOVERY, AND
this method of analysis to a number of pathogenic species for which a GENOMIC PROFILING
method of genetic manipulation was not previously available. By using As mechanisms of microbial pathogenicity, acquisition of virulence,
transposable elements with unique genetic tags, negative selection can and drug resistance are revealed, pathogen detection, strain identifica-
be applied to a pool of random mutants in a relevant model of patho- tion, and pathogen discovery assume increasing importance in the
genesis. This approach, known as signature-tagged mutagenesis, has practice of clinical infectious diseases.32 It is already apparent that
identified a number of genes in gram-negative and gram-positive studies of microbial pathogenicity at the molecular level have made
bacteria that are essential for virulence.89,90 substantial contributions to our understanding of the epidemiology,
Other methods for the identification of virulence-associated genes clinical manifestations, diagnosis, treatment, and prevention of infec-
are based on the regulation of such genes by the transitions between tious diseases. Even the fundamental issue of disease causation and the
10
possible role of microorganisms and microbial communities in chronic of ribosomal RNA gene sequences by amplifying them directly from
diseases of uncertain etiology must be reexamined in light of newer digested, infected human tissue.99 Reliable evolutionary relationships
experimental methods and insight. of a putative organism can then be established from these amplified
Infectious disease epidemiology hinges on a clear definition of the sequences. A number of organisms resistant to cultivation or propaga-
clinical problem under study and, moreover, precise identification of tion have been identified with non–culture-based methods.101,102 Some
the etiologic agent. Molecular techniques provide for the sensitive and of the same molecular methods are used to explore diversity within the
specific detection of putative pathogens and supply a means for estab- indigenous microbial communities that populate the skin and mucosal
lishing relationships among multiple isolates of the same species. As a surfaces of the human body.
result, seemingly unrelated cases occurring during an outbreak have Conceptual advances in our understanding of microbial virulence,
been connected; similarly, geographically or temporally distinct out- revolutionary developments in our technical means, and emerging
breaks have been linked to the same pathogenic clone. Molecular tech- challenges from a rapidly changing environment around us suggest a
niques have been used in other epidemiologic investigations to study number of future scenarios and goals. First, we should focus our efforts
transmission mechanisms and the role of avirulent microbial variants on the identification and characterization of pathogens directly from
in the spread of disease. Whole-genome sequencing sometimes pro- clinical specimens and infected hosts, using cultivation-independent
vides the only clue that a group of cases are related, that is, that an approaches. Manipulation and genome-wide characterization of single
outbreak of disease has occurred, as well as the relationships of the bacterial cells is now entirely feasible.103 Deep sequencing–based
outbreak strain to other strains.97 Morphologic and general metabolic pathogen identification from clinical specimens is also a reality.104,105
features often fail to indicate the important genetic diversity found We should expect to be able to measure genome-wide microbial tran-
within strains. script abundance and metabolic activity directly from human speci-
Nucleic acid amplification techniques have had a far-reaching mens as well. Second, the composition and function of the indigenous
impact on the study of microbial pathogenesis and the diagnosis of microbial communities can be assessed using metagenomic and other
infectious diseases. For a variety of reasons, some of them economic community-wide postgenomic technologies.6,8,106 By combining assess-
(reimbursement structure for clinical testing) and some of them tech- ments of community and human response, we stand to gain novel
nical (suboptimal sample preparation methods, and because of insuf- insights into the nature of chronic inflammatory disorders of skin and
ficient attention to the complexity of specimen matrix), specific mucosa.107 Third, it is now time to fully embrace the importance of
polymerase chain reaction assays for microbial pathogens have failed host genetic variation in differential susceptibility to infection and
to penetrate the clinical workplace to a thorough degree.98 Current subsequent disease.108 Fourth, genomic and postgenomic technologies
methods for the identification of microbial pathogens rely heavily on enable us to measure and interpret patterns of human gene and protein
cultivation or propagation in the laboratory. Molecular pathogen dis- expression associated with the response to infectious disease; these
covery methods avoid these dependencies and have spawned new patterns may serve as the basis for signatures, enabling early recogni-
searches for microorganisms that might play important causal roles in tion and classification of patients on the basis of agent or future disease
a wide variety of poorly explained acute and chronic diseases.32,99,100 By course.32,109-112 As virulence factors for essential steps in pathogenesis
providing a speedy and specific microbiologic diagnosis, these methods are identified, it should be possible to interfere with their function. As
will encourage the development and deployment of targeted therapeu- they become better characterized, manipulation of global virulence
tics, and dampen the empirical use of broad-spectrum antimicrobials, regulatory systems may have therapeutic value. New acellular or
thereby reducing selection for resistant organisms. The principle recombinant live-attenuated vaccines and vaccine candidates have
behind these methods is reliance on molecular signatures to identify already resulted from the identification of immunoprotective antigens
or classify a previously unrecognized pathogen; the most commonly through molecular and genomic approaches.12,13 The result of these
used signature is the genomic sequence, but other small molecules may efforts should be a more informed and effective approach to the detec-
prove useful. One of these methods targets highly conserved regions tion, treatment, and prevention of infectious diseases.
Key References 23. Finlay BB, McFadden G. Anti-immunology: evasion of the

host immune system by bacterial and viral pathogens. Cell.
diversity in structure and function. FEMS Microbiol Rev.
2012;36:1046-1082.
2006;124:767-782. 82. Basler M, Ho BT, Mekalanos JJ. Tit-for-tat: type VI secre-
The complete reference list is available online at Expert Consult.
25. Rasko DA, Rosovitz MJ, Myers GS, et al. The pangenome tion system counterattack during bacterial cell-cell interac-
structure of Escherichia coli: comparative genomic analysis tions. Cell. 2013;152:884-894.
1. Dethlefsen L, McFall-Ngai M, Relman DA. An ecological
of E. coli commensal and pathogenic isolates. J Bacteriol. 88. Broz P, Monack DM. Molecular mechanisms of inflamma-
and evolutionary perspective on human-microbe mutual-
2008;190:6881-6893. some activation during microbial infections. Immunol Rev.
ism and disease. Nature. 2007;449:811-818.
28. Jones KE, Patel NG, Levy MA, et al. Global trends in 2011;243:174-190.
2. Tremaroli V, Bäckhed F. Functional interactions between
emerging infectious diseases. Nature. 2008;451:990-993. 94. Evans AS. Causation and disease: the Henle-Koch postu-
the gut microbiota and host metabolism. Nature. 2012;489:
34. Pace NR. Mapping the tree of life: progress and prospects. lates revisited. Yale J Biol Med. 1976;49:175-195.
242-249.
Microbiol Mol Biol Rev. 2009;73:565-576. 95. Falkow S. Molecular Koch’s postulates applied to microbial
5. Yatsunenko T, Rey FE, Manary MJ, et al. Human gut micro-
43. Moodley Y, Linz B, Yamaoka Y, et al. The peopling of the pathogenicity. Rev Infect Dis. 1988;10:S274-S276.
biome viewed across age and geography. Nature. 2012;486:
Pacific from a bacterial perspective. Science. 2009;323: 96. Fredricks DN, Relman DA. Sequence-based identification
222-227.
527-530. of microbial pathogens: a reconsideration of Koch’s postu-
6. The Human Microbiome Project Consortium. Structure,
45. Moran NA. Microbial minimalism: genome reduction in lates. Clin Microbiol Rev. 1996;9:18-33.
function and diversity of the healthy human microbiome.
bacterial pathogens. Cell. 2002;108:583-586. 97. Reuter S, Ellington MJ, Cartwright EJ, et al. Rapid bacterial
Nature. 2012;486:207-214.
49. Groisman EA, Ochman H. Pathogenicity islands: bacterial whole-genome sequencing to enhance diagnostic and
9. Dethlefsen L, Relman DA. Incomplete recovery and indi-
evolution in quantum leaps. Cell. 1996;87:791-794. public health microbiology. JAMA Intern Med. 2013;173:
vidualized responses of the human distal gut microbiota to
56. Parkinson JS. Signal transduction schemes of bacteria. Cell. 1397-1404.
repeated antibiotic perturbation. Proc Natl Acad Sci U S A.
1993;73:857-871. 100. Pallen MJ, Loman NJ, Penn CW. High-throughput sequenc-
2011;108(suppl 1):4554-4561.
59. Bina J, Zhu J, Dziejman M, et al. ToxR regulon of ing and clinical microbiology: progress, opportunities and
10. Merrell DS, Falkow S. Frontal and stealth attack strategies
Vibrio cholerae and its expression in vibrios shed by challenges. Curr Opin Microbiol. 2010;13:625-631.
in microbial pathogenesis. Nature. 2004;430:250-256.
cholera patients. Proc Natl Acad Sci U S A. 2003;100: 101. Relman DA, Loutit JS, Schmidt TM, et al. The agent of
11. Falkow S. The microbe’s view of infection. Ann Intern Med.
2801-2806. bacillary angiomatosis. An approach to the identification of
1998;129:247-248.
60. Bassler BL. Small talk. Cell-to-cell communication in bac- uncultured pathogens. N Engl J Med. 1990;323:1573-1580.
13. Relman DA. Microbial genomics and infectious diseases.
teria. Cell. 2002;109:421-424. 106. Smith MI, Yatsunenko T, Manary MJ, et al. Gut microbi-
N Engl J Med. 2012;365:347-357.
64. Rutherford ST, Bassler BL. Bacterial quorum sensing: its omes of Malawian twin pairs discordant for kwashiorkor.
17. Pizarro-Cerda J, Cossart P. Bacterial adhesion and entry
role in virulence and possibilities for its control. Cold Science. 2013;339:548-554.
into host cells. Cell. 2006;124:715-727.
Spring Harb Perspect Med. 2012;2:a012427. 107. Jostins L, Ripke S, Weersma RK, et al. Host-microbe inter-
19. Baxt LA, Garza-Mayers AC, Goldberg MB. Bacterial sub-
69. Thannasi DG, Bliska JB, Christie PJ. Surface organelles actions have shaped the genetic architecture of inflamma-
version of host innate immune pathways. Science. 2013;340:
assembled by secretion systems of gram-negative bacteria: tory bowel disease. Nature. 2012;491:119-124.
697-701.
10.e1
References 32. Relman DA. New technologies, human-microbe interac-
tions, and the search for previously unrecognized patho-
gonococci of colony type 1 or type 4. J Infect Dis. 1981;
143:413-422.
1. Dethlefsen L, McFall-Ngai M, Relman DA. An ecological
gens. J Infect Dis. 2002;186(suppl 2):S254-S258. 67. Borst P. Antigenic variation and allelic exclusion. Cell.
and evolutionary perspective on human-microbe mutual-
33. Relman DA. Mining the natural world for new pathogens. 2002;109:5-8.
ism and disease. Nature. 2007;449:811-818.

Am J Trop Med Hyg. 2002;67:133-134. 68. Meier JT, Simon MI, Barbour AG. Antigenic variation is
2. Tremaroli V, Bäckhed F. Functional interactions between
34. Pace NR. Mapping the tree of life: progress and prospects. associated with DNA rearrangements in a relapsing fever
the gut microbiota and host metabolism. Nature. 2012;
Microbiol Mol Biol Rev. 2009;73:565-576. Borrelia. Cell. 1985;41:403-409.
489:242-249.
35. Hugenholtz P. Exploring prokaryotic diversity in the 69. Thannasi DG, Bliska JB, Christie PJ. Surface organelles
3. Flint HJ, Scott KP, Louis P, Duncan SH. The role of the gut
genomic era. Genome Biol. 2002;3:REVIEWS0003. assembled by secretion systems of gram-negative bacteria:
microbiota in nutrition and health. Nat Rev Gastroenterol
36. Maiden MC, Bygraves JA, Feil E, et al. Multilocus sequence diversity in structure and function. FEMS Microbiol Rev.
Hepatol. 2012;9:577-589.
typing: a portable approach to the identification of clones 2012;36:1046-1082.
4. Dethlesen L, Eckburg PB, Bik EM, et al. Assembly of the
within populations of pathogenic microorganisms. Proc 70. Sibley LD. Invasion and intracellular survival by protozoan
human gastrointestinal microbiota. Trends Ecol Evol. 2006;
Natl Acad Sci U S A. 1998;95:3140-3145. parasites. Immunol Rev. 2011;240:72-91.
21:517-523.
37. Achtman M. Evolution, population structure, and phylo- 71. Mengaud J, Ohayon H, Gounon P, et al. E-cadherin is the
5. Yatsunenko T, Rey FE, Manary MJ, et al. Human gut micro-
geography of genetically monomorphic bacterial patho- receptor for internalin, a surface protein required for entry
biome viewed across age and geography. Nature. 2012;486:
gens. Annu Rev Microbiol. 2008;62:53-70. of L. monocytogenes into epithelial cells. Cell. 1996;84:
222-227.
38. Daubin V, Moran NA, Ochman H. Phylogenetics and the 923-932.
6. The Human Microbiome Project Consortium. Structure,
cohesion of bacterial genomes. Science. 2003;301:829-832. 72. Amieva MR, Vogelmann R, Covacci A, et al. Disruption of
function and diversity of the healthy human microbiome.
39. Falkow S. The evolution of pathogenicity in Escherichia, the epithelial apical-junctional complex by Helicobacter
Nature. 2012;486:207-214.
Shigella, and Salmonella. In: Neidhardt FC, ed. Escherichia pylori CagA. Science. 2003;300:1430-1434.
7. Haiser HJ, Gootenberg DB, Chatman K, et al. Predicting
coli and Salmonella typhimurium. Washington, DC: Ameri- 73. Cao W, Henry MD, Borrow P, et al. Identification of
and manipulating cardiac drug inactivation by the human
can Society for Microbiology Press; 1996:2723-2729. alpha-dystroglycan as a receptor for lymphocytic chorio-
gut bacterium Eggerthella lenta. Science. 2013;341:295-298.
40. Roumagnac P, Weill FX, Dolecek C, et al. Evolutionary meningitis virus and Lassa fever virus. Science. 1998;282:
8. Turnbaugh PJ, Hamady M, Yatsunenko T, et al. A core gut
history of Salmonella typhi. Science. 2006;314:1301-1304. 2079-2081.
microbiome in obese and lean twins. Nature. 2009;457:
41. Johnson JR, Manges AR, O’Bryan TT, et al. A disseminated 74. Kenny B, DeVinney R, Stein M, et al. Enteropathogenic
480-484.
multidrug-resistant clonal group of uropathogenic Esche- E. coli (EPEC) transfers its receptor for intimate adherence
9. Dethlefsen L, Relman DA. Incomplete recovery and indi-
richia coli in pyelonephritis. Lancet. 2002;359:2249-2251. into mammalian cells. Cell. 1997;91:511-520.
vidualized responses of the human distal gut microbiota to
42. Chen SL, Wu M, Henderson JP, et al. Genomic diversity 75. Paton AW, Manning PA, Woodrow MC, et al. Translocated
repeated antibiotic perturbation. Proc Natl Acad Sci U S A.
and fitness of E. coli strains recovered from the intestinal intimin receptors (Tir) of Shiga-toxigenic Escherichia coli
2011;108(suppl 1):4554-4561.
and urinary tracts of women with recurrent urinary tract isolates belonging to serogroups O26, O111, and O157
10. Merrell DS, Falkow S. Frontal and stealth attack strategies
infection. Sci Transl Med. 2013;5:184ra60. react with sera from patients with hemolytic-uremic syn-
in microbial pathogenesis. Nature. 2004;430:250-256.
43. Moodley Y, Linz B, Yamaoka Y, et al. The peopling of the drome and exhibit marked sequence heterogeneity. Infect
11. Falkow S. The microbe’s view of infection. Ann Intern Med.
Pacific from a bacterial perspective. Science. 2009;323: Immun. 1998;66:5580-5586.
1998;129:247-248.
527-530. 76. Goldberg MB. Actin-based motility of intracellular micro-
12. Medini D, Serruto D, Parkhill J, et al. Microbiology in the
44. Fleischmann RD, Adams MD, White O, et al. Whole- bial pathogens. Microbiol Mol Biol Rev. 2001;65:595-626.
post-genomic era. Nat Rev Microbiol. 2008;6:419-430.
genome random sequencing and assembly of Haemophilus 77. Blocker A, Komoriya K, Aizawa S. Type III secretion
13. Relman DA. Microbial genomics and infectious diseases.
influenzae Rd. Science. 1995;269:496-512. systems and bacterial flagella: insights into their function
N Engl J Med. 2012;365:347-357.
45. Moran NA. Microbial minimalism: genome reduction in from structural similarities. Proc Natl Acad Sci U S A. 2003;
14. Relman DA, Falkow S. The meaning and impact of the
bacterial pathogens. Cell. 2002;108:583-586. 100:3027-3030.
human genome sequence for microbiology. Trends Micro-
46. Parkhill J, Sebaihia M, Preston A, et al. Comparative analy- 78. Pallen MJ, Chaudhuri RR, Henderson IR. Genomic analy-
biol. 2001;9:206-208.
sis of the genome sequences of Bordetella pertussis, Borde- sis of secretion systems. Curr Opin Microbiol. 2003;6:
15. Winter SE, Thiennimitr P, Winter MG, et al. Gut inflam-
tella parapertussis and Bordetella bronchiseptica. Nat Genet. 519-527.
mation provides a respiratory electron acceptor for Salmo-
2003;35:32-40. 79. Silverman JM, Brunet YR, Cascales E, Mougous JD. Struc-
nella. Nature. 2010;467:426-429.
47. Cummings CA, Brinig MM, Lepp PW, et al. Bordetella ture and regulation of the type VI secretion system. Annu
16. Casadevall A, Pirofski LA. The damage-response frame-
species are distinguished by patterns of substantial gene Rev Microbiol. 2012;66:453-472.
work of microbial pathogenesis. Nat Rev Microbiol. 2003;1:
loss and host adaptation. J Bacteriol. 2004;186:1484-1492. 80. Kubori T, Sukhan A, Aizawa SI, et al. Molecular character-
17-24.
48. Cole ST, Eiglmeier K, Parkhill J, et al. Massive gene decay ization and assembly of the needle complex of the Salmo-
17. Pizarro-Cerda J, Cossart P. Bacterial adhesion and entry
in the leprosy bacillus. Nature. 2001;409:1007-1011. nella typhimurium type III protein secretion system. Proc
into host cells. Cell. 2006;124:715-727.
49. Groisman EA, Ochman H. Pathogenicity islands: bacterial Natl Acad Sci U S A. 2000;97:10225-10230.
18. Kolenbrander PE, Palmer RJ, Periasamy S, et al. Oral mul-
evolution in quantum leaps. Cell. 1996;87:791-794. 81. Hardt WD, Chen LM, Schuebel KE, et al. S. typhimurium
tispecies biofilm development and the key role of cell-cell
50. Guiney DG. Regulation of bacterial virulence gene expres- encodes an activator of Rho GTPases that induces mem-
distance. Nature Rev Microbiol. 2010;8:471-480.
sion by the host environment. J Clin Invest. 1997;99: brane ruffling and nuclear responses in host cells. Cell.
19. Baxt LA, Garza-Mayers AC, Goldberg MB. Bacterial sub-
565-569. 1998;93:815-826.
version of host innate immune pathways. Science. 2013;
51. Roszak DB, Colwell RR. Survival strategies of bacteria in 82. Basler M, Ho BT, Mekalanos JJ. Tit-for-tat: type VI secre-
340:697-701.
the natural environment. Microbiol Rev. 1987;51:365-379. tion system counterattack during bacterial cell-cell interac-
20. Young D, Hussell T, Dougan G. Chronic bacterial infec-
52. Lipp EK, Huq A, Colwell RR. Effects of global climate on tions. Cell. 2013;152:884-894.
tions: living with unwanted guests. Nat Immunol. 2002;3:
infectious disease: the cholera model. Clin Microbiol Rev. 83. Ramakrishnan L. Revisiting the role of the granuloma in
1026-1032.
2002;15:757-770. tuberculosis. Nat Rev Immunol. 2012;12:352-366.
21. Bowie AG, Unterholzner L. Viral evasion and subversion
53. Skaar EP. The battle for iron between bacterial pathogens 84. Eisele NA, Ruby T, Jacobson A, et al. Salmonella require the
of pattern-recognition receptor signalling. Nat Rev Immu-
and their vertebrate hosts. PLoS Path. 2010;6:e1000949. fatty acid regulator PPARδ for the establishment of a meta-
nol. 2008;8:911-922.
54. Merrell DS, Goodrich ML, Otto G, et al. pH-regulated gene bolic environment essential for long-term persistence. Cell
22. Best SM. Viral subversion of apoptotic enzymes: escape
expression of the gastric pathogen Helicobacter pylori. Host Microbe. 2013;14:171-182.
from death row. Annu Rev Microbiol. 2008;62:171-192.
Infect Immun. 2003;71:3529-3539. 85. Ferenczy A, Franco E. Persistent human papillomavirus
23. Finlay BB, McFadden G. Anti-immunology: evasion of the
55. Herbst K, Bujara M, Heroven AK, et al. Intrinsic thermal infection and cervical neoplasia. Lancet Oncol. 2002;3:
host immune system by bacterial and viral pathogens. Cell.
sensing controls proteolysis of Yersinia virulence regulator 11-16.
2006;124:767-782.
RovA. PLoS Pathog. 2009;5:e1000435. 86. Joshi SG, Francis CW, Silverman DJ, et al. Nuclear factor
24. Welch RA, Burland V, Plunkett G 3rd, et al. Extensive
56. Parkinson JS. Signal transduction schemes of bacteria. Cell. kappa B protects against host cell apoptosis during Rickett-
mosaic structure revealed by the complete genome
1993;73:857-871. sia rickettsii infection by inhibiting activation of apical and
sequence of uropathogenic Escherichia coli. Proc Natl Acad
57. Mitrophanov AY, Groisman EA. Signal integration in effector caspases and maintaining mitochondrial integrity.
Sci U S A. 2002;99:17020-17024.
bacterial two-component regulatory systems. Genes Dev. Infect Immun. 2003;71:4127-4136.
25. Rasko DA, Rosovitz MJ, Myers GS, et al. The pangenome
2008;22:2601-2611. 87. Shisler JL, Senkevich TG, Berry MJ, et al. Ultraviolet-
structure of Escherichia coli: comparative genomic analysis
58. Merrell DS, Butler SM, Qadri F, et al. Host-induced epi- induced cell death blocked by a selenoprotein from a human
of E. coli commensal and pathogenic isolates. J Bacteriol.
demic spread of the cholera bacterium. Nature. 2002;417: dermatotropic poxvirus. Science. 1998;279:102-105.
2008;190:6881-6893.
642-645. 88. Broz P, Monack DM. Molecular mechanisms of inflamma-
26. Smillie CS, Smith MB, Friedman J, et al. Ecology drives a
59. Bina J, Zhu J, Dziejman M, et al. ToxR regulon of Vibrio some activation during microbial infections. Immunol Rev.
global network of gene exchange connecting the human
cholerae and its expression in vibrios shed by cholera 2011;243:174-190.
microbiome. Nature. 2011;480:241-244.
patients. Proc Natl Acad Sci U S A. 2003;100:2801-2806. 89. Hensel M, Shea JE, Gleeson C, et al. Simultaneous identi-
27. Jonathan R, Davis JR, Lederberg J, eds. Emerging Infectious
60. Bassler BL. Small talk. Cell-to-cell communication in bac- fication of bacterial virulence genes by negative selection.
Diseases from the Global to the Local Perspective: A
teria. Cell. 2002;109:421-424. Science. 1995;269:400-403.
Summary of a Workshop of the Forum on Emerging Infec-
61. Donlan RM, Costerton JW. Biofilms: survival mechanisms 90. Cummins J, Gahan CG. Signature tagged mutagenesis in
tions. Board on Global Health. Washington, DC: Institute
of clinically relevant microorganisms. Clin Microbiol Rev. the functional genetic analysis of gastrointestinal patho-
of Medicine, National Academy Press; 2001.
2002;15:167-193. gens. Gut Microbes. 2012;3:93-103.
28. Jones KE, Patel NG, Levy MA, et al. Global trends in
62. Novick RP, Geisinger E. Quorum sensing in staphylococci. 91. Angelichio MJ, Camilli A. In vivo expression technology.
emerging infectious diseases. Nature. 2008;451:990-993.
Annu Rev Genet. 2008;42:541-564. Infect Immun. 2002;70:6518-6523.
29. Eckburg PB, Lepp PW, Relman DA. Archaea and their
63. Zhu J, Mekalanos JJ. Quorum sensing–dependent biofilms 92. Valdivia RH, Falkow S. Fluorescence-based isolation of
potential role in human disease. Infect Immun. 2003;71:
enhance colonization in Vibrio cholerae. Dev Cell. 2003;5: bacterial genes expressed within host cells. Science. 1997;
591-596.
647-656. 277:2007-2011.
30. Lepp PW, Brinig MM, Ouverney CC, et al. Methanogenic
64. Rutherford ST, Bassler BL. Bacterial quorum sensing: its 93. Goodman AL, Wu M, Gordon JI. Identifying microbial
Archaea and human periodontal disease. Proc Natl Acad
role in virulence and possibilities for its control. Cold fitness determinants by insertion sequencing using
Sci U S A. 2004;101:6176-6181.
Spring Harb Perspect Med. 2012;2:a012427. genome-wide transposon mutant libraries. Nat Protoc.
31. Vianna ME, Holtgraewe S, Seyfarth I, et al. Quantitative
65. Simon M, Zieg J, Silverman M, et al. Phase variation: evolu- 2011;6:1969-1980.
analysis of three hydrogenotrophic microbial groups,
tion of a controlling element. Science. 1980;209:1370-1374. 94. Evans AS. Causation and disease: the Henle-Koch postu-
methanogenic archaea, sulfate-reducing bacteria, and
66. McGee ZA, Johnson AP, Taylor-Robinson D. Pathogenic lates revisited. Yale J Biol Med. 1976;49:175-195.
acetogenic bacteria, within plaque biofilms associated
mechanisms of Neisseria gonorrhoeae: observations on 95. Falkow S. Molecular Koch’s postulates applied to microbial
with human periodontal disease. J Bacteriol. 2008;190:
damage to human fallopian tubes in organ culture by pathogenicity. Rev Infect Dis. 1988;10:S274-S276.
3779-3785.
10.e2
96. Fredricks DN, Relman DA. Sequence-based identification of uncultured pathogens. N Engl J Med. 1990;323: 107. Jostins L, Ripke S, Weersma RK, et al. Host-microbe inter-
of microbial pathogens: a reconsideration of Koch’s postu- 1573-1580. actions have shaped the genetic architecture of inflamma-
lates. Clin Microbiol Rev. 1996;9:18-33. 102. Chang Y, Cesarman E, Pessin MS, et al. Identification of tory bowel disease. Nature. 2012;491:119-124.
97. Reuter S, Ellington MJ, Cartwright EJ, et al. Rapid bacterial herpesvirus-like DNA sequences in AIDS-associated 108. Quintana-Murci L, Alcaïs A, Abel L, et al. Immunology in
whole-genome sequencing to enhance diagnostic and Kaposi’s sarcoma. Science. 1994;266:1865-1869. natura: clinical, epidemiological and evolutionary genetics
public health microbiology. JAMA Intern Med. 2013;173: 103. Lasken RS. Genomic sequencing of uncultured micro of infectious diseases. Nat Immunol. 2007;8:1165-1171.
1397-1404. organisms from single cells. Nat Rev Microbiol. 2012;10: 109. Boldrick JC, Alizadeh AA, Diehn M, et al. Stereotyped and
98. Baron EJ, Miller JM, Weinstein MP, et al. A guide to the 631-640. specific gene expression programs in human innate
utilization of the microbiology laboratory for diagnosis 104. Kistler AL, Gancz A, Clubb S, et al. Recovery of divergent immune responses to bacteria. Proc Natl Acad Sci U S A.
of infectious diseases: 2013 recommendations by the avian bornaviruses from cases of proventricular dilatation 2002;99:972-977.
Infectious Diseases Society of America (IDSA) and the disease: identification of a candidate etiologic agent. Virol 110. Woods CW, McClain MT, Chen M, et al. A host transcrip-
American Society for Microbiology. Clin Infect Dis. 2013; J. 2008;5:88. tional signature for presymptomatic detection of infection
57:e22-e121. 105. Loman NJ, Constantinidou C, Christner M, et al. in humans exposed to influenza H1N1 or H3N2. PLoS One.
99. Relman DA. The search for unrecognized pathogens. A culture-independent sequence-based metagenomics 2013;8:e52198.
Science. 1999;284:1308-1310. approach to the investigation of an outbreak of Shiga- 111. Whitney AR, Diehn M, Popper SJ, et al. Individuality and
100. Pallen MJ, Loman NJ, Penn CW. High-throughput sequenc- toxigenic Escherichia coli O104 : H4. JAMA. 2013;309: variation in gene expression patterns in human blood. Proc
ing and clinical microbiology: progress, opportunities and 1502-1510. Natl Acad Sci U S A. 2003;100:1896-1901.
challenges. Curr Opin Microbiol. 2010;13:625-631. 106. Smith MI, Yatsunenko T, Manary MJ, et al. Gut microbi- 112. Popper SJ, Gordon A, Liu M, et al. Temporal dynamics of
101. Relman DA, Loutit JS, Schmidt TM, et al. The agent of omes of Malawian twin pairs discordant for kwashiorkor. the transcriptional response to dengue virus infection in
bacillary angiomatosis. An approach to the identification Science. 2013;339:548-554. Nicaraguan children. PLoS Negl Trop Dis. 2012;6:e1966.

Capitulo 1 Mandell

Uploaded by

Copyright:

Available Formats

Capitulo 1 Mandell

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Capitulo 1 Mandell

Uploaded by

Copyright:

Available Formats

1

Basic Principles in the

A Molecular Perspective of Microbial

Chapter 1 A Molecular Perspective of Microbial Pathogenicity

it is just “passing through” and of little

Chapter 1 A Molecular Perspective of Microbial Pathogenicity

Chapter 1 A Molecular Perspective of Microbial Pathogenicity

TABLE 1-3 Examples of Bacterial Virulence Regulatory Systems

Chapter 1 A Molecular Perspective of Microbial Pathogenicity

lipase C. Once in the cytoplasm, Listeria replicates and induces its

SUBVERSION OF HOST CELLULAR

Chapter 1 A Molecular Perspective of Microbial Pathogenicity

Key References 23. Finlay BB, McFadden G. Anti-immunology: evasion of the

Chapter 1 A Molecular Perspective of Microbial Pathogenicity

You might also like