Section A  microbial pathogenesis

1
A Molecular Perspective of
Microbial Pathogenicity
DAVID A. RELMAN*  |  STANLEY FALKOW

The Diversity of Human-Microbe atic, and while it may be accompanied by subtle signs of pathophysiol-
ogy, the host is generally better off for their encounter with the
Relationships microorganism. Such is the usual outcome of most host-microbe
Beginning immediately after birth, humans are colonized by a myriad interactions. The term infectious disease applies when an interaction
of microorganisms that assemble into complex stereotypic communi- with a microbe causes damage to the host, and the associated damage
ties, creating a beneficial indigenous microbiota. The result is a “super- or altered physiology results in clinical signs and symptoms of disease.
organism” in which microbial symbionts outnumber human cells by A pathogen is usually defined as any microorganism that has the capac-
tenfold. Most currently available information about the human indig- ity to cause disease. It is a medical definition; it is not a biological defi-
enous microbiota concerns the bacterial component; although they are nition, and certainly not all pathogens have an equal probability of
by no means the only important members, bacteria are the focus of causing disease. Virulence provides a quantitative measure of pathoge-
the following discussion as well. nicity or the likelihood of causing disease. For example, encapsulated
In contrast to the relatively rare harmful encounters with pathogens, pneumococci are more virulent than nonencapsulated pneumococci,
human-microbe relationships in which either microbe or host benefits and Escherichia coli strains that express Shiga-like toxins are more viru-
without causing harm (commensal relationships) and relationships in lent than those that do not express these toxins. Virulence factors refer
which both benefit (mutualistic relationships) are the dominant forms to the properties (i.e., gene products) that enable a microorganism to
of interaction and are fundamentally important to human biology. establish itself and replicate on or within a specific host species, and
Coevolution, coadaptation and co-dependency are features of our that enhance the microbe’s potential to cause disease.
relationship with our indigenous microbiota.1 The human microbiota It is useful to distinguish “principal” pathogens, which regularly
facilitates nutrient acquisition and energy extraction from food, pro- cause disease in some proportion of susceptible individuals with
motes terminal (postnatal) differentiation of mucosal structure and apparently intact defense systems, from other potentially pathogenic
function, and stimulates both the innate and adaptive immune systems. microorganisms, such as Pseudomonas aeruginosa. This microorgan-
By so doing, it helps to maintain epithelial boundary function and ism does not usually cause disease in individuals with intact host
integrity, as well as to “educate” our innate immune defenses, provides defense systems; yet it causes devastating disease in many immuno-
“colonization resistance” against pathogen invasion, and provides compromised patients. Many microorganisms with a capacity for sus-
small amounts of human accessory growth factors.2 The rules and tained multiplication in humans, including members of the indigenous
features of community assembly are fundamentally important, but so microbiota, cause disease more readily in individuals with underlying
far, are poorly understood.3 In the neonatal period, the community chronic disease or in those who are otherwise compromised. The
assembly process is especially dynamic and is influenced by early envi- common term opportunist suits this category of pathogen well.
ronmental (in particular, maternal) exposures and stochastic effects. An emerging concept of microbial disease causation, with origins in
The composition of the indigenous microbiota evolves in a generally the field of ecology, is the notion of “community as pathogen,” in
orderly fashion, as diet, hormonal environment, other environmental which a conserved broad feature of the microbial community contrib-
contacts, and occasional ecologic disturbances play out their effects on utes to pathology, rather than any one specific member or component.
a distinct, albeit diverse human genetic background. This concept may be relevant to a wide variety of chronic inflamma-
Bacterial diversity in the indigenous communities of the human tory processes of skin and mucosa, including inflammatory bowel
body is striking in its richness of distinct species and strains, but also disease and chronic periodontitis. It suggests that studies of pathogen-
noteworthy for the limited number of phyla commonly found. Despite esis consider general properties of microbial communities, such as
exposure to more than 100 bacterial phyla in the surrounding environ- resilience, or conserved functional interactions, such as syntrophy,
ment, members of the phyla Firmicutes, Bacteroidetes, Actinobacteria, rather than the role of single microbes in isolation, especially for devel-
Proteobacteria, and Fusobacteria dominate the human habitats, sug- opment of novel approaches for maintaining or restoring health.
gesting a role for strong selective forces and diversification over hun- The difficulty therefore is that the distinctions between a commen-
dreds of thousands of years of co-evolution with their host. Within the sal, an opportunist, and a “principal” pathogen can be blurred at times,
domain Archaea, diversity in the human body is limited to a handful in part because some commensals cause disease, albeit usually in
of methanogen species: Methanobrevibacter smithii is commonly found immunocompromised hosts, and some of the most feared pathogens
in the healthy distal gut, and Methanobrevibacter-related species are can persist in humans for a lifetime without causing disease symptoms,
found in the inflamed subgingival crevice in some patients with mod- and because microbial pathogenesis involves synergies between organ-
erate or severe chronic periodontitis. Interestingly, patterns of bacte- isms (as well as between gene products), each of which may be insuf-
rial diversity in humans display individual-specific features. Although ficient alone in causing disease in most settings. For example, several
there is evidence for shared functional capabilities among the intestinal members of the human health-associated nasopharyngeal microbi-
microbial communities of different healthy humans, host genetics is a ota—including Streptococcus pneumoniae, Neisseria meningitidis, Hae-
prominent source of variation in the make-up of the human indige- mophilus influenzae type B, and Streptococcus pyogenes—regularly
nous microbiota.4 cause well-defined, well-known human diseases. Immunization against
Infection is simply the establishment of a microorganism on or these microbes not only protects against disease, but also prevents (in
within a host; it may be short lived. Infection is most often asymptom- an antigen-specific fashion) their ability to colonize the host (reviewed
in Segal and Pollard5). Such “commensal pathogens” persist in a sig-
nificant proportion of the human population, the vast majority of
*All material in this chapter is in the public domain, with the exception whom are asymptomatic carriers. Are they pathogens or are these
of any borrowed figures or tables. organisms members of the indigenous microbiota that have evolved
3
4 Part I  Basic Principles in the Diagnosis and Management of Infectious Diseases

to live in a perilous location—associated with respiratory tract lym- host niche. Clearly, the state of the host plays as important a role as
phatic tissue where they regularly come into contact with elements of the pathogen in determining outcome.10
an immune system that hold them at bay most of the time, but occa- An initial step required of a pathogen is to gain access to the host
sionally fail to do so, resulting in disease? in sufficient numbers. Such access requires that the microorganism not
An understanding of the definition of a pathogen is not required only make contact with an appropriate surface, but also then reach its
when a clinician is faced with an infected patient who needs treatment. unique niche or microenvironment on or within the host. This require-
However, if we are to understand disease-associated microbes and ment is not trivial. Some pathogens must survive for varying periods
discover effective therapies, we will also need to appreciate their fun- in the external environment. Others have evolved an effective and
damental biology and the ecological setting in which they secure a suitable means of transmission. To accomplish this goal, the infecting
niche. And it is important to be reminded that antibiotics don’t always microbe may make use of motility, chemotactic properties, and adhe-
(are increasingly less likely to) work against many pathogens, that sive structures (or adhesins) that mediate binding to specific eukaryotic
antibiotics incur a cost (in terms of resistance and collateral damage), cell receptors or to other microorganisms (see Chapter 2).11-13 Patho-
and that we still lack effective vaccines against a multitude of infectious gens that persist at the surface of skin or mucosa usually rely upon
agents that are encountered in everyday medical practice. multiple, redundant adhesins and adherence mechanisms. If the
Thus, the capacity of certain microorganisms to cause disease in adhesin is immunogenic, expression is usually regulated; in addition,
healthy, uncompromised human hosts on a regular basis should reflect antigenic variants may arise (see following section, “Regulation of
fundamental biological differences in their virulence capabilities from Bacterial Pathogenicity”). Preexisting microorganisms, the indigenous
those of opportunists and commensal species that rarely, if ever, cause microbiota, provide competition against establishment of the new-
disease. In the following sections we address this issue and discuss how comer; furthermore, the latter must adapt, at least temporarily, to the
insight into pathogenesis has been applied to the practice of contem- particular nutrient environment in which it now finds itself. For
porary infectious disease medicine. example, intracellular Neisseria gonorrhoeae use their outer Opa cell
membrane to capture active host pyruvate kinase which is required for
The Attributes of Microbial gonococcal intracellular growth.14
Normal host defense mechanisms pose the most difficult set of
Pathogens obstacles for pathogens and commensals in establishing themselves in
What are the distinguishing characteristics of microbes that live in a host. For any set of specific host defenses, an individual pathogen
humans? A successful pathogen or commensal must do the following: will have a unique and distinctive counterstrategy. Some of the best
(1) enter the human host; (2) become established; (3) acquire nutri- known mechanisms that pathogenic microbes use for countering host
ents; (4) avoid or circumvent the host’s innate defenses and a powerful defenses include the use of an antiphagocytic capsule and the elabora-
immune system; (5) above all, replicate; (6) disseminate if necessary tion of toxins and microbial enzymes that act on host immune cells
to a preferred site; and eventually (7) be transmitted to a new suscep- and/or destroy anatomic barriers. Microorganisms also use subtle bio-
tible host. chemical mechanisms to avoid, subvert, or even manipulate host
Whether a pathogen or a commensal, a microorganism must also defenses. These strategies include the elaboration of immunoglobulin-
possess an interactive group of complementary genetic properties, specific proteases, iron sequestration mechanisms, coating themselves
sometimes coregulated, that promote its interaction with the human with host proteins to confuse the immune surveillance system, or
host. For a given microorganism, the genetic traits define unique attri- causing host cells to signal inappropriately, leading to dysregulation of
butes that enable it to follow a common sequence of steps used in host defenses or even host cell death. Examples of these mechanisms
establishing infection or in some cases subsequent disease.6,7 include the production of immunoglobulin A1 protease by the menin-
Elegant molecular and genetic techniques now permit the identifica- gococci, the use of receptors for iron-saturated human transferrin and
tion, isolation, and characterization of many of these genes and their lactoferrin by N. gonorrhoeae, and the coating of Treponema pallidum
products. We now also possess the complete genome sequences of with human soluble fibronectin. Yersinia, Mycobacterium, and Borde-
virtually every major pathogenic bacterial species. This information tella induce host cell production of interleukin-10, which is a potent
provides important clues and insight into the potential of a microor- immunosuppressive cytokine, thereby downregulating important ele-
ganism for causing disease and facilitates new experimental strategies ments of the innate immune defense. Antigenic variation and intracel-
for understanding pathogens and commensals alike.8 The availability lular invasion are other common strategies used by successful
of the host (e.g., human) genome sequence also enables multiple syn- pathogens to avoid immune detection.15,16 The intimacy of the rela-
ergistic approaches for understanding virulence, including the identi- tionships between viral pathogens and host is reflected in the fre-
fication of host susceptibility traits, genome-wide assessments of host quency with which these pathogens co-opt host molecules and
response, and clues about the mechanisms of host defense and patho- pathways for subverting host defenses (see “Subversion of Host Cel-
gen counterdefense.9 lular Processes and Immune Defenses”).17-20
These genomic analyses have at last underscored the working The ability to multiply is a characteristic of all living organisms.
hypothesis of almost a half-century of research—that the distinguish- Whether the pathogen’s niche in the relevant host be intracellular or
ing characteristic of microorganisms that regularly cause disease is a extracellular, mucosal or submucosal, within the bloodstream or
set of special genetic traits that provide them with the capacity to within another privileged anatomic site, pathogens have evolved a
breach intact host anatomic, cellular, or biochemical barriers that ordi- distinct set of biochemical tactics to achieve this goal. The ultimate
narily prevent entry by other microorganisms into sterile tissue sites. success of a pathogen, indeed, of any microorganism, is measured by
Thus, pathogens “go where other microbes dare not.” In addition, the degree to which it can multiply. The pathogen must not only rep-
many pathogens, such as Mycobacterium tuberculosis, Treponema pal- licate sufficiently to establish itself in a host on reaching its specific
lidum, Chlamydia trachomatis, Salmonella typhi, and Helicobacter niche, but it also must replicate sufficiently at some point in its life
pylori have the capacity to establish persistent (usually asymptomatic) cycle to ensure its potential transmission to a new susceptible host. The
infection in the human host, and have evolved the extraordinary rate of pathogen multiplication is appreciated by a clinician in terms
capacity to live in the inner sanctums of our innate and adaptive of a characteristic incubation period spanning the time of exposure to
immune defenses. A distinction, then, between primary pathogen and the appearance of signs and symptoms of disease.
opportunist is that the pathogen has an inherent ability to breach the The degree to which the pathogen has perpetuated itself and the
host barriers that ordinarily restrict other microbes, whereas the nature of the relationship it has established with its host define the
opportunist requires some underlying defect or alteration in the host’s extent of altered host physiology and tissue damage. Infectious disease,
defenses, whether it be genetic, ecologic (altered microbiota), or in one sense, is simply a by-product of the method and site chosen by
caused by underlying disease, to establish itself in a usually privileged pathogens for replication and persistence; disease per se is not a
 1 A Molecular Perspective of Microbial Pathogenicity 5

measure of microbial success. Disease, in part reflects the status of the TABLE Examples of Plasmid- and Phage-Encoded
host as much as it does the virulence characteristics of the offending 1-1 Virulence Determinants
microorganism. Death of the host is fortunately a rare event, and one Organism Virulence Factor Biologic Function
that must be viewed with the dispassion of biology as being detrimental
Plasmid encoded
to both parties involved! The usual rules of host-pathogen engagement
Enterotoxigenic Heat-labile, Activation of adenylate/guanylate
most often produce a tie: sufficient multiplication of the pathogen to Escherichia coli heat-stable cyclase in the small bowel,
ensure its establishment within the host (transient or long-term infec- enterotoxins which leads to diarrhea
tion) and to ensure its successful transmission to a new susceptible CFA/I and CFA/II Adherence/colonization factors
host, while at the same time, no more than is tolerated by the host as Extraintestinal E. Hemolysin Cytotoxin
it gains immunity from further incursion by the same and even related coli
pathogens. Shigella spp. and Gene products Induces internalization by
Why do some pathogens cause disease more readily than others? enteroinvasive involved in intestinal epithelial cells
E. coli invasion
The strategy used for multiplication on or within the host (i.e., its
Yersinia spp. Adherence factors Attachment/invasion
ability to overcome host barriers) often defines fundamental differ- and gene products
ences between pathogens that commonly cause acute disease symp- involved in
toms and those that do not. An organism that can reach and multiply invasion
in privileged anatomic sites away from the competitive environment Bacillus anthracis Edema factor, lethal Edema factor has adenylate
of skin and mucosal surfaces is more likely to disrupt homeostasis in factor, and cyclase activity; lethal factor is
protective antigen a metalloprotease that acts on
the host and cause disease than one that chooses a different strategy. host signaling molecules
If a microorganism has evolved a means to nullify or destroy phago- Staphylococcus Exfoliative toxin Causes toxic epidermal necrolysis
cytic cells to multiply successfully, it is more likely to be found in aureus
deeper compartments and associated with acute disease. Commensal Clostridium tetani Tetanus neurotoxin Blocks the release of inhibitory
or mutualistic organisms are content to multiply just enough, in the neurotransmitter, which leads
midst of competing microbiota, to persist but not damage the host’s to muscle spasms
self-preserving homeostatic and innate immunity mechanisms. It is Phage encoded
important to emphasize that a microorganism equipped to multiply Corynebacterium Diphtheria toxin Inhibition of eukaryotic protein
efficiently in a human may be exceptional in the biological sense, but diphtheriae synthesis
unexceptional as a pathogen in the medical sense, and only infre- Streptococcus Erythrogenic toxin Rash of scarlet fever
pyogenes
quently, if ever, a cause of clinically manifested disease.
Clostridium Botulism neurotoxin Blocks synaptic acetylcholine
Some organisms such as P. aeruginosa are “only” opportunists in botulinum release, which leads to flaccid
humans despite their impressive array of virulence factors. These viru- paralysis
lence factors work well in some plant hosts and in predators it encoun- Enterohemorrhagic Shiga-like toxin Inhibition of eukaryotic protein
ters in the environment. However, for Pseudomonas, these same E. coli synthesis
pathogenic determinants usually fail to overcome the average human’s Vibrio cholerae Cholera toxin Stimulates adenylate cyclase in
defenses. A microorganism has no presupposition about the state of host cells
the host or its defenses. For opportunistic pathogens, the state of the CFA, colonization factor antigen.
Data from Elwell LP, Shipley PL. Plasmid-mediated factors associated with virulence
host is the major determinant of whether disease is the outcome of of bacteria to animals. Annu Rev Microbiol. 1980;34:465-496; Cheetham BR, Katz ME. A
their interaction with the host. Many opportunistic infectious agents role for bacteriophages in the evolution and transfer of bacterial virulence determinants.
in healthy humans are simply transients, “just passing through.” Not Mol Microbiol. 1995;18:201-208.
so in the human compromised by a defect in some aspect of host
defense. Commensals and mutualists, for example, which are the usual
cause of opportunistic infections, may be very adept at colonization,
but because of their preferred growth locale (e.g., at the mucosal packets of information have also been shared among bacteria by
surface) and preferred growth conditions (e.g., a microaerophilic envi- genetic transfer. The lateral inheritance of large blocks of genes, called
ronment), they may have limited growth opportunities outside their pathogenicity islands, is often the key to the expression of pathogenicity
restricted niche in an unimpaired individual. Little more than 50 years in bacteria. Many of these virulence determinants acquired by lateral
ago, there was a prevalent view that pathogens had undergone retro- gene transfer have several features, including distinct chromosomal
grade evolution and caused disease because they were little more than nucleotide composition and association with plasmids or phages, sug-
parasites. Pathogens were then viewed as organisms, often unadapted gesting that their ancestry derives from an unrelated microbe. Because
to their hosts, that elaborated potent toxins or other powerful aggres- of the rapidly expanding capabilities and availability of genome
sive factors that caused the signs and symptoms of disease. However, sequencing, it is possible to recognize genes that arose by lateral gene
bacterial pathogenicity has been redefined over the past quarter transfer by simple inspection of genome sequences. One surprising
century using the tools of molecular genetics. We can now directly finding is that the amount of acquired DNA associated with virulence
address the question: why are some bacteria pathogenic for humans in many bacteria can be substantial. For example, uropathogenic,
while other (closely related) bacteria are not? enterohemorrhagic, and extraintestinal types of E. coli all display
We understand now that pathogenic bacteria are indeed often mosaic genome structure, with hundreds of gene islands distinct to
exquisitely adapted microbes that use sophisticated biochemical strate- each type, comprising as much as 40% of the overall gene content in
gies to interfere with, or manipulate for their own benefit, the normal each of these strains.21 Each pathotype is as distinct from each other
function(s) of the host cell. They are impressive human cell biologists! as each is from a nonpathogenic laboratory strain of E. coli. Con-
It is now also quite clear that these sophisticated biochemical proper- versely, no more than half of the combined gene set is common to all
ties that distinguish pathogens from their nonpathogenic brethren E. coli strains. From this and other similar findings arises the concept
derive from specialized genes possessed by pathogens that are absent of the “pan-genome” or the complete set of genes for a species. E. coli
from nonpathogens. The driving force for the inheritance of patho- has an “open” pan-genome, in that with every new genome sequence,
genic traits is not slow adaptation to the host, but rather a more a new set of approximately 300 unique genes is discovered, suggesting
dynamic process of horizontal (lateral) gene transfer via mobile genetic ongoing evolution of this species by gene acquisition.22 Bacillus anthra-
elements. Hence, the genes for many specialized “bacterial” products, cis and other pathogens with a restricted environmental habitat instead
like toxins and adhesins, actually reside on transposons (“jumping display a “closed” pan-genome, and a much greater fraction of shared
genes”) and bacterial viruses (bacteriophages) (Table 1-1). Larger genes.
6 Part I  Basic Principles in the Diagnosis and Management of Infectious Diseases

Hence, we can conclude that in most cases, human adapted patho- The Clonal Nature of
gens have virulence genes not present in nonpathogenic relatives, and
the distribution of these genes suggests that bacteria evolve to become
Bacterial Pathogens
pathogens by acquiring virulence determinants and not by the gradual As noted previously, pathogenicity is not a microbial trait that has
loss of genes. This is not to say that over time, some pathogens do not appeared by chance. Instead, particular microbial strains and species
dispense with some genes that are no longer useful for a pathogenic have evolved to carry very specific arrays of virulence-associated genes.
lifestyle, but rather that the fundamental evolutionary push to patho- By examining the genetic organization of pathogens, opportunists, and
genicity results from gene acquisition. This is not simply a mechanism nonpathogenic bacteria, one can begin to understand the origins of
that microbes use to become pathogenic, but instead, a general strategy pathogenicity. Also, even within a pathogenic species, we need to
for specialization and success in some environmental niches that are understand why some are more pathogenic or more successful than
highly competitive. Why bacteria have adapted this tactic to maximize their peers.
their diversity and to increase their opportunity for continuing evolu- Techniques used in the study of genetic relatedness include primary
tion is most likely a reflection of their haploid state, and their need to protein or nucleic acid sequence comparisons and DNA hybridization
conserve fundamental characteristics, such as the ability to live on a methods, including DNA microarray-based approaches.8 Some genetic
mucosal surface, while still being able to try new combinations of sequences, such as those of the small- and large-subunit ribosomal
genes. The sharing of genes among seemingly disparate micoroorgan- RNAs, have been used as reliable evolutionary clocks.30,31 Comparative
isms occupying the same niche in a sense provides these microbes with analysis of these sequences allows one to infer phylogenetic relation-
an endless number of combinations of genes for evolutionary experi- ships among all known cellular life, but these sequences provide only
mentation as it were, within a niche like the human host. Overall, limited resolution between strains and limited insight into organismal
across the bacterial world, the number of such successful experiments, biology and function. The increasing ease with which primary genomic
resulting in the emergence of a pathogen appears to have been quite sequence information can be acquired, differences quantified, and
rare (see “The Clonal Nature of Bacterial Pathogens”). Yet, when suc- these data shared, has led to more precise methods of strain charac-
cessful, these experiments are surprisingly efficient, at least from the terization such as multilocus sequence typing.32 Today, with the revo-
perspective of the microbe. lutionary advances in DNA sequencing technology, full genome
Some infectious diseases occur predominantly in dramatic epidemic sequences and genome-wide single nucleotide polymorphism analysis
form, which argues against the evolution of a balanced host-parasite are increasingly feasible on a widescale basis, and offer the greatest
relationship; however, in many such epidemics, mitigating circum- insight into evolutionary relationships and population biology of
stances involving herd immunity and other underlying social, eco- microbes. All of these sequence-based approaches avoid the classic
nomic, and political issues impinge on this relationship. So-called comparisons of phenotypes (i.e., gross observable characteristics of a
emerging infectious diseases reflect various aspects of imbalance in the microbe), which can be unreliable. When these sequence-based tech-
relationships between host, pathogen, and environment.23 Many of the niques are used, a consistent finding emerges concerning the popula-
most serious and feared infectious diseases occur when humans are tion structure of microorganisms: most natural populations of
infected by microorganisms that prefer, and are better adapted to, microorganisms consist of a number of discrete clonal lineages.33
another mammalian host. In fact, most emerging infectious diseases The finding of a clonal population structure implies that the rates
in humans are of zoonotic origin.24 As seen in many zoonotic diseases, of recombination of chromosomal genes between different strains of
the rules of engagement between the host and the pathogen are blurred, the same species and between different bacterial species are very low.
often to the detriment of both the host and the microbe. It is often an Clonal organization has been substantiated by the concordance
evolutionary dead end for both parties. between evolutionary trees derived from unrelated chromosomal
Given the increasingly frequent and unexpected emergence of previ- sequences.34 Even though bacteria possess well-established, naturally
ously unrecognized pathogens, it is appropriate to question how well we occurring genetic exchange mechanisms, they retain their individual-
appreciate the true diversity and distribution of extant microorganisms ity. We might have thought that in light of unmistakable gene shuffling
capable of causing human disease. Although most emerging pathogens among and between bacteria, we might see homogenization of bacte-
are zoonotic agents and already adapted to a different host, the question rial species and little specialization. In fact, the opposite is true. Bacte-
also concerns a more basic uncertainty about how often, in what phylo- rial species have remained discrete and distinct taxonomic entities,35
genetic backgrounds, and through what mechanisms virulence for because the bacterial chromosome is a highly integrated and coadapted
humans among microbes can arise. Pathogenicity appears to have entity that has, in general, resisted rearrangement.
arisen on multiple occasions throughout the domain Bacteria but only It is intriguing that analysis of natural populations of microorgan-
in a small fraction of the overall phyla. Although there are currently no isms with pathogenic potential has revealed the prominent representa-
known traditional pathogens within the domain Archaea, the methano- tion of a relatively few clones. In fact, most cases of serious disease may
gens, through a synergistic interaction with other microbes known as be caused by only a few of the many extant clones that constitute a
syntrophy, may contribute to pathology in certain clinical settings.25-27 pathogenic bacterial species. For example, one sees this in meningo-
Finally, before considering several facets of pathogenicity in more coccal disease where there is a clear predominance of a particular clone
detail, three further points should be considered: (1) pathogen detec- in large areas worldwide. In contrast, in the case of the typhoid bacillus,
tion and identification remain suboptimal, in part because of continu- there is only one major clone worldwide, although antibiotic resistance
ing dependence on cultivation methods, and therefore a number of may be forcing diversity.36 Indeed, in some extreme cases, all members
novel pathogens may have gone undetected28; (2) some potential of a species, such as Shigella sonnei or Bordetella pertussis, belong to
pathogens may not have had adequate contact with humans to have the same clonal type or small group of closely related types. Not all
made themselves known (yet)29; and (3) dominant ideas of microbial pathogenic bacterial species reveal this pattern of clonal organization.
disease causation (e.g., a single pathogenic agent in a susceptible host) Two notable exceptions are N. gonorrhoeae and Helicobacter pylori,
may be too restrictive. As mentioned earlier, some microbial diseases which appear to use chromosomal recombination to increase their
may require a consortium of agents or “pathogenic community struc- genetic diversity. It may be that organisms like N. gonorrhoeae and H.
ture,” thereby posing challenges for pathogen identification. If we pylori, which are quite specialized in their preference for discrete ana-
define success for a microbial pathogen without a requirement for tomic sites where they rarely encounter related species, must resort to
long-term survival, a much larger number of organisms may qualify, constant recombination and genetic reassortment as a means of
in being able to cause devastating human disease but only over a spreading the mutations that accumulate within their populations and
limited number of generations. These matters have obvious relevance increasing their diversity. Thus, genetic variability among gonococcal
to the troubling issue of bioterrorism and the potential malevolent use isolates from discrete geographic locations suggests that this organism
and genetic manipulation of microorganisms. is essentially sexual.
 1 A Molecular Perspective of Microbial Pathogenicity 7

Clonal analysis has generated other important conclusions concern- provides a dramatic example of evolution through both acquisition
ing the evolution of bacterial species and pathogenic strains in particu- and loss of genes. It is estimated that Y. pestis evolved from the entero-
lar. Study of E. coli populations in the human intestinal tract indicates pathogenic Yersinia pseudotuberculosis only 2000 to 20,000 years ago.
that only a small number of clonal lineages persist, while numerous All pathogenic yersiniae species harbor a 70-kb virulence plasmid
unrelated cell lines appear and disappear.33 E. coli urinary tract patho- (pYV) needed for toxicity and to overcome host immune system; but
gens causing symptomatic disease in humans are even less genetically there are two Y. pestis–specific plasmids that were recently acquired by
diverse than E. coli strains found in the intestinal flora or those that horizontal gene transfer. One encodes a plasminogen activator (a
cause asymptomatic urinary tract colonization.37 Perhaps the evolu- surface molecule that provides proteolytic, adhesive, and invasive
tion of these E. coli strains to live in a more specialized epithelial niche functions) and facilitates dissemination from an intradermal site of
results in constraints on recombination that preserve their added infection. The other plasmid encodes a capsular antigen (blocks
degree of specialization. Pathogens have even taught us about human phagocytosis) and murine toxin needed for survival in the flea. Thus,
prehistory: sequence-based population analysis of human-restricted this organism evolved to establish a distinct mammalian reservoir,
and human-adapted bacterial pathogens, such as H. pylori, has clari- ensure its transmission by a flea, and gained attributes that permitted
fied important aspects of human migration and human population it to spread to systemic sites in its preferred murine host and with
structure.38 obvious devastating effect in a human host. In the process, it rear-
ranged its chromosome and inactivated genes that were relics of its
Genomics and the Evolution previous gastrointestinal life. That a microorganism can accomplish
this remarkable feat of evolution in what is a relative blink of the eye,
of Pathogenicity in evolutionary terms, should be a cautionary lesson for what the
The first complete genome sequence for a free-living organism, future may hold in store for any living entity that is host to microbes.
H. influenzae, was described in 1995.39 Since then, more than 840 Although genomic analysis provides us with a fascinating story of
bacterial and archaeal genome sequences have been completed and how pathogens evolved by genetic acquisition of specialized secretion
released to public databases (see http://www.ncbi.nlm.nih.gov/ systems and the role of these systems in exporting a variety of genes
genomes/lproks.cgi). Despite the obvious value of a primary genomic that provide the microbe with extraordinary properties to survive in a
blueprint, it is increasingly clear that genetic, genomic, and epidemio- specific host, we still remain ignorant of the precise origins of these
logic approaches provide complementary advantages. Each contrib- and other virulence-associated systems. It seems likely, however, that
utes to the search for new chromosomal determinants of virulence. pathogenicity is an old and honorable bacterial trait that can be traced
As noted earlier, comparisons of pathogenic and nonpathogenic to their need for avoiding predation as more sophisticated organisms
representatives of a single genus or species usually demonstrate the evolved, such as free-living amoebae, nematodes, and a host of other
nonpathogens to be relatively devoid of functional genetic sequences equally invisible creatures that use microbes for food.
encoding the pathogenic trait or traits. Inactive mutational variants or
portions of virulence-associated genes infrequently occur in non-
pathogenic strains of the same species. In general, as bacteria evolve
Regulation of Bacterial Pathogenicity
from free-living organisms with multiple habitats to obligate patho- If an organism possesses specialized gene products for its virulence, it
gens, host-restricted organisms, endosymbionts, or obligate intracel- must be able to use them when needed, but not squander its metabolic
lular organisms, their genomes appear to become reduced in size or energy producing them aimlessly or risk having them detected by host
they accumulate inactive or defective genes (pseudogenes), or both.8,40 defenses and prematurely neutralized. Consequently, regulating the
For example, the evolution of B. pertussis as a host-specific, human- expression of virulence factors is an additional, yet essential complica-
adapted pathogen from a Bordetella bronchiseptica–like ancestor has tion of a pathogenic microbe’s life.46 The host presents an array of
been accompanied by extensive gene loss and gene inactivation (3816 conditions strikingly distinct from those of the outside environment,
coding sequences, versus 5007 for B. bronchiseptica; 9.4% of coding conditions that are not easily reproduced in the laboratory. In fact,
sequences are pseudogenes, versus 0.4% for B. bronchiseptica).41 In this laboratory culture conditions bias our understanding of microbial
case, a highly restricted host range has meant loss of genetic diversity. adaptation to natural environments. This bias is reflected in the
In contrast to B. bronchiseptica, which infects multiple animal hosts concept of a “viable but nonculturable state” for bacteria in their
and can survive in the environment, B. pertussis varies little in gene natural external environment.47 V. cholerae, for example, is thought to
content among different strains isolated over the past 50 years and persist in this state in brackish estuaries and other saline aquatic envi-
across several continents.42 Bacillus anthracis, which exists predomi- ronments, sometimes associated with the chitinous exoskeleton of
nantly as an inactive spore, and Mycobacterium tuberculosis, which various marine organisms.48 Transition from this milieu to the con-
exists primarily in a latent phase in human granulomas, also exhibit trasting environment of the human small intestinal lumen must be
limited genomic diversity. Mycobacterium leprae displays an extreme accompanied by substantial genetic regulatory events.
degree of gene decay.43 The microbial cell is relatively simple, yet it possesses the means to
Not uncommonly, virulence-specific sequences are bounded by detect, often simultaneously, changes in temperature, ionic conditions,
repeated DNA segments, some of which represent known insertion oxygen concentration, pH, and calcium, iron, and other metal concen-
elements, which suggests that these virulence genes were once associ- trations that might appear to be subtle signals, but which are essential
ated with a mobile genetic element or that these genes formerly occu- for the precise mobilization of virulence determinants. Similarly, envi-
pied another chromosomal locale in either the same species or another ronmental regulatory signals prepare the microbe for its transition
microorganism altogether. Acquisition of an adhesin, toxin, or serum from an extracellular to an intracellular state. For example, iron is a
resistance factor might lead a previously nonpathogenic organism to critical component of many cell metabolic processes; therefore, it is
cause disease in a host that had previously been nonsusceptible. not surprising that animals rely on high-affinity iron-binding and
This concept has been supported by the discovery of “pathogenicity storage proteins to deprive microorganisms from access to this nutri-
islands.”44 Pathogenicity islands contain clusters of virulence-associ- ent, especially at the mucosal surface. In turn, most pathogens sense
ated genes that encode specialized secretion systems, secreted effector iron availability and induce or repress various iron acquisition systems
molecules, adhesins, and regulatory proteins. Salmonella typhimurium accordingly.49 Indeed, many microorganisms possess toxins which are
is believed to have begun evolving as a pathogen from a common regulated by iron such that low iron concentrations trigger toxin bio-
ancestor that it shares with E. coli approximately 130 million years ago synthesis. For the gastric pathogen H. pylori, pH may be a critical
through the sequential acquisition of at least two pathogenicity islands, signal. The H. pylori response to low pH involves changes in transcript
one of which mediates internalization within host cells and the other, abundance for 7% of its genes and is associated with increased motility,
survival and replication within an intracellular vacuole.45 Yersinia pestis perhaps as a means for penetrating the gastric mucous layer.50
8 Part I  Basic Principles in the Diagnosis and Management of Infectious Diseases

Reversible regulation of the expression of virulence genes by tem- TABLE

perature is a feature common to many pathogens, including entero- 1-2 Examples of Bacterial Virulence Regulatory Systems
pathogenic and uropathogenic E. coli (K-88 and K-99 fimbriae, Regulatory Environmental
pyelonephritis-associated pilus fimbriae, and K-1 capsular antigen), Organism Gene(s) Stimuli Regulated Functions
Shigella spp. (invasiveness and Shiga toxin), and Yersinia spp. (viru- Escherichia coli drdX Temperature Pyelonephritis-associated
lence-associated determinants, including a low-calcium response and pili
outer membrane proteins). Changes in DNA topology, messenger fur Iron concentration Shiga-like toxin,
RNA conformation, and, in the case of the heat shock response, siderophores
protein stability mediate thermal regulation of these diverse virulence Bordetella bvgAS Temperature, ionic Pertussis toxin,
determinants.51 pertussis conditions, filamentous
nicotinic acid hemagglutinin,
The number of well-characterized virulence regulatory systems is adenylate cyclase,
rapidly increasing, in part because of the development of rapid others
methods for screening gene expression on a genome-wide basis (e.g., Vibrio cholerae toxR Temperature, Cholera toxin, pili,
with the use of DNA microarrays). At the same time, relatively little is osmolarity, pH, outer-membrane
known about both the specific environmental signals to which these amino acids proteins
systems respond in vivo and the exact role of these responses in the Yersinia spp. lcr loci Temperature, Secretion of effector
calcium proteins
course of human infection.
virF Temperature Adherence, invasiveness
One common mechanism for bacterial transduction of environ-
Shigella spp. virR Temperature Invasiveness
mental signals involves two-component regulatory systems that act on
gene expression, usually at the transcriptional level.52,53 Such systems Salmonella pag genes pH Virulence, macrophage
typhimurium survival
make use of similar pairs of proteins; one protein of the pair spans the Staphylococcus agr genes Cell density α-, β-Hemolysins; toxic
cytoplasmic membrane, contains a transmitter domain, and may act aureus shock syndrome
as a sensor of environmental stimuli, whereas the other is a cytoplas- toxin-1, protein A
mic protein (“response regulator”) with a receiver domain that regu- Data from Miller JF, Mekalanos JJ, Falkow S. Coordinate regulation and sensory
lates responsive genes or proteins. Sensor proteins are often kinases transduction in the control of bacterial virulence. Science. 1989;243:916-922; Mekalanos
that phosphorylate themselves at a conserved histidine residue. These JJ. Environmental signals controlling the expression of virulence determinants in
bacteria. J Bacteriol. 1992;174:1-7.
high-energy intermediates then transfer their phosphate groups to a
conserved aspartate residue within the receiver domain of the response
regulator proteins. Competing dephosphorylases determine an overall
phosphorylation state of these response regulators and hence their from the two-component sensory transduction system. The combina-
level of activity. Many of these regulators are DNA-binding proteins tion of these features into one protein may lead to increased specificity
that regulate transcription of multiple gene targets. Systems of this type of action. Vibrios in cholera stools may be hyperinfectious and pre-
control, for example, the permeability properties of the E. coli cell pared for enhanced transmission.54 The transcriptional profile of these
envelope in response to osmotic stimuli (EnvZ/OmpR), motor control organisms as they exit patients suggests recent nutrient deprivation,
involved in E. coli chemotaxis (CheA/CheY, CheB), the switch from iron limitation, downmodulated toxin expression, and reduced che-
vegetative growth to sporulation by Bacillus subtilis (KinA/SpoOF, motactic activity.54,55
SpoOA), and even the ability of the soil bacterium Agrobacterium Quorum sensing is a means by which bacteria keep track of their
tumefaciens to induce tumors in susceptible plant cells in response to cell density and regulate their behavior accordingly.56 It is inextricably
phenols found within plant wound exudates (VirA/VirG). involved in the formation of complex community structures called
The coordinated control of pathogenicity incorporates the impor- biofilms by bacteria on environmental surfaces for long-term persis-
tant concept of a regulon. A regulon is a group of operons or individual tence and resistance to host defenses. Gram-negative organisms secrete
genes controlled by a common regulator, usually a protein activator and respond to acylated homoserine lactones as a means of cell-cell
or repressor. This regulator may, in some cases, be the second com- communication. Production of light by marine vibrios and tissue-
ponent of a two-component system. A regulon provides a means by degrading enzymes by P. aeruginosa is activated by these autoinducing
which many genes can respond in concert to a particular stimulus. At compounds when they reach sufficient concentration.57 Gram-positive
other times, the same genes may respond independently to other organisms such as S. aureus use peptide autoinducers and repressors
signals. Global regulatory networks are a common feature of microbial to sense cell density and regulate toxin expression. The ability of bacte-
virulence as well as basic microbial physiology (Table 1-2). The rial pathogens to take their own census enables precise choreography
complexity of virulence regulation in a single microbial pathogen is of virulence factor production during the course of growth in a vigilant
magnified by the coexistence of multiple interacting (“cross- host. For example, in the early stages of a developing soft tissue abscess,
talking”) systems and by regulons within regulons. P. aeruginosa, for S. aureus turns on antiphagocytic toxins just as the bacteria reach
example, contains genes for 55 sensors and 89 response regulators, numbers sufficient to draw the attention of neutrophils.58 V. cholerae
whereas H. pylori contains genes for 4 and 7, respectively. Perhaps the relies on quorum sensing to regulate biofilm formation on marine
more restricted numbers and types of microenvironments occupied by plankton and mediate release from these biofilms upon entry into a
the latter organism reduce the number of cues that it must recognize. human host.59 Quorum factors sometimes exhibit strain specificity,
Given the limited sensitivity of in vitro models, it appears that some and might serve as targets for novel immunoprophylactic approaches.60
but not all regulatory systems are essential for virulence. Some microbial pathogens (e.g., N. gonorrhoeae, Borrelia recurrentis,
Regulation of the expression of virulence determinants by V. chol- and Trypanosoma brucei) periodically vary prominent antigenic com-
erae illustrates the use of a global regulatory protein that in this case ponents of their surface and, by so doing, avoid the host immune
serves a dual function. The toxR gene product is a transmembrane, response. Antigenic variations in S. typhimurium and N. gonorrhoeae
DNA-binding protein that regulates expression of cholera toxin, toxin- provide examples of alternative molecular mechanisms (i.e., DNA
coregulated pili, and specific outer-membrane proteins. The ToxR rearrangements) that mediate regulation of the expression of virulence
protein is thought to sense a variety of environmental regulatory factors. S. typhimurium varies an immunodominant antigen by alter-
signals, including osmolarity, amino acid concentration, temperature, nating between the expression of two different flagellin genes, H1 and
and pH. ToxR directs expression of these genes indirectly by activating H2. The mechanism for this form of variation involves inversion of a
transcription of toxT, whose product is a member of the AraC family 995-bp chromosomal DNA sequence containing a promoter.61 By
of transcriptional regulators. At the level of amino acid sequence, the altering expression of flagellin, S. typhimurium avoids the host anti-
ToxR protein contains features of both sensor and regulator proteins body response directed against it.
 1 A Molecular Perspective of Microbial Pathogenicity 9

Pili are essential for virulence of the gonococcus in the human host, proteins during the earliest stages of entry help create conditions nec-
probably as a result of their role in adherence to the mucosal target essary for growth and development of the pathogen.
surface.62 They also elicit a specific local and systemic host antibody Some pathogenic microorganisms seem to regulate when and where
response. Intermittent production of pili, as well as variation in the they enter host cells by using preexistent host signaling pathways.13
antigenic type of pilus, may be strategies used by the gonococcus to Among the receptors that recognize pathogens and mediate entry are
avoid the host immune response. The molecular mechanisms behind integrins (Yersinia spp.), tight junction apparatus cadherins (Listeria
these strategies are complex. In general terms, phase and antigenic monocytogenes)67 and ZO-1 (H. pylori),68 dystroglycans (arenavi-
variation result from DNA rearrangements that move pilin-related ruses),69 and growth factor receptors (S. typhimurium). In some of
sequences scattered around the gonococcal chromosome (in silent pilS these cases, the pathogens do not depend on only one receptor family
loci) to the expression site (pilE locus). Numerous different pilus types for cellular entry. In addition, cell or organ tropism may be determined
may be expressed by derivatives of a single N. gonorrhoeae strain. by recognition of different members of the same family.
Among other microbial pathogens, DNA rearrangements account for Signaling events at the surface of the host cell, between pathogens
the antigenic variation of variant surface glycoproteins of T. brucei63 of the same type, and between pathogen and host cell indicate a
and the antigenic variation of variable major proteins in Borrelia spp.64 complex, highly evolved process of coadaptation and co-optation.6,15,56
Proper presentation of certain virulence-associated gene products Many of these signals induce rearrangement of host cell cytoskeleton
on the microbial surface is now recognized to be as important to to the advantage of the pathogen. In a particularly dramatic example,
pathogenicity as the initial expression of these genes. Presentation enteropathogenic E. coli induces the effacement of normal epithelial
entails export pathways, association with other periplasmic or surface cell surface architecture and the formation of a specialized structure
factors, and sometimes macromolecular assembly at the surface and is containing reorganized actin that protrudes from the host cell surface
also subject to regulation. Among bacterial pathogens, shared homol- and is called a “pedestal” or pseudopod (Fig. 1-1). Pedestals facilitate
ogy is apparent among families of proteins involved in these processes. intimate attachment but not entry of enteropathogenic E. coli to the
One family consists of proteins that are known as chaperones and host cell; attachment is mediated by the bacterial adhesin intimin and
ushers, concepts first proposed in a model for the assembly of uroep- a receptor, Tir, that is secreted by enteropathogenic E. coli into the host
ithelium-adherent E. coli P pili.65 Periplasmic chaperones such as PapD cell and then localized to the host cell membrane at the apical surface
escort protein subunits from the cytoplasmic to the outer membrane of the pedestal.70 These events require a specialized secretion system
and assist in their proper folding. Outer-membrane ushers such as (see later) that delivers not only Tir but also effector proteins that
PapC target these complexes to a surface assembly site. Folding, trans- direct host cell phosphorylation of Tir and stimulate other signaling
port, and assembly enable a microorganism to present a specific array pathways. All these factors are encoded by genes found within a patho-
of surface molecules necessary for eukaryotic cell tropism, intoxica- genicity island known as the locus for enterocyte effacement; this chro-
tion, or entry.6 A precise configuration of microbial surface molecules mosomal island is also found in some strains of Shiga toxin–producing
might be viewed as an “attack complex,” with properties not found in E. coli. Tir from Shiga toxin–producing E. coli is immunogenic in
any of the individual components. humans and exhibits sequence and antigenic diversity among different
isolates.71
Microbial Pathogens as Other forms of cytoskeletal rearrangements are essential to the
process by which Salmonella, Shigella, and other intracellular patho-
Intracellular Parasites gens enter host cells. Salmonella induces “ruffling” of the host cell
Despite their capacity for an extracellular existence, a wide variety of membrane, which then engulfs the bacterium and leads to internaliza-
bacterial and protozoal pathogens have evolved the means to enter,
survive, multiply, and even persist within host eukaryotic cells. By so
doing, a microorganism avoids host immune defenses and gains access
to what are otherwise restricted nutrients. These advantages impose a
strong selective evolutionary pressure that is dramatically reflected in
the refined strategies developed by microbial pathogens for life within
a host cell. These strategies include molecular mimicry, coercion, and
intimate adaptation to eukaryotic cellular processes.
To a large degree, the mechanisms used by a microorganism to
adhere to a eukaryotic cell dictate whether and how it enters the cell
and its subsequent intracellular fate.13 Most, if not all, intracellular
pathogens have multiple means for attachment to a eukaryotic cell
surface; the particular combination of microbial attachment factors
and cognate host receptors favors selection of one of several entry
pathways and predetermines basic features of the intracellular vacuole.
However, in a general sense, it is unclear to what extent microbial
pathogens accept preprogrammed pathways dictated by phagocytic
(e.g., complement and Fc receptors) and nonphagocytic receptors, and
to what extent they may be able to modify or exploit these pathways.
Toxoplasma gondii invades and replicates within all types of nucleated
mammalian cells. After entry and through unidentified receptors, T. Figure 1-1  Scanning electron micrograph depicting pseudopod or
gondii resides within a parasitophorous vacuole that is permanently “pedestal” formation by enteropathogenic Escherichia coli (EPEC)
incapable of fusion with other intracellular organelles, including lyso- as it interacts with the surface of an epithelial cell. This form of
somes. Parasite survival within this vacuole depends on the accompa- intimate adherence requires a bacterial adhesin, intimin; a receptor
nying lack of acidification, exclusion of lysosomal contents, and of bacterial origin, Tir, that is injected into the host cell; and a series of
specific mechanisms for nutrient acquisition and environmental EPEC-initiated signaling events. Disruption of normal absorptive func-
tion results in diarrhea. Other bacterial pathogens are also capable of
sensing. However, when this organism is directed to enter eukaryotic inducing pedestal formation on intestinal epithelial cells. (From Rosen-
cells by means of an alternative pathway (i.e., mediated by receptors shine I, Ruschkowski S, Stein M, et al. A pathogenic bacterium triggers
for the constant region of immunoglobulin G, Fc), this vacuole fusion epithelial signals to form a functional bacterial receptor that mediates
block is overcome.66 Presumably, parasite-directed modifications of actin pseudopod formation. EMBO J. 1996;15:2613-2624. Courtesy of
the surrounding vacuolar membrane and exclusion of certain host BB Finlay.)
10 Part I  Basic Principles in the Diagnosis and Management of Infectious Diseases

tion through macropinocytosis. This response by nonphagocytic cells in macrophages and dendritic cells but not epithelial cells. Although
is similar to that provoked by growth factors. Salmonella Sip proteins the induction of cell death and frank apoptosis is a common shared
and the related Shigella Ipa proteins are secreted into host cells after strategy of many pathogens, each accomplishes this outcome through
surface contact and are each necessary for the cytoskeletal responses different mechanisms and with a different precise temporal program.15,18
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 replication. microbial pathogens. Because they establish dependent relationships
Some pathogens remain within a vacuole (e.g., Toxoplasma, Salmo- with host cells, viruses often manipulate host cells in dramatic fashion.
nella), and some lyse the initial phagosomal membrane and replicate Human papillomaviruses and other animal viruses induce expansion
within the host cell cytoplasm (Shigella, Listeria, Trypanosoma, some of their preferred host niche by interfering with critical cell-cycle con-
Rickettsia species). Maintenance of specific and favorable vacuolar trols.78 In an interesting analogy, Rickettsia rickettsii blocks a host cell
conditions may entail inhibition of phagolysosomal fusion and acidi- apoptosis defensive strategy to prolong the life of the infected cell,
fication (Toxoplasma), association of eukaryotic organelles with bac- facilitate rickettsial replication, and then spread to other host cells.79
teria-containing vacuoles (Legionella, Salmonella), and regulation of Molluscum contagiosum virus protects its host cell from oxidative or
pH (Salmonella). ultraviolet-induced damage by expressing a glutathione peroxidase–
Early escape from the vacuole is essential for the growth and virulence like selenoprotein that acts as a scavenger of toxic oxygen metabo-
of some intracellular pathogens. L. monocytogenes relies on several lites.80 Other opportunistic strategies of viral pathogens include
molecules for lysis of the early phagosome, including a pore-forming suppression of viral antigen presentation by host cells and interference
hemolysin (listeriolysin O) and two forms of phospholipase C. Once in with host cytokine, complement, and interferon activities.17-20
the cytoplasm, Listeria replicates and induces its own movement A wide variety of intracellular pathogens and their patterned molec-
through a remarkable process of host cell actin polymerization and ular components are recognized by members of the NOD (nucleotide-
formation of microfilaments within a comet-like tail. Shigella also lyses binding oligomerization)-like family of receptors, which then leads to
the phagosomal vacuole and induces the formation of similar structures activation of a cytosolic protein complex known as the “inflamma-
for the purpose of intracytoplasmic movement and cell-cell spread. In some” and activation of caspase-1, a protease that in turns promotes
both cases, bacterial and host factors involved in actin polymerization activation of proinflammatory cytokines and host cell death.81 The
have been identified.72 In the same way that microbial pathogens fare inflammasome can be viewed as a kind of ancient record of the long-
differently in their interactions with phagocytic cells, the outcome of standing interplay between phagocytes and microbes. The scope and
intracellular parasitism for the host cell also varies considerably, sophistication of the means by which pathogens overcome the host
depending on the specific host cell and pathogen involved. barrier to their establishment, replication, persistence, and subse-
quently exit and transmission to a new host must be seen as one of the
Subversion of Host Cellular most impressive examples of evolutionary diversity and adaptation.
We cannot hope to do justice to the topic here, but we do hope to
Processes and Immune Defenses inspire appreciation and respect among clinicians for their micro-
Pathogens can be distinguished from commensal microorganisms by scopic adversaries.
their ability to subvert host cellular processes to their own advan-
tage.13,15 Enhanced adherence or internalization of the pathogen, inhi- Identification and Characterization
bition of host cell antimicrobial activity, altered inflammatory
responses, enhanced multiplication, and death are potential outcomes
of Virulence Genes
for the host cell and goals for the pathogen. As mentioned earlier, one Characterization of microbial pathogenicity at the molecular level has
common mechanism by which bacterial pathogens alter or subvert the traditionally begun with the identification of a virulence-associated
host cell involves specialized secretion systems known as the type III phenotype. Such identification may come from clinical observation,
or contact-dependent secretion pathway and the type IV secretion epidemiologic investigation, or the use of a model system that reliably
pathway.73,74 Type III secretion systems from diverse bacterial patho- reproduces the microbial phenotype in a manner similar to that seen
gens share structural and functional features that suggest an evolution- in natural infection. Traditionally, a virulent strain was compared with
ary relationship with the bacterial flagellar apparatus.73 These systems a naturally occurring avirulent variant. Such variants, however, may
are encoded by blocks of genes that are usually located within patho- have complex genotypic alterations involving multiple genetic loci.
genicity islands. Using a supramolecular structure that spans the entire Today, increasingly sophisticated computational tools and ever more-
cell wall and resembles a hypodermic syringe,75 pathogens secrete easily acquired complete genome sequences have led to predictions of
effector molecules directly across host cell membranes. Whereas Sal- phenotype and identification of candidate virulence genes from the
monella and Shigella use type III secretion systems (Salmonella patho- sequence. The inspection of genome sequences from multiple strains,
genicity island 1 [SPI-1] and invasion plasmid systems, respectively) each with variant virulence phenotypes, provides a powerful starting
to mediate entry into host cells, Salmonella relies on a second type III point for subsequent hypothesis testing using specific engineered
system (SPI-2) for successful replication within an intracellular mutant strains, when the microbial species is genetically tractable.
vacuole; this second system is expressed only when the organism occu- Some pathogens and many commensals, however, remain genetically
pies this privileged niche. intractable and resistant to laboratory cultivation. For these organisms,
Type III secreted effector molecules mediate diverse tasks. Salmo- emerging methods offer the possibility of examining their behavior
nella SopE is secreted by the SPI-1 system and binds directly to and putative properties directly from clinical specimens without the
members of the Rho small-molecular-weight guanosine triphospha- need for laboratory propagation (see “Molecular Microbiology at the
tase protein family in the host cell cytoplasm; this action activates Bedside: Pathogen Detection, Pathogen Discovery, and Genomic
membrane ruffling.76 SopE also stimulates mitogen-activated protein Profiling”).
kinases and, thus, nuclear factor κB (NF-κB) and activator protein 1 Some of the now-standard approaches for identifying virulence-
(AP-1)–mediated nuclear transcriptional responses. The Yersinia associated genes include the use of insertional elements (e.g., transpo-
YopH effector protein is a potent protein tyrosine phosphatase, viru- sons) as mutational agents for generating isogenic mutant strains.
lence factor, and antiphagocytic factor. YopJ induces apoptosis in acti- Transposons have the advantage of marking the mutagenized genetic
vated macrophages, suppresses tumor necrosis factor-α production, locus with a new selectable phenotype, typically antibiotic resistance,
and is critical for Yersinia translocation from Peyer’s patches to lym- but the disadvantage of possible pleiotropic effects on cotranscribed
phoid tissue and replication in the spleen.77 A number of pathogens, genes or on overall microbial fitness. The development of broad-host-
including Shigella and Salmonella, are capable of inducing cell death range plasmid vectors carrying well-defined transposons has extended
 1 A Molecular Perspective of Microbial Pathogenicity 11

this method of analysis to a number of pathogenic species for which a practice of clinical infectious diseases.28 It is already apparent that
method of genetic manipulation was not previously available. By using studies of microbial pathogenicity at the molecular level have made
transposable elements with unique genetic tags, negative selection can substantial contributions to our understanding of the epidemiology,
be applied to a pool of random mutants in a relevant model of patho- clinical manifestations, diagnosis, treatment, and immunoprophylaxis
genesis. This approach, known as signature-tagged mutagenesis, has of infectious diseases. Even the fundamental issue of disease causation
identified a number of genes in gram-negative and gram-positive bac- and the possible role of microorganisms in chronic diseases of uncer-
teria that are essential for virulence.82,83 tain etiology must be reexamined in light of newer experimental
Other methods for the identification of virulence-associated genes methods and insight.89
are based on the regulation of such genes by the transitions between Infectious disease epidemiology hinges on a clear definition of the
external and internal host and cellular environments. Two approaches clinical problem under study and, moreover, precise identification of
allow one to select for genes and promoters that are preferentially the etiologic agent. Molecular techniques provide for the sensitive and
expressed by a microbial pathogen within a host cell or within a host specific detection of putative pathogens and supply a means for estab-
organ. These approaches rely on specially designed vectors into which lishing relationships among multiple isolates of the same species. As a
a complete library of chromosomal genes are cloned such that when result, seemingly unrelated cases occurring during an outbreak have
the promoters for these genes are activated they turn on the expression been connected; similarly, geographically or temporally distinct out-
of factors that can be easily selected, either by expression of antibiotics, breaks have been linked to the same pathogenic clone. Molecular
by complementation of an engineered growth-attenuating mutation techniques have been used in other epidemiologic investigations to
in the pathogen, or by expression of a fluorescent protein. In the first study transmission mechanisms and the role of avirulent microbial
approach, the application of in vivo expression technology to V. cholerae variants in the spread of disease. Molecular strain typing data some-
and S. typhimurium has clarified the conditions encountered by patho- times provide the only clue that a group of cases are related, that is,
gens in vivo as well as their regulatory responses.84 By using the second that an outbreak of disease has occurred.90 Morphologic and metabolic
approach termed differential fluorescence induction, one identifies pro- features often fail to indicate the important genetic diversity found
moters that are selectively induced within host cells or tissues by fusing within strains.
random fragments of a pathogen’s genome to the gene encoding green Nucleic acid amplification techniques have had a far-reaching
fluorescent protein and then applying fluorescence-activated flow impact on study of microbial pathogenesis and the diagnosis of infec-
cytometry to a pool of recombinant organisms bearing these reporter tious diseases. For a variety of reasons, some of them economic (reim-
gene fusions.85 bursement structure for clinical testing) and some of them technical
Broad-based, nonselective approaches for screening an entire (suboptimal sample preparation methods, insufficient attention to the
genome and its complement of expressed genes are now quite feasible complexity of specimen matrix), specific PCR assays for microbial
with subtractive hybridization, full genome sequences, and DNA pathogens have failed to penetrate the clinical workplace to a thorough
microarray technology. Methods based on subtractive hybridization degree.91 Current methods for the identification of microbial patho-
techniques facilitate the detection of subtle differences between two gens rely heavily on cultivation or propagation in the laboratory.
populations of DNA or RNA. These differences can be amplified by Molecular pathogen discovery methods provide alternative approaches
polymerase chain reaction (PCR).86 DNA microarrays are high-density and have spawned new searches for microorganisms that might play
grids of probes displayed on a solid surface; tens or hundreds of thou- important causal roles in a wide variety of poorly explained acute and
sands of probes can be arrayed in an area of 1 cm2. By displaying probes chronic diseases.28,92,93 The principle behind these methods is reliance
for every gene of a given pathogen on a microarray and hybridizing on molecular signatures to identify or classify a previously unrecog-
labeled genomic DNA or complementary DNA, one can obtain a com- nized pathogen; the most commonly used signature is the genomic
plete gene content or expression profile, respectively, for the pathogen sequence, but other small molecules may prove useful. One of these
under any desired condition. Comparative genomic hybridization methods targets highly conserved regions of ribosomal DNA sequences
analysis of multiple strains of pathogens reveals localized regions of by amplifying them directly from digested, infected human tissue.92
frequent gene gain and loss (“regions of difference,” equivalent in Reliable evolutionary relationships of a putative organism can then be
many cases to pathogenicity islands) and can provide insight into established from these amplified ribosomal DNA sequences. A number
pathogen evolution as well as novel mechanisms of virulence.42 With of organisms resistant to cultivation or propagation have been identi-
these approaches, genes and their products are incriminated by their fied with non–culture-based methods.94,95 Some of the same molecular
relationship with a disease-associated process. Final proof, however, methods are used to explore diversity within the indigenous microbial
that a gene is associated with pathogenicity requires that certain crite- communities that populate the skin and mucosal surfaces of the
ria be met. In a manner analogous to Koch’s original postulates, these human body.1,96
criteria must include return of the putative causal agent (the cloned Conceptual advances in our understanding of microbial virulence,
virulence-associated gene mutated or intact) to the host of origin. revolutionary developments in our technical means, and emerging
Unless one can demonstrate an effect on pathogenicity by this kind of challenges from a rapidly changing environment around us suggest a
controlled genetic manipulation, causality with respect to virulence number of future scenarios and goals. First, we should focus our efforts
has not been proved. Just as the original Henle-Koch postulates have on the identification and characterization of pathogens directly from
provided a reference point for later revised criteria of microbial causal- clinical specimens and infected hosts, using cultivation-independent
ity,87 a molecular form of Koch’s postulates88 can serve as a guideline approaches. Manipulation and genome-wide characterization of single
for an experimental approach to the molecular genetic basis of patho- bacterial cells is now entirely feasible.97 Deep sequencing–based and
genicity. These postulates continue to coevolve in conjunction with microarray-based pathogen identification from clinical specimens is
emerging insights into microbial virulence and rapidly improving also a reality.98 We should expect to be able to measure genome-wide
experimental approaches and technologies. microbial transcript abundance and metabolic activity directly from
human specimens as well. Second, the composition and behavior of
the indigenous microbial communities can be assessed using metage-
Molecular Microbiology at the nomic and other community-wide postgenomic technologies.4,99 By
Bedside: Pathogen Detection, Pathogen combining assessments of community and human response, we stand
to gain novel insights into the nature of chronic inflammatory disor-
Discovery, and Genomic Profiling ders of skin and mucosa.100 Third, it is now time to fully embrace the
As mechanisms of microbial pathogenicity, acquisition of virulence, importance of host genetic variation in differential susceptibility to
and drug resistance are revealed, pathogen detection, strain identifica- infection and subsequent disease.101 Fourth, genomic and post-
tion, and pathogen discovery assume increasing importance in the genomic technologies enable us to measure and interpret patterns of
12 Part I  Basic Principles in the Diagnosis and Management of Infectious Diseases

human gene and protein expression associated with the response to tion of global virulence regulatory systems may have therapeutic value.
infectious disease; these patterns may serve as the basis for signatures, New acellular or recombinant live-attenuated vaccines and vaccine
enabling early recognition and classification of patients on the basis of candidates have already resulted from the identification of immuno-
agent or future disease course.28,102-104 As virulence factors for essential protective antigens through molecular and genomic approaches.8 The
steps in pathogenesis are identified, it should be possible to interfere result of these efforts should be a more informed and effective approach
with their function. As they become better characterized, manipula- to the detection, treatment, and prevention of infectious diseases.

REFERENCES
1. Dethlefsen L, McFall-Ngai M, Relman DA. An ecological and 31. Hugenholtz P. Exploring prokaryotic diversity in the genomic 63. Borst P. Antigenic variation and allelic exclusion. Cell.
evolutionary perspective on human-microbe mutualism and era. Genome Biol. 2002;3:REVIEWS0003. [Epub 2002 Jan 29]. 2002;109:5-8.
disease. Nature. 2007;449:811-818. 32. Maiden MC, Bygraves JA, Feil E, et al. Multilocus sequence 64. Meier JT, Simon MI, Barbour AG. Antigenic variation is associ-
2. Bäckhed F, Ley RE, Sonnenburg JL, et al. Host-bacterial mutual- typing: A portable approach to the identification of clones ated with DNA rearrangements in a relapsing fever Borrelia. Cell.
ism in the human intestine. Science. 2005;307:1915-1920. within populations of pathogenic microorganisms. Proc Natl 1985;41:403-409.
3. Dethlesen L, Eckburg PB, Bik EM, et al. Assembly of the human Acad Sci U S A. 1998;95:3140-3145. 65. Jones CH, Jacob-Dubuisson F, Dodson K, et al. Adhesin presen-
gastrointestinal microbiota. Trends Ecol Evol. 2006;21:517-523. 33. Achtman M. Evolution, population structure, and phylogeogra- tation in bacteria requires molecular chaperones and ushers.
4. Turnbaugh PJ, Hamady M, Yatsunenko T, et al. A core phy of genetically monomorphic bacterial pathogens. Annu Rev Infect Immun. 1992;60:4445-4451.
gut microbiome in obese and lean twins. Nature. Microbiol. 2008;62:53-70. 66. Sinai AP, Joiner KA. Safe haven: The cell biology of non­fusogenic
2009;457:480-484. 34. Daubin V, Moran NA, Ochman H. Phylogenetics and the cohe- pathogen vacuoles. Annu Rev Microbiol. 1997;
5. Segal S, Pollard AJ. Vaccines against bacterial meningitis. Br Med sion of bacterial genomes. Science. 2003;301:829-832. 51:415-462.
Bull. 2004;72:65-81. 35. Falkow S. The evolution of pathogenicity in Escherichia, Shigella, 67. Mengaud J, Ohayon H, Gounon P, et al. E-cadherin is the recep-
6. Merrell DS, Falkow S. Frontal and stealth attack strategies in and Salmonella. In: Neidhardt FC, ed. Escherichia coli and tor for internalin, a surface protein required for entry of
microbial pathogenesis. Nature. 2004;430:250-256. Salmonella typhimurium. Washington, DC: ASM Press; 1996: L. monocytogenes into epithelial cells. Cell. 1996;84:923-932.
7. Falkow S. The microbe’s view of infection. Ann Intern Med. 2723-2729. 68. Amieva MR, Vogelmann R, Covacci A, et al. Disruption of the
1998;129:247-248. 36. Roumagnac P, Weill FX, Dolecek C, et al. Evolutionary history epithelial apical-junctional complex by Helicobacter pylori CagA.
8. Medini D, Serruto D, Parkhill J, et al. Microbiology in the post- of Salmonella typhi. Science. 2006;314:1301-1304. Science. 2003;300:1430-1434.
genomic era. Nat Rev Microbiol. 2008;6:419-430. 37. Johnson JR, Manges AR, O’Bryan TT, et al. A disseminated 69. Cao W, Henry MD, Borrow P, et al. Identification of alpha-
9. Relman DA, Falkow S. The meaning and impact of the human multidrug-resistant clonal group of uropathogenic Escherichia dystroglycan as a receptor for lymphocytic choriomeningitis
genome sequence for microbiology. Trends Microbiol. coli in pyelonephritis. Lancet. 2002;359:2249-2251. virus and Lassa fever virus. Science. 1998;282:2079-2081.
2001;9:206-208. 38. Moodley Y, Linz B, Yamaoka Y, et al. The peopling of the Pacific 70. Kenny B, DeVinney R, Stein M, et al. Enteropathogenic E. coli
10. Casadevall A, Pirofski LA. The damage-response framework of from a bacterial perspective. Science. 2009;323:527-530. (EPEC) transfers its receptor for intimate adherence into mam-
microbial pathogenesis. Nat Rev Microbiol. 2003;1:17-24. 39. Fleischmann RD, Adams MD, White O, et al. Whole-genome malian cells. Cell. 1997;91:511-520.
11. Jones GW, Isaacson RE. Proteinaceous bacterial adhesins and random sequencing and assembly of Haemophilus influenzae 71. Paton AW, Manning PA, Woodrow MC, et al. Translocated
their receptors. Crit Rev Microbiol. 1983;10:229-260. Rd. Science. 1995;269:496-512. intimin receptors (Tir) of Shiga-toxigenic Escherichia coli iso-
12. Kolenbrander PE, Andersen RN, Blehert DS, et al. Communica- 40. Moran NA. Microbial minimalism: Genome reduction in bacte- lates belonging to serogroups O26, O111, and O157 react with
tion among oral bacteria. Microbiol Mol Biol Rev. rial pathogens. Cell. 2002;108:583-586. sera from patients with hemolytic-uremic syndrome and exhibit
2002;66:486-505. 41. Parkhill J, Sebaihia M, Preston A, et al. Comparative analysis of marked sequence heterogeneity. Infect Immun. 1998;66:5580-
13. Boyle EC, Finlay BB. Bacterial pathogenesis: Exploiting cellular the genome sequences of Bordetella pertussis, Bordetella paraper- 5586.
adherence. Curr Opin Cell Biol. 2003;15:633-639. tussis and Bordetella bronchiseptica. Nat Genet. 2003;35:32-40. 72. Goldberg MB. Actin-based motility of intracellular microbial
14. Williams JM, Chen GC, Zhu L, et al. Using the yeast two-hybrid 42. Cummings CA, Brinig MM, Lepp PW, et al. Bordetella species pathogens. Microbiol Mol Biol Rev. 2001;65:595-626.
system to identify human epithelial cell proteins that bind gono- are distinguished by patterns of substantial gene loss and host 73. Blocker A, Komoriya K, Aizawa S. Type III secretion systems and
coccal Opa proteins: Intracellular gonococci bind pyruvate adaptation. J Bacteriol. 2004;186:1484-1492. bacterial flagella: Insights into their function from structural
kinase via their Opa proteins and require host pyruvate for 43. Cole ST, Eiglmeier K, Parkhill J, et al. Massive gene decay in the similarities. Proc Natl Acad Sci U S A. 2003;100:3027-3030.
growth. Mol Microbiol. 1998;27:171-186. leprosy bacillus. Nature. 2001;409:1007-1011. 74. Pallen MJ, Chaudhuri RR, Henderson IR. Genomic analysis of
15. Hornef MW, Wick MJ, Rhen M, et al. Bacterial strategies for 44. Groisman EA, Ochman H. Pathogenicity islands: Bacterial evo- secretion systems. Curr Opin Microbiol. 2003;6:519-527.
overcoming host innate and adaptive immune responses. Nat lution in quantum leaps. Cell. 1996;87:791-794. 75. Kubori T, Sukhan A, Aizawa SI, et al. Molecular characterization
Immunol. 2002;3:1033-1040. 45. Groisman EA, Ochman H. How Salmonella became a pathogen. and assembly of the needle complex of the Salmonella
16. Young D, Hussell T, Dougan G. Chronic bacterial infections: Trends Microbiol. 1997;5:343-349. typhimurium type III protein secretion system. Proc Natl Acad
Living with unwanted guests. Nat Immunol. 2002;3:1026- 46. Guiney DG. Regulation of bacterial virulence gene expression by Sci U S A. 2000;97:10225-10230.
1032. the host environment. J Clin Invest. 1997;99:565-569. 76. Hardt WD, Chen LM, Schuebel KE, et al. S. typhimurium
17. Bowie AG, Unterholzner L. Viral evasion and subversion of 47. Roszak DB, Colwell RR. Survival strategies of bacteria in the encodes an activator of Rho GTPases that induces membrane
pattern-recognition receptor signalling. Nat Rev Immunol. natural environment. Microbiol Rev. 1987;51:365-379. ruffling and nuclear responses in host cells. Cell.
2008;8:911-922. 48. Lipp EK, Huq A, Colwell RR. Effects of global climate on infec- 1998;93:815-826.
18. Best SM. Viral subversion of apoptotic enzymes: escape from tious disease: The cholera model. Clin Microbiol Rev. 77. Monack DM, Mecsas J, Bouley D, et al. Yersinia-induced apop-
death row. Annu Rev Microbiol. 2008;62:171-192. 2002;15:757-770. tosis in vivo aids in the establishment of a systemic infection of
19. Roy CR, Mocarski ES. Pathogen subversion of cell-intrinsic 49. Crosa JH. Signal transduction and transcriptional and posttran- mice. J Exp Med. 1998;188:2127-2137.
innate immunity. Nat Immunol. 2007;8:1179-1187. Erratum in: scriptional control of iron-regulated genes in bacteria. Microbiol 78. Ferenczy A, Franco E. Persistent human papillomavirus infec-
Nat Immunol. 2008;9:105. Mol Biol Rev. 1997;61:319-336. tion and cervical neoplasia. Lancet Oncol. 2002;3:11-16.
20. Finlay BB, McFadden G. Anti-immunology: evasion of the host 50. Merrell DS, Goodrich ML, Otto G, et al. pH-regulated gene 79. Joshi SG, Francis CW, Silverman DJ, et al. Nuclear factor kappa
immune system by bacterial and viral pathogens. Cell. expression of the gastric pathogen Helicobacter pylori. Infect B protects against host cell apoptosis during Rickettsia rickettsii
2006;124:767-782. Immun. 2003;71:3529-3539. infection by inhibiting activation of apical and effector caspases
21. Welch RA, Burland V, Plunkett G III, et al. Extensive mosaic 51. Hurme R, Rhen M. Temperature sensing in bacterial gene and maintaining mitochondrial integrity. Infect Immun.
structure revealed by the complete genome sequence of uro- regulation—What it all boils down to. Mol Microbiol. 2003;71:4127-4136.
pathogenic Escherichia coli. Proc Natl Acad Sci U S A. 1998;30:1-6. 80. Shisler JL, Senkevich TG, Berry MJ, et al. Ultraviolet-induced cell
2002;99:17020-17024. 52. Parkinson JS. Signal transduction schemes of bacteria. Cell. death blocked by a selenoprotein from a human dermatotropic
22. Rasko DA, Rosovitz MJ, Myers GS, et al. The pangenome struc- 1993;73:857-871. poxvirus. Science. 1998;279:102-105.
ture of Escherichia coli: comparative genomic analysis of E. coli 53. Mitrophanov AY, Groisman EA. Signal integration in bacterial 81. Mariathasan S, Monack DM. Inflammasome adaptors and
commensal and pathogenic isolates. J Bacteriol. 2008;190:6881- two-component regulatory systems. Genes Dev. 2008;22: sensors: intracellular regulators of infection and inflammation.
6893. 2601-2611. Nat Rev Immunol. 2007;7:31-40.
23. Jonathan R, Davis JR, Lederberg J, eds. Emerging Infectious Dis- 54. Merrell DS, Butler SM, Qadri F, et al. Host-induced epidemic 82. Hensel M, Shea JE, Gleeson C, et al. Simultaneous identification
eases from the Global to the Local Perspective: A Summary of a spread of the cholera bacterium. Nature. 2002;417:642-645. of bacterial virulence genes by negative selection. Science.
Workshop of the Forum on Emerging Infections. Board on Global 55. Bina J, Zhu J, Dziejman M, et al. ToxR regulon of Vibrio cholerae 1995;269:400-403.
Health. Washington, DC: Institute of Medicine, National and its expression in vibrios shed by cholera patients. Proc Natl 83. Chiang SL, Mekalanos JJ, Holden DW. In vivo genetic analysis
Academy Press; 2001. Acad Sci U S A. 2003;100:2801-2806. of bacterial virulence. Annu Rev Microbiol. 1999;53:129-154.
24. Jones KE, Patel NG, Levy MA, et al. Global trends in emerging 56. Bassler BL. Small talk. Cell-to-cell communication in bacteria. 84. Angelichio MJ, Camilli A. In vivo expression technology. Infect
infectious diseases. Nature. 2008;451:990-993. Cell. 2002;109:421-424. Immun. 2002;70:6518-6523.
25. Eckburg PB, Lepp PW, Relman DA. Archaea and their potential 57. Donlan RM, Costerton JW. Biofilms: Survival mechanisms of 85. Valdivia RH, Falkow S. Fluorescence-based isolation of bacterial
role in human disease. Infect Immun. 2003;71:591-596. clinically relevant microorganisms. Clin Microbiol Rev. genes expressed within host cells. Science. 1997;277:2007-2011.
26. Lepp PW, Brinig MM, Ouverney CC, et al. Methanogenic 2002;15:167-193. 86. Lisitsyn N, Wigler M. Cloning the differences between two
Archaea and human periodontal disease. Proc Natl Acad Sci 58. Novick RP, Geisinger E. Quorum sensing in staphylococci. Annu complex genomes. Science. 1993;259:946-951.
U S A. 2004;101:6176-6181. Rev Genet. 2008;42:541-564. 87. Evans AS. Causation and disease: The Henle-Koch postulates
27. Vianna ME, Holtgraewe S, Seyfarth I, et al. Quantitative analysis 59. Zhu J, Mekalanos JJ. Quorum sensing–dependent biofilms revisited. Yale J Biol Med. 1976;49:175-195.
of three hydrogenotrophic microbial groups, methanogenic enhance colonization in Vibrio cholerae. Dev Cell. 2003;5: 88. Falkow S. Molecular Koch’s postulates applied to microbial
archaea, sulfate-reducing bacteria, and acetogenic bacteria, 647-656. pathogenicity. Rev Infect Dis. 1988;10:S274-S276.
within plaque biofilms associated with human periodontal 60. Camara M, Williams P, Hardman A. Controlling infection by 89. Fredricks DN, Relman DA. Sequence-based identification of
disease. J Bacteriol. 2008;190:3779-3785. tuning in and turning down the volume of bacterial small-talk. microbial pathogens: A reconsideration of Koch’s postulates.
28. Relman DA. New technologies, human-microbe interactions, Lancet Infect Dis. 2002;2:667-676. Clin Microbiol Rev. 1996;9:18-33.
and the search for previously unrecognized pathogens. J Infect 61. Simon M, Zieg J, Silverman M, et al. Phase variation: Evolution 90. Bender JB, Hedberg CW, Besser JM, et al. Surveillance by molec-
Dis. 2002;186(Suppl 2):S254-S258. of a controlling element. Science. 1980;209:1370-1374. ular subtype for Escherichia coli O157:H7 infections in Minne-
29. Relman DA. Mining the natural world for new pathogens. Am J 62. McGee ZA, Johnson AP, Taylor-Robinson D. Pathogenic mecha- sota by molecular subtyping. N Engl J Med. 1997;337:388-394.
Trop Med Hyg. 2002;67:133-134. nisms of Neisseria gonorrhoeae: Observations on damage to 91. Persing DH, Tenover FC, Versalovic J, et al. Molecular Microbiol-
30. Pace NR. A molecular view of microbial diversity and the bio- human fallopian tubes in organ culture by gonococci of colony ogy: Diagnostic Principles and Practice. Washington, DC: ASM
sphere. Science. 1997;276:734-740. type 1 or type 4. J Infect Dis. 1981;143:413-422. Press; 2004.
 1 A Molecular Perspective of Microbial Pathogenicity 13

92. Relman DA. The search for unrecognized pathogens. Science. 97. Marcy Y, Ouverney C, Bik EM, et al. Dissecting biological “dark 101. Quintana-Murci L, Alcaïs A, Abel L, et al. Immunology in
1999;284:1308-1310. matter” with single-cell genetic analysis of rare and uncultivated natura: clinical, epidemiological and evolutionary genetics of
93. Nikkari S, Lopez FA, Lepp PW, et al. Broad-range bacterial TM7 microbes from the human mouth. Proc Natl Acad Sci infectious diseases. Nat Immunol. 2007;8:1165-1171.
detection and the analysis of unexplained death and critical U S A. 2007;104:11889-11894. 102. Boldrick JC, Alizadeh AA, Diehn M, et al. Stereotyped and spe-
illness. Emerg Infect Dis. 2002;8:188-194. 98. Kistler AL, Gancz A, Clubb S, et al. Recovery of divergent avian cific gene expression programs in human innate immune
94. Relman DA, Loutit JS, Schmidt TM, et al. The agent of bacillary bornaviruses from cases of proventricular dilatation disease: responses to bacteria. Proc Natl Acad Sci U S A. 2002;99:
angiomatosis. An approach to the identification of uncultured identification of a candidate etiologic agent. Virol J. 2008; 972-977.
pathogens. N Engl J Med. 1990;323:1573-1580. 5:88. 103. Nau GJ, Richmond JF, Schlesinger A, et al. Human macrophage
95. Chang Y, Cesarman E, Pessin MS, et al. Identification of herpes- 99. Gill SR, Pop M, Deboy RT, et al. Metagenomic analysis of the activation programs induced by bacterial pathogens. Proc Natl
virus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. human distal gut microbiome. Science. 2006;312:1355-1359. Acad Sci U S A. 2002;99:1503-1508.
Science. 1994;266:1865-1869. 100. Peterson DA, Frank DN, Pace NR, et al. Metagenomic approaches 104. Whitney AR, Diehn M, Popper SJ, et al. Individuality and varia-
96. Turnbaugh PJ, Ley RE, Hamady M, et al. The human microbi- for defining the pathogenesis of inflammatory bowel diseases. tion in gene expression patterns in human blood. Proc Natl Acad
ome project. Nature. 2007;449:804-810. Cell Host Microbe. 2008;3:417-427. Sci U S A. 2003;100:1896-1901.