To access the private area of this site, please log in.

Username: 

Password:  

 

Chapter 149

The Evolution of Pathogenicity in Escherichia, Shigella, and Salmonella

STANLEY FALKOW

 

INTRODUCTION

The pathogenic gram-negative enteric bacilli, particularly the Escherichia, Shigella, and Salmonella spp., are important causes of global suffering and death. Evolutionary rate estimates from 5S and 16S rRNA sequence analysis suggest that the Escherichia and Salmonella spp. diverged from a common ancestor about 120 to 160 million years ago, around the time of the origin of mammals (see chapters 147 and 148 in this volume). The Shigella spp., which are considered clonal lineages of Escherichia coli, arose somewhat later, about 80 million years ago, presumably coinciding with the evolution of the early primate species. Commensal E. coli prefers the mammalian colon and ferments lactose (milk sugar); however, phylogenetic analysis shows that most pathogenic E. coli types are relatively distinct clusters of essentially clonal populations that occupy unique anatomic niches outside the colon. Typically, Salmonella spp. parasitize reptiles, birds, and mammals, although certain clonal types show a remarkable degree of host-species specificity and host-cell tropism. The Salmonella spp. have the capacity to cross the mucosal barrier and invade and replicate within a restricted number of host cell types to cause chronic infection and long-term carriage, as well as, in some cases, systemic disease. The primate is the only reservoir of Shigella spp. While long-term carriage by the host species is commonly seen for the Escherichia or Salmonella spp., the Shigella spp. seem to sustain themselves through an extraordinarily high rate of transmission from host to host. The Shigella spp. readily invade host cells but are generally restricted to the superficial epithelial layers.

The chapters that follow include an in-depth consideration of the bacterial properties that are known to contribute to the capacity of these bacteria to cause infection and disease. This chapter focuses on the common themes that have arisen from the study of these traits, interwoven with a fanciful view of the evolution of pathogenicity in these bacterial descendants of a common ancestral clone.

 

THE MEANING OF PATHOGENICITY

A pathogen has been defined as any microorganism capable of causing disease (10, 14). This is a practical definition based on the reality that we are subject to the onslaught of a myriad of microorganisms. Historically, most human misery and death can be attributed to infectious diseases (11). From a strict bacteriological viewpoint, pathogenicity is simply another facet of the extraordinary metabolic versatility of microbes and represents a form of bacterial specialization, in which replication on or within another host is essential for the long-term survival of a particular microbial clonal population. Pathogens replicate at the expense of host cellular integrity; although in most cases the damage is not serious (subclinical in humans), a proportion of hosts can be expected to suffer overt disease or even be killed. The outcome is as dependent upon the properties of the clone of the microorganism as it is upon the state of the host animal. Thus, a bacterial pathogen must enter a host, find a unique niche, and circumvent competing microbes and host defense barriers to replicate to a sufficient number to be transmitted to a new susceptible host.

Can we envision how the evolution of pathogenicity took place for the enteric species that are the subject of this monograph? To begin, it may be useful to speculate about the niche occupied by the ancestor of current enteric species. While E. coli is arguably the best known and the most common facultative microorganism of the mammalian gastrointestinal tract, numerically it is one of the minority species of the more than 500 species of normal intestinal bacteria. Thus, one can suppose the first niche occupied by the aboriginal enteric species was a location that was advantageous for a facultative species. Was this in the mucus gel overlying the blood-rich epithelium? Was the ancestral species a pathogen? One cannot know, of course. It is perhaps noteworthy that the bacteria we now know as members of the family Enterobacteriaceae are more likely to translocate across the intestinal mucosal barrier than are the predominant bowel anaerobic species (34). Presumably, life in the large bowel is a constant struggle for existence. The basic genes of the ancestral facultative microbe were likely to be focused on the continuity of the lineage; learning to live in this unique niche as a commensal was possibly enough selective pressure without the necessity of becoming pathogenic.

If we examine what is known about the pathogenic traits of modern-day enteric bacilli, one suspects that an early step in the evolution of pathogenicity was the expansion of certain variant enteric clones into relatively privileged sites with less microbial competition, such as the small bowel and specific mucosal surfaces adjacent to the large bowel. There is also one other correlative theme that is strikingly clear from the analysis of the virulence traits of modern-day enteric bacilli. All pathogenic E. coli, Shigella, and Salmonella strains have at least one (and in some cases, the majority) of their essential virulence determinants on an extrachromosomal element (reviewed in reference 14 and in following chapters).

 

PLASMIDS AND PATHOGENICITY

For over 20 years, it has been recognized that the principal determinants of pathogenicity in strains causing enterotoxigenic infection and neonatal diarrheal disease are carried on plasmids. Hence, the average enterotoxigenic E. coli (ETEC) isolated from traveller’s diarrhea in humans, or from sick young animals, harbors at least two plasmids. One plasmid encodes adherence pili, which are often host-species specific. Another plasmid encodes one or more enterotoxins (chapters 150 and 153). Similarly, the enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) possess a plasmid species that encodes an adhesin called bundle-forming pili (BFP) (8; chapter 150). The invasive phenotype of the Shigella spp. is carried on a large plasmid (chapter 151). Less appreciated is the fact that the capacity of the Salmonella spp. to cause systemic disease (with the notable exception of the typhoid and paratyphoid bacilli) is dependent upon the presence of a plasmid species that is interchangeable along many distinct Salmonella strains (chapter 152).

What seems striking to me about the correlation of plasmids with pathogenicity is that all of the essential virulence plasmids of enteric species belong to the F-incompatibility complex of transferable or mobilizable plasmids. These data are consistent with the idea that certain clonal populations of the ancestral enteric bacilli inherited adapted plasmid species that conferred upon them the capacity to adhere to privileged sites in the host by distinct pili or nonfimbrial adhesins. Yet, the idea that these self-transmitted plasmids might be transmitted promiscuously among enteric species is not sustained by modern-day observations. The mystery remains: What is the origin and how do/did they spread? We are reminded that due to the immense population sizes and short generation times involved, evolutionary changes in bacteria can be very rapid (chapter 146).

The importance of bacterial plasmids to the evolution of pathogenicity is unquestioned, as is their general role in a haploid world as seminal effectors of metabolic diversity and specialization. However, while a plasmid or bacteriophage may play an essential role in the expression of the pathogenic phenotype, it would be most unusual to discover that the transfer of a single plasmid or phage, even a very large plasmid or phage, would be sufficient to convert a nonpathogenic commensal microbe into a pathogenic microbe without a multitude of other contributing factors.

 

PATHOGENICITY ISLANDS

We now accept the concept that bacterial pathogenicity is a multifactorial process which involves a myriad of genes (both chromosomal and extrachromosomal) that are choreographed together through complex regulatory circuits (2; chapter 154). The success of an emerging pathogen presumably required the husbanding of virulence genes against indiscriminate genetic transfer and recombination (10). Thus, the analysis of pathogenic strains isolated from intestinal disease, such as traveller’s diarrhea and infant diarrhea, and from extraintestinal infection, particularly urinary tract infection, reveals that they are more likely to belong to distinct subsets of E. coli, rather than reflecting membership in the random fecal flora (chapter 148).

One of the most striking observations of the nature of pathogenic determinants in enteric bacilli is that certain genes, like those encoding alpha-hemolysin (chapter 153) and pyelonephritis-associated pili (Pap pili) (chapter 150) are commonly found in isolates from clinical disease, but these genes, despite their chromosomal origin, are absent from "ordinary" fecal isolates of the same species (3, 10, 25). Also, certain uropathogenic strains deleted certain virulence-associated determinants with a shockingly high frequency (17).

In the past several years, the genetic and molecular analysis of virulence genes in uropathogenic E. coli and EPEC has led to the concept of "pathogenicity islands" called Pais, which are specific regions of chromosomal DNA (3, 25). Pais represent large fragments of DNA that include a number of virulence genes. For example, two pathogenicity islands, PaiI (70 kb) and PaiII (190 kb), are known in the uropathogenic strain 536 (3, 25). This strain carries two hemolysin determinants as well as two distinct Pap-related genes. Pais have also been described for other enteric bacilli including EPEC (22) and Yersinia pestis (12). While the Pais of Yersinia strains are flanked by insertion sequences (IS), remarkably both PaiI of the uropathogenic strain 536 and the 35-kb locus of enterocyte effacement (LEE) of EPEC isolate E2348/69 were found to be flanked by tRNA loci (3, 22).

LEE and PaiI are clearly different loci. Not only do they differ in size, but they encode different pathogenicity functions as well. Yet both are bound at one end at a position 16 bp downstream of the selC (selenocysteine) tRNA locus, which also is the site of insertion of the reverse-transcriptase-encoding E. coli retronphage φR73 (33). While PaiI and φR73 are flanked by direct repeats and the regions surrounding them are colinear with the E. coli K-12 genome, one of the junctions of LEE differs from that in E. coli K-12.

DNA probes prepared from LEE have been employed to show that this locus hybridizes to enterohemorrhagic E. coli and pathogens of other genera (Citrobacter freundii and Hafnia alvei) (22). These microorganisms, like EPEC, elicit damage to the microvilli and subapical cytoskeleton of epithelial cells at sites of bacterial attachment (attachment and effacement [AE] lesions) (8, 28). The conservation of this region among divergent bacterial species and their exclusive association with AE are consistent with the idea that a block of virulence genes were, and may still be, spread horizontally among seemingly unrelated bacteria. The striking similarity of one junction of PaiI and LEE suggests that the insertion of these distinct virulence islands is mediated by a similar genetic process. It has been suggested that the selC region may act as a "slot" in the E. coli chromosome into which "cartridges" of virulence determinants are inserted as discrete units (22). There is no direct evidence to indicate that Pais or LEE are, or were, ever contained on a mobile genetic element, but the circumstantial evidence is intriguing. These findings permit us to view the evolution of pathogenicity of certain E. coli pathogenic clones in discrete steps. For example, one can suppose that the inheritance of a plasmid encoding BFP and the inheritance of LEE (not necessarily in that order) into the right genetic background was the groundwork for the eventual emergence of the E. coli clones we now call EPEC. It has been proposed that the enterohemorrhagic E. coli clone O157:H7 emerged when an O55:H7 progenitor capable of producing AE was lysogenized by a bacteriophage containing Shiga-like toxin genes (chapter 148).

One disquieting aspect of these exciting discoveries is that the base compositions of the virulence DNA insertions are strikingly different from the overall composition of the bacterial chromosome, which implies that the pathogenicity islands come from a markedly different microbial or viral chromosome. This is often the finding, as well, for virulence genes found on plasmids and phage; the virulence loci seem alien to the current host species and even to the replicative machinery of the accessory genetic element. Assuming that the Pais of pathogenic E. coli represent former plasmid or phage-specific fragments of chromosome inserted into the bacterial chromosome, what was the ancestral source? Can the newer methods of finding and amplifying small bits of unique DNA sequences in the vast microbial universe help us discover the source? Is transfer still occurring at some measurable rate? It is worthwhile to suppose that many members of the "normal" human microbial flora (many of which likely have escaped culture with current technology) that are permanent bowel residents or the transients we ingest in food, were, or are, the origin of these gene blocks. We also should believe that there are many more blocks of virulence specialization in the chromosomes of these microbes than we have currently recognized.

The study of PaiI and PaiII has permitted other insights into the evolution of pathogenicity and its regulation. As noted, the Pais are unstable. Derivatives of uropathogenic strain 536 devoid of both Pais have been isolated (3, 25). The phenotype of these mutants was not expected. The wild-type strain is virulent and serum resistant and grows under anaerobic conditions; it also produces two hemolysins and three distinct adhesins—Pap pili, S-pili, and common (type 1) pili—as well as synthesizing the iron chelator enterobactin and flagella. Pai-negative bacteria have reduced anaerobic growth and have lost the capacity to produce the hemolysin, all adhesins, flagella, and enterobactin. It is clear that not only do the Pais have an impact on virulence because of the action of directly encoded genes like those for the hemolysins and P-pili, but they also influence virulence by encoding positive regulators of S-fimbria expression. Now it has been shown that the Pais modulate virulence gene expression through the action of particular tRNAs (27). As noted, selC, which is adjacent to the site of PaiI insertion, encodes selenocysteine incorporation into protein. The enzyme formate dehydrogenase, which plays a key role in the mixed acid fermentation, contains selenocysteine and is involved in the decomposition of formate to carbon dioxide and molecular hydrogen. The disruption of the selC locus in PaiI-deficient mutants abolishes formate dehydrogenase activity and leads to impaired anaerobic growth (27). Another tRNA, produced by the gene leuX, is part of PaiII. Although leucine can be transferred by six different tRNA species, leuX-specific tRNA stimulates the production of at least three important virulence factors, common pili, flagella, and enterobactin, which lie outside of the pathogenicity island of the uropathogenic strain (27). In addition, leuX contributes to serum resistance and virulence as measured by animal survival in infection models. Thus, leuX represents a form of global regulation influencing the expression of a number of complex gene clusters. This finding emphasizes the interdependency of microbial pathogenicity and metabolic activities in pathogenic bacteria.

The most recent example of a pathogenicity island is a 40-kb region on the Salmonella chromosome that is near min 59 of the chromosome of Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) (24) and is not present on the E. coli chromosome. It is known that a large number of genes required for bacterial entry into nonphagocytic cells are located in this region of the chromosome. Moreover, this region is unstable in certain Salmonella serotypes, which leads to the frequent occurrence of deletions and the loss of pathogenicity. The instability has been ascribed to the presence of IS3-like elements adjacent to the border of this Pai (chapter 151).

 

SHARED GENETIC STRATEGIES FOR PATHOGENICITY IN THE ENTERIC BACILLI

It is not clear whether there is any real distinction between a virulence gene found on a huge block of inserted genes in the chromosome of Salmonella or EPEC, or a virulence gene found on a large, dedicated plasmid that encodes similar functions, as found in Shigella and enteroinvasive E. coli. What has become eminently clear, however, as will be discussed in detail by Galan and Sansonetti (chapter 151), is that Salmonella, Shigella, and EPEC share a number of homologous gene sequences that code for components of a dedicated secretion machinery, which is referred to as type III secretion. It is equally striking that similar homologous sequences are present in plant pathogens, are part of the flagellar assembly machinery of other bacteria, and are seen as components of filamentous phage assembly, as well as being present on plasmids in virulent Yersinia spp. (see chapter 151 for a detailed comparative view of these findings). The similarities between EPEC virulence attributes, and Salmonella invasion genes being more than homologous genes associated with secretion, are beginning to be appreciated (7).

The hallmark of EPEC infection is the AE lesion, which includes degenerated microvilli and "pedestals" of densely clustered cytoskeletal proteins (including filamentous actin) that protrude from the apical membrane and intimately cup individual bacteria (8). Salmonella and Shigella also induce a marked rearrangement in the host cell cytoskeleton that is manifested as cellular ruffling leading to engulfment of the bacteria and their internalization (2). Both Salmonella and EPEC infection trigger a host signal pathway (or pathways) marked by protein phosphorylation and a rise in the level of intracellular Ca²⁺ and inositol phosphates (13; chapter 151). One EPEC gene, eaeB, is necessary for intimate attachment, cytoskeletal rearrangement, and stimulation of host cell signal transduction. Presumably, other genes will become known now that the LEE pathogenicity island has been defined. It appears that at least one Salmonella protein, InvJ, shows homology to the eaeB product, although its full effect on host cells has yet to be determined (7). How similar are the Pais of EPEC and Salmonella and the clusters of Shigella plasmid virulence genes? It is clear that some of their shared secretion determinants can substitute for one another in the laboratory. It is equally likely that they will have surprisingly similar shared functional determinants of pathogenicity.

A common virulence strategy seems to be the synthesis of an "attack complex" (2) or invasome (chapter 151), in which there is a contact-dependent assembly of proteins on the bacterial surface that acts in concert to bring about a productive interaction with the host cell, one of the initial, essential steps in the pathogenesis of infection. This is a common strategy for EPEC, Salmonella, Yersinia, and Shigella. Each of these microbes has a specific strategy, but they must now be viewed in the context of a general, shared organization of genes with a significant degree of shared structural similarity. Perhaps it is a sign of the commonwealth of bacterial genes that microbes carefully husband their individuality in most chromosomal genes and certain plasmids, but exchange and share a variety of transmissible genetic elements including plasmids, bacterial viruses, and transposable elements.

Does the widespread sharing of a similar dedicated protein secretion system and some virulence genes mean that there was only a limited assemblage of ancestral virulence genes available in nature that permitted gram-negative prokaryotes to interact with the surface of eukaryotic cells? There is evidence that at least the pathogenicity islands of Salmonella and the virulence plasmids of Shigella come from diverse sources. Thus, even though the structure and order of the shared secretory spa genes of Shigella and Salmonella are conserved, the nucleotide content of Salmonella spa is 47% G+C (the overall Salmonella chromosome is about 52%), while the Shigella plasmid genes are only 35% G+C, much lower than the Shigella chromosome (chapter 148). Is this a reflection of an alien origin as noted above? Or could it be that there was a common ancestral pathogenicity island of lower overall G+C composition distributed among ancestral enteric clones? Could current differences in overall nucleic acid composition of the pathogenicity islands reflect the result of selection for a functionally distinct region of the genome that is restricted in its recombination, by its sharp difference in composition, from the incursion of the rare horizontal transfer from related microorganisms?

 

SHARED CELLULAR TARGETS AMONG ENTERIC BACILLI

As noted earlier, most of the pathogenic enteric bacilli specifically target a niche that is distinct from the more numerous commensals of the bowel. The basis for enterotoxigenic E. coli and uropathogenic E. coli cellular targeting and attachment is detailed in a subsequent chapter (chapter 150). Only the behavior of the invasive pathogens Shigella and Salmonella will be considered here.

A number of cultured mammalian cell lines have been used as in vitro models to study the interaction of invasive enteric species (9; chapter 151). Salmonella, Shigella, and most enteropathogenic E. coli lines readily adhere to and enter such cells with little regard to the host origin of the cells or even the cellular type (2, 9; chapter 151). The essential cellular features of invasion, membrane ruffling for Salmonella and Shigella and AE lesions for most EPEC species, are observed in these models. However, in actual human infection, the pathology of disease and study of biopsy material lead to the conclusion that for each pathogenic "species," the cellular target is an epithelial cell that is distinctive in its location or surface characteristics. The apparent paradox between in vivo cellular specificity and in vitro permissiveness of these bacteria is reasonably explained when one considers that the host is rather more than cells in culture dishes in the laboratory.

A growing body of data suggests that many invasive enteric bacterial and viral agents, including Shigella, host-adapted Salmonella, and some EPEC strains, use a terminally differentiated epithelial cell, the M cell of the Peyer’s patch in the terminal ileum and in other gut-associated lymphoid tissue, as a portal of entry into the host (21, 26). M cells found in Peyer’s patches are thought to internalize luminal contents for delivery to underlying antigen-presenting cells. These specialized epithelial cells, which have yet to be cultured in vitro, represent about 10% of the total cells in the organized patch and are characterized by shortened microvilli and active endocytosis; however, they possess a smaller than usual number of lysosomes. The Peyer’s patch is mostly populated by enterocytes, although there is a paucity of goblet cells. Consequently, there is only a sparse mucus layer in this anatomic region. Possibly this latter feature, or fewer lysosomes, made the M cell a prime target for upwardly mobile, aspiring bacterial pathogens.

S. typhimurium preferentially invades murine M cells when introduced into a ligated intestinal loop (6, 19). As seen with cultured cells, these organisms trigger membrane ruffling as they enter. The M cell is killed by the invasion process and is extruded into the bowel lumen. The invading bacteria do not seem to replicate to any extent within M cells but subsequently interact with the underlying mononuclear cell population, including resident macrophages, as well as with adjacent enterocytes. It seems significant that two independent S. typhimurium mutants, selected because they no longer invaded cultured epithelial cells, could not cross the M cell barrier in the ligated loop model, and were avirulent (19). These same mutants, when injected interperitoneally, were perfectly virulent. Salmonella typhi (official designation, S. enterica serovar Typhi) can also enter the murine M cell with a ruffling flourish, although it usually does not kill the M cell, nor can it productively interact with or replicate within other cellular components (20). It is not clear whether non-host-adapted Salmonella serovars enter via the M cell route. I think that it is possible that they do and that the subsequent outcome of the infection reflects their nonspecific interaction with a host. Host-adapted salmonellae have a relatively prolonged incubation period that gives rise to a mononuclear response and invokes cellular immunity. In many cases, infection leads to asymptomatic, long-term shedding of the microbe or to severe systemic disease. Recovery from systemic disease after a relatively prolonged incubation period often may be followed by a carrier state. In contrast, infection of an immunologically intact human with nontyphoidal Salmonella gives rise to an acute inflammatory disease that is in general self- limiting and less likely to lead to chronic carriage. Entry in either case could take place through the M cell. In one case, the subsequent events reflect restricted access to only adjacent enterocytes and the ensuing infiltration of polymorphonuclear leukocytes in response to this cellular damage. In the host-adapted case, however, a host-parasite relationship honed by evolution leads to interaction with a more limited repertoire of cellular types and the capacity of the invading bacterium to modulate the host immune system more precisely to achieve its long-term goals. Host-adapted infection productively leads to spread from individual to individual, whereas Salmonella gastroenteritis, typically spread by contaminated food, leads to few secondary cases (18). Thus, one clonal population is sustained within the species, while the other is short-lived. Hence, the human host is not the primary reservoir of infection, nor, presumably, of replication for nontyphoidal Salmonella spp. We still do not understand the genetic and molecular basis of bacterial host specificity; many host-adapted strains possess seemingly identical determinants and their virulence plasmids can even be exchanged without affecting host preference (1; chapter 152).

In the modern era, at least in humans, the host-adapted clonal populations, like the typhoid bacillus, have decreased dramatically with the increase of sanitation and public health measures (18); this was obvious even before the introduction of effective vaccines and antimicrobial treatment. In contrast, there has been a striking increase in the incidence of Salmonella gastroenteritis in human populations (18); it is a veritable measure of the technological state of food handling and mass distribution of food products in any human population. Are we seeing another phase in the evolution of Salmonella for humans? I suppose the ancestral form of S. typhi came from the interaction of paleolithic humans with a Salmonella-infected animal. S. typhi is no longer so successful, but our populations are increasingly meeting diverse Salmonella serovars in our foodstuffs. Will there be a Salmonella better adapted to the exigencies of modern life with the ability to spread among humans as a result of acute gastroenteritis?

Based on studies done in ligated monkey and rabbit loops, there is growing evidence that M cells are also the major entry site at early stages of Shigella infection (29; chapter 151). One is immediately faced with the reality that host-adapted salmonellae preferentially exploit the M cells of the small bowel, while shigellae are largely restricted to the large bowel. But M cells do vary. Colonic and rectal M cells display glycosylation patterns distinct from those of the Peyer’s patch M cells and are characterized by the presence of terminal galactose residues (4, 5, 16). There are morphological differences as well. Even within a single Peyer’s patch, subpopulations of M cells can be distinguished by distinct fucose-specific probes (4, 16). It is not clear whether the invasion of colonic M cells occurs through an active process that invokes cellular ruffling or whether it "simply" exploits the normally increased endocytic activity of these cells. A specific adhesin would be expected, but it has not been described. After M-cell entry by Shigella, the resident macrophages appear to be invaded, and, surprisingly, they die by apoptosis (35). As described in a later chapter by Galan and Sansonetti (chapter 151), the resulting induction of an acute inflammatory response is thought to provoke the destabilization of epithelial cell integrity at the mucosal surface, leading to a massive exposure of the basolateral aspect of the colonic epithelia and the increased entry and replication of Shigella cells in the superficial cells of the colon. Moreover, Shigella cannot invade ordinary enterocytes through the apical pole; they must reach the basolateral side of cells (chapter 151). The resulting acute inflammatory response and dysentery (diarrhea with blood and mucus), so important in human morbidity and mortality, is seen as only a strategy to increase the efficiency of bacterial replication.

The use of M cells as a portal of entry by Shigella spp. is quite distinct from that of host-adapted Salmonella spp., although there are some analogies in the clinical picture to that seen for Salmonella gastroenteritis. Do the shared secretory network and the growing number of shared functional determinants of virulence suggest that the basic early entry strategies of Shigella and Salmonella are, or were, essentially identical? The shared homology of certain aspects of the secretory pathway of Yersinia with Salmonella and Shigella also has its analogy in its use of M cells for entry. In a similar vein, certain, but not all, EPEC adhere to Peyer’s patch M cells, as do the enteroaggregative E. coli (20). The extent that EPEC target the enterocytes of the Peyer’s patch is not certain, although in most cases they seem to prefer the small bowel as a niche. This is not true of all AE organisms, since infectious hyperplasia of mice is caused by a C. freundii strain that possesses a related pathogenicity island and causes typical AE lesions of the colon (22, 30). Of course, it is hardly surprising that the capacity to enter M cells and other early strategies of cellular attachment and entry entail more than a single large block of shared genes. Yet it is perhaps equally surprising that such a divergent group of microbes have chosen to exploit a single cellular target of the host and that they have such fundamental similarities. Does this explain the types of niches available to facultative organisms, or does this explain the basic repertoire of genes of the original facultative gut commensal?

Shigella, like the host-adapted Salmonella, is spread by individual contact, as well as by contaminated food and water. As is the case for host-adapted Salmonella, the incidence of clinically apparent shigellosis in technologically advanced nations is decreasing. However, in some developed countries, the incidence of the less virulent and more urban Shigella sonnei clonal population is increasing, even while the global epidemic strains remain the more virulent clonal types (chapter 148).

 

SHARED STRATEGIES FOR INTRACELLULAR SURVIVAL

Once past the epithelial barrier of the gut, enteric bacteria face a potential barrage of phagocytic cells and innate host defense mechanisms. The successful pathogen does not dare incite the host too quickly; that is, it must replicate to a sufficient extent to survive and be transmitted to a new susceptible host before the inevitable full measure of host defense factors come into play. At times, as is apparently the case for Shigella, it is necessary to deliberately inflame the host defense in order to ensure adequate opportunities for replication. Shigella has the added advantage that it requires transmission of relatively few microorganisms to incite a subsequent infection (32). Other enteric bacilli have devised more subtle mechanisms to survive and replicate within host cells. The shared features of attachment and entry for enteric pathogens are obvious from genetic and molecular analysis. After entry, the bacterial strategies for replication beyond the epithelial border seem more distinctive. Some organisms like Salmonella have learned to exploit the intracellular trafficking pathway of host cells to become lodged in a privileged niche safe from the phagosome-lysosomal fusion pathway that can be quickly fatal to commensals (2, 15; chapter 151). Initially, shigellae probably enter a host cell vacuole in a similar manner to the salmonellae but quickly exit to the nutrient-rich, nonthreatening cytoplasm of the cell (chapter 151). The specifics of these strategies for intracellular survival, the progression of these microbes to cause systemic disease, and the ensuing immune response are discussed in considerable detail in a following chapter (chapter 152). It is worth noting, however, that the initial phase of interaction after passage through the epithelial barrier seems to be with resident macrophages.

Each microorganism must learn to deal with this next cellular hurdle. Shigella causes the macrophage to commit cellular suicide. Salmonella boldly enters the macrophage, again with a ruffle, and has the temerity to replicate there and to kill the cell. It is likely that Salmonella goes undetected by the macrophage in the early stages of host-adapted infection possibly because the bacterium is sequestered within a vacuole that is most likely used for other purposes but now is modified for the bacterium’s use. As a counterpoint, Yersinia sticks to the outside of the macrophage and injects catalytic agents that quickly neutralize the capacity of the macrophage either to phagocytose or to signal a warning (2). In each instance, the invading bacterium is again faced with a cellular barrier, be it the plasma membrane or the membrane of an endocytic vacuole in the macrophage. In each case, there is evidence that the microorganism responds to similar environmental cues that include temperature, iron, and pH, to name a few, to deal with these cellular barriers (23; chapter 154). Will we learn that these apparently distinct and unique modes of intracellular life have a shared evolutionary linkage? The first clues may come from sequence data showing conserved homology, just as we learned recently about the conserved type III secretory pathways found on pathogenicity islands and virulence plasmids. Will there be another set of conserved genes or another pathogenicity island shared among enteric species that has evolved to take the measure of macrophages and other host functions?

We currently know less about the genetic and molecular basis of intracellular life than we do about bacterial entry into cells. For bacteria that live within vacuoles, such as Salmonella and Mycobacterium, there are a good deal of data that indicate they live in a niche that is acidic but secure from normal host cell trafficking, which leads to potentially dangerous lysosomal products (31). For microbes that escape from vacuoles to live in the cytoplasm, such as Shigella, Listeria, and some Rickettsia spp., there is a shared ability to exploit host cell cytoskeletal components to ensure their cell-to-cell transmission (9; chapter 151).

How does one view the evolution of these remarkably similar strategies among such widely divergent microorganisms? No obvious homology exists in the genes from each that are known to encode for life within the cytoplasm and the cell. Simply, there are a limited number of ways that prokaryotes can utilize host cell building blocks. Is there possibly a single evolutionary thread that links them?

It seems to me that considerable insight into these questions will come from the investigation of natural populations of bacteria in their native habitat, so to speak. With tools such as nucleic acid amplification and the discovery of ways to make very sensitive reporter genes, we soon will be able to monitor microbial activity in an animal or even in the "wild." We are bound to learn more about the biology of these microorganisms from their encounters in the real world, not just from guesses in a simulated laboratory growth medium or within the confines of a tissue culture flask. We still only believe that we know what cell types are actually encountered by an enteric pathogen within the animal. Much of what we believe is based on conventional wisdom gleaned from the pathology of terminally ill animals (and genetically hypersusceptible animals in most cases at that), autopsy findings, and, less frequently, biopsy material.

Our view of pathogenicity has been based on disease, which is as much host response as it is microbial action. Rather, we need to understand more fully the parameters of infection. For example, for all of our effort toward understanding the pathogenesis of infection and disease by Salmonella and Shigella, we still don’t know very much about the E. coli genes that function to allow it to establish itself in the mammalian bowel and replicate there. This is possibly the most fundamental attribute of this microorganism. It was the foundation of the basis for the evolution of pathogenicity by today’s pathogens. By the next edition of this monograph, while we may not know very much more about the evolution of pathogenicity, it is certain that we will know a great deal more about the genetic and molecular basis of pathogenicity as an expression of bacterial specialization and its role in the biology of microorganisms.

ACKNOWLEDGMENTS

I thank the members of my laboratory for their comments. In particular, Joan Mecsas and Evi Strauss provided useful comments and suggestions about the more fanciful parts of the manuscript. Both Roy Curtiss and Brett Finlay provided me with valuable commentary about several of my views supported by wishful thinking rather than data. Sara Fisher, as always, brought organization to the chaos.