Modulation of the enterohemorrhagic E. coli virulence program through the human gastrointestinal tract.

Enteric pathogens must not only survive passage through the gastrointestinal tract but must also coordinate expression of virulence determinants in response to localized microenvironments with the host. Enterohemorrhagic Escherichia coli (EHEC), a serious food and waterborne human pathogen, is well equipped with an arsenal of molecular factors that allows it to survive passage through the gastrointestinal tract and successfully colonize the large intestine. This review will explore how EHEC responds to various environmental cues associated with particular microenvironments within the host and how it employs these cues to modulate virulence factor expression, with a view to developing a conceptual framework for understanding modulation of EHEC's virulence program in response to the host. In vitro studies offer significant insights into the role of individual environmental cues but in vivo studies using animal models as well as data from natural infections will ultimately provide a more comprehensive picture of the highly regulated virulence program of this pathogen.

Introduction

Infection with enterohemorrhagic Escherichia coli (EHEC) is a leading cause of bloody diarrhea and hemorrhagic colitis, occasionally resulting in life-threatening systemic complications including hemolytic uremic syndrome (HUS)., This food and waterborne zoonotic agent has been associated with numerous outbreaks worldwide and constitutes a serious public health threat. Of over 380 EHEC serotypes, the O157:H7 serotype is the one most highly associated with outbreaks and severe disease in North America., However, the non-O157 serotypes also represent a significant public health concern, and are frequently associated with HUS, particularly in Latin America, Europe, and Australia.,

EHEC infection typically begins with watery diarrhea, vomiting and abdominal cramping that then progresses to bloody diarrhea. In the majority of infected individuals, the infection resolves within a week to 10 d. However, in 5–7% of infected individuals, the infection leads to a systemic, sometimes life-threatening complication known as hemolytic uremic syndrome (HUS). HUS is characterized by thrombocytopenia, hemolytic anemia, and acute renal failure. Currently, treatment consists primarily of supportive therapy including rehydration.- Conventional antibiotic treatment is generally not recommended although there is no clear consensus on this matter. In vitro studies have shown that, at least for EHEC O157, sublethal doses of antibiotics, particularly trimethoprim, the quinolones or furazolidone, promote the production and release of Shiga toxins, a development that constitutes a risk factor for progression to HUS.- A number of clinical studies have also shown that patients on antibiotic therapy for hemorrhagic colitis have a higher risk of developing HUS.,,- However, it should be noted that these studies are often limited by small sample size as well as the advanced stage of illness of the patients and the findings may be more relevant for certain EHEC seropathotypes. There is evidence that antibiotic treatment of EHEC O104:H4 does not promote Shiga toxin releaseand two clinical studies found that antibiotic treatment of EHEC O104:H4 infection did not enhance the risk of HUS., Other agents often used to treat bacterial infection including antimotility agents, narcotics and non-steroidal anti-inflammatory medication are also not recommended for EHEC infection. Given the increasing number of EHEC outbreaks and HUS complications and the lack of available therapeutic strategies, there is a significant, critical need for new approaches to the prevention and treatment of EHEC infection. Recent research on toxin antibodies, novel peptides and small molecule drugs as well as zinc-based salts have shown some promise in the quest for effective preventative and/or very early treatment strategies.-

EHEC Virulence Factors

EHEC was first identified as the pathogen responsible for colitis-mediated HUS by Karmali et al. who found that patients presenting with diarrhea and HUS were positive for a toxin capable of inducing irreversible cytotoxicity in cultured Vero cells. The toxin, originally referred to as Verotoxin, was later shown to be structurally and antigenically similar to the toxin produced by Shigella dysenteriaie type 1, a finding which resulted in the name, Shiga toxin (Stx). Stx contains two major structural subunits, A and B, the former which has RNA N-glycosidase activity against 28S rRNA, resulting in protein synthesis inhibition and the latter which binds to globotriaosylceramide-3 (Gb3) on the surface on endothelial cells, permitting toxin dissemination and toxin-mediated tissue damage.- Human renal glomerular endothelial tissue express high levels of surface Gb3 which may explain why Stx production results in acute renal failure.,

EHEC pathogenesis is not however limited to toxin-mediated effects. Hemorrhagic colitis, an earlier event in the infection, is thought to be promoted by a number of virulence factors that include fimbrial and nonfimbrial adhesins, flagella, Stx, and the Type III secretion system. The primary adhesin, intimin, a bacterial outer membrane protein encoded in the chromosomal pathogenicity island, LEE (locus for enterocyte effacement), promotes bacterial adhesion to the columnar epithelial cells lining the terminal ileum and transverse colon through binding to its own injected receptor, Tir (translocated intimin receptor), as well as binding to host cell proteins, integrin and nucleolin.- However, intimin mutants still bind to host epithelial cells, providing persuasive evidence of the involvement of other adhesins., A number of other non-fimbrial EHEC adhesins have been implicated in adhesion including the plasmid-encoded toxB, the chromosomal genetic locus, efa1 (EHEC factor for adherence), and the chromosomally-encoded adhesins, Iha (Vibrio cholerae IrgA homolog), Cah (calcium-binding antigen 43 homolog), and OmpA (outer membrane protein A).- Fimbrial structures that have been implicated in host adhesion include two long polar fimbriae, F9 (a type 1 pilus homolog), two type IV pili (HCP in EHEC O157 and TFP in a non-O157 EHEC), the sorbitol-fermenting EHEC O157:H-plasmid-encoded fimbriae, SFP and ECP (E. coli common pilus), a pilus structure produced by both pathogenic and nonpathogenic E. coli . The long polar fimbriae of EHEC O157:H7 appear to play a role in sheep and pig colonization although some studies have suggested they may also play complementary roles in adhesion to human cells. By contrast, F9 fimbriae may actually prevent or disrupt adhesion since F9 mutants show increased adhesion to cultured epithelial cells. HCP are recognized by the sera of HUS patients and may function in a synergistic manner with the adhesion-receptor pair, intimin-Tir., ECP may play a role in colonization of pathogenic and commensal E. coli strains, promoting interbacterial interactions in biofilm communities., Flagella have also been reported to function as adhesins, mediating bacterial adherence to mucins, the primary component of the mucous coat in the gastrointestinal tract. Finally, Stx has also been shown to promote EHEC adhesion to host epithelial cells by upregulating surface expression of two receptor candidates, phosphatidylethanolamine and nucleolin.,

Other virulence factors include the LEE-encoded type III secretion system translocation (TTSS) and effector proteins, as well as a variety of non-LEE encoded effector proteins, all of which contribute to the modulation of host cell signaling to support bacterial replication and survival, host colonization and the development of disease. Host cell changes include marked cytoskeletal rearrangements, disruption of intestinal barrier function, downregulation of the host inflammatory response, and induction of host cell apoptosis. Although intimin and Tir are the primary mediators of human intestinal adhesion, other LEE-encoded proteins, including EspA, EspH, Map, Esp,F and EspG, also promote EHEC colonization.,

Expression of Virulence Factors

It is well established that EHEC virulence factor expression is influenced by numerous experimental conditions including temperature, culture media, pH, bile salts, and even host cell factors.- Clearly, the pathogen successfully survives passage through the human gastrointestinal tract (GIT), but recent research suggests that it may also use exposure to different GIT environments to regulate expression and function of virulence factors. The idea that environmental conditions may cue temporal-spatial expression of virulence factors in a pathogen has been garnering significant attention recently., In this review, we will examine key host environmental cues that influence EHEC virulence factor expression with a view to understanding how they may play a role in the temporal-spatial regulation of EHEC virulence during passage through the human GIT.

Acid Stress

Depending on intestinal motility and the location within the food bolus, bacteria must be able to survive up to 2.5 h at pH values ranging from 2–6, in order to successfully transit from the human stomach. Both nonpathogenic and pathogenic E. coli encode four different acid resistance systems that provide protection against exposure to pH as low as 2–2.5., Since the infectious dose of EHEC is typically very low (50–100 organisms), acid tolerance and resistance are critical virulence traits. The acid resistance systems are dependent on culture conditions and growth phase and are employed differentially by EHEC for survival in foods vs. the intestinal tract., The glutamate-dependent acid-resistance system (Gad; AR3) is one of three amino acid decarboxylase systems (Ar2–Ar4) and is thought to offer the best protection below pH 3. The AR1 system which employs the stationary phase alternative sigma factor, RpoS, and the global regulatory protein, cAMP receptor protein (CRP), provides an acid adaptation or tolerance response (ATR) that permits E. coli exposed to sublethal pH values (pH 5) to survive subsequent exposure to lower pH values (pH 2.5). Since the pH of the human stomach can increase to pH 6 after a large meal before dropping to pH 2, the acid adaptation response may be physiologically relevant in the survival of ingested EHEC. However, the acid resistance/adaptation response can also be triggered prior to ingestion, either in the bovine intestinal tract or within acidic foods. Studies have further revealed that EHEC O157:H7 that are already expressing acid resistance remain acid resistant for at least a month during refrigeration and that no further induction upon encountering pH 2.5 is required. These findings suggest that EHEC in contaminated foods are well prepared to survive the acid stress of gastric passage.

In addition to the expression of acid resistance systems, exposure to acid can trigger induction or repression of specific virulence genes and sets of genes in EHEC. In a DNA microarray study, investigators examined the gene expression profiles of EHEC O157 that had been acid stressed and then neutralized relative to the same unstressed strain. There were significant expression changes in virulence factors associated with adhesion, motility and type III secretion including genes encoding known and putative adhesins, fimbria, flagella, and curli as well as many of the LEE-encoded type III translocation and effector proteins. These changes correlated with changes in virulence properties including enhanced motility and host cell adhesion following acid stress and neutralization. The TTSS genes whose products mediate colonization and infection in the large intestine were downregulated following acid stress and this is consistent with their negative regulation by two regulators, GadE and H-NS, under acid conditions., Adhesin expression profiles were more variable, depending on the nature and duration of the acid stress with several known adhesins including intimin showing little change and a few novel adhesins showing significant upregulation, suggesting that acid stress alters the adhesin profile. Interestingly, adhesion of acid-stressed EHEC to human epithelial cells is increased and at least one of the novel adhesins appears to play a role in the acid-induced adhesion. Flagellar synthesis genes were also upregulated under acute acid stress along with a modest increase in motility, a response which may offer a defense strategy against acute acid. Interestingly, there was no change in stx gene expression and no increase in Stx-mediated cytotoxicity after acid stress which is consistent with the fact that Stx-induced cytotoxicity is generally associated with large intestine. Collectively, these findings suggest that acid stress serves to arm EHEC with defensive strategies including acid resistance and enhanced motility for escape as well as downregulation of genes whose proteins are typically involved in colonization and subsequent infection of the large intestine.

Recent studies have revealed that EHEC also employs the transcriptional regulator SdiA to coordinate the transcription of the LEE genes needed for A/E lesion formation and the gad genes required acid resistance, at least in cattle.- SdiA is a member of the LuxR family of transcriptional regulators and it senses acyl-homoserine lactones (AHL) produced by other bacteria. Based on these studies, it has been proposed that SdiA senses AHL, upregulates gad genes required for acid resistance, and downregulates LEE genes required for colonization. These findings support a model for modulation of the EHEC virulence program in the bovine intestinal tract, where EHEC resides as a commensal organism.

Bile

Bile resistance, a critical virulence property of gastric pathogens, is generally achieved through active efflux using a variety of resistance nodulation division (RND) efflux systems and altered outer membrane permeability often achieved through modifications of lipopolysaccharide layer.- Studies have shown that bile also serves an environmental cue for a number of enteric bacteria including Salmonella, enteropathogenic E. coli, and EHEC by modulating the expression of specific virulence factors.,,- DNA microarray analysis of EHEC O157:H7 treated with bile salts showed upregulation of genes encoding the AcrAB efflux pump, a two component signal transduction system (basRS/pmrAB) and a lipid A modification pathway (arnBCADTEF and ugd). Interestingly, this correlated with bile salt-induced resistance to the antimicrobial agent, polymyxin B, which was basS- and arnT-dependent. ArnT encodes the enzyme that transfers L-Ara4N to lipid A, a modification that decreases the negative charge of the lipopolysaccharide and has been shown to enhance resistance to several cationic antimicrobial peptides.,, The authors also reported that bile salt treatment did not enhance Shiga toxin-mediated cytotoxicity, a finding that was consistent with the downregulation of stx2 genes after bile salt treatment. Finally, expression of several other well established virulence factors including those encoded on the LEE pathogenicity island, was not altered by bile salt treatment. These findings suggest that bile secreted into the small intestine serves an environmental cue for EHEC, signaling changes that result in protective modifications of the bacterial outer membrane, thereby enhancing successful migration of the pathogen through the small intestine while at the same time suppressing the expression of virulence factors required for subsequent colonization and infection of the large intestine.

Ethanolamine

The constant turnover of intestinal epithelial cells and commensal flora in the human gastrointestinal tract generates a large number of membrane lipid metabolites including the breakdown product of phosphatidylethanolamine, ethanolamine (EA). Through degradation to ammonia and acetaldehyde, EA can serve as a source of nitrogen and occasionally carbon for some bacteria. Recent studies indicate that several GI pathogens including Clostridium, Listeria, Enterococcus, EHEC, enteropathogenic E. coli (EPEC), and Salmonella possess genes necessary to catabolize EA and that EA utilization (Eut) may be a possible virulence determinant. In Salmonella enterica, the global virulence regulators, Fis and CsrA, have been found to regulate eut genes and mutations in those genes triggered a loss of virulence in a mouse model of infection., Recent studies have also shown that EA serves as a source of nitrogen but not carbon for EHEC grown under conditions that mimic the intestinal environment.

Interestingly, the source of host-generated EA, phosphatidylethanolamine, has also been found to play a role in EHEC pathogenesis. EHEC preferentially binds to phosphatidylethanolamine (PE) in the host epithelial cell membrane and induces apoptosis in the host cell, resulting in the upregulation of outer leaflet PE levels and increased adhesion to the apoptotic cells., Studies with the related attaching and effacing pathogen, EPEC, showed that PE binding by EPEC modulated host phospholipid metabolism, leading to increased outer leaflet PE and similar to EHEC, increased adhesion to the host cell.- These data suggest that the elevated host outer-leaflet PE levels triggered by pathogen binding to epithelial cells in the large intestine may be providing a critical source of EA for the pathogen.

Recent studies by Kendall et al. now indicate that EA may also serve as an environmental cue to EHEC to modulate virulence factor expression. EHEC cultured in minimal media containing EA showed increased expression of genes encoding virulence regulators (Ler, QseE, and QseC) as well as Shiga toxin (Stx2a) and also showed increased characteristic attaching and effacing (A/E) lesions on host epithelial cells. However, the nature of that regulation is still not fully understood. While the eut genes are upregulated by culture in EA, the increased expression of virulence genes appears to be independent of the Eut catabolic enzymes. A positive transcriptional regulator of the eut locus, EutR, appears to partially regulate expression of the virulence genes under certain growth conditions in EA but the data also suggest the involvement of a second as yet unidentified regulator. Nevertheless these data provide evidence that EA in the microenvironment of the intestinal lumen may be acting as an environmental cue for virulence modulation in EHEC to assist in colonization of the large intestine.

Microbial Flora Metabolites

During passage through and colonization of the human GIT, EHEC encounters the highly complex microflora and the metabolites that they produce. These metabolities can include simple metabolic byproducts such as short chain fatty acids (SCFA) as well as metabolites that allow the microflora populations to modulate their metabolism according to the size of their populations. The three principal SCFAs present in the intestine are acetate, propionate and butyrate and the concentrations of these acids vary through the ileum and the colon.- High concentrations of SCFAs (above 50 mM) more typical of that found in the proximal and distal colon have been shown to inhibit the growth of EHEC while low concentrations, particularly butyrate, (from 6.25 to 25 mM) more typical of the distal ileum enhance expression of virulence genes involved in motility, adhesion and induction of A/E lesion formation,, suggesting that SCFAs may be cueing EHEC migration and adhesion to the distal ileum. However, another study reported that high concentrations of SCFAs (172 mM) more typical of the distal colon were associated with increased expression of the gene encoding the adhesin Iha suggesting that at least this adhesin is promoting host adherence in the colon. Since Iha can also function as an iron siderophore and, in this study, was upregulated along with TonB, the outer membrane ferrichrome transport protein, it is also possible that SCFAs may be cueing the pathogen to increase its iron-scavenging capacity in the colonic environment. EHEC is also able to sense the quorum signaling molecule AI-3, secreted by commensal flora, using the histidine kinase-response regulator two component signaling system, QseCB.- EHEC responds to AI-3 with increased flagellar synthesis and motility and it is thought that increased motility permits the pathogen to more closely approach the mucosal epithelium at the site of colonization. Collectively, these studies suggest that EHEC employs certain molecular cues secreted by commensal flora to upregulate virulence factors that enhance motility, adhesion and iron-scavenging, all of which promote the establishment of infection.

However there are other molecular structures secreted by the normal gut microbiota that may protect the host against infection. De Sablet et al. showed that one or more factors secreted by a complex human gut microbiota including the predominant species, Bacteroides thetaiotaomicron, repressed stx2 mRNA expression in a manner independent of SdiA, QseA, QseC, or AI-3. This is consistent with other studies that show that pure cultures of several probiotic strains inhibit stx2 transcription in laboratory media. It is also well established that certain commensal bacteria exert generalized antibacterial effects against enteric pathogens through the production of antimicrobial proteins such as colicins, lantibiotics, and microcins, which typically result in inhibited pathogen growth. These data point to the positive benefit of normal gut microbiota in protecting against EHEC infection, leading to the speculation that disruptions in gut microbiota may enhance risk of EHEC infection.

In the final analysis, the impact of normal gut microbiota on EHEC infection must be contextualized within our enhanced understanding of mucosal microbial populations based on recent, in-depth investigations. As we begin to fully appreciate the extensive variation in the microbial community structure along the entire length of the human gastrointestinal tract, we recognize the need for further research to better understand the roles of the gut microbiota within specific microenvironments in the modulation of the EHEC virulence program.

Host Hormones, Epinephrine, and Norepineprine

EHEC, along with a number of other disease-causing organisms including ETEC, Salmonella enterica, Vibrio parahemolyticus, and Edwardsiella tarda, have been shown to use host-generated hormones epinephrine and norepinephrine as signals for differential regulation of virulence factors mediating invasion, motility, and in the case of EHEC and EPEC, A/E lesion formation.,,- EHEC uses the histidine sensor kinases QseC and QseE as sensors of the two hormones. QseC, via its cognate response regulator QseB regulates flagellar and motility genes and through QseF, QseC is able to activate production of Stx., Through another response regulator, KdpE, QseC also upregulates LEE genes. Not surprisingly, deletion of qseC attenuates EHEC virulence. The second sensor, QseE, responds to epinephrine as well as to phosphate and sulfate and now appears to regulate the LEE genes and nleA (nonLEE-encoded effector A) negatively. However, this negative regulation is mediated indirectly through transcriptional inhibition of the response regulator, RcsB, which is a positive regulator of the LEE. Regulation of EHEC virulence by epinephrine and norepinephrine appears to be quite complex and is still not fully resolved. Nevertheless, that data collectively suggest that EHEC coordinates a temporal response to the microbial flora-produced AI-3 and the two host-derived hormones epinephrine and norepinephrine to assist in cueing the site of colonization and to enhance approach to the epithelial layer through increased motility and increased A/E lesion formation.,

Low Oxygen

The environment of the intestinal tract is characterized by variable oxygen levels and a number of studies on other pathogens report that oxygen levels do modulate pathogen virulence.,, While the lumen of the intestinal tract is relatively anaerobic, there is a zone of relative oxygenation adjacent to the mucosal surface that is generated by diffusion from the microvilli capillary network., E. coli can sense changes in oxygen availability and switch from aerobiosis to either anerobiosis or microaerobiosis, a process which is governed by two global regulators, Fnr (anaerobiosis) and ArcA (microaerobiosis).- Studies reveal that E. coli are alternatively dependent on microaerobic and anaerobic respiration and that this respiratory flexibility is a key determinant in their ability to successfully colonize the human intestine. Despite our understanding of this respiratory flexibility in E. coli, there is very little known about how varying oxygen concentrations modulate EHEC virulence. A recent study examined a model of EHEC infection of the apical side of polarized epithelial cells under oxygen concentrations of 1–2% atmospheric pressure (considered microaerobic) and found that EHEC-host adhesion was increased and that expression and translocation of EHEC TTSS effector proteins were also increased. The increased adhesion appeared to be mediated primarily by the TTSS translocon, EspA, while other potential adherence factors including flagella and the E. coli common pilus were only minimally expressed. These results suggest that the microaerobic environment adjacent to the intestinal microvilli may upregulate expression of EHEC virulence factors that promote successful colonization of the large intestine. They also point to the need for further study on the role of oxygen availability in modulating EHEC virulence.

Temporal-Spatial Cueing

We have yet to put together the picture of how EHEC adapts to each of the successive environments of the human GIT and responds to these various cues in a temporal-spatial fashion. By assembling a sequential list of environmental cues encountered by the pathogen along with data on the modulation of virulence factors and properties, one can begin to envision a model for the temporal-spatial regulation of the EHEC virulence program (Table 1). Passage through each of the local GIT environments appears to differentially arm EHEC with specific protective mechanisms appropriate to the local environments including enhanced resistance to acid, bile, and cationic antimicrobial proteins along with increased motility which could mediate escape from stressful environments. Exposure to different microenvironmental cues also alters expression of virulence factors and properties that may provide EHEC with selective advantages in current or upcoming local environments including AI-3-induced acid tolerance possibly to upcoming SCFA stress, and flagella-mediated motility toward the gut epithelium. As EHEC approaches the site of colonization and infection, exposure to environmental cues in the ileum and colon including short chain fatty acids, quorum signals, ethanolamine, host hormones, and changes in oxygen levels is accompanied by the upregulation of virulence factors that promote adhesion, A/E lesion formation, and cytotoxicity, all of which promote colonization and the establishment of infection. While the picture is beginning to emerge, there are still many gaps and some inconsistencies. What is still missing is the integration of signals delivered in a sequential but not necessarily spatially isolated fashion. Large scale studies using molecular genomics, genetics, and proteomic approaches have generated huge amounts of information but determining the physiological relevance of these data remains a challenge. Natural infections can provide us with retrospective information but again the data are difficult to evaluate in the absence of appropriate controls. Animal models will likely provide us with the most useful insight but it is still difficult to appreciate the value of each cue or sets of cues or sequence of cues in a definitive manner. Expression of EHEC virulence factors, both the timing and the level of expression, is highly regulated by the environment and to more fully understand this regulation will involve well-designed animal infection models and more data from infection outbreaks.

Table 1. Modulation of EHEC virulence program by microenvironmental cues in the human gastrointestinal tract

Local GIT Environment	Cue	Regulons Involved	Virulence factors: expression changes	Virulence modulation	References
Stomach	Low pH	RpoS, CRP, H-NS, GadE	↑ AR1–4, ↑ Flagella and motility genes, ↑ novel adhesins↓ LEE genes	↑ acid resistance, ↑ motility↑ adhesion	66, 73 and 77
Duodenum	Bile	BasRS, PhoP?	↑ arnBCADTEF↑ acrAB↓ stx2	LPS modification,↑ Bile and CAMP resistance	69 and 89
Ileum	AI-3(quorum sensing)	QseCB, SdiA	↑ gad genes, flagella	↑motility, ↑acid resistance (to SCFAs?)	81 and 115
	SCFA (< 25 mM)		↑ LEE, flagella	↑ adhesion, ↑ motility	112,113
Colon	EA	EutR, Ler, QseE, QseC	↑ Stx2a	↑ cytotoxicity	70
	SCFA (> 50 mM)		↑ Iha	↑ adhesion, ↑ iron scavenging	114
	Low oxygen	Fnr, AcrA	↑ EspA, ↑ TTSS effectors(at microaerobic oxygen levels)	↑ adhesion, A/E lesion	132 and 133
	Epinephrine, norepinephrine	QseCB, QseCF, QseC/KdpE	↑ flagella↑ LEE genes↑ Stx	↑ motility,↑ A/E lesion↑ cytotoxicity	117
	Epinephrine, phosphate, sulfate	QseE	Inhibits RcsB,↓ LEE?	↓ A/E lesion?	79

Cues encountered at various locations within the GIT are provided along with associated changes in the expression of specific virulence factors and properties (increased, ↑, or decreased, ↓). Regulons reported to be involved in the responses are also provided.