Virulence Gene Regulation in <b><i>Escherichia coli</i></b>
JAY L. MELLIES* AND ALEX M. S. BARRON
[SECTION EDITORS: EDUARDO GROISMAN AND JAMES B. KAPER]
Posted June 6, 2006
Biology Department, Reed College, 3203 SE Woodstock Boulevard, Portland, OR 97202
*Corresponding author. Phone: (503) 517-7964, Fax: (503) 777-7773, E-mail:
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A commensal inhabitant of the human gastrointestinal tract, the facultative anaerobe Escherichia coli, normally causes us no harm. This gram-negative bacterium produces vitamins utilized by its host and colonizes the colonic mucosa, helping to prevent colonization of this region by pathogenic organisms. Three general clinical syndromes are caused by these normally harmless bacteria, however: diarrheal disease, urinary tract infections, and sepsis/meningitis. Six well-defined categories of diarrheal pathogens now affect humans: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) (177). EIEC pathogens also cause diarrhea as well as dysentery and are closely related to Shigella spp., both genetically and biochemically and in pathogenic mechanisms. Regulation of virulence genes of Shigella spp. is the subject of Chapter Virulence Gene Regulation in Shigella; since no differences in regulation between EIEC and Shigella spp. have been described, EIEC will not be discussed separately in this chapter. The majority of urinary tract infections in individuals with normal anatomy are caused by uropathogenic E. coli (UPEC), and an increasing number of extraintestinal E. coli infections are caused by the meningitis-associated strains (MNEC), mostly in newborn infants. The clinical symptoms and pathogenesis of E. coli-caused diseases and mechanisms of how these pathogens disrupt host cell processes to cause disease have been reviewed elsewhere (39, 125, 132).
What distinguishes the E. coli pathogens from those that normally inhabit the human intestinal mucosa? There are two fundamental differences. First, the pathogens possess unique genetic elements, most of which are thought to be acquired by horizontal transfer, which enable them to cause damage to the human host. Though some pathogens share conserved virulence determinants, e.g., the type III secretion systems encoded by the locus of enterocyte effacement (LEE) pathogenicity islands (PAIs) of EPEC and EHEC, fimbrial adhesins, toxins, autotransporter proteins, etc., profound as well as subtle genetic differences enable these bacteria to colonize specific niches and cause specific diseases. Pathogens must possess and properly regulate virulence genes to cause disease. Regulation plays a critical role in the ability of bacteria to accomplish the four steps necessary to cause disease: attachment, colonization, evasion of the host immune system, and infliction of damage. This second fundamental difference between pathogens and commensal bacteria is the subject of this chapter.
EPEC was the first type of E. coli pathogen to be described and causes watery diarrhea in infants, primarily under the age of one. It continues to be a significant cause of morbidity and mortality in developing countries (177). A hallmark of EPEC infection is its ability to form attaching and effacing (AE) lesions on intestinal epithelial cells whereby the bacteria intimately attach to the host cell, forming a "pedestal" cupping individual bacteria (164).
The mechanism of causing diarrhea is complex and is linked to the LEE-encoded type III secretion system (TTSS) (for a review of TTSSs of gram-negative pathogens, see reference 112). The TTSS causes disruption of host cell signaling events via injection of bacterial effector molecules into the epithelial cell cytoplasm, leading to cytoskeletal rearrangement, intimate attachment, and pedestal formation. More than 20 proteins are now known to be secreted through the LEE TTSS (81) and many of these, e.g., Tir, EspF, EspG, EspH, EspZ, and Map, have been shown to be translocated into the host cell. Several non-LEE-encoded proteins, including NleA/EspI, EspG2, EspJ, and Cif, are also translocated by the LEE TTSS into host epithelial cells (50, 81). Presumably secretion of all of these effector molecules is coordinately controlled, responding to environmental conditions similar to those found within the human intestinal tract. Destruction of the microvilli in the formation of AE lesions results in loss of absorptive tissue and alteration of host cell signaling events causing active ion secretion, increased intestinal permeability, loosening of tight junctions, and inflammation at the site of infection, resulting in diarrhea.
In addition to those found in the EPEC LEE, several virulence factors have been described. LifA is a large 385-kDa protein that inhibits lymphocyte activation (139) (termed Efa1 in EHEC), and EspC is an enterotoxin encoded within a second EPEC PAI that causes alterations in short-circuit current in Ussing chambers (160, 259). The bundle-forming pilus (BFP) is encoded on the E. coli attachment factor (EAF) virulence plasmid.
It is clear that EPEC must respond properly to its surrounding environment, in particular, temperature (133, 134, 275), to coordinate virulence gene expression and to survive in its specific niche, the small intestine. Rosenshine et al. (217) demonstrated that activation of EPEC at 37°C in tissue culture medium was necessary for the formation of AE lesions on human epithelial cells in culture. AE lesions did not form if the bacteria were incubated at 28°C prior to infecting host cells at 37°C, and these lesions were formed more readily by cultures in the early exponential phase of growth. Elucidation of the kinetics of AE lesion formation demonstrated this complex phenotype was tightly regulated in response to environmental conditions, growth phase, and interaction with host cells.
Similarly, studies have shown that environmental and media conditions regulate protein secretion via the type III apparatus (Fig. 1). Consistent with the temperature control of AE lesion formation, protein secretion occurs maximally at host body temperature in tissue culture medium such as Dulbecco’s modified Eagle’s medium (DMEM), at pH 7 and at physiological osmolarity (133, 134). Secretion of EspA, EspB, EspC, and Tir proteins was also stimulated in the presence of iron and sodium bicarbonate, whereas it was inhibited by ammonium chloride or by omitting calcium from the growth medium (117).
Studies to elucidate transcriptional control of the TTSS were assisted by characterization of the genetic organization of the EPEC LEE (66, 159, 224). The LEE1, LEE2, and LEE3 operons encode the type III apparatus components that span the inner and outer membranes, including EscC, the outer membrane porin, and EscN, the ATPase of the system. The LEE4 operon encodes the EspA protein, the monomer that forms the molecular syringe necessary for injection of effector molecules into the host cell cytoplasm (142), EspB, EspD, and EspF. The LEE5 operon encodes Tir and the intimin proteins, which are necessary for intimate attachment to the host epithelium, and CesT, a chaperone for Tir (66, 153).
The LEE1-located Ler (LEE-encoded regulator) has emerged as a key regulator of the AE phenotype, exerting its effect at the level of transcription (67, 159) (Fig. 1). The predicted 15.1-kDa Ler protein exhibits amino acid sequence similarity with the H-NS family of DNA-binding proteins, and shows greater similarity to the C terminus of H-NS, predicted to be a DNA-binding domain (253). Ler is clearly distinct from H-NS because all other members of this protein family identified to this date repress transcription, either directly or indirectly. Ler activates transcription of the LEE2, LEE3, LEE4, and LEE5 operons as well as espG, escD, and map of the LEE (32, 67, 96, 152, 159, 224, 253, 275). Ler also activates expression of espC (67, 152); since this gene is not located within the LEE PAI Ler is therefore a global regulator of EPEC virulence. Ler also has been shown to repress transcription of the LEE1 operon, and thus the ler gene is autoregulated (15). Ler exerts tight control over the genes of the LEE as a ler mutant of the prototypical EPEC strain E2348/69 is severely diminished in its ability to form AE lesions on epithelial cells in culture (67, 76). Leverton and Kaper (150) monitored the temporal expression of LEE operons during infection of HEp-2 cells in culture, presenting evidence that Ler expression is only necessary during the early stage of the infection process. By real-time PCR they demonstrate that transcription of LEE3, LEE4, and LEE5 operons increase over 3 h postinfection, while the expression of LEE1 encoding ler decreased during the same period. The EspA, EspB, EspD, and Tir proteins were visualized by immunofluorescent microscopy 5 h postinfection, indicating that Ler is not necessary for maintenance of these proteins once they are expressed. In follow-up studies using DNA microarray analysis, Ler regulated a number of EPEC and EHEC genes in addition to those already known to be regulated by Ler and these investigations are ongoing (J. Smart and J. B. Kaper, unpublished data).
Several lines of evidence suggest that the mechanism of Ler action is to disrupt an H-NS-mediated nucleoprotein complex involving both upstream and downstream repressing regions (32, 96, 224, 253, 275). To date there is no evidence that Ler activates transcription in the classical sense, by direct interaction with RNA polymerase, and thus it may be more appropriate to term Ler as an antirepressor as opposed to a transcriptional activator.
As with many virulence systems of gram-negative pathogens, H-NS plays an important role in the repression of genes of the LEE and is responsive to multiple environmental signals and regulatory proteins (Fig. 1). Umanski et al. (275) demonstrated that LEE1, encoding Ler, was repressed by H-NS at 27°C and activated at 37°C. In a cascade fashion Ler then activated transcription at the LEE2, LEE3, LEE4, LEE5 operons, and espG; hence, both increased temperature and the Ler protein are necessary for activation of these loci. It was shown that H-NS binds directly to LEE1, LEE2, and LEE3 regulatory DNA (275), whereas Ler binds directly to LEE2 and LEE5 regulatory DNA (96, 253). Thus both of these regulatory proteins apparently act directly on the LEE. H-NS and Ler act at the same regions upstream of the LEE2 and LEE5 promoters (32, 96, 253); this observation combined with the Kd of H-NS and Ler binding being approximately 1 μM and 200 nM, respectively (275; A. Carmona and J. L. Mellies, unpublished data), may indicate that Ler acts to disrupt repression by abrogating H-NS binding. Ler is, however, not a general antagonist of H-NS. Regulation of LEE genes is specific because this protein does not increase expression of the H-NS-regulated proU operon (67).
The EAF plasmid-encoded regulators PerABC control the AE lesion formation and localized adherence (LA) (see below) phenotypes. Early experiments indicated that an eae-phoA fusion in EPEC was influenced by the EAF plasmid (119), and it was later demonstrated that the perABC genes located on this plasmid affected the expression of eae reporter gene fusion (80). Intimin is downregulated upon contact with host epithelial cells (141), a process that involves the EAF plasmid.
It is now known that regulation of intimin by PerC is indirect; PerC activates transcription of LEE1, and in a cascade of events Ler then increases transcription of the LEE5 operon encoding eae (intimin) (32, 159, 203) (Fig. 1). The predicted PerB and PerC proteins originally showed no significant identity with any nucleotide sequences currently in the database (80). Just recently, five predicted open reading frames (ORFs) found within prophage of a EHEC serotype showed amino acid sequence similarity to PerC of EPEC and three of these ORFs, designated pch, were found to control LEE gene expression (118) (see "EHEC," below).
Evidence indicates the existence of a second, non-H-NS repressor of the LEE5 operon that functions at host body temperature ( 96, 224, 275), adding an additional layer of control on the genes necessary for intimate attachment to the host epithelium. Our data indicate that a RecA-LexA-dependent, DNA-damage-associated signal represses LEE5 transcription at 37°C (K. Haack and J. L. Mellies, unpublished data). Significantly, in Staphylococcus aureus it was recently demonstrated that the DNA-damaging antibiotic ciprofloxacin induces fibronectin binding in a RecA-LexA-dependent manner (19), and in E. coli β-lactam antibiotics induce the SOS response and mitigate the lethal effects of drugs on these bacteria (162). In EHEC, Stx2 production is enhanced by the presence of DNA-damaging antibiotics; this point will be discussed further below. Surely these recent findings will have an important impact on the use of antibiotics as chemotherapeutic agents.
Integration host factor (IHF) directly affects the expression of the LEE1 operon and thus expression of Ler (76) (Fig. 1). The observation that purified IHF protein did not bind to LEE2 regulatory DNA in this study supported earlier conclusions concerning the Ler regulatory cascade. Fis and BipA have also been demonstrated to regulate LEE1 transcription and thus indirectly control the AE phenotype (85, 89). Finally, the LEE1 operon is also subject to quorum-sensing regulation (254). Data demonstrating that Ler expression is controlled by multiple regulatory proteins and multiple environmental signals illustrate the importance of this protein in the complex control of EPEC virulence.
Clinical identification of EPEC strains has classically included their ability to attach to cultured epithelial cells in what has been termed localized adherence (229). This phenotype requires the type IV BFP (58, 83, 248, 249, 260) and is indicated by the formation of microcolonies: in general, between 5 and about 200 individual bacteria in three-dimensional clusters on the surface of the epithelial cells. The genes encoding the BFP are located on the 70- to 90-kb EAF virulence plasmid. EPEC strains deleted for bfpA, the gene encoding the major subunit of the BFP, were attenuated for virulence in human volunteers (18). Some studies indicate that BFP is the initial attachment factor conferring adherence to the human intestinal epithelia and tissue specificity (83), but other studies have indicated that EPEC can adhere to intestinal epithelial cells and form AE lesions in the absence of BFP (105), that intimin protein confers tissue tropism or that perhaps an initial adhesin is yet to be identified.
Similar to observations on the regulation of the AE phenotype, the LA phenotype is influenced by environmental conditions, carbon source, and phase of growth. EPEC showed increased adherence to Hep-2 human intestinal epithelial cells in culture in DMEM as opposed to rich media (83, 207, 280, 283), adhering as microcolonies effectively in the presence of glucose but not in the presence of galactose (280). Expression of BFP occurs maximally at 37°C during the exponential phase of growth (207). Transcription of bfpA is also subject to ammonium ion regulation; the cation NH4 + represses transcription of this gene (31, 32, 156, 207). Though NH4 + exists within the gut, in higher concentrations in the distal versus the proximal small intestine, the significance of this finding in terms of EPEC pathogenesis remains to be determined.
PerA regulates bfp transcription and is encoded on the EAF plasmid adjacent to the bfp operon (267). Several studies have indicated that PerA is required for expression of BFP (32, 156, 267), and indeed mutation in perA renders EPEC unable to display the LA phenotype. (PerA has also been called BfpT.) PerA shows amino acid sequence similarity to the AraC family of transcriptional regulators, and it is most closely related to the VirF protein of Shigella flexneri, a primary activator of virulence genes in this pathogen (62). PerA is also similar to the Rns transcriptional activator in ETEC (169). Transcriptional regulation of bfp by PerA is direct as this protein was shown to bind to DNA regulatory sequences upstream of bfpA in vitro (203, 267) and cis-acting sequences between positions –85 and –46 were required for activation (31). The perA gene is subject to autoregulation and binding regions upstream of the perA and bfp promoters share significant sequence similarity (116, 156).
The bfp and per operons are also subject to H-NS repression, similar to that observed for the genes of the LEE (203) (Fig. 1). It is perhaps not surprising that most, if not all of the EPEC virulence determinants described to date are repressed by this nucleoid-associated protein. Regulatory observations in EPEC and the E. coli pathogens discussed herein may indicate a common strategy for proper regulation of horizontally acquired virulence determinants and this notion is discussed below (see "Common Themes," below).
Clinically, strains possessing the EAF virulence plasmid have been termed typical EPEC and those lacking the plasmid have been termed "atypical." In human volunteer studies, the BFP and intimin protein have been identified as virulence factors (18, 59). The perABC locus controls expression of both of these factors, in a direct and indirect manner, respectively, and thus it seems clear that the perABC locus is important for EPEC virulence (18). Okeke et al. (192) found variation in the perA gene among clinical isolates. The genes perB and perC were apparently invariant in those strains tested, but frameshift mutations at nucleotide G114 were found in perA of two clinical isolates that would result in predicted truncated, nonfunctional proteins. Two mechanisms of elimination of BFP expression, either loss of the entire BFP plasmid or frameshift mutation in the essential PerA regulator, may indicate that loss and subsequent gain of BFP function is a requisite aspect of EPEC pathogenesis. The E2348/69-derived strain JPN15 lacks the EAF plasmid because of spontaneous curing during a volunteer study (120). Similarly, some clinical isolates from children infected with EPEC would be predicted not to express BFP because of the absence of the PerA regulator (192). A string of G nucleotides exist in the 5' region of the perA structural gene (G114-G120) and may indicate that frameshift mutations could occur by a slip strand mispairing mechanism at these sequences, resulting in phase variation of BFP via the PerA regulator (B. Bart and J. L. Mellies, unpublished data). In a recent study both typical and atypical EPEC strains were isolated from healthy children in Germany and Australia, and all but one of the 26 strains of EPEC were in fact atypical (17). In addition to not forming microcolonies, strains lacking the EAF plasmid are also attenuated for their ability to rearrange cytoskeletal actin and thus form AE lesions. Certainly the transmission of EPEC disease, including the loss and subsequent gain of key virulence factors and/or regulators, is more complex than our current understanding would indicate and will most likely be an important area for future inquiry.
The EAF plasmid-located perABC locus is subject to higher-order regulation. GadX, an activator of glutamate decarboxylase genes involved in acid tolerance, represses the expression of perABC (243). Consistent with this observation, because PerC regulates expression of Ler, a mutation in gadX resulted in increased translocation of Tir into host epithelial cells. GadX is a member of the XylR/AraC family of transcriptional regulators and its expression is increased in DMEM culture medium and under acidic conditions, pH 5.5. GadX was deemed a transcriptional regulator based on gel shift assays showing that this protein binds directly to perABC and gadA and gadB regulatory sequences. GadX may function to properly regulate acid tolerance and virulence gene expression in response to environmental cues within the gastrointestinal tract.
A few studies have addressed the regulation of motility, a phenotype thus far not linked directly to EPEC pathogenesis. The flagellar master regulators flhDC are controlled in EPEC and EHEC (discussed below) by the QseBC two-component system (257). The recently described HosA protein, a member of the SlyA family of transcription, is a positive regulator of motility (72). Regulation is direct as purified HosA protein binds to the fliC promoter region. HosA protein is subject to autoregulation, with maximal expression occurring at 37°C and pH 6.0. Yona-Nadler et al. (302) demonstrated that IHF represses expression of flagella in EPEC. At 37°C in DMEM medium wild-type EPEC are nonmotile, whereas an ihfA::kan mutant in the identical conditions was highly motile. Interestingly, this regulation was different in EPEC versus laboratory strains; the observed regulation did not occur in K-12 strains W3110 and N99 and appeared to be indirect in EPEC.
Mutation in bipA also results in a hypermotile phenotype (89). BipA protein shares amino acid sequence similarity with eukaryotic ribosome-binding elongation factor G. It possesses both GTPase and ATPase activity, is autophosphorylated at a tyrosine, involved in resistance to the cationic host defense peptide bactericidal/permeability-increasing protein (BPI), regulation of actin pedestal formation (71), and is essential for growth at low temperatures in K-12, EPEC, and EHEC strains (199). Control of motility occurs by a Ler-independent mechanism; however, data suggest control of transcription of LEE genes by BipA is indirect, through Ler (89). BipA is found in both soluble and particulate fractions in immunoblot analysis, and how this protein regulates resistance to host-derived defenses, motility, and pedestal formation by EPEC will be of great interest.
Enterohemorrhagic E. coli (EHEC) was first recognized as a serious, distinct class of E. coli pathogen in 1983 (129, 210). A cause of bloody and nonbloody diarrhea, in a small percentage of cases EHEC can cause the life-threatening complication known as hemolytic-uremic syndrome (HUS). In many patients with HUS, long-term renal damage occurs, often requiring dialysis or kidney transplantation. The main reservoir for EHEC is thought to be cattle, and the disease is spread by consumption of undercooked hamburger as well as contamination of uncooked food items, such as cantaloupe and radish sprouts, and recreational and municipal water sources, person-to-person contact, and petting zoos (158). Spread of the disease is aided by a low infectious dose, estimated to be less than 100 bacteria. The serious aspects of EHEC disease, bloody diarrhea resulting from damage to the colon, the site of infection, and damage to the renal endothelial cells, which can lead to HUS, result from production of the Shiga (Stx) toxin.
EHEC also possesses the LEE PAI, conferring the ability of the bacterium to form AE intestinal lesions on epithelial cells. Unlike for EPEC, however, the EHEC LEE cloned into a multicopy plasmid is not able to confer the AE phenotype on human epithelial cells in culture (68), suggesting that either factors and/or regulatory proteins necessary for this phenotype exist outside the LEE. Indeed the TccP (Tir-cytoskeleton-coupling protein) and EspFu (a protein with 24% amino acid identity to EspF) are encoded within the CP-933U cryptic prophage in EHEC, are translocated through the TTSS, and are necessary for actin accumulation and thus AE lesion formation (33, 82).
In addition to the EspA-containing TTSS translocon and intimin protein, other adherence factors have been postulated to facilitate initial attachment to host cells (125). These include the pO157 plasmid-encoded 362-kDa ToxB protein (263), which shares amino acid sequence similarity to the large Clostridium toxin family; the EPEC LifA protein (139); and the Efa-1 protein that is thought to be involved in adhesion of non-O157:H7 EHEC strains (184). The outer membrane protein OmpA is also implicated in initial attachment (269). Other pO157 virulence factors include the hemolysin-like repeats in toxin (RTX) toxin, the EspP autotransported serine protease, a catalase, and the StcE protein that cleaves the C1 esterase inhibitor of the complement pathway (146). Using both classical and genomics-based approaches, investigators have identified several regulatory proteins controlling EHEC virulence factors.
As with EPEC, Ler is a key regulator of EHEC virulence genes (Fig. 2). Elliott et al. (67) demonstrated that EHEC strain 86-24 deleted for the gene encoding this protein did not form AE lesions on cultured human intestinal epithelial cells by fatty acid synthase assay and that the EHEC ler can functionally substitute for EPEC ler. Ler regulates expression of the EHEC EspA, EspB, EspD, Tir, intimin, and EspG proteins targeted for secretion (53, 67), and by lacZ fusions Ler regulated transcriptional activity of the LEE2, LEE3, and LEE5 operons. Deletion of ler also caused expression of novel fimbriae in EHEC. The pO157 virulence plasmid-located stcE (tagA) gene, encoding a metalloprotease capable of cleaving C1 esterase inhibitor, is regulated by Ler at the level of transcription (67, 146, 152).
Another study establishing the importance of Ler in EHEC pathogenesis found that a clinical isolate containing an amino acid substitution in the putative oligomerization domain (Ile57Thr) did not adhere or form AE lesions on HEp-2 human epithelial cells in culture (191). Intimin expression was not induced in the presence of the mutated Ler, and the mutated protein exhibited a dominant negative effect on wild-type Ler in the strain EDL933 containing the mutated ler gene on a multicopy plasmid. These initial experiments firmly established Ler as a global regulator of virulence-associated genes of EHEC.
Although Ler is a key direct regulator of virulence genes, other regulatory pathways indirectly control virulence factor expression by coordinating expression of Ler (Fig. 2). Cell density, or quorum sensing (QS), regulation of EHEC and EPEC LEE-encoded virulence factors was first reported in Sperandio et al. (254). A fundamental difference between EHEC and EPEC infection is that EHEC colonizes the large bowel, containing a high density of commensal bacteria, whereas EPEC colonizes the proximal small intestine, which contains comparatively fewer bacterial inhabitants. Thus it has been proposed that QS regulation of the LEE may contribute to this differential colonization (254). The LEE1 operon, encoding Ler, shows the greatest activation by this regulatory pathway. The LEE2 operon is also directly regulated by QS signals, and the LEE3, LEE4, and LEE5 operons are regulated by QS indirectly through Ler. Additionally, the alternate sigma factor RpoS plays a role in cell density-dependent signaling in EHEC (254). (For further review of QS signaling in pathogenic bacteria, see references 126 and 281.)
An important distinction in the study of QS in EHEC and EPEC bacteria is that E. coli apparently produces no N-acyl-L-homoserine lactone (AHL) signaling molecules, as the E. coli genome does not contain a LuxI homolog, encoding the AHL synthase (301). In both K-12 and EHEC genetic backgrounds, sterile filtered culture supernatants from the EHEC strain 86-24h11 containing non-AHL autoinducer (AI) molecules activated expression of LEE1-lacZ and LEE2-lacZ reporter gene fusions (254). Thus the signaling molecule necessary for QS regulation is not unique to E. coli pathogens, but rather is most likely produced by all E. coli. Consistent with this observation, the laboratory strain DH5α contains a point mutation in the luxS gene responsible for production of autoinducer-2 (AI-2) (it is also void of AI-3 production; see discussion below) and thus quorum sensing signaling is absent from this laboratory strain (261, 262). In EPEC, a mutation in luxS eliminated QS signaling of the LEE1 and LEE2 operons (254). The same conditions that stimulate QS activation of the LEE1 operon, high osmolarity, temperature of 37°C, and culturing in DMEM, also stimulate maximal protein secretion by the TTSS, solidifying an important role for LuxS-dependent, cell density signaling in EHEC and EPEC bacteria.
To further investigate the scope of luxS-dependent quorum sensing signaling in EHEC investigators used DNA array technology to compare transcriptional profiles of cDNA isolated from strain 86-24 and an isogenic luxS mutant (255). Approximately 10% of the 4,290 K-12 array genes showed differential expression: 169 genes showed decreased and 235 genes showed increased transcription in the wild-type versus mutant strain. Genes showing decreased expression included those involved in cell division, tRNA and ribosome biosynthesis, and the wild-type strain grew slower than the isogenic luxS mutant in DMEM. Genes showing increased expression in the wild type compared with the mutant strain included those involved in flagella biosynthesis, motility, and chemotaxis. These results were confirmed by transcriptional reporter gene fusion assays, demonstrating altered expression of class I, II, and III flagella genes, and by Western immunoblot and electron microscopy analyses. Predictably, the luxS mutant strain showed decreased motility on semisolid agar.
As for the flagella-encoding genes, those involved in the SOS response were also differentially expressed in the array analysis (255); recA, uvrA, and sulA all showed at least 20-fold increased expression in the wild-type versus luxS mutant strain. Because the SOS response induces expression of lambdoid phage genes upon cleavage of the cI repressor, and the Stx2 toxin is encoded within the BP-933W prophage, these investigators hypothesized that expression of stx2 would also be responsive to QS signaling. Transcriptional of the stx2 gene is under control of a Q-dependent, late phage gene promoter, and indeed this promoter showed increased activity by lacZ reporter gene fusion in the wild-type versus luxS mutant strain, and this result was confirmed by Western immunoblot analysis. Thus Stx2 production responds to cell density-dependent regulation.
The quorum-sensing regulatory cascade of EHEC has been further elucidated by the description of a LysR-type molecule and a two-component regulatory system that control expression of the TTSS and flagellar motility, respectively (252, 257). The K-12 ORF b3243, also present in EHEC strain 86-24, was shown to increase LEE1, and to a lesser extent LEE2 transcription, and was thus renamed QseA, for quorum-sensing E. coli regulator A (252, 255). QseA is a member of the LysR family of transcriptional regulators with similarity to the AphB and PtxR regulators of Vibrio cholerae and Pseudomonas aeruginosa, respectively. In EHEC, a qseA mutant was impaired in its ability to secrete Tir, EspA, and EspB but was still able to form AE lesions on epithelial cells in culture. Similarly, expression of LEE genes and secretion of TTSS components are diminished in mutant derivatives of EPEC strain E2348/69 deleted for luxS or qseA, though these mutants are still able to form AE lesions on HeLa cells (244). QseA expression is reduced by mutation in luxS (255), it is autoregulated, and data suggest that QseA acts directly on the ler-encoding LEE1 operon (V. Sperandio, personal communication). Only the more distal LEE1 promoter, corresponding to the LEE1 promoter of EPEC (159), was responsive to QseA regulation. Neither flagellar motility nor Stx production was affected by mutation of qseA (252).
E. coliORFs b3025 and b3026 have been designated QseB and QseC, respectively, because of their role as a two-component regulatory system involved in EHEC QS signaling (257). QseB, the response regulator, and QseC, the sensor kinase, are encoded within a single operon (38) and share amino acid sequence similarity with the PmrAB two-component regulatory system of Salmonella enterica serovar Typhimurium. Mutation of qseC in strain 86-24 reduced flagellin subunit production, decreased transcription of flhD, encoding a flagella master regulator, and reduced expression of motA and fliA (257). Additionally, a FliA (σ28)-dependent consensus sequence was identified as the flhDC promoter, and indeed transcription of an flhDC-lacZ fusion was reduced by deletion of the fliA gene. Mutation in qseC also resulted in reduced motility in soft agar assays. By deletion analysis, electrophoretic mobility shift and DNase I protection assays, it was demonstrated that purified, phosphorylated QseB protein bound to two sites, one high and one low affinity, upstream of the flhDC promoter (38). Thus, QseBC controls flagellum production by activating transcription of the flhDC master regulators, and this regulation is complex, involving a class 2 flagella gene encoding σ28. QseBC is subject to autoregulation, and identically with flhDC, QseB activates qseBC transcription by binding to high- and low-affinity sites upstream of a QseBC-responsive promoter (37). Thus, as for the QseA regulator, the QseBC two-component system is autoregulated and present in both E. coli K-12 and EHEC strains. Finally, because QseBC does not control certain QS-associated phenotypes, such as type III secretion and Stx2 production, it was hypothesized that additional, uncharacterized regulatory systems must also respond to the AI-2 and/or other signaling molecules.
Perhaps the most intriguing discovery to date concerning cell-to-cell signaling in EHEC bacteria is a luxS-dependent AI-3-signaling molecule, which cross talks with the mammalian hormone epinephrine and activates LEE and flagella genes (256). The AI-2 autoinducer is a furanosyl borate diester that also depends on LuxS for biosynthesis (36, 262), but it is now known that AI-2 does not activate expression of EHEC virulence genes (256). Activation of LEE1 and flhDC transcription in a luxS mutant can be restored by providing exogenous, partially purified AI-3 or by the addition of epinephrine or norepinephrine (256). The AI-3 molecule is <1 kDa and is methanol soluble, consistent with its being similar to epinephrine, but the exact identity is yet to be established. AI-3 is apparently not produced by HeLa epithelial cells in culture, but rather it is found in the fetal bovine serum added to the culture medium. The AI-3-dependent signaling can be blocked by the addition of α- or β-adrenergic antagonists. The QseBC two-component system may respond directly to the AI-3/epinephrine signal as a qseC mutant is blind to both AI-3 and epinephrine (257). The exact mechanism of the sensor kinases involved in quorum sensing signaling in both EHEC and EPEC is currently under intense investigation.
Though no AHL signaling molecules exist in E. coli the LuxR homolog SdiA directly represses the EspD- and intimin-encoding operons LEE4 and LEE5, respectively (124). It has been hypothesized that the biological function of SdiA, which activates transcription of the ftsQAZ operon encoding proteins essential for cell division, (4, 286) is to detect the presence of other species of bacteria that do produce AHL signaling molecules (301). Thus, these data, and our current understanding of quorum sensing in EHEC and EPEC bacteria, indicate that cell-to-cell communication plays a significant role in activating LEE genes in EHEC when this bacterium reaches the densely colonized large intestine.
Until recently no Per-like regulators had been identified in EHEC, perhaps consistent with the importance of QS signaling to this pathotype upon entering the large intestine. However, the question of whether Per-like molecules exist in EHEC was addressed by two independent research groups (118, 202). Iyoda and Watanabe (118) screened a genomic library of the EHEC O157:H7 Sakai strain for genes that controlled expression of LEE fusions. They identified a DNA fragment containing a gene with similarity to perC of EPEC. Upon closer inspection of the genomic DNA sequences they identified five perC-like sequences but no sequences with similarity to either perA or perB of EPEC. Thus they termed these five loci pch for perC homologs A through E. Deletions in either pchA (also termed perC1-1) or pchB (perC1-2), or double deletions in pchA and pchB or pchA and pchC (perC1-3) reduced the expression of EspA, EspB, and EspD proteins and adherence to HEp-2 cells in culture. This group postulated that multiple pch loci were necessary for full expression of the LEE and found that these loci exerted their effect through transcription activation of the LEE1 operon encoding Ler (Fig. 2).
PerC-like molecules exist not only in the E. coli O157:H7 strain Sakai (PerC1-1, PerC1-2, PerC1-3, PerC2, PerC3), but also in the O157:H7 strain EDL933 (PerC1, PerC2, PerC3-1, PerC3-2), UPEC (PerC2, PerC3, YfdN), in the S. enterica serovar Typhimurium ST64B phage (YfdN), the S. flexneri SfV phage (YfdN), and the E. coli laboratory strain MG1655 (YfdN) (202). Many of the genes encoding the PerC-like molecules are found with lambdoid phage. Porter et al. (202) found that the PerC1, but not the PerC2 proteins of EHEC strains could activate expression of LEE1 from both organisms, though in in vitro assays they could not demonstrate binding of purified PerC protein to LEE1 regulatory fragments. They hypothesized that PerC molecules may bind in the presence of IHF, also a positive regulator of LEE1 transcription, and perhaps other unidentified proteins to activate transcription. The latter possibility now seems likely, as PerC did not bind even in the presence of purified IHF. It was also demonstrated that the PerC-responsive promoters of EHEC and EPEC are similarly located in these bacteria, approximately 170 bp upstream of the start codon of lerof LEE1.
As with PerC of EPEC, the nucleoid-binding protein IHF positively regulates EHEC LEE gene expression through Ler (302), whereas Hha negatively regulates Ler expression (238) (Fig. 2). Deletion of the gene hha in EHEC resulted in increased expression of Ler, and adherence to HEp-2 epithelial cells in culture, and the purified 8.5-kDa Hha protein, shown previously to regulate α-hemolysin (185), bound to the LEE1 regulatory DNA, demonstrating that this regulation is direct (237).
Through comprehensive mutagenesis of the LEE PAI of the mouse pathogen Citrobacter rodentium a positive regulator, ORF11, of EspB and Tir expression was identified (53). The orf11 gene from C. rodentium EHEC and EPEC complemented the activity when expressed from a plasmid in trans, and therefore it was concluded that this positive regulator was functionally equivalent in these three AE pathogens. Data presented suggested that ORF11, renamed GrlA (for global regulator of LEE activator), acted at the level of transcription upstream of Ler. A subsequent study in C. rodentium indicated that Ler acts directly to control GrlA expression and is part of a positive regulatory loop whereby optimal Ler expression depends on GrlA, thus controlling spatiotemporal transcription of the genes of the LEE (12). GrlA shares 37% and 23% identity to Sgh protein of Salmonella and CaiF of Enterobacteriaceae, respectively. ORF10, most likely expressed from the same operon as ORF11, or GrlA, had a repressing effect on LEE1, LEE2, and LEE5 transcription but a more dramatic effect on EspB and Tir expression, and was thus renamed GrlR (for global regulator of LEE repressor) (51). This repressor was postulated to act upstream of Ler as well (Fig. 2). Repression by GrlR (L0044) of EspB and Tir expression in a Ler-dependent manner was recently confirmed in EHEC (ATCC 43888) (153).
Control of secretion of translocator molecules, e.g., EspB, and effectors, e.g., Tir, is complex, involving posttranscriptional and posttranslational regulation. Heterogeneous secretion of EspD protein in human and bovine O157:H7 isolates lead investigators to study the underlying mechanism regulating this observation (216). They found that strains possessing EspA translocons, visualized by immunofluorescence, correlated with high levels of EspD secretion and that the observed variations in secretion were not controlled at the level of transcription demonstrated by reporter gene fusions. By Northern analysis, secretion of translocon proteins was inversely related to espADB mRNA transcript levels. Evidence suggested that sepL, espA, espD, and espB are transcribed as a single polycistronic operon, but that mRNA processing must occur separating the transcript into two fragments, an approximate 1.2-kb sepL-containing transcript and a 2.8-kb espADB transcript. These investigators clearly established that posttranscriptional regulation is involved in EspA filament, or translocon formation, though the exact mechanism of control remains to be determined. They postulate that the inability to detect the espADB transcripts in the high-secretor strains may be explained by engagement of the molecule in the type III apparatus prior to secretion, a coupling of translation and secretion as proposed for flagella assembly in S. enterica serovar Typhimurium (128).
One issue that has remained controversial in the study of type III systems is whether contact-dependent secretion occurs (42). Certainly environmental signals can cue secretion; Ebel et al. (63) established that temperature and media conditions affect secretion of EHEC polypeptides. Calcium-deficient conditions signal secretion of effector Yops of Yersinia spp. and secretion of Ipa effector proteins of S. flexneri can be triggered by the dye Congo red (194), but whether these environmental conditions and/or chemical cues actually relate to contact-dependent secretion remains to be determined. Some studies have indicated that rapid increase in EHEC LEE gene expression occurs upon contact with host epithelial cells; transcriptional activity from an espADB-lacZ fusion increased upon contact with HeLa cells (14) and tir-egfp and map-egfp fusion activity increased upon contact with bovine intestinal epithelial cells (215). Beltrametti et al. (14) also demonstrated that the addition of Ca2+ and Mn2+ but not Mg2+ increased the expression of the espADB-lacZ fusion. In a recent, elegant study Deng et al. (52) revealed that SepD and SepL, encoded by the LEE2 and LEE4 operons, respectively, constitute a molecular switch controlling secretion of translocators and effector molecules, and began to clarify the role of calcium in these processes. Beginning in C. rodentium, but expanding their studies to EHEC and EPEC, they found that low-calcium conditions inhibit the secretion of translocators, such as EspA, EspD, and EspB, but enhance the secretion of effectors such as Tir and NleA. This phenotype was similar to that observed for sepD and sepL nonpolar deletion mutants. SepD and SepL proteins interact with each other and are localized to the inner membrane. These authors proposed a model whereby a high calcium concentration, about 2.5 mM in the mammalian extracellular fluid (41), stimulates the secretion of translocator proteins necessary for assembly of the translocon and then, once connected to the host cell, the low-calcium environment, 100 to 300 nM as most calcium is sequestered in the endoplasmic reticulum, triggers secretion of the effector proteins. How the calcium signal is perceived from the host cell cytoplasm and sensed by the bacterium remains to be determined; not surprisingly, in vitro SepD/SepL protein complexes were not affected by alterations in CaCl2 concentration (52). Thus SepD and SepL were termed "gatekeepers," controlling the switch releasing effector proteins into the host cell cytoplasm.
To better understand the regulation of EHEC adherence, Torres and Kaper (269) screened a transposon mutant library for increased adherence on HeLa cells, finding that insertional inactivation of the tdcA gene regulated this phenotype (Fig. 2). By reporter gene fusion tdcA modestly repressed the expression of an ompA-lacZ transcriptional fusion. The gene tdcA encodes a transcriptional activator of the tdc operon involved in anaerobic degradation of L-threonine. Through this study these authors implicated OmpA as an adherence factor mediating binding to human epithelial cells in culture, confirming their findings on the more relevant Caco-2 colorectal adenocarcinoma cell line. Tatsuno et al. (264) performed transposon mutagenesis to find EHEC loci involved in adherence to Caco-2 cells. They identified two novel regulators of EHEC adherence, YhiF and YhiE. These proteins share amino acid identity (23%) and are members of the LuxR family of transcriptional regulators, which include portions of two-component regulatory systems in Xanthomonas axonopodis pv. citri, Pseudomonas putida, and the gram-positive S. aureus. YhiE and YhiF control the secretion of EspB, EspD, and Tir and were shown to exert transcriptional control on the LEE2 and LEE4 operons, but not ler. Insertional inactivation of the yhiE locus caused increased shedding of the O157:H7 Sakai strain in a mouse model, implicating this gene in the regulation of EHEC colonization. Another adhesin implicated in the ability of EHEC to attach to epithelial cells in culture, the long polar fimbria (Lpf), is regulated by growth phase and medium conditions, showing maximal expression in late exponential phase (strain 86-24) and greater expression in DMEM versus LB (268).
Beyond classical, informative genetic approaches and coincident with the completion of EHEC genomic DNA sequences (98, 196), researchers are beginning to use DNA array analysis to study regulatory phenomena. A recent work used a DNA array analysis to determine differential gene expression as a function of attachment to red blood cells (RBCs) (45). (RBCs had been used previously to model attachment to host cells mediated by the type III secretion system of EHEC, whereby EspA filaments are responsible for binding and EspD and Tir are localized to the plasma membrane [183, 239, 240] and EHEC can induce hemolysis.) Maximum adherence by EHEC occurred 5 h postinoculation in DMEM, though the mean number of bacteria adhering to individual RBCs was low, three; differential gene expression comparing mRNA transcripts isolated from EHEC grown in DMEM versus those adherent to the plasma membrane of RBCs was analyzed by using a DNA microarray. Of the 404 genes differentially expressed, 299 were repressed and 105 were induced by attachment to RBCs. The most notable result from this study was that the genes of the LEE were generally repressed at the 5-h time point; this result was confirmed by real-time PCR, with the exception of the LEE2 operon. These results were consistent with the observations that both Intimin and EspA disappear from the bacterial cell surface 6 h postinfection of intestinal epithelial cells (141, 142). We have also observed downregulation of the LEE1 operon of EPEC upon attachment to intestinal epithelial cells in culture (R. Green and J. L. Mellies, unpublished results), but one cautionary note is that the native lac operon was also repressed, which may indicate a general downregulation of transcription upon attachment. The gene feoAB, encoding an iron transport system, was also repressed in the recent DNA array study (45). Lastly, Dahan et al. (45) found that the putative two-component system regulators YhiE and YhiF and YhiX (GadX), which may be involved in acid tolerance, were upregulated in response to RBC attachment.
DNA microarrays and classical transcriptional fusion assays were used to identify regulators of the LEEs that are found in a second cryptic type III secretion system of the Sakai 813 strain (304). Deletion of either of the regulatory genes etrA or eivF from this second nonfunctional type III system led to increased secretion of EspA, EspB, Tir, TagA, and EspP of the LEE by immunoblot analysis and adherence to Int-407 cultured epithelial cells (Fig. 2). Overexpression of EtrA or EivF repressed secretion in a high-secreting O26:H− strain, emphasizing these proteins’ role in repression of LEE gene expression. Reporter gene fusions of the five major LEE operons suggested that EtrA and EivF exerted their effect at the level of transcription. This study is of special interest because it illustrates the concept that the functionality of regulatory genes may outlast the functionality of a decayed gene cluster in which they are located, and that they may regulate distally located genes, a phenomenon dubbed the "Cheshire cat effect" after the disappearing cat in Alice in Wonderland. This may be a particularly apt metaphor since it is increasingly apparent that proper regulation of attachment factors is essential for colonization of bacterial pathogens, including EHEC, and one sees the Cheshire cat’s teeth, i.e., the regulators, when he disappears in this classic children’s tale.
Herold et al. (104) recently used a DNA microarray approach to monitor changes in transcriptional activity in the presence of low levels of the DNA gyrase inhibitor norfloxacin. Several studies have demonstrated that production of Stx can be modulated by antibiotics (91, 138, 285, 305) and that Stx production is linked to the Stx phage growth cycle (181, 284) (Fig. 2). The Shiga toxins exist in two subgroups, Stx1 and Stx2, and are encoded within the prophage (196), though to date only the prophage BP-933W encoding Stx2 has been shown to produce infectious phage particles. Expression of Stx2 is under transcriptional control of a late phage promoter (284). Norfloxacin induced DNA damage, and the SOS response was indicated by elongated bacterial cells visualized by electron microscopy (104). Phage titer and Stx2 production dramatically increased approximately 2 h after the addition of norfloxacin. Of the 118 spots showing increased expression in the presence of the antibiotic, 85 were phage borne; 52 were from the Stx phages BP-933W and CP-933V. The most strongly induced genes were found within the BP-933W phage; stxA 2 was induced 158-fold. Regulation of stx 2 and recA genes was confirmed by real-time reverse transcriptase (RT)-PCR. Other induced genes included putative phage integrases not found in prophages, some stress-induced proteins, including the heat shock protein IbpB, and DNA-damage-inducible proteins associated with the SOS response, including DinB. Several genes of the EHEC LEE were downregulated by norfloxacin, but this result was not confirmed by the alternate methodology.
There is an intimate connection between DNA damage and phage induction, and hence in the case of EHEC the release of the Shiga toxin so important in causing serious disease. Several papers have suggested that DNA damage may play a role in bacterial virulence signaling (9, 19), and we predict this to be an area of intensive investigation because of the implications for treating bacterial infections. Exposure to ciprofloxacin and ampicillin has been shown to induce the SOS response (162), and it seems likely that the treatment of E. coliinfections by antibiotics may have unintended negative consequences; the prevailing thought that for the treatment of EHEC infections antibiotics are contraindicated is certainly consistent with this idea. In addition to simply upregulating virulence genes, the error-prone replication machinery activated by the SOS response may provide the genetic variability necessary for generation and selection of spontaneously resistant bacterial strains. To add insult to injury, Gamage et al. (79) demonstrated that Stx phage conversion of intestinal commensal E. coli led to increased production of the Stx2 toxin in a mouse model, and that ciprofloxacin, also a DNA gyrase inhibitor and a commonly prescribed antibiotic in the United States, led to lysis of phage-infected host E. coli and release of the toxin. There was indeed a 40-fold increase in Stx2 production when the susceptible commensal E. coli were infected with the 933W phage, indicating that the Shiga toxin phage regulate amplification of the toxin upon phage induction, known to occur in the presence of DNA-damaging agents.
A common cause of childhood diarrhea in developing countries (177), ETEC causes a watery diarrhea that in most cases is self-limiting but can cause more severe disease. ETEC is the etiological agent of diarrhea in travelers visiting the developing world, though outbreaks occasionally occur in developed nations. It is also an important cause of diarrhea in military troops deployed in developing countries (97, 115). Intestinal secretion leading to diarrhea is mediated by two groups of enterotoxins, the heat-labile enterotoxins (LTs) and the heat-stable enterotoxins (STs). ETEC strains can express either just an LT, just an ST, or both types of toxins in combination. The LTs are AB-type toxins related in structure and function to the cholera enterotoxin (CT) of V. cholerae, whereas the STs are small single-peptide toxins (for a review, see reference 177). ETEC colonizes the small bowel and a number of colonization factors are involved in this process. Most of these colonization factors (CFs) are fimbriae, or fimbria-like, and are characterized as CF antigens (CFAs), coli surface antigens (CSs), or putative CFs (PCFs) followed by a number designation. CFs are often flanked by insertion sequences, indicating that most likely they were acquired by horizontal gene transfer.
CFA/I, CFA/II, and CFA/IV are the most prevalent CFAs found to be associated with human disease (299) and have been extensively studied both biochemically and genetically. CFA/II comprises CS1, CS2 and CS3, whereas CFA/IV comprises CS4, CS5, and CS6. The three antigenically distinct CFA/II fimbriae CS1, CS2, and CS3 are plasmid associated (26, 43, 166, 245). Early experiments indicated that fimbriae of ETEC are regulated by temperature, as expression occurs at temperatures above 25°C (246) (Fig. 3). Specifically, the CFA/I genes are temperature regulated, but this temperature-dependent regulation is absent in strains mutated for H-NS (78). The genes required for production of the 15-kDa structural subunit and assembly of the CFA/I fimbriae are located on a large virulence plasmid, which also encodes the ST toxin, and expression requires two unlinked, separated regions of the plasmid (228, 245). Region 1 encodes the major fimbrial subunit CfaB and the fimbrial tip subunit CfaE, while region 2 encodes the AraC-like regulator CfaR (also termed CfaD) necessary for CFA/I elaboration (34, 228). Spontaneous loss of CfaR results in no expression of CFA/I, while providing this positive-acting factor on a plasmid in trans restores expression (90, 250). CFA/I expression is repressed by H-NS (122) and the presence of iron (127).
Positive regulators of fimbriae such as CfaR control the expression of multiple ETEC colonization factors (Fig. 3). For example, expression of CFA/I, CS4, CS19, PCFO159 (also called CS12), and PCFO166 fimbrial adhesins can be expressed by transformation of ETEC strains, pathogenic to both humans and porcine, containing these adhesins, with a CfaR-encoding plasmid (90, 251, 297). Similarly, CS20 of the human ETEC clinical isolate H271Ab is regulated by CfaR (277). (CfaR can also substitute for other members of the AraC family of transcriptional regulators as this protein can complement a null mutation in the virF gene of Shigella [204].) CS20 is produced at either 22°C or 37°C when CfaR is present while it is only produced at 37°C when CfaR is absent, suggesting that CfaR can override repression at lower temperatures. Correspondingly, the ETEC transcriptional regulator Rns can override temperature regulation at its own promoter (77).
Similar to CfaR, Rns is an AraC-like protein required for expression of CFA/II fimbrial antigens CS1 and CS2 (34) (Fig. 3) and can functionally substitute for other positive regulators of fimbrial expression (35, 48, 169, 204, 297, 298). AraC-like molecules similar to Rns include RhaR and RhaS, which regulate the rhamnose operon in E. coli (34), PerA of EPEC (80), AggR of EAEC, and ToxT of V. cholerae (106). Rns is the best-characterized regulator of ETEC fimbriae to this date and is itself plasmid encoded (34), but not on the same plasmid that encodes the CS1 or CS2 antigens.
Rns directly regulates the coo operon encoding CS1 (170) where coo comprises four genes, cooB, cooA, cooC, and cooD (Fig. 3). The genes cooB, cooC, and cooD are required for CS1 assembly (77, 236) and cooA encodes the fimbriae structural subunit (195). The gene cooD encodes a tip adhesin that is part of the fimbrial structure. Munson and Scott (170) demonstrated that Rns binds upstream of the coo promoter at two locations designated site I, spanning positions –129 to –93, and site II, spanning positions –63 to –23, in relation to the P2 promoter. Because the sites were not palindromic, though located on the same side of the DNA helix, and both were required for full expression, these investigators hypothesized that Rns binds as an asymmetric monomer. A similar mechanism of monomeric binding has been proposed for the PerA protein of EPEC. In this case, PerA binding is to single regions spanning positions –75 to –47 and –83 to –55 upstream of the perA and bfpA promoters, respectively (116).
Expression of Rns is autoregulated and this protein functions in a unique manner at its own promoter (77, 171). Regulation is direct as purified protein binds to positions centered at –227, +43, and +82 in relation to the start of transcription. Sites 1 and 3 centered at –227 and +82 were necessary for rns activation in vivo, and in an in vitro assay the addition of purified Rns protein facilitated open complex formation (171). Thus Rns activates transcription by binding both upstream and downstream of the Prns promoter. An unknown negative regulator also acts at regions both upstream and downstream of Prns , and based on the important role of H-NS in virulence gene regulation and its ability to bind to regions flanking promoters (see "Common Themes," below), it is tempting to speculate that H-NS also regulates rns.
Evidence suggests that Rns activates transcription, at least in part, through disrupting repression by H-NS at the coo operon (172) (Fig. 3). This prediction comes from genetic data demonstrating increased transcription at Pcoo in an hns mutant background and identification of a downstream negative regulatory element extending from positions +7 to +929, within the cooBA structural genes. It is not known, however, whether H-NS binds over this region. A Pcoo regulatory fragment lacking this downstream region is severely deregulated for β-galactosidase activity (172). Downstream negative-acting regions have been identified in many H-NS-regulated operons (32, 73, 96, 123, 193, 231) and may represent a common mechanism of H-NS function. Positive-acting AraC-like proteins, such as CfaD and Rns, function by interacting directly with RNA polymerase and/or disrupting H-NS-mediated repression. For example, at Pcoo Rns binds at site II between positions –63 and –23 and at this position most likely interacts directly with RNA polymerase to affect transcriptional activity, but evidence suggests that it may also disrupt repression by H-NS. In contrast, Rns binds at a position far upstream of RNA polymerase binding, −227, as well as a downstream site centered at +82 at the Prns promoter (171). As these authors point out, the mechanism of transcriptional activation by Rns at the Pcoo and Prns promoters clearly is different, though a commonality may be that promoter fragments lacking regulatory elements fused to reporter genes are severely deregulated for transcriptional activity (77, 171), indicating that repression is the key form of regulation at both promoters. It will be interesting to know whether H-NS also represses rns expression and, if so, the DNA sequences necessary for H-NS function.
The LT toxin is transcriptionally regulated with maximal expression occurring at 37°C and this temperature regulation is eliminated in a strain mutated for H-NS (272) (Fig. 3). Consistent with observations of H-NS repression of many other virulence- and non-virulence-associated operons of E. coli, a downstream negatively regulatory element (DRE) was described for the LT A-subunit DNA. Deletion of the DRE resulted in elevated levels of LT mRNA at 18°C. Further investigation indicated that H-NS represses the LT-encoding eltAB operon by binding to two regions spanning positions +31 to +110 and +460 to +556 downstream of the promoter (300).
Though this review focuses on human pathogens, one particular study of the regulation of the porcine ETEC fimbrial adhesin 987P has provided important clues concerning expression in response to environmental signals. Expression of the 987P fimbriae responds to carbon source availability as mutations in either cya or crp reduced expression (64). Consistent with this observation, expression was greater in minimal medium containing glycerol versus LB at 37°C. FasA is the major subunit of the fimbria, and transcription from a fasA-blaM fusion was repressed at 20°C; this regulation required H-NS. FasA and FasH, the AraC-like transcriptional activator of fasA, responded to nitrogen availability and were activated in the presence of glycerol and ammonia but not activated in the presence of glycerol and glutamine or glutamate. Thus, Edwards and Schifferli (64) present a working model of how ETEC strains respond to carbon and nitrogen sources within the small intestine.
All ETEC fimbrial adhesins discussed thus far are regulated by AraC-like molecules. In contrast, the CS18, 987P-like fimbriae possess no known associated AraC-like regulator and are subject to phase variation (109). A 312-bp region containing the promoter for fotA, the structural subunit, is flanked by 16-bp (inverted repeat) IR elements and is invertible. Inversion is controlled by two site-specific recombinases encoded by fotS and fotT that are divergently transcribed from fotA. Both FotS and FotT can perform inversion, though FotT is more effective at placing the promoter in the OFF position.
Enteroaggregative E. coli is an emerging pathotype of diarrhea-causing E. coli. First recognized by a 1987 prospective study on childhood diarrhea in Chile (178), EAEC is a causative agent of traveler’s diarrhea, endemic pediatric diarrhea in both developing and more developed countries, and a chronic diarrheal syndrome in individuals living with human immunodeficiency virus (HIV). EAEC has also been associated with a persistent pediatric diarrheal syndrome lasting longer than 2 weeks (177). This E. coli pathotype colonizes both the small and large bowel, and secretion of enterotoxins is an important part of EAEC disease. The toxins are both plasmid and chromosomally located and include the Shigella enterotoxin 1 (ShET1), the plasmid-encoded toxin (Pet), the mucinase Pic (101), and the enteroaggregative ST-like toxin (EAST1) (44, 161). EAEC can cause damage to the intestinal mucosa, which appears to be most severe in colon sections. (For a recent review of pathogenesis of EAEC, see reference 175.)
As the name of the pathotype indicates, EAEC forms thick aggregative structures, akin to biofilms on mucosal surfaces and human epithelial cells in culture (176, 274, 282), and a number of bacterial adhesins have been described. Aggregative adherence fimbriae (AAF)/I and II are encoded on large, ~100-kb virulence plasmids termed pAA (65, 227). In the prototypical strain 042 adherence to colonic mucosa requires expression of AAF/II and thus this structure is thought to be a virulence factor important for disease in humans (44). AAF/I form flexible bundles and individual fimbriae are from 2 to 3 nm in diameter, while AAF/II form semirigid bundles and are 5 nm in diameter; the predicted structural subunits of the these fimbriae, AggA and AafA, respectively, share approximately 25% amino acid sequence identity and are related to the Dr family of adhesins that bind the Dra blood group antigen. An additional allelic variant, AAF/III, was recently described (16). The AAF antigens are found in only a minority of EAEC isolates (16, 44), but this finding may be misleading because loss and subsequent gain of either a pAA plasmid, or fimbrial expression by genetic regulation, such as, for example, phase variation, may be essential for EAEC disease ecology (see the discussion of the PerA regulator of EPEC). A surface protein of EAEC termed dispersin was found to facilitate dispersal of these bacteria across the mucosal surface, apparently acting by neutralization of the negative charge of the lipopolysaccharide and thus allowing extension of the positively charged fimbriae away from the bacterial outer membrane (241). Dispersin (encoded by the aap gene) is transported to the cell surface by an ABC transporter complex (aat); both loci are encoded on the pAA plasmid though in unlinked locations.
The most important regulator of EAEC virulence factors described to date is AggR, encoded on the pAA virulence plasmid (65, 179, 180) (Fig. 4). The 29.4-kDa AggR protein is a member of the AraC family of transcriptional regulators, and like many of these proteins is subject to autoregulation (77). In EAEC strain 17-2 expression of the AAF/I fimbriae requires two unlinked regions of the pAA plasmid (227), and deletion of the aggR gene eliminates autoagglutination, hemagglutination, and aggregative adherence on HEp-2 and Caco2 epithelial cells in culture (180). As predicted, AggR exerts its effect at the level of transcription, demonstrated by positive control of an aggA-phoA fusion. Expression of the aggA-phoA fusion was maximal at pH 5.5 and was responsive to several growth conditions, including temperature, osmolarity, media components, oxygen tension, and iron availability. Under all conditions tested, the presence of AggR resulted in increased transcription, except for when strains were grown at either 27°C or 42°C. Thus, temperature is a strong signal governing the expression of aggA in an aggR-independent manner at non-host-body temperatures. In a heterologous experiment, the AraC-like regulator CfaR of ETEC complemented deletion of the EAEC aggR in the HEp-2 adherence assay.
In the 042 strain, deletion of aggR also resulted in loss of AAF/II fimbrial expression—regulation occurs at the level of transcription (65). By Northern blot analysis AggR affected transcription of the aafD and aafA genes, encoding the chaperone and pilin structural subunit, respectively. Though aafDA apparently form a polycistronic operon in region 1 of the AAF/II gene cluster, the aafA gene was found in greater abundance than aafD in the presence of AggR. This may suggest greater stability of the aafA transcript, which may be supported by a predicted hairpin structure located just downstream of aafA, or that a second, separate, as yet unidentified promoter drives expression of aafA. The aap gene encoding dispersin and aat gene cluster encoding the ABC transporter necessary for transport to the outer membrane are coregulated by AggR (188, 242). Significantly, the presence of aggR correlates with the presence of the aat gene cluster in EAEC clinical isolates (188). Recent observations also suggest that AggR activates a chromosomal island in EAEC strains, though the role of this island is as yet uncharacterized (E. Dudley and J. Nataro, personal communication).
Sheikh et al. (242) used transposon mutagenesis to identify genes, one of which being aggR, which are necessary for EAEC strain 042 biofilm formation (Fig. 4). Transposon insertion into AAF/II biogenesis genes located on the pAA2 plasmid as well as those in chromosomally located genes prevented biofilms from forming. Thus AAF/II elaboration was necessary for biofilm formation, though biofilms were also produced by strains not expressing AAF/II, which suggests at least two distinct genetic pathways for biofilm formation. The authors also identified transposon insertions in non-plasmid-encoded genes included fis, dsbA, and yafK. Fis increases transcription of aagR, aafD, and to a lesser extent aafA, by qualitative RT-PCR (qRT-PCR). The 11.4-kDa Fis nucleoid-associated protein is involved in DNA inversion events and growth phase regulation of several E. coli genes. The DsbA protein is required for disulfide bond formation of AafA, the structural subunit of the AAF/II, and thus mutation in dsbA prevents elaboration of the fimbriae. Similar to the action of Fis, the predicted 28-kDa YafK protein controls AAF/II expression at the level of transcription, also determined by qRT-PCR (242). Recent experiments suggest that expression of the aggR promoter is activated by Fis and AggR itself, and repressed by downstream binding of H-NS (R. Sohoni and J. Nataro, personal communication). The regulation of aggR and aafD by YafK is most likely indirect because the predicted YafK protein possesses a sec-dependent N-terminal signal sequence and is predicted to be localized to the periplasm or to the outer membrane (Fig. 4). The nature and mechanism of signal transduction through YafK remains to be elucidated.
Similar to those observed on mucosal tissue, EAEC forms biofilms on abiotic surfaces, including glass and polystyrene plastic, and these structures are responsive to environmental cues (242). In this study, maximum biofilm formation in vitro occurred in Dulbecco’s minimal essential medium in the presence of 0.4% glucose at 37°C, while this medium supplemented with 0.1% glucose and 0.2% NaCl also supported robust biofilm formation. Dulbecco’s minimal essential medium supported biofilm formation in the presence of 0.4% of a number of other sugars, including maltose, lactose, mannose, or fructose, albeit to a slightly lesser extent than for 0.4% glucose. The authors were not able to produce biofilms when culturing EAEC in L broth, even in the presence of glucose, amino acids, or other components of the minimal essential medium. Medium pH influenced biofilms, as a pH of 5.2 promoted more robust biofilm formation than did a pH of 5.7 or 6.0. Thus carbon source and low pH are strong signals for EAEC biofilm formation on abiotic surfaces.
In a novel approach, Ruiz-Perez et al. (218) used a continuous-flow anaerobic culture system mimicking the human colonic environment to investigate the expression of aagR and two aggR-regulated genes, aafD and aap, in the presence of fecal bacteria from healthy human volunteers. They observed that aggR expression increased at low pH, 5.5 to 6.0 versus 7.0, in the presence of human normal biota by using qRT-PCR. Similarly, they found that aggR expression was greater under low-salt conditions, 0 to 30 mM versus 200 to 400 mM added NaCl, in the presence of human microbiota. Paradoxically, they observed the opposite effect in response to pH and salt in the absence of commensal bacteria—aggR expression was maximal in neutral pH and 200 to 400 mM NaCl. The genes aafD and aap were coregulated with aggR under all conditions tested. Of the normal biota in the continuous-flow system, coculture experiments showed that strains of Enterococcus and Clostridium increased whereas Lactobacillus and Veillonella decreased expression of aggR (Fig. 4). The coculture signaling was apparently not LuxS dependent because preconditioned media prepared from EPEC and EHEC, known to possess intact luxS genes (254), did not stimulate transcription from an aggR-lacZ fusion.
Similar to AggR, expression of the ShET1 enterotoxin, encoded within the pic/set virulence locus of EAEC, is strongly activated in the simulated human intestinal microbial ecosystem (13) (Fig. 4). The setA gene encoding the putative 20-kDa catalytic A subunit and the setB gene encoding the B subunit of the ShET1 toxin are transcribed as a complementary ~2.5-kb transcript within the pic gene. The PsetB promoter is separated from the setAB genes by a ~1.5-kb DRE that dampens setAB transcription. This repression is relieved by coculture with human intestinal bacteria, determined by using a setB-lacZ fusion strain.
The pic gene, encoding a high-molecular-weight mucinase with serine protease activity, which is secreted by an autotransporter mechanism (101), may also be subject to complex regulation because three promoters were identified upstream of its translational start codon (13). The Ppic2 promoter is most active of the three and is maximally expressed during the exponential phase of growth at 37°C. In contrast to pic, the plasmid-encoded toxin Pet, also a serine protease, autotransported protein, is not regulated by temperature (69).
Urinary tract infections (UTIs) are among the most common of bacterial diseases. Affecting mostly healthy women, approximately 130 to 175 million cases of cystitis are reported worldwide each year (222), and in one survey the lifetime risk of contracting a UTI was 60% (75). In the United States, UPEC isolates are responsible for 70 to 90% of the 7 million cases of acute cystitis and 250,000 cases of pyelonephritis reported each year. Versatile pathogens, extraintestinal UPEC strains are able to harmlessly inhabit the human intestine or cause disease by entering the urinary tract, blood, or cerebrospinal fluid. This dual lifestyle is an important aspect of UPEC disease. The genome sequence of strain CFT073 (288), isolated from the blood of a woman with acute pyelonephritis (121), contains 1,827 genes in 247 islands that differ from the genome sequence of the reference E. coli strain MG1655 (21). These islands, acquired by horizontal transfer, are thought to enable UPEC to infect the urinary tract and bloodstream while evading host immune defenses but not affect ability of these strains to harmlessly inhabit the intestinal tract (288).
Several well-studied UPEC virulence factors have been described (reviewed in references 5, 93, and 168). Several adhesive organelles are elaborated on the surface of the bacterium that facilitate attachment to host cell surfaces and are thought to mediate tissue tropism. (Interaction of UPEC with the host epithelium is reviewed in reference 130.) The best understood of these structures are the type 1 and P fimbriae, though S fimbriae, Curli, antigen 43, and Dr adhesins have also been described. Both the type 1 and P fimbriae are assembled via a chaperone-usher pathway and possess an adhesive subunit on their distal end. The FimH adhesin of the type 1 fimbriae binds to mannose moieties, recognizing mannosylated residues on uroplakin proteins present on bladder epithelial cells (114, 167). The P fimbriae possess the PapG adhesin, which binds to globoside present on human kidney cells, and PapG is necessary for pyelonephritis-associated disease (57). The S fimbria, often associated with UTI and newborn meningitis isolates, is enconded by the sfa operon, but apparently does not possess a fibrillar tip subunit (11). SfaA is the major subunit, and the S fimbrial adhesin has been shown to bind to host receptor molecules that contain α-sialic acid (143). Not surprisingly, the completed genome sequence of strain CFT073 revealed genes for ten chaperone-usher pilus systems, two type IV pili, and seven predicted autotransporter proteins, perhaps consistent with complex duality of the UPEC lifestyle (288).
Autotransporter proteins can play a variety of roles in bacterial pathogens, including those of adhesin, toxins, serum resistance factors, proteases, and invasion and motility mediators (102). Three UPEC autotransporter proteins have been described to date: Sat, Pic, and Tsh. These proteins are members of the serine protease autotransporters in E. coli or SPATEs (103). Sat is toxic to urinary tract cells in vitro (94), producing extreme vacuolization in the cytoplasm of host cells. This vacuolization is also observed in the cytoplasm of infected bladder and kidney cells in a mouse model, along with severe histological damage of the kidney (95). The autotransported Pic and Tsh proteins possess serine protease activity and, in general, are associated with the pyelonephritis strains of UPEC.
At least two additional toxins are expressed by UPEC (though by analysis of the CFT073 genome sequence UPEC does not possess phage-encoded or plasmid-encoded toxins, and unlike EPEC and EHEC pathogens does not encode a type III secretion system [288])—α-hemolysin protein HlyA, an RTX protein that forms pores in a variety of host cell membranes, and cytotoxic necrotizing factor 1 (CNF1), which targets the Rho family of GTP-binding proteins and thus affects the host cell cytoplasm (163).
Assisting in survival within the nutrient-limited environment of the urinary tract, UPEC produces several siderophore systems dedicated to iron acquisition, including aerobactin and yersiniabactin ( 208, 233). Aerobactin and yersiniabactin have been associated with virulence of extraintestinal E. coli pathogens (232, 271, 296). The iroN gene, encoding a siderophore receptor, and ireA, which may be involved in siderophore transport, have also been described in extraintestinal E. coli (219, 220, 223). Lastly, the ChuA outer membrane receptor enables E. coli to use heme as a source of iron (270).
The type 1 fimbriae are necessary for colonization of the urinary tract (for a review, see reference 166a) and are one of four UPEC virulence factors for which molecular Koch’s postulates have been satisfied (40, 131), the other three factors being DegS, TonB, and the Dr fimbriae of chronic pyelonephritis infections (86, 209, 271). The fimACDFGH operon encodes components necessary for biosynthesis and assembly of the type 1 fimbriae, including the major subunit encoded by fimA. Production of the type 1 fimbria is subject to phase variation, whereby a 314-bp invertible element controls transcription of the fim genes (1). Switching of the element occurs by two recombinases, FimB and FimE (140, 157). FimB facilitates flipping from phase OFF to phase ON, and phase ON to phase OFF, whereas FimE is thought to mainly switch from phase ON to phase OFF. Phase variation and transcriptional regulation of the type 1 fimbriae respond to several environmental signals (Fig. 5) and have been linked to specific clinical syndromes and sites of infection.
Gunther et al. (92) developed a PCR-based assay to monitor the orientation of the invertible element controlling fim phase variation during experimental infection in mice. They found that for cystitis UPEC isolates in mouse urine a higher percentage (85%) had their invertible element in the ON position than for pyelonephritis isolates (34%). Indeed the percentage of the population with the fim regulatory region in the ON position in urine correlated with the respective CFU per gram of bladder tissue but not with the CFU per gram of kidney tissue. During the entire course of infection it was found that the cystitis-causing isolates were more likely to have the invertible element in the ON position than those isolates that caused pyelonephritis.
Transcriptional regulation of the fim operon in response to changes in environmental conditions has also been investigated. Using fim-lacZ transcriptional fusions Schwan et al. (235) found that growth in acidic conditions slightly reduced expression from the fimA, fimB, and fimE promoters and that increased osmolarity in acidic conditions repressed fimA and fimB, but increased fimE expression. When fusion strains were cultured in human urine, they observed, similarly for observations in acidic high-osmolarity conditions, that fimB transcription decreased, whereas fimE increased. The authors confirmed these results by using limited-dilution RT-PCR on clinical isolates. Investigating fim phase variation, the OFF orientation was favored in low pH and high osmolarity, whereas neutral pH and low osmolarity favored the ON orientation in clinical isolate NU149. Immunoassay showed that fewer type 1 fimbriae were present under low-pH, high-osmolarity conditions, consistent with their regulatory discoveries. Results also indicated that OmpR and H-NS most likely play a role in fim phase variation.
The pH of urine from the kidneys is lower than that of the bladder, and osmolarity is higher in the kidneys as well. Thus results showing a greater propensity for the OFF fim orientation in high-osmolarity, low-pH conditions may be consistent with the study demonstrating that the ON orientation is favored by cystitis-causing isolates in vivo (92).
P fimbria, encoded by the 11-gene pap operon (113), is also subject to phase variation (155), and expression is controlled at the level of transcription (Fig. 5). Leucine-responsive regulatory protein (Lrp), DNA adenine methylase (Dam), and the PapI regulatory protein, which is encoded by the papI gene divergently transcribed from the papBA promoter, are all necessary for pap transcription and thus fimbrial biogenesis (28, 279). PapB is a regulatory protein and papA encodes the major structural subunit of the P fimbria. Differential methylation by the Dam at sites proximal (GATCprox) and distal (GATCdist) to the papBA promoter control phase variation (23, 28, 189, 278, 291). Both the proximal and distal sites are methylated in an lrp null mutant at 37°C (27). Binding of Lrp at GATCprox blocks methylation at the proximal site and is sufficient to repress transcription from the papBA promoter (291). Conversely, when Lrp binds at GATCdist and blocks methylation at the distal site, Lrp activates transcription of pap fimbrial genes and they are thus phase ON. The PapI protein is required for methylation protection by Lrp at the GATCdist site (190).
Catabolite activator protein (CAP) acts independently of Lrp to control transcription of the pap operon (Fig. 5); cAMP-CAP binding is centered at position –215.5 upstream of the papBA promoter, while activation by Lrp required binding at positions –180 to –149 (290). (The GATCprox and GATCdist DNA methylation sites are located at positions −53 and −102 upstream of the papBA promoter [292]). CAP is also required for expression from the divergently transcribed papI promoter (87). Data are consistent with papBA being a CAP class 1 promoter, whereby activation of transcription was eliminated by mutation in the activating region 1 (AR1) of CAP, but not AR2. Consistent with this observation the C-terminal domain of the α-subunit of RNA polymerase appears to be involved in transcriptional activation. The helical phase of the CAP-binding site in relation to the papBA promoter was essential for pap phase variation (290).
At 26°C and below, neither pap expression (88, 294) nor phase variation (24, 295) occurs. H-NS is required for repression under these conditions, whereby this nucleoid-associated protein blocks DNA methylation (292). The PapI and PapB regulatory proteins are not required for the thermoregulation. H-NS acts to prevent Lrp binding and thus does not allow activation of pap transcription to occur. The PapB protein binds to the papI-papBA intergenic and acts to increase papBA expression; at high concentrations PapB represses papBA transcription and thus acts as an autoregulatory protein (74).
Disruption of the rimJ gene encoding the N-terminal acetyltransferase of ribosomal protein S5 eliminated thermoregulation, allowing transcription of papAB to occur at low temperatures (294, 295). RimJ modulates papAB expression in response to multiple environmental signals as disruption of rimJ also relieved repression by LB medium and glucose (293). RimJ represses the rate of transition from phase OFF to ON, and also regulates transcription of papI.
Expression of a third, S fimbrial adhesin-encoding operon, sfa, is regulated by environmental conditions including temperature, osmolarity, and glucose availability (230) and the regulatory proteins SfaB and SfaC act positively to influence transcription (Fig. 5). The sfaC gene is divergent to sfaB. The sfaBADEFGSH operon is also subject to posttranscriptional mRNA processing that controls stoichiometry of the components necessary for elaborating this fimbria. Specifically, terminators and mRNA-processing sites control mRNA stability of the sfa transcript (11).
RfaH is a transcriptional antiterminator that regulates several virulence determinants in UPEC (8) (Fig. 5). The mechanism of RfaH regulation can be understood by how this protein functions at the hlyCABD operon encoding α-hemolysin. The RfaH protein acts at the Rho-independent terminator that resides between the hlyA and hlyB genes of the hlyCABD operon. The terminator has a polar effect on transcription, resulting in transcription of hlyCA, but minor expression of the hlyCABD transcript. RfaH prevents termination at the Rho-independent terminator, increasing production of the hlyBD-containing transcript necessary for secretion of the HlyA toxin (7, 8, 147, 148). Messenger RNA stability plays a role in the balance of the truncated and full-length transcript (289), and RfaH also acts to increase transcription at the Phly promoter.
In the ascending model of UTI, deletion of the rfaH gene in strain 536 resulted in severely reduced virulence, abolished serum resistance, and reduction in the number of recoverable bacteria from the bladder and kidney (174). Thus inactivation of the rfaH gene resulted in the inability of the bacteria to colonize the upper urinary tract. Along with reduced expression of the hemin receptor ChuA (173), loss of RfaH function resulted in an altered lipopolysaccharide phenotype and reduced expression of K-15 capsule, as well as α-hemolysin (174).
The CNF1 toxin is chromosomally encoded downstream to the hlyCABD operon, separated by a 943-bp intergenic sequence (igs), and is regulated by RfaH (145). Coregulation of the CNF1 toxin and α-hemolysin (20, 144) led investigators to ask whether cnf1 transcription depended on the upstream Phly promoter. Indeed cnf1 transcription required activity of the hly promoter and depended on RfaH antitermination to produce its transcript (Fig. 5). Insertional inactivation of rfaH resulted in 100-fold reduction in cytoplasmic levels of CNF1 compared with the isogenic J96 parent strain (145).
A second type of posttranscriptional regulation controls expression of CNF1. Using translational cnf1-lacZ fusions, deletion constructs, and site-directed mutagenesis, an anti-Shine-Dalgarno sequence was identified contained within codons 45 to 48 and controlled CNF1 synthesis (145). Repression occurs by a foldback inhibition (fbi) mechanism, whereby the hlyCABD igs cnf1 transcript forms a loop structure to inhibit translation from the cnf1 ribosome-binding site (70). Evidence also suggests that a promoter located just upstream of the cnf1 gene is repressed by the fbi mechanism.
Production of the HlyA α-hemolysin is subject to complex regulation, including response to environmental signals and global regulatory proteins. The Phly promoter is repressed by high osmolarity, low temperature, but activated by anaerobic growth (165) (Fig. 5). Maximal expression of hly occurs during mid-exponential phase. The nucleoid-associated proteins H-NS and Hha repress hly transcription, forming a nucleoprotein complex to govern thermo-osmotic regulation by altering DNA topology (10, 186, 187).
Efficient expression of the α-hemolysin requires the leuX-encoded alternate tRNA5 Leu. The level of rfaH and hha transcripts as well as expression of the RfaH and Hha proteins was higher in strains deleted for leuX. Thus regulation of hly was partially indirect, as increased expression resulted from decreased levels of the Hha repressor in the leuX deletion strain (55) (Fig. 5). The tRNA5 Leu is a minor tRNA encoded adjacent to the PAIII of strain 536 and regulates a number of virulence traits (22, 54, 212, 213). Not surprisingly, the minor tRNA5Leu is regulated differently than the major leucyl-tRNA gene leuV, responding to several environmental signals, including temperature, osmolarity, the stationary phase of growth, and the heat-shock sigma factor RpoH (56).
Zhang and Normark (303) studied the effect of P-pilus attachment to epithelial cells on the induction of virulence genes. They identified a gene encoding a sensor-regulator, AirS (also called BarA), that was transcriptionally activated upon P-pilus attachment (Fig. 5), and found that this protein was essential for the iron starvation response. Using differential-display PCR, Schwan and colleagues ( 234) demonstrated that the kps operon in UPEC strain NU149 is downregulated when the bacteria attach via type 1 fimbriae to mannose-coated sepharose beads This result was confirmed by RT-PCR, and by showing that transcription of the kpsFEDUCS capsular assembly operon was decreased by using Northern blot analysis and lacZ fusion assay. These authors postulated that upon entering the bladder UPEC bacteria are capsulated and form loose attachments to the bladder epithelial cells via the type 1 fimbriae. Once attached, downregulation of the capsule assembly genes is advantageous because the extracellular polysaccharide would inhibit tight attachment and cellular invasion, as previously demonstrated in both Klebsiella pneumoniae and Neisseria meningitidis (49, 225).
Russo and colleagues (219, 220, 221) previously investigated alterations in gene expression that resulted from culturing UPEC in human urine and other body fluids. Expression of the ure1 (urine-responsive element) gene was strongly increased in urine and was repressed by the presence of glucose (220). The gene ure1 was unresponsive to iron, amino acids, or nucleotides, so it was concluded that this locus was not involved in the uptake of these nutrients. They also identified guaA and argC genes to be regulated by urine and found that urine was limited for arginine and guanine. Expression of the periplasmic arginine-binding protein ArtJ was increased in urine, and these data suggested that synthesis and/or acquisition of arginine is important for survival within the human bladder (220).
Snyder et al. (247) monitored differential expression of UPEC genes in the ascending mouse model. CBA/J mice were experimentally infected transurethrally, and urine samples were collected 1 to 10 days postinoculation. Transcription of the 5,611 ORFs were compared for strain CFT073 inoculated into mice and grown in LB medium. The authors found that 303 genes were upregulated and 207 were downregulated during growth in vivo. The most highly expressed genes in vivo were those encoding translational machinery, indicating that UPEC is actively growing within the mouse. Five iron-uptake systems were also highly upregulated, confirming that the urinary tract is indeed iron-limiting. Genes involved in elaboration of the type 1 fimbria, capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion were increased. Expression of genes encoding other adhesins, including two copies of the P fimbriae, F1C, Curli, and antigen 43, were repressed in vivo. This study also provided an exciting glimpse of the environmental conditions within the urinary tract of the mouse—iron and nitrogen limiting, high osmolarity, and moderate oxygenation—indicated by downregulation of the Fnr-regulated genes frdABCD, glpABC, and aspA and unchanged transcriptions of the key regulators of respiration arcA and fnr. They also consistently found that expression of capsule, microcin secretion, and iron uptake genes was increased in human urine as compared with expression in LB medium.
The acquisition of iron in the iron-depleted urinary tract is an important aspect of UPEC pathogenesis, as multiple groups have identified iron transport systems to play a role in virulence in the mouse model (219, 271). The transport of iron by siderophores or heme depends on TonB, which is anchored to the cytoplasmic membrane and provides energy to outer membrane receptors. A laboratory strain of E. coli deleted for tonB failed to use heme as an iron source or to use enterobactin or aerobactin siderophores (30); deletion of tonB in strain CFT073 resulted in reduced virulence in a mouse ascending model of UTI (271). All iron transport systems described in E. coli strains to this date are controlled by the master regulator Fur (29).
Using a TnphoA mutant library of the extraintestinal pathogen CP9, a member of the J96-like clonal group, Russo et al. (219) identified the ireA gene (iron-responsive element) that showed reduced ability to colonize the bladder in the mouse model. Expression of ireA was increased in the presence of human urine, ascites, and blood in comparison with LB medium. Amino acid similarities to siderophore receptors and the predicted IreA protein and increased expression of an ireA::TnphoA fusion in iron-depleted minimal medium suggest that IreA is involved in iron transport. The iroN gene of E. coli encodes a catecholate siderophore receptor, and its expression is also increased when cultured in human urine, ascites, or blood as compared with growth in LB medium. Transcription of iroN is repressed by exogenous iron and urine pH of 5.0.
By RT-PCR expression of pic and tsh encoding autotransporter proteins was greater when UPEC bacteria were cultured at 37°C than at 25°C (100) (Fig. 5), consistent with regulation of expression of the Pic and Tsh proteins in EAEC (101) and avian pathogenic E. coli (258). These investigators also found that pic and tsh of UPEC were upregulated when the bacteria were isolated from urine of transurethrally infected mice as compared with bacteria grown in urine from uninfected animals.
A gene cluster containing eight genes with significant DNA sequence identity to the pap fimbrial operon was recently described (149). In this locus, designated pix, genes encoding homologs of PapB, PixB, were identified, but a homolog to PapI was absent. Instead, the gene R6 of the pyelonephritis-causing strain CFT073, encoding a protein with similarity to transposases, was identified in the pix operon adjacent to pixA. The R6 protein regulated the pix operon at the level of transcription. The pix gene was mainly expressed during exponential growth, repressed by culturing at 26°C, and did not respond to exogenous glucose.
The BarA/UvrY two-component system of UPEC controls carbon metabolism and affects long-term survival in competition assays (197). When grown on carbon sources that feed directly into glycolysis, such as glucose or glycerol, the wild-type UPEC has a growth advantage over strains with mutations in uvrY. Conversely, when grown on carbon sources downstream of glycolysis, such as fumarate, acetate, or pyruvate, strains containing knockouts in uvrY showed a growth advantage over the wild-type, isogenic UPEC strain J96. In LB medium strains with mutations in either uvrY or barA demonstrated a growth advantage over the wild-type strain. Homologs of the BarA/UvrY system linked to virulence have been described for several bacterial pathogens, including Erwinia, Pseudomonas, Vibrio, and Salmonella (3, 99, 198). Taken together, these data suggest a role for the BarA/UvrY two-component regulatory system in UPEC’s ability to switch between different metabolic pathways in response to a variety of carbon sources, and thus may play a role in adaptation and survival within the human host.
Finally, exposure to the pharmacological agents clofibric and ethacrynic acid, used to treat hypertriglyceridemia and used as a diuretic, respectively, were shown to increase resistance to a number of antibiotics (9). The resistance phenotype was primarily due to induction of the marRAB operon encoding multiple drug resistances, and alterations of the outer membrane permeability properties by repression of the porin protein OmpF.
E. coliisolates are the most common cause of gram-negative neonatal meningitis and are associated with infections occurring in the first three weeks of life (226). Bacterial meningitis is a devastating disease, resulting in a great deal of morbitity and mortality. Approximately half of the survivors develop long-term medical complications, including neurological sequelae, developmental delay, or hearing loss (276). The mode of infection can be either transplacental or by aspiration or inhalation of the pathogen (200). Meningitis-causing, extraintestinal isolates of E. coli must translocate from the intestinal lumen to the bloodstream, sustain high levels of bacteremia, and then pass through the blood-brain barrier (BBB) followed by invasion and growth in the arachnoidal space (25, 84).
Isolates of E. coli expressing the K1 polysaccharide capsule are the most predominant etiological agent causing meningitis and sepsis (MNEC) (137). The infection process of MNEC is complex, involving attachment to the gut epithelium, invasion and translocation into deeper tissues, followed by bacteremia and crossing of the BBB. Many unanswered questions remain, e.g., how do K1-encapsulated E. coli cross the BBB? This continues to be an intensive area of research, and several virulence-associated factors have been described for this pathotype of E. coli. The K1 capsule is necessary for crossing the BBB, prevents phagocytic activities, and is necessary for survival in the bacteremia and systemic stages of infection (2, 108, 151, 214). The outer membrane porin OmpA contributes to the ability of K1 strains to cross the BBB (206), and to serum resistance (273). The outer membrane protein IbeA and CNF-1 are also involved in crossing of the BBB (6, 111). The traJ gene, with significant sequence identity to traJ of the tra operon contained within F-like plasmids, was demonstrated to be involved in MNEC invasion of the central nervous system, in the intracellular aspects of the systemic stage of the disease, and in meningitis in the neonatal rat model (6, 107). Nègre et al. (182) demonstrated that the IroN siderophore receptor plays a role in the bacteremic stage of the disease.
Another active area of investigation has been directed at understanding initial binding events that lead to invasion and translocation of cultured human brain microvascular endothelial cells (HBMEC), which are an in vitro model of the BBB. The OmpA outer membrane protein is involved in binding to these endothelial cells in culture (136). Evidence suggests that the type 1 fimbriae are involved, but there are conflicting reports on whether the S fimbriae play a role in attachment to HBMEC (205, 266, 287).
Though several virulence factors have been described for MNEC, few studies exist that describe the regulation of these factors. Teng and colleagues (266) demonstrated that the type 1 fimbriae play an important role in binding to and invasion of HBMEC, and by DNA microarray analysis that E. coli K1 associated with the endothelial cells showed significantly higher expression of fim genes than bacteria that were not associated with HBMEC. E. coli K1 bacteria associated with HBMEC were predominantly found in phase ON for fim expression.
By gentamicin protection assay the addition of exogenous glucose to minimal medium enhanced the ability of E. coli K1 bacteria to invade HBMEC (110). The addition of exogenous cAMP blocked the stimulatory effect of glucose, and galactose did not enhance invasion, suggesting that carbon source plays a regulatory role in crossing the BBB.
Khan and Isaacson (135) used in vivo expression technology (IVET) to identify genes of strain i484 that were expressed during infection of a mouse model of septicemia. By isolating bacteria from infected mice, these investigators identified differentially expressed genes required for amino acid metabolism, anaerobic respiration, the heat shock response, DNA repair, a repressor of the SOS response, and an aerobactin biosynthetic gene. Deletion mutations of five of the identified genes, including bglG encoding an enzyme involved in β-glucoside metabolism, resulted in attenuation in the mouse model of infection.
Several common themes have emerged through the study of virulence gene regulation in E. coli. First, AraC-like transcription regulators in EPEC, ETEC, and EAEC control expression of fimbrial adhesins. In EPEC PerA regulates BFP, while in ETEC CfaR and Rns regulate the CFA/I and CS1 fimbriae, respectively. In EAEC AggR is the key AraC-like molecule controlling expression of AAF/I and II. All of these regulators, with the exception of AggR, appear to be dedicated to the regulation of fimbrial adhesins, and all control expression in response to environmental signals. These regulators can override several environmental cues. For example, Rns and CfaR can override temperature regulation in ETEC, and AggR can override osmolarity, oxygen tension and iron signals in EAEC, but cannot override temperature at the aggA promoter. Many, but not all of them are functional in heterologous systems. Whether these AraC-like molecules bind to signal molecules, or metabolites as in the AraC protein binding to arabinose, and the precise mechanism of action remain to be determined. Another commonality of the virulence genes regulated by AraC-like molecules is that most, if not all are repressed by the nucleoid-associated protein H-NS. To date, it has been determined that H-NS regulates the bfp operon of EPEC, the cfa and coo operons of ETEC, and the pap operon of UPEC.
Studies concerning the mechanism of E. coli virulence gene regulators have also furthered our understanding of the mechanism of H-NS-mediated repression at these loci. It has been known for some time that H-NS can bind in regions that flank promoters, and that H-NS can facilitate DNA looping (for recent reviews on H-NS, see references 60, 61, 211, and 265). For example, H-NS binds over extended positions both upstream and downstream of the proU and bgl promoters of E. coli K-12 and the virF promoter of EIEC. Similarly, H-NS-dependent repressing DNA sequences exist both upstream and downstream of the Ler-activated LEE2 and LEE5 promoters, and Ler binds to the same region upstream of the LEE2 and LEE5 promoters to which H-NS binds in EPEC. DNA looping by H-NS has also been demonstrated for the ure operon of the UTI pathogen Proteus mirabilis, which is positively regulated by UreR, an AraC-like molecule (201). A unifying mechanism emerges from these data, suggesting that the unrelated AraC-like regulators and H-NS-like protein Ler activate transcription by disruption of H-NS-dependent nucleoprotein complexes requiring H-NS binding that flanks promoter sequences. Dame and colleagues (46, 47) have presented striking atomic force microscopy (AFM) images whereby H-NS represses the rrnB P1 promoter of E. coli K-12 by forming a collarlike structure, holding RNA polymerase in an open complex and thus preventing transcription elongation. We have proposed a similar mechanism of H-NS-mediated repression at the LEE2 and LEE5 promoters of EPEC (96), and this may be a common theme in H-NS control of virulence gene expression in the Enterobacteriaceae.
Another important idea, which recently reappeared in the literature (60), is that H-NS may play an important role in the regulation of horizontally transferred DNA. Because foreign DNA in gram-negative bacteria tends to be low G+C, i.e., high A-T content in comparison with the recipient’s genome and H-NS preferentially binds to AT-rich sequences, we and others hypothesize that H-NS may endogenously silence horizontally transferred DNA elements, which when expressed inappropriately might be deleterious to the bacterium. Subsequently, bacteria must then activate expression of these virulence determinants. Regulatory proteins that counteract H-NS repression may be acquired along with the horizontally transferred DNA, or may already exist in the recipient, either located on the chromosome or a plasmid. Several mobile DNA elements encode proteins that either interact with or inactivate the function of H-NS (51, 66, 154). For example, the protein encoded by gene 5.5 of phage T7 disrupts H-NS function resulting in expression of both host and viral genes (154). Consistent with this idea, to properly express the horizontally acquired type III secretion system of EPEC and EHEC the H-NS, LEE-encoded Ler protein disrupts the repressing activity of endogenous H-NS to respond to host-associated environmental cues.
Phase variation, including that observed for the well-characterized type 1 and pap fimbriae, also plays an important, and perhaps underappreciated role in E. coli virulence. Studies to correlate phase variability with cystitis and pyelonephritis-associated strains of UPEC illustrate the dynamic nature of the infection process and the remarkable adaptive capabilities of these pathogens. Phase variation for the 987P-like fimbriae of ETEC may indicate that this type of gene regulation is more common in E. coli pathogens than we initially thought.
E. colipathogens are remarkably versatile in their ability to cause intestinal as well as extraintestinal infections. They colonize specific niches within the intestine and urinary tract; some can translocate from the intestinal lumen into the bloodstream, then pass through the BBB, invade and colonize the arachnoidal space. Diffusible toxins and secreted effector molecules, as well as the colonization process itself, can cause damage to host tissues and organs. Genes unique to each pathotype, those shared by pathotypes, and those common to all E. coli contribute to pathogenesis. The study of the regulation of these genes and the related virulence phenotypes contribute to our understanding of E. coli disease and will ultimately lead to the development of effective vaccines and chemotherapeutic agents. These studies are aided by the availability of genomic DNA sequences now available for EPEC strain E2348/69, EHEC strains EDL933 (http://www.genome.wisc.edu/sequencing/o157.htm), RIMD 0509952 isolated from the Sakai outbreak (http://genome.gen-info.osaka-u.ac.jp/bacteria/o157), EAEC strain 042 (http://www.sanger.ac.uk/Projects/Escherichia_Shigella/), and UPEC strain CFT073 (http://www.genome.wisc.edu/sequencing/upec.htm).
How these pathogens alter gene expression in response to environmental cues, host tissue contact, and innate as well as adaptive immune defenses strike at the heart of the bacterium-host interaction. In recent years researchers have developed technologies to study the regulation of virulence determinants in vivo. The IVET, differential display, signature-tagged mutagenesis (STM), PCR-based assays to monitor phase variation, microarray analyses, and quantitative real-time PCR (pRT-PCR) have given us a more accurate understanding of gene regulation that occurs inside an animal host than more traditional in vitro assays. These methodologies, and additional molecular techniques targeted to the bacterium, e.g., a procedure to quantify virulence gene expression on the single-cell level (215) guarantee to greatly expand our understanding of E. coli virulence gene regulation, and thus host-microbe interactions in the coming years.
We thank Jim Kaper, Alfredo Torres, Jim Nataro, Harry Mobley, Eileen Barry, and Ken Haack for critical comments on the chapter. Research in the laboratory of J.L.M. is funded by the National Institutes of Health, the Defense Advanced Research Projects Agency (DARPA), and the Howard Hughes Medical Institute.
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Module8.9.1
