Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS). Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton. Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains. In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 beta-lactamase. Translocation was detected directly in living host cells by using the fluorescent beta-lactamase substrate CCF2/AM. We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells. Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner. A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation. In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation. In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells.     

Enteropathogenic Escherichia coli inhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level

Enteropathogenic Escherichia coli (EPEC), a food-borne human pathogen, is responsible for infantile diarrhea, especially in developing countries. The pathophysiology of EPEC-induced diarrhea, however, is not completely understood. Our recent studies showed modulation of Na+/H+ and Cl-/HCO3- exchange activities in Caco-2 cells in response to EPEC infection. We hypothesized that intestinal short-chain fatty acid absorption mediated by monocarboxylate transporter 1 (MCT1) might also be altered by EPEC infection. The aim of the current studies was to examine the effect of EPEC infection on butyrate uptake. Caco-2 cells were infected with wild-type EPEC, various mutant strains, or nonpathogenic E. coli HS4, and [14C]butyrate uptake was determined. EPEC, but not nonpathogenic E. coli, significantly decreased butyrate uptake. Infection of cells with strains harboring mutations in escN, which encodes a putative ATPase for the EPEC type III secretion system (TTSS), or in the espA, espB, or espD genes encoding structural components of the TTSS, had no effect on butyrate uptake, indicating the TTSS dependence. On the other hand, strains with mutations in the effector protein genes espF, espG, espH, and map inhibited butyrate uptake, similar to the wild-type EPEC. Surface expression of MCT1 decreased considerably after EPEC but not after nonpathogenic E. coli infection. In conclusion, our studies demonstrate inhibition of MCT1-mediated butyrate uptake in Caco-2 cells in response to EPEC infection. This inhibition was dependent on a functional TTSS and the structural proteins EspA, -B, and -D of the translocation apparatus.     

Enteropathogenic Escherichia coli Tir translocation and pedestal formation requires membrane cholesterol in the absence of bundle-forming pili

Enteropathogenic Escherichia coli (EPEC) is a significant cause of paediatric diarrhoea worldwide. Virulence requires adherence to intestinal epithelial cells, mediated in part through type IV bundle-forming pili (BFP), and the EPEC protein Tir. Tir is inserted into the enterocyte plasma membrane (PM), resulting in the formation of actin-rich pedestals. Tir is translocated by the type III secretion system (TTSS), through a pore comprised of EPEC proteins inserted into the PM. Here, we demonstrate that in the absence of BFP, EPEC adherence, effector translocation and pedestal formation are dependent on lipid rafts. Lipid raft disruption using methyl-beta-cyclodextrin (MbetaCD) decreased adherence by an EPEC BFP-deficient strain from 85% to 1%. Translocation of the effectors Tir and EspF was blocked by MbetaCD treatment, although the TTSS pore still formed. MbetaCD treatment after Tir delivery decreased pedestal formation by EPEC from 40% to 5%, but not by the related pathogen E. coli O157:H7 which uses a different Tir-based mechanism. In contrast, EPEC expressing the BFP can circumvent the requirement for membrane cholesterol. This suggests that lipid rafts play a role in virulence of this medically important pathogen.     

The enteropathogenic E. coli effector EspB facilitates microvillus effacing and antiphagocytosis by inhibiting myosin function

Enteropathogenic Escherichia coli (EPEC) destroys intestinal microvilli and suppresses phagocytosis by injecting effectors into infected cells through a type III secretion system (TTSS). EspB, a component of the TTSS, is also injected into the cytoplasm of host cells. However, the physiological functions of EspB within the host cell cytoplasm remain unclear. We show that EspB binds to myosins, which are a superfamily of proteins that interact with actin filaments and mediate essential cellular processes, including microvillus formation and phagocytosis. EspB inhibits the interaction of myosins with actin, and an EspB mutant that lacks the myosin-binding region maintained its TTSS function but could not induce microvillus effacing or suppress phagocytosis. Moreover, the myosin-binding region of EspB is essential for Citrobacter rodentium, an EPEC-related murine pathogen, to efficiently infect mice. These results suggest that EspB inhibits myosin functions and thereby facilitates efficient infection by EPEC.     

Enteropathogenic Escherichia coli effector EspF interacts with host protein Abcf2

Enteropathogenic Escherichia coli (EPEC) is a major causative agent of infant diarrhoea in developing countries. The EspF effector protein is injected from EPEC into host cells via a type III secretion system and is involved in the disruption of host intestinal barrier function. In addition, EspF is sorted to mitochondria and has a role in initiating the mitochondrial death pathway. To clarify the manner in which EspF affects host cells, we sought to identify eukaryotic EspF-binding proteins using affinity purification. Abcf2, a protein of unknown function and member of the ABC-transporter family, bound EspF in this assay. An interaction between EspF and Abcf2 was confirmed in a yeast two-hybrid system, by colocalization and by co-immunoprecipitation from EPEC-infected cells. Levels of Abcf2 were decreased in cells infected with EPEC in an EspF dose-dependent manner. Knock-down of Abcf2 expression by RNA interference increased EspF-induced caspase 9 and caspase 3 cleavage. In addition, Abcf2-knocked down cells showed increased caspase 3 cleavage upon treatment with the apoptosis inducing agent staurosporine. These results indicate that EspF induces or facilitates host cell death by targeting and interfering with the putative protective function of Abcf2.     

Caveolae-mediated entry of Salmonella typhimurium in a human M-cell model

Intestinal M cells in Peyer’s patches, the specialized antigen-sampling cells of the mucosal immune system, are exploited by Salmonella and other pathogens as a route of invasion. Thus, M cells have attracted lots of attention as a major target of the mucosal immune system. Here, we report that caveolin-1 plays a crucial role in the entry of Salmonella into M cells. We established an in vitro M-like cell model in which polarized enterocyte-like Caco-2 cells created after co-culturing with the Raji B cell line that underwent a phenotypic switch to a form that morphologically and functionally resembles the specialized antigen-transporting M cells. Caveolin-1 was highly expressed in the M-like cells, while not in Caco-2 cells, and a great number of Salmonella infected caveolin-1-expressing M-like cells. To elucidate the role of caveolin-1 in the entry of Salmonella, we downregulated caveolin-1 expression by siRNA and analyzed the level of Salmonella transcytosis across the M-like cells. Transcytosis of Salmonella was markedly reduced by downregulation of caveolin-1 in the M-like cells. These results suggest that caveolin-1 is implicated in the gateway of microbial pathogens through M cells, and, thus, provides a new target of mucosal immunity.     

Designed Coiled-Coil Peptides Inhibit the Type Three Secretion System of Enteropathogenic Escherichia coli

Background

Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry.

Methods/Principal Findings

We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled–coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments.

Conclusions

Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis.     

Translocation of Yersinia entrocolitica across reconstituted intestinal epithelial monolayers is triggered by Yersinia invasin binding to beta1 integrins apically expressed on M-like cells

Yersinia enterocolitica cross the intestinal epithelium via translocation through M cells, which are located in the follicle-associated epithelium (FAE) of Peyer's patches (PP). To investigate the molecular basis of this process, studies were performed using a recently developed in vitro model, in which the enterocyte-like cell line Caco-2 and PP lymphocytes are co-cultured in order to establish FAE-like structures including M cells. Here, we demonstrate that Y. enterocolitica does not adhere significantly to the apical membrane of differentiated enterocyte-like Caco-2 cells that express binding sites for Ulex europaeus agglutinin (UEA)-1. In contrast, Y. enterocolitica adhered to, and was internalized by, cells that lacked UEA-1 binding sites and displayed a disorganized brush border. These cells were considered to be converted to M-like cells. Further analysis revealed that part of these cells expressed β1 integrins at their apical surface and, as revealed by comparison of wild-type and mutant strains, interacted with invasin of Y. enterocolitica . Consistently, anti-β1 integrin antibodies significantly inhibited internalization of inv -expressing yersiniae. Experiments with Yersinia mutant strains deficient in YadA or Yop secretion revealed that these virulence factors play a minor role in this process. After internalization, yersiniae were transported within LAMP-1-negative vacuoles to, and released at, the basal surface. Internalization and transport of yersiniae was inhibited by cytochalasin D, suggesting that F-actin assembly is required for this process. These results provide direct evidence that expression of β1 integrins at the apical surface of M cells enables interaction with the invasin of Y. enterocolitica , and thereby initiates internalization and translocation of bacteria.     

Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells

A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.     

A bacterial effector targets host DH‐PH domain RhoGEFs and antagonizes macrophage phagocytosis

Bacterial pathogens often harbour a type III secretion system (TTSS) that injects effector proteins into eukaryotic cells to manipulate host processes and cause diseases. Identification of host targets of bacterial effectors and revealing their mechanism of actions are crucial for understating bacterial virulence. We show that EspH, a type III effector conserved in enteric bacterial pathogens including enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli and Citrobacter rodentium, markedly disrupts actin cytoskeleton structure and induces cell rounding up when ectopically expressed or delivered into HeLa cells by the bacterial TTSS. EspH inactivates host Rho GTPase signalling pathway at the level of RhoGEF. EspH directly binds the DH‐PH domain in multiple RhoGEFs, which prevents their binding to Rho and thereby inhibits nucleotide exchange‐mediated Rho activation. Consistently, infection of mouse macrophages with EPEC harbouring EspH attenuates phagocytosis of the bacteria as well as FcγR‐mediated phagocytosis. EspH represents the first example of targeting RhoGEFs by bacterial effectors, and our results also reveal an unprecedented mechanism used by enteric pathogens to counteract the host defence system.     

Enteropathogenic E. coli-induced barrier function alteration is not a consequence of host cell apoptosis

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that perturbs intestinal epithelial function. Many of the alterations in the host cells are mediated by effector molecules that are secreted directly into epithelial cells by the EPEC type III secretion system. The secreted effector molecule EspF plays a key role in redistributing tight junction proteins and altering epithelial barrier function. EspF has also been shown to localize to mitochondria and trigger membrane depolarization and eventual host cell death. The relationship, if any, between EspF-induced host cell death and epithelial barrier disruption is presently not known. Site-directed mutation of leucine 16 (L16E) of EspF impairs both mitochondrial localization and consequent host cell death. Although the mutation lies within a region critical for type III secretion, EspF(L16E) is secreted efficiently from EPEC. Despite its inability to promote cell death, EspF(L16E) was not impaired for tight junction alteration or barrier disruption. Consistent with this, the pan-caspase inhibitor Q-VD-OPH, despite reducing EPEC-induced host cell death, had no effect on infection-mediated barrier function alteration. Thus EPEC alters the epithelial barrier independent of its ability to induce host cell death.     

Enteropathogenic Escherichia coli EspF is targeted to mitochondria and is required to initiate the mitochondrial death pathway

Enteropathogenic Escherichia coli (EPEC) is a causative agent of infant diarrhoea in developing countries. The EspF protein is the product of the espF gene found on the locus of enterocyte effacement, the key pathogenicity island carried by EPEC and enterohemorrhagic E. coli. EspF is injected from adherent EPEC into host cells via a type III secretion system and was previously shown to induce apoptotic cell death and to be required for disruption of host intestinal barrier function. In this work, we show by immunofluorescence and fractionation studies that EspF is targeted to host mitochondria. The N-terminal region of EspF serves as a mitochondrial import signal and, when expressed within cells, can target hybrid green fluorescent protein to mitochondria. Assessment of mitochondrial membrane potential in infected epithelial cells indicated that EspF plays a role in the mitochondrial membrane permeabilization induced by EPEC infection. Furthermore, EspF was associated with the release of cytochrome c from mitochondria into the cytoplasm and with caspase-9 and caspase-3 cleavage. These findings indicate a role for EspF in initiating the mitochondrial death pathway.     

Peptidoglycan recognition protein expression in mouse Peyer's Patch follicle associated epithelium suggests functional specialization

Mammalian Peyer's Patches possess specialized epithelium, the follicle associated epithelium (FAE), and specialized cells called M cells which mediate transcytosis of antigens to underlying lymphoid tissue. To identify FAE specific genes, we used TOGA gene expression profiling of microdissected mouse Peyer's Patch tissue. We found expression of laminin beta3 across the FAE, and scattered expression of peptidoglycan recognition protein (PGRP)-S. Using the M cell specific lectin Ulex europaeus agglutinin 1 (UEA-1), PGRP-S expression was nearly exclusively co-localized with UEA-1+ M cells. By contrast, the related gene PGRP-L was expressed among a subset of UEA-1 negative FAE cells. Expression of these proteins in transfected cells demonstrated distinct subcellular localization. PGRP-S showed a vesicular pattern and extracellular secretion, while PGRP-L showed localization to both the cytoplasm and the cell surface. The potential function of these PGRP proteins as pattern recognition receptors and their distinctive cellular distribution suggests a complex coordination among specialized cells of the FAE in triggering mucosal immunity and innate immune responses.     

Enteropathogenic and enterohaemorrhagic Escherichia coli deliver a novel effector called Cif, which blocks cell cycle G2/M transition

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. Both use a type III secretion system (TTSS) encoded by the 'locus of enterocyte effacement' (LEE) to subvert and attach to epithelial cells through the injection of a repertoire of effector molecules. Here, we report the identification of a new TTSS translocated effector molecule called Cif, which blocks cell cycle G2/M transition and induces the formation of stress fibres through the recruitment of focal adhesions. Cif is not encoded by the LEE but by a lambdoid prophage present in EPEC and EHEC. A cif mutant causes localized effacement of microvilli and intimately attaches to the host cell surface, but is defective in the ability to block mitosis. When expressed in TTSS competent LEE-positive pathogens, Cif is injected into the infected epithelial cells. These cells arrested at the G2/M phase displayed accumulation of inactive phosphorylated Cdk1. In conclusion, Cif is a new member of a growing family of bacterial cyclomodulins that subvert the host eukaryotic cell cycle.     

Interaction of Listeria monocytogenes with the intestinal epithelium

Listeria monocytogenes is a food-borne pathogen that must cross the intestinal epithelial barrier to reach its target organs. We have investigated the importance of M cells in translocation using an experimental mouse model and a novel, recently described in vitro coculture system that mimics the follicle-associated epithelium (FAE). Our data demonstrate that L. monocytogenes does not require, nor specifically use, M cells of the FAE to cross the gut. We also show that bacterial translocation is rapid and L. monocytogenes can attach very efficiently to exposed basal lamina of the small intestine indicating an important role for extracellular matrix proteins.     

SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD

Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two-hybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.     

Macrophage migration inhibitory factor plays a role in the regulation of microfold (M) cell-mediated transport in the gut

It has been shown previously that certain bacteria rapidly (3 h) up-regulated in vivo microfold cell (M cell)-mediated transport of Ag across the follicle-associated epithelium of intestinal Peyer's patch. Our aim was to determine whether soluble mediators secreted following host-bacteria interaction were involved in this event. A combination of proteomics and immunohistochemical analyses was used to identify molecules produced in the gut in response to bacterial challenge in vivo; their effects were then tested on human intestinal epithelial cells in vitro. Macrophage migration inhibitory factor (MIF) was the only cytokine produced rapidly after in vivo bacterial challenge by CD11c(+) cells located beneath the M cell-rich area of the follicle-associated epithelium of the Peyer's patch. Subsequently, in vitro experiments conducted using human Caco-2 cells showed that, within hours, MIF induced the appearance of cells that showed temperature-dependent transport of microparticles and M cell-specific bacterium Vibrio cholerae, and acquired biochemical features of M cells. Furthermore, using an established in vitro human M cell model, we showed that anti-MIF Ab blocked Raji B cell-mediated conversion of Caco-2 cells into Ag-sampling cells. Finally, we report that MIF(-/-) mice, in contrast to wild-type mice, failed to show increased M cell-mediated transport following in vivo bacterial challenge. These data show that MIF plays a role in M cell-mediated transport, and cross-talk between bacteria, gut epithelium, and immune system is instrumental in regulating key functions of the gut, including M cell-mediated Ag sampling.     

Role of EspF in host cell death induced by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in children in developing countries. Many EPEC genes involved in virulence are contained within the locus of enterocyte effacement (LEE), a large pathogenicity island. One of the genes at the far righthand end of the LEE encodes EspF, an EPEC secreted protein of unknown function. EspF, like the other Esps, is a substrate for secretion by the type III secretory system. Previous studies found that an espF mutant behaved as wild type in assays of adherence, invasion, actin condensation and tyrosine phosphorylation. As EPEC can kill host cells, we tested esp gene mutants for host cell killing ability. The espF mutant was deficient in host cell killing despite having normal adherence. The addition of purified EspF to tissue culture medium did not cause any damage to host cells, but expression of espF in COS or HeLa cells caused cell death. The mode of cell death in cells transfected with espF appeared to be pure apoptosis. EspF appears to be an effector of host cell death in epithelial cells; its proline-rich structure suggests that it may act by binding to SH3 domains or EVH1 domains of host cell signalling proteins.     

EspJ of enteropathogenic and enterohaemorrhagic Escherichia coli inhibits opsono-phagocytosis

A key strategy in microbial pathogenesis is the subversion of the first line of cellular immune defences presented by professional phagocytes. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) remain extracellular while colonizing the gut mucosa by attaching and effacing mechanism. EPEC use the type three secretion system effector protein EspF to prevent their own uptake into macrophages. EPEC can also block in trans the internalization of IgG-opsonized particles. In this study, we show that EspJ is the type three secretion system effector protein responsible for trans -inhibition of macrophage opsono-phagocytosis by both EPEC and EHEC. While EspF plays no role in trans -inhibition of opsono-phagocytosis, espJ mutants of EPEC or EHEC are unable to block uptake of opsonized sheep red blood cells (RBC), a phenotype that is rescued upon complementation with the espJ gene. Importantly, ectopic expression of EspJ^EHEC in phagocytes is sufficient to inhibit internalization of both IgG- and C3bi-opsonized RBC. These results suggest that EspJ targets a basic mechanism common to these two unrelated phagocytic receptors. Moreover, EspF and EspJ target independent aspects of the phagocytic function of mammalian macrophages in vitro .     

Balance of bacterial pro- and anti-inflammatory mediators dictates net effect of enteropathogenic Escherichia coli on intestinal epithelial cells

Enteropathogenic Escherichia coli (EPEC) virulence requires a type III secretion system (TTSS) to deliver effector molecules in host cells. Although the TTSS is crucial to EPEC pathogenesis, its function in EPEC-induced inflammation is not known. The aim of this study was to investigate the role of the TTSS in EPEC-induced inflammation. HT-29 intestinal epithelial cells were infected with wild-type (WT) EPEC or select mutant strains or exposed to corresponding filter-sterilized supernatants (SN), and interleukin-8 (IL-8) secretion was determined by ELISA. EPEC SN stimulated significantly greater IL-8 production than EPEC organisms. Flagellin, as well as a TTSS-independent >50-kDa nonflagellin protein, was found to significantly contribute to this response. Dose-response studies showed that increasing concentrations of WT SN proportionally increased IL-8, whereas increasing multiplicity of infection of EPEC inversely correlated with IL-8 secretion, suggesting that EPEC dampens this host response. Infection with DeltaescN (nonfunctional TTSS) markedly increased IL-8 compared with WT, indicating that a functional TTSS is required for this anti-inflammatory property; complementation of escN restored the attenuated response. Mutation of espB also enhanced the IL-8 response, and complementation returned IL-8 to near WT levels, suggesting involvement of this effector. The anti-inflammatory effect extends to both bacterial and host-derived proinflammatory stimuli, since prior infection with EPEC suppressed the IL-8 response to tumor necrosis factor-alpha, IL-1beta, and enterohemorrhagic E. coli flagellin. These findings indicate that EPEC-induced inflammation is a balance between pro- and anti-inflammatory proteins; extracellular factors, including flagellin and an unidentified TTSS-independent, >50-kDa protein, trigger inflammation while intracellular TTSS-dependent factors, including EspB, attenuate this response.