E. coli secreted protein F promotes EPEC invasion of intestinal epithelial cells via an SNX9‐dependent mechanism

Enteropathogenic Escherichia coli (EPEC) infection requires the injection of effector proteins into intestinal epithelial cells (IECs) via type 3 secretion. Type 3‐secreted effectors can interfere with IEC signalling pathways via specific protein–protein interactions. For example, E. coli secreted protein F (EspF) binds sorting nexin 9 (SNX9), an endocytic regulator, resulting in tubulation of the plasma membrane. Our aim was to determine the mechanism of EspF/SNX9‐induced membrane tubulation. Point mutation of the SNX9 lipid binding domains or truncation of the EspF SNX9 binding domains significantly inhibited tubulation, as did inhibition of clathrin coated pit (CCP) assembly. Although characterized as non‐invasive, EPEC are known to invade IECs in vitro and in vivo. Indeed, we found significant invasion of Caco‐2 cells by EPEC, which, like tubulation, was blocked by pharmacological inhibition of CCPs. Interestingly, however, inhibition of dynamin activity did not prevent tubulation or EPEC invasion, which is in contrast to Salmonella invasion, which requires dynamin activity. Our data also indicate that EPEC invasion is dependent on EspF and its interaction with SNX9. Together, these findings suggest that EspF promotes EPEC invasion of IECs by harnessing the membrane‐deforming activity of SNX9.     

Endocytosis in the epithelium of the mouse oviduct

We studied the pathway of serum protein transport into the lumen of the mouse oviduct by localizing several tracer proteins in the oviduct after intravenous injection on days 1, 5, and 11 of pregnancy. Fluorescent proteins were observed in the lamina propria and in vesicles in the lumenal epithelial cells mainly in the preampulla segment on days 5 and 11 of pregnancy. In the isthmus, there was much less fluorescence in the lamina propria and no fluorescent vesicles in lumenal epithelial cells. This is similar to previous observations on day 1 and indicates that the uptake of serum proteins into lumenal epithelial cells in the preampulla is not limited to the time when embryos are present in the oviductal lumen. Horseradish peroxidase (HRP) was present in the lamina propria of the preampulla on days 1 and 5, but direct tracer movement into the oviductal lumen was blocked by the epithelial junctional complexes. Within the epithelial cells, HRP was localized in endocytic vesicles along the basolateral membrane, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. Ferritin was also used as a tracer and was observed in the same locations as HRP. Acid phosphatase in the epithelial cells of the preampulla on day 1 was localized in mvb and bdb, indicating that these structures are lysosomes. It appeared that HRP and ferritin followed two pathways after basolateral endocytosis by the epithelial cells in the preampulla: 1) they were transported to apical vesicles that may release their contents into the oviductal lumen, or 2) they were transported to lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of intestinal alkaline phosphatase on intestinal epithelial cells,macrophages and chronic colitis in mice

Aims

Intestinal alkaline phosphatase (IAP) is an intestinal brush border enzyme that is shown to function as a gut mucosal defense factor, but its defensive mechanism remains unclear. The aims of this study were to evaluate the effect of IAP on intestinal epithelial cells and macrophages, and on chronic colitis in interleukin-10-deficient (IL-10^−/−) mice.

Main methods

Human intestinal epithelial cells COLO 205 and peritoneal macrophages from IL-10^−/− mice were pretreated with IAP and then stimulated with lipopolysaccharide (LPS). IL-8 secretion from COLO205 cells and TNF-α, IL-6, IL-12 from peritoneal macrophages were measured by ELISA. Electrophoretic mobility shift assay was used to assess the DNA binding activity of NF-κB and IκBα phosphorylation/degradation was evaluated by immunoblot assay in COLO 205. For the in vivo study, colitis was induced in IL-10^−/− mice with piroxicam, the mice were then treated with 100 or 300 units of IAP by oral gavage for 2 weeks. Colitis was quantified by histopathologic scoring, and the phosphorylation of IκBα in the colonic mucosa was assessed using immunohistochemistry.

Key findings

IAP significantly inhibited LPS-induced inflammatory cytokine production in both IECs and peritoneal macrophages. IAP also attenuated LPS-induced NF-κB binding activity and IκBα phosphorylation/degradation in IECs. Oral administration of IAP significantly reduced the severity of colitis and down-regulated colitis-induced IκBα phosphorylation in IL-10^−/− mice.

Significance

IAP may inhibit the activation of intestinal epithelial cells and peritoneal macrophages, and may attenuate chronic murine colitis. This finding suggests that IAP supplementation is a potential therapeutic option for inflammatory bowel disease.     

Immunization of mice with Lactobacillus casei expressing intimin fragments produces antibodies able to inhibit the adhesion of enteropathogenic Escherichia coli to cultivated epithelial cells

Enteropathogenic Escherichia coli (EPEC) are frequently isolated as a cause of infantile diarrhea in developing countries. Its pathogenicity is distinguished by histopathological alterations at the site of infection, known as attaching and effacing (A/E) lesions, in which bacterial virulence factors and host proteins participate. Intimin, a bacterial adhesin expressed by all EPEC described to date, is responsible for the intimate adherence of the bacteria to host cells and is essential for the formation of A/E lesions. Mucosal vaccination may represent an efficacious intervention to prevent EPEC infection and lower morbidity and mortality rates. Strategies for mucosal vaccinations that use lactic acid bacteria for the delivery of heterologous antigens rely on their safety profile and ability to stimulate the immune system. In the present work, we have constructed Lactobacillus casei strains expressing different fragments of intimin beta, a subtype that is frequently expressed by EPEC strains. Mucosal immunization of mice with L. casei expressing intimin fragments induced specific systemic and mucosal antibodies. These antibodies were able to recognize native intimin on the surface of EPEC and to inhibit in vitro EPEC binding to epithelial cells.     

The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen that adheres to epithelial cells of the small intestine and uses a type III secretion system to inject effector proteins into host cells. EPEC infection leads to disruption of host intestinal tight junctions that are important for maintaining intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Here we show that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. Using the drug Brefeldin A, we demonstrate that the effect of NleA on tight junction integrity is related to its inhibition of host cell protein trafficking through COPII-dependent pathways. These results suggest that NleA's striking effect on virulence is mediated, at least in part, via its role in disruption of intestinal barrier function.     

O-Antigen Delays Lipopolysaccharide Recognition and Impairs Antibacterial Host Defense in Murine Intestinal Epithelial Cells

Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella.     

Enteropathogenic Escherichia coli (EPEC) attachment to epithelial cells: exploiting the host cell cytoskeleton from the outside

Enteropathogenic Escherichia coli (EPEC), a leading cause of human infantile diarrhoea, is the prototype for a family of intestinal bacterial pathogens that induce attaching and effacing (A/E) lesions on host cells. A/E lesions are characterized by localized effacement of the brush border of enterocytes, intimate bacterial attachment and pedestal formation beneath the adherent bacteria. As a result of some recent breakthrough discoveries, EPEC has now emerged as a fascinating paradigm for the study of host–pathogen interactions and cytoskeletal rearrangements that occur at the host cell membrane. EPEC uses a type III secretion machinery to attach to epithelial cells, translocating its own receptor for intimate attachment, Tir, into the host cell, which then binds to intimin on the bacterial surface. Studies of EPEC-induced cytoskeletal rearrangements have begun to provide clues as to the mechanisms used by this pathogen to subvert the host cell cytoskeleton and signalling pathways. These findings have unravelled new ways by which pathogenic bacteria exploit host processes from the cell surface and have shed new light on how EPEC might cause diarrhoea.     

Epithelial p38α Controls Immune Cell Recruitment in the Colonic Mucosa

Intestinal epithelial cells (IECs) compose the first barrier against microorganisms in the gastrointestinal tract. Although the NF-κB pathway in IECs was recently shown to be essential for epithelial integrity and intestinal immune homeostasis, the roles of other inflammatory signaling pathways in immune responses in IECs are still largely unknown. Here we show that p38α in IECs is critical for chemokine expression, subsequent immune cell recruitment into the intestinal mucosa, and clearance of the infected pathogen. Mice with p38α deletion in IECs suffer from a sustained bacterial burden after inoculation with Citrobacter rodentium. These animals are normal in epithelial integrity and immune cell function, but fail to recruit CD4⁺ T cells into colonic mucosal lesions. The expression of chemokines in IECs is impaired, which appears to be responsible for the impaired T cell recruitment. Thus, p38α in IECs contributes to the host immune responses against enteric bacteria by the recruitment of immune cells.     

Polymorphonuclear leukocyte migration across model intestinal epithelia enhances Salmonella typhimurium killing via the epithelial derived cytokine,IL-6

The host response to Salmonella typhimurium involves movement of polymorphonuclear leukocytes (PMN) across the epithelium and into the intestinal lumen. Following their arrival in the lumen, the PMN attempt to combat bacterial infection by activating antimicrobial defenses such as granule release, oxidative burst, phagocytosis, and cell signaling. We sought to examine PMN-S. typhimurium interaction following PMN arrival in the lumenal compartment. Here, for the first time, we demonstrate that PMN that have transmigrated across model intestinal epithelia have an enhanced ability to kill S. typhimurium. Our data provide evidence to indicate that the extracellular release of the primary and secondary granules of PMN, myeloperoxidase and lactoferrin, respectively, is correlated with enhanced bacterial killing. Furthermore, epithelial cells, during PMN transmigration, release the cytokine IL-6. IL-6 is known to increase intracellular stores of Ca(2+), and we have determined that this epithelial released cytokine is not only responsible for priming the PMN to release their granules, but also stimulating the PMN to kill S. typhimurium. These results substantiate the pathway in which PMN transmigration activates the epithelial release of IL-6, which in turn increases intracellular Ca(2+) storage. Our results, herein, extend this pathway to include an enhanced PMN granule release and an enhanced killing of S. typhimurium.     

Proteomic analysis of the intestinal epithelial cell response to enteropathogenic Escherichia coli

We present the first large scale proteomic analysis of a human cellular response to a pathogen. Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much childhood morbidity and mortality worldwide. EPEC uses a type III secretion system (TTSS) to inject bacterial proteins into the cytosol of intestinal epithelial cells, resulting in diarrhea. We analyzed the host response to TTSS-delivered EPEC effector proteins by infecting polarized intestinal epithelial monolayers with either wild-type or TTSS-deficient EPEC. Host proteins were isolated and subjected to quantitative profiling using isotope-coded affinity tagging (ICAT) combined with electrospray ionization tandem mass spectrometry. We identified over 2000 unique proteins from infected Caco-2 monolayers, of which approximately 13% are expressed differentially in the presence of TTSS-delivered EPEC effector proteins. We validated these data in silico and through immunoblotting and immunofluorescence microscopy. The identified changes extend cytoskeletal observations made in less relevant cell types and generate testable hypotheses with regard to host proteins potentially involved in EPEC-induced diarrhea. These data provide a framework for future biochemical analyses of host-pathogen interactions.     

Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia coli effector‐mediated subversion of host innate immune pathways

Enteric bacterial pathogens commonly use a type III secretion system (T3SS) to successfully infect intestinal epithelial cells and survive and proliferate in the host. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC; EHEC) colonize the human intestinal mucosa, form characteristic histological lesions on the infected epithelium and require the T3SS for full virulence. T3SS effectors injected into host cells subvert cellular pathways to execute a variety of functions within infected host cells. The EPEC and EHEC effectors that subvert innate immune pathways – specifically those involved in phagocytosis, host cell survival, apoptotic cell death and inflammatory signalling – are all required to cause disease. These processes are reviewed within, with a focus on recent work that has provided insights into the functions and host cell targets of these effectors.     

Bacterial translocation from the intestines

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal (GI) tract through the mucosal epithelium to other sites, such as the mesenteric lymph nodes, spleen, liver and blood. This paper reviews results from animal models utilized to obtain information concerning the defense mechanisms operating in the healthy host to confine bacteria to the GI tract. Gnotobiotic and antibiotic-decontaminated mice colonized with particular bacteria demonstrated that the indigenous GI flora maintains an ecologic equilibrium to prevent intestinal bacterial overgrowth and translocation from the GI tract. Studies with athymic (nu/nu) mice, thymus-grafted (nu/nu) mice, neonatally thymectomized mice, and mice injected with immunosuppressive agents demonstrated that the host immune system is another defense mechanism inhibiting bacterial translocation from the GI tract. Ricinoleic acid given orally to mice disrupted the intestinal epithelial barrier allowing indigenous bacteria to translocate from the GI tract. Thus, bacterial translocation from the GI tract of healthy adult mice is inhibited by: (a) an intact intestinal epithelial barrier, (b) the host immune defense system, and (c) an indigenous GI flora maintaining ecological equilibrium to prevent bacterial overgrowth. Deficiencies in host defense mechanisms act synergistically to promote bacterial translocation from the GI tract as demonstrated by animal models with multiple alterations in host defenses. Bacterial translocation occurred to a greater degree in mice with streptozotocin-induced diabetes, mice receiving nonlethal thermal injury, and mice receiving the combination of an immunosuppressive agent plus an oral antibiotic than in mice with only a primary alteration in host defenses. The study of bacterial translocation in these complex models suggests that opportunistic infections from the GI tract occur in discrete stages. In the healthy adult animal, bacterial translocation from the GI tract either does not occur or occurs at a very low level and the host immune defenses eliminate the translocating bacteria. Bacterial translocation does take place if one of the host defense mechanisms is compromised, such as a deficiency in the immune response, bacterial overgrowth in the intestines, or an increase in the permeability of the intestinal barrier. In this first stage, the bacteria usually translocate in low numbers to the mesenteric lymph node, and sometimes spleen or liver, but do not multiply and spread systemically.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proteomic analysis of the binding partners to enteropathogenic Escherichia coli virulence proteins expressed in Saccharomyces cerevisiae

Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much worldwide morbidity and mortality. EPEC uses a type III secretion system to inject bacterial proteins into the cytosol of intestinal epithelial cells to cause diarrheal disease. We are interested in determining the host proteins to which EPEC translocator and effector proteins bind during infection. To facilitate protein enrichment, we created fusions between GST and EPEC virulence proteins, and expressed these fusions individually in Saccharomyces cerevisiae. The biology of S. cerevisiae is well understood and often employed as a model eukaryote to study the function of bacterial virulence factors. We isolated the yeast proteins that interact with individual EPEC proteins by affinity purifying against the GST tag. These complexes were subjected to ICAT combined with ESI-MS/MS. Database searching of sequenced peptides provided a list of proteins that bound specifically to each EPEC virulence protein. The dataset suggests several potential mammalian targets of these proteins that may guide future experimentation.     

The cell death response to enteropathogenic Escherichia coli infection

Given the critical roles of inflammation and programmed cell death in fighting infection, it is not surprising that many bacterial pathogens have evolved strategies to inactivate these defences. The causative agent of infant diarrhoea, enteropathogenic Escherichia coli (EPEC), is an extracellular, intestinal pathogen that blocks both inflammation and programmed cell death. EPEC attaches to enterocytes, remains in the gut lumen and utilizes a type III secretion system (T3SS) to inject multiple virulence effector proteins directly into the infected cell, many of which subvert host antimicrobial processes through the disruption of signalling pathways. Recently, T3SS effector proteins from EPEC have been identified that inhibit death receptor‐induced apoptosis. Here we review the mechanisms used by EPEC T3SS effectors to manipulate apoptosis and promote host cell survival and discuss the role of these activities during infection.     

Real-Time Sensing of Enteropathogenic E. coli-Induced Effects on Epithelial Host Cell Height,Cell-Substrate Interactions,and Endocytic Processes by Infrared Surface Plasmon Spectroscopy

Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell''s height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.     

Enteropathogenic and enterohaemorrhagic Escherichia coli : more subversive elements

Enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide. Both pathogens colonize the intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the 'attaching and effacing' (A/E) lesion. Although EPEC was the first E. coli to be associated with human disease in the 1940s and 1950s, it was not until the late 1980s and early 1990s that the mechanisms and bacterial gene products used to induce this complex brush border membrane lesion and diarrhoeal disease started to be unravelled. During the past few months, there has been a burst of new data that have revolutionized some basic concepts of the molecular basis of bacterial pathogenesis in general and EPEC pathogenesis in particular. Major breakthroughs and developments in the genetic basis of A/E lesion formation, signal transduction, protein translocation, host cell receptors and intestinal colonization are highlighted in this review.     

双歧杆菌粘附体外肠上皮细胞的钙信号传递的研究

本文采用钙荧光探剂 Ｆｌｕｏ—３／ＡＭ染色法,定量研究了双歧杆菌１０２７株、肠致病性大肠杆菌（ＥＰＥＣ）对体外肠上皮细胞Ｌｏｖｏ细胞株粘附的钙信号传递机制。结果表明,双歧杆菌１０２７株粘附可引起Ｌｏｖｏ细胞内Ｃａ~＋２随时间延长而梯度升高,但双歧杆菌１０２７株的作用远不如ＥＰＥＣ明显。同时发现双歧杆菌粘附引起Ｌｏｖｏ。细胞内Ｃａ~２＋升高主要源于细胞外Ｃａ~２＋内流所致,这与ＥＰＥＣ粘附引起宿主细胞内Ｃａ~２＋升高主要源于细胞内Ｃａ~２＋储池的Ｃａ~２＋释放不同。ＥＰＥＣ粘附引起宿主细胞内Ｃａ~２＋大幅度升高是其致病的重要信号传递基础;而双歧杆菌粘附仅引起宿主细胞内Ｃａ~２＋轻度升高,可能是其作为生理性细菌与肠上皮细胞和谐共生的信号传递基础。     

Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation-mimicking SEPT9 mutant rescued these effects. Collectively, this study provides the first global analysis of phosphorylation-mediated processes during infection with an extracellular, diarrheagenic bacterial pathogen.Diarrheagenic E. coli are a major global health burden and cause much morbidity and mortality worldwide. Enteropathogenic E. coli (EPEC)¹ is the causative agent of potentially fatal infantile diarrhea and remains an endemic health threat for children in developing countries. EPEC and the closely related enterohemorrhagic E. coli (EHEC) belong to the group of attaching and effacing (A/E) pathogens that form distinct A/E lesions on the surface of intestinal epithelial cells causing the loss of the characteristic intestinal brush border architecture (1).Upon attachment to intestinal cells, EPEC uses a syringe-like molecular apparatus, the type III secretion system (T3SS), to inject at least 25 unique bacterial effector proteins into the host cell (2–4). Once translocated into mammalian cells, these effectors manipulate a wide range of host signaling pathways, thereby subverting host cell function and promoting virulence (5). The bacterial translocated intimin receptor (Tir) is one of the first and most abundant effectors injected into the host cell: it mediates intimate attachment of EPEC to the enterocyte apical surface via its interaction with the bacterial surface adhesin intimin (6, 7). In concert with other effectors, Tir also provokes an expansive cytoskeletal rearrangement leading to the formation of actin-rich protrusions, termed pedestals, beneath the site of bacterial attachment (8). Besides altering the host cell cytoskeleton, EPEC effectors also manipulate cellular trafficking, host immune response and ion and water homeostasis to cause disease (5). Although significant effort in recent years has led to the identification of multiple key players in both the host and the pathogen, the complex interactions between EPEC and the epithelial host cell, and the underlying molecular mechanisms, are still collectively not well understood.There is increasing evidence that hijacking host post-translational mechanisms such as protein phosphorylation is a key strategy for bacterial pathogens to efficiently subvert host cell function (9) and there are several indications that this may be the case for EPEC. For example, Tir is phosphorylated upon insertion into the host cell membrane and this event plays a role in the rearrangement of the actin cytoskeleton (10). Another EPEC-encoded effector, NleH, contains a functional kinase domain suggesting the potential of directly phosphorylating host cell targets (11). Moreover, the phosphorylation profiles of a few specific host proteins such as cortactin, CT10 regulator of kinase (CRK) adaptors, focal adhesion kinase (FAK) and mitogen-activated protein kinase 1 (MAPK1), as well as alterations in tyrosine phosphorylation of host proteins, are impacted in an EPEC effector-dependent manner (12–18). These are selective observations though. Thus, a more comprehensive, system-level analysis is needed to better understand how and to what extent EPEC hijacks host cell phosphorylation to cause disease.Recent advances in quantitative phosphoproteomics have made it possible to successfully profile the changes in host protein phosphorylation following infection by the invasive, diarrheagenic bacterial pathogens Shigella and Salmonella (19–21). To our knowledge, no such analysis has been reported for a noninvasive, diarrheagenic bacterial pathogen such as EPEC. In this study, we applied a stable isotope labeling by amino acids in cell culture (SILAC)-based (22) quantitative phosphoproteomics approach to assess the impact of EPEC infection on the host cell phosphoproteome. The integration of time course experiments and the use of an EPEC mutant deficient in type III secretion (T3S) provided further insights into the dynamics as well as the effector dependence of these processes. This experimental approach enabled identification of both stable and transient interactions between EPEC bacterial effectors and host proteins. Additional infection studies focusing on a newly identified host target, septin-9, further emphasizes the biological significance of the manipulation of host protein phosphorylation in EPEC pathogenesis.