Attenuation of a DNA cruciform by a conserved regulator directs T3SS1 mediated virulence in Vibrio parahaemolyticus.

Pathogenic Vibrio species account for 3-5 million annual life-threatening human infections. Virulence is driven by bacterial hemolysin and toxin gene expression often positively regulated by the winged helix-turn-helix (wHTH) HlyU transcriptional regulator family and silenced by histone-like nucleoid structural protein (H-NS). In the case of Vibrio parahaemolyticus, HlyU is required for virulence gene expression associated with type 3 Secretion System-1 (T3SS1) although its mechanism of action is not understood. Here, we provide evidence for DNA cruciform attenuation mediated by HlyU binding to support concomitant virulence gene expression. Genetic and biochemical experiments revealed that upon HlyU mediated DNA cruciform attenuation, an intergenic cryptic promoter became accessible allowing for exsA mRNA expression and initiation of an ExsA autoactivation feedback loop at a separate ExsA-dependent promoter. Using a heterologous E. coli expression system, we reconstituted the dual promoter elements which revealed that HlyU binding and DNA cruciform attenuation were strictly required to initiate the ExsA autoactivation loop. The data indicate that HlyU acts to attenuate a transcriptional repressive DNA cruciform to support T3SS1 virulence gene expression and reveals a non-canonical extricating gene regulation mechanism in pathogenic Vibrio species.

Tyrosine Phosphorylation as a Widespread Regulatory Mechanism in Prokaryotes.

Phosphorylation events modify bacterial and archaeal proteomes, imparting cells with rapid and reversible responses to specific environmental stimuli or niches. Phosphorylated proteins are generally modified at one or more serine, threonine, or tyrosine residues. Within the last ten years, increasing numbers of global phosphoproteomic surveys of prokaryote species have revealed an abundance of tyrosine-phosphorylated proteins. In some cases, novel phosphorylation-dependent regulatory paradigms for cell division, gene transcription, and protein translation have been identified, suggesting that a wide scope of prokaryotic physiology remains to be characterized. Recent observations of bacterial proteins with putative phosphotyrosine binding pockets or Src homology 2 (SH2)-like domains suggest the presence of phosphotyrosine-dependent protein interaction networks. Here in this minireview, we focus on protein tyrosine phosphorylation, a posttranslational modification once thought to be rare in prokaryotes but which has emerged as an important regulatory facet in microbial biology.

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence.

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.

The Transcriptional Regulator HlyU Positively Regulates Expression of exsA, Leading to Type III Secretion System 1 Activation in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a marine bacterium that is globally recognized as the leading cause of seafood-borne gastroenteritis. V. parahaemolyticus uses various toxins and two type 3 secretion systems (T3SS-1 and T3SS-2) to subvert host cells during infection. We previously determined that V. parahaemolyticus T3SS-1 activity is upregulated by increasing the expression level of the master regulator ExsA under specific growth conditions. In this study, we set out to identify V. parahaemolyticus genes responsible for linking environmental and growth signals to exsA gene expression. Using transposon mutagenesis in combination with a sensitive and quantitative luminescence screen, we identify HlyU and H-NS as two antagonistic regulatory proteins controlling the expression of exsA and, hence, T3SS-1 in V. parahaemolyticus Disruption of hns leads to constitutive unregulated exsA gene expression, consistent with its known role in repressing exsA transcription. In contrast, genetic disruption of hlyU completely abrogated exsA expression and T3SS-1 activity. A V. parahaemolyticushlyU null mutant was significantly deficient for T3SS-1-mediated host cell death during in vitro infection. DNA footprinting studies with purified HlyU revealed a 56-bp protected DNA region within the exsA promoter that contains an inverted repeat sequence. Genetic evidence suggests that HlyU acts as a derepressor, likely by displacing H-NS from the exsA promoter, leading to exsA gene expression and appropriately regulated T3SS-1 activity. Overall, the data implicate HlyU as a critical positive regulator of V. parahaemolyticus T3SS-1-mediated pathogenesis.IMPORTANCE Many Vibrio species are zoonotic pathogens, infecting both animals and humans, resulting in significant morbidity and, in extreme cases, mortality. While many Vibrio species virulence genes are known, their associated regulation is often modestly understood. We set out to identify genetic factors of V. parahaemolyticus that are involved in activating exsA gene expression, a process linked to a type III secretion system involved in host cytotoxicity. We discover that V. parahaemolyticus employs a genetic regulatory switch involving H-NS and HlyU to control exsA promoter activity. While HlyU is a well-known positive regulator of Vibrio species virulence genes, this is the first report linking it to a transcriptional master regulator and type III secretion system paradigm.

Feed Supplementation with Red Seaweeds, Chondrus crispus and Sarcodiotheca gaudichaudii, Reduce Salmonella Enteritidis in Laying Hens.

Salmonella Enteritidis is vertically transmitted to eggs from laying hens through infected ovaries and oviducts. S. Enteritidis can also penetrate the eggshell from contaminated feces. Reducing S. Enteritidis in laying hens is vital to provide safer eggs and minimize the spread of salmonellosis to humans. Antibiotics have been widely used to control bacterial diseases in broilers and laying hens. However, there is a major concern that the use of antibiotics leads to the development of antibiotic resistance and adverse effects on microbiota of the treated birds. Thus, there is an interest in developing alternatives to antibiotics, such as dietary prebiotics. In the present study, feed supplemented with the red seaweeds: Chondrus crispus (CC) or Sarcodiotheca gaudichaudii (SG), was offered to laying hens late in production to control S. Enteritidis. Diets contained one of the following; 2% or 4% Chondrus crispus (CC2, and CC4, respectively) or Sarcodiotheca gaudichaudii (SG2 and SG4, respectively). Chlortetracycline was used in the positive control diet. During week-4, 48 birds were orally challenged with 2 × 109 CFU/mL of S. Enteritidis. Eggs and fecal samples were collected 1, 3, 5, and 7 days' post inoculation. Birds were euthanized and organs (ceca, ovary, liver, and spleen) were sampled and analyzed for the presence of S. Enteritidis, 7 days' post inoculation. Results showed that seaweed reduced the negative effect on body weight and egg production in S. Enteritidis-challenged laying hens. Analysis of fecal samples showed that the antibiotic (CTC) reduced S. Enteritidis in the intestinal tract and fecal samples, 3 days' post inoculation. Fecal samples from Chlortetracycline and CC4 supplemented birds tested negative for S. Enteritidis on days 5 and 7 post inoculation (lowest detection limit = 10-1). S. Enteritidis colonization in the ceca was also significantly reduced in birds fed CC (4%) and Chlortetracycline. Blood serum profiles revealed that there were no significant differences in serum aspartate aminotransferase (AST) and sodium. However, the level of serum immunoglobulin (IgA) was higher in the CC4 treatment. The relative abundance of Lactobacillus acidophilus was significantly higher in CC4 while, the abundance of the pathogenic bacteria, Clostridium perfringens and Salmonella Enteritidis were reduced compared to control. Results indicate that feed supplemented with 4% CC is effective in providing protection against Salmonella Enteritidis colonization in laying hens.

Early Changes in Microbial Community Structure Are Associated with Sustained Remission After Nutritional Treatment of Pediatric Crohn's Disease.

BACKGROUND: Clinical remission achieved by exclusive enteral nutrition (EEN) is associated with marked microbiome changes. In this prospective study of exclusive enteral nutrition, we employ a hierarchical model of microbial community structure to distinguish between pediatric Crohn's disease patients who achieved sustained remission (SR) and those who relapsed early (non-SR), after restarting a normal diet. METHODS: Fecal samples were obtained from 10 patients (age 10-16) and from 5 healthy controls (age 9-14). The microbiota was assessed via 16S rRNA sequencing. In addition to standard measures of microbial biodiversity, we employed Bayesian methods to characterize the hierarchical community structure. Community structure between patients who sustained remission (wPCDAI <12.5) up to their 24-week follow-up (SR) was compared with patients that had not sustained remission (non-SR). RESULTS: Microbial diversity was lower in Crohn's disease patients relative to controls and lowest in patients who did not achieve SR. SR patients differed from non-SR patients in terms of the structure and prevalence of their microbial communities. The SR prevalent community contained a number of strains of Akkermansia muciniphila and Bacteroides and was limited in Proteobacteria, whereas the non-SR prevalent community had a large Proteobacteria component. Their communities were so different that a model trained to discriminate SR and non-SR had 80% classification accuracy, already at baseline sampling. CONCLUSIONS: Microbial community structure differs between healthy controls, patients who have an enduring response to exclusive enteral nutrition, and those who relapse early on introduction of normal diet. Our novel Bayesian approach to these differences is able to predict sustained remission after exclusive enteral nutrition.

The Gut Microbiome of Pediatric Crohn's Disease Patients Differs from Healthy Controls in Genes That Can Influence the Balance Between a Healthy and Dysregulated Immune Response.

BACKGROUND: Exclusive enteral nutrition (EEN) is a first-line therapy in pediatric Crohn's disease (CD) thought to induce remission through changes in the gut microbiome. With microbiome assessment largely focused on microbial taxonomy and diversity, it remains unclear to what extent EEN induces functional changes that thereby contribute to its therapeutic effect. METHODS: Fecal samples were collected from 15 pediatric CD patients prior to and after EEN treatment, as well as from 5 healthy controls. Metagenomic data were obtained via next-generation sequencing, and nonhuman reads were mapped to KEGG pathways, where possible. Pathway abundance was compared between CD patients and controls, and between CD patients that sustained remission (SR) and those that did not sustain remission (NSR). RESULTS: Of 132 KEGG pathways identified, 8 pathways differed significantly between baseline CD patients and controls. Examination of these eight pathways showed SR patients had greater similarity to controls than NSR patients in all cases. Pathways fell into one of three groups: 1) no prior connection to IBD, 2) previously reported connection to IBD, and 3) known roles in innate immunity and immunoregulation. CONCLUSIONS: The microbiota of CD patients and controls represent alternative ecological states that have broad differences in functional capabilities, including xenobiotic and environmental pollutant degradation, succinate metavolism, and bacterial HtpG, all of which can affect barrier integrity and immune regulation. Moreover, our finding that SR patients were more similar to healthy controls suggests that community microbial function, as inferred from fecal microbiomes, could serve as a valuable diagnostic tool.

Red Seaweeds Sarcodiotheca gaudichaudii and Chondrus crispus down Regulate Virulence Factors of Salmonella Enteritidis and Induce Immune Responses in Caenorhabditis elegans.

Red seaweeds are a rich source of unique bioactive compounds and secondary metabolites that are known to improve human and animal health. S. Enteritidis is a broad range host pathogen, which contaminates chicken and poultry products that end into the human food chain. Worldwide, Salmonella outbreaks have become an important economic and public health concern. Moreover, the development of resistance in Salmonella serovars toward multiple drugs highlights the need for alternative control strategies. This study evaluated the antimicrobial property of red seaweeds extracts against Salmonella Enteritidis using the Caenorhabditis elegans infection model. Six red seaweed species were tested for their antimicrobial activity against S. Enteritidis and two, Sarcodiotheca gaudichaudii (SG) and Chondrus crispus (CC), were found to exhibit such properties. Spread plate assay revealed that SG and CC (1%, w/v) significantly reduced the growth of S. Enteritidis. Seaweed water extracts (SWE) of SG and CC, at concentrations from 0.4 to 2 mg/ml, significantly reduced the growth of S. Enteritidis (log CFU 4.5-5.3 and log 5.7-6.0, respectively). However, methanolic extracts of CC and SG did not affect the growth of S. Enteritidis. Addition of SWE (0.2 mg/ml, CC and SG) significantly decreased biofilm formation and reduced the motility of S. Enteritidis. Quantitative real-time PCR analyses showed that SWE (CC and SG) suppressed the expression of quorum sensing gene sdiA and of Salmonella Pathogenesis Island-1 (SPI-1) associated genes sipA and invF, indicating that SWE might reduce the invasion of S. Enteritidis in the host by attenuating virulence factors. Furthermore, CC and SG water extracts significantly improved the survival of infected C. elegans by impairing the ability of S. Enteritidis to colonize the digestive tract of the nematode and by enhancing the expression of C. elegans immune responsive genes. As the innate immune response pathways of C. elegans and mammals show a high degree of conservation, these results suggest that these SWE may also impart beneficial effects on animal and human health.

Transcriptional profiling of Vibrio parahaemolyticus exsA reveals a complex activation network for type III secretion.

Vibrio parahaemolyticus (Vp) is a marine halophilic bacterium that is commonly associated with oysters and shrimp. Human consumption of contaminated shellfish can result in Vp mediated gastroenteritis and severe diarrheal disease. Vp encodes two type 3 secretion systems (T3SS-1 and T3SS2) that have been functionally implicated in cytotoxicity and enterotoxicity respectively. In this study, we profiled protein secretion and temporal promoter activities associated with exsA and exsB gene expression. exsA is an AraC-like transcriptional activator that is critical for activating multiple operons that encode T3SS-1 genes, whereas exsB is thought to encode an outer membrane pilotin component for T3SS-1. The exsBA genetic locus has two predicted promoter elements. The predicted exsB and exsA promoters were individually cloned upstream of luxCDABE genes in reporter plasmid constructs allowing for in situ, real-time quantitative light emission measurements under many growth conditions. Low calcium growth conditions supported maximal exsB and exsA promoter activation. exsB promoter activity exhibited high basal activity and resulted in an exsBA co-transcript. Furthermore, a separate proximal exsA promoter showed initial low basal activity yet eventually exceeded that of exsB and reached maximal levels after 2.5 h corresponding to an entry into early log phase. exsA promoter activity was significantly higher at 30°C than 37°C, which also coincided with increased secretion levels of specific T3SS-1 effector proteins. Lastly, bioinformatic analyses identified a putative expanded ExsA binding motif for multiple transcriptional operons. These findings suggest a two wave model of Vp T3SS-I induction that integrates two distinct promoter elements and environmental signals into a complex ExsA activation framework.

Properdin provides protection from Citrobacter rodentium-induced intestinal inflammation in a C5a/IL-6-dependent manner.

Citrobacter rodentium is an attaching and effacing mouse pathogen that models enteropathogenic and enterohemorrhagic Escherichia coli in humans. The complement system is an important innate defense mechanism; however, only scant information is available about the role of complement proteins during enteric infections. In this study, we examined the impact of the lack of properdin, a positive regulator of complement, in C. rodentium-induced colitis. Following infection, properdin knockout (P(KO)) mice had increased diarrhea and exacerbated inflammation combined with defective epithelial cell-derived IL-6 and greater numbers of colonizing bacteria. The defect in the mucosal response was reversed by administering exogenous properdin to P(KO) mice. Then, using in vitro and in vivo approaches, we show that the mechanism behind the exacerbated inflammation of P(KO) mice is due to a failure to increase local C5a levels. We show that C5a directly stimulates IL-6 production from colonic epithelial cells and that inhibiting C5a in infected wild-type mice resulted in defective epithelial IL-6 production and exacerbated inflammation. These outcomes position properdin early in the response to an infectious challenge in the colon, leading to complement activation and C5a, which in turn provides protection through IL-6 expression by the epithelium. Our results unveil a previously unappreciated mechanism of intestinal homeostasis involving complement, C5a, and IL-6 during bacteria-triggered epithelial injury.

Reprint of "GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC)".

Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures. Here, we apply a solution-phase electrophoretic platform (GELFrEE) followed by MS to characterize the secreted proteome of a wild type and mutant strain of EPEC (ΔsepD), exhibiting enhanced secretion of effector proteins. Through peptide-level analysis, a total of 363 and 155 proteins were identified from the wild type and mutant strains, respectively. Semi-quantitative analysis of the MS data reveals the effector proteins EspB, EspC, and EspD appear in a relatively greater abundance from wild type EPEC, while two major virulence factors in EPEC, Tir and NleA appear in greater abundance from the secreted proteome of the mutant strain. Additionally, intact proteins may further be characterized following GELFrEE with MS to improve throughput of analysis. This study demonstrates the application of GELFrEE-MS to differentiate wild type and mutant strains of EPEC. This platform is therefore a powerful tool to study exotoxin secretion from pathogenic bacteria.

GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC).

Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures. Here, we apply a solution-phase electrophoretic platform (GELFrEE) followed by MS to characterize the secreted proteome of a wild type and mutant strain of EPEC (ΔsepD), exhibiting enhanced secretion of effector proteins. Through peptide-level analysis, a total of 363 and 155 proteins were identified from the wild type and mutant strains, respectively. Semi-quantitative analysis of the MS data reveals the effector proteins EspB, EspC, and EspD appear in a relatively greater abundance from wild type EPEC, while two major virulence factors in EPEC, Tir and NleA appear in greater abundance from the secreted proteome of the mutant strain. Additionally, intact proteins may further be characterized following GELFrEE with MS to improve throughput of analysis. This study demonstrates the application of GELFrEE-MS to differentiate wild type and mutant strains of EPEC. This platform is therefore a powerful tool to study exotoxin secretion from pathogenic bacteria.

A novel C-terminal region within the multicargo type III secretion chaperone CesT contributes to effector secretion.

The enteropathogenic Escherichia coli (EPEC) multicargo chaperone CesT interacts with at least 10 effector proteins and is central to pathogenesis. CesT has been implicated in coordinating effector hierarchy, although the mechanisms behind this regulation are poorly understood. To address this question, we set out to functionally characterize CesT with respect to roles in (i) effector binding, (ii) effector recruitment to the type III secretion system (T3SS), and (iii) effector translocation into host cells. A CesT variant expression library was screened in EPEC using a newly developed semi-high-throughput secretion assay. Among many deficient CesT variants, a predominant number were localized to a novel CesT C-terminal region. These CesT C-terminal variants exhibited normal effector binding yet reduced effector secretion levels. Structural correlation and thermal spectroscopy analyses of purified CesT variants implicated multiple surface-exposed residues, a terminal helix region, and a flexible C-terminal triple-serine stretch in effector secretion. Site-directed mutagenesis of the flexible CesT C-terminal triple-serine sequence produced differential effector secretion, implicating this region in secretion events. Infection assays further indicated that the C-terminal region of CesT was important for NleA translocation into host cells but was dispensable for Tir translocation. The findings implicate the CesT C terminus in effector secretion and contribute to a model for multiple-cargo chaperone function and effector translocation into host cells during infection.

Characterization of the type III secretion associated low calcium response genes of Vibrio parahaemolyticus RIMD2210633.

Vibrio parahaemolyticus is a significant human pathogen associated with gastroenteritis. Two V. parahaemolyticus type 3 secretion systems, T3SS-1 and T3SS-2, secrete effector proteins and have been implicated in host-cell cytotoxicity and enterotoxicity, respectively. Vibrio parahaemolyticus T3SS-1 substrates have been identified, although many predicted substrates (based on homologies) remain undetected in secreted fractions and therefore uncharacterized. We have experimentally developed and optimized a secretion assay protocol allowing for reliable and reproducible detection of V. parahaemolyticus T3SS-1 secreted proteins within culture supernatants. The presence of magnesium and absence of calcium were critical factors in promoting type III secretion of protein substrates. Proteomic approaches identified known V. parahaemolyticus secreted effectors in addition to previously unidentified proteins. Isogenic mutants in putative low calcium response genes were generated, and experiments further implicated the genes in secretion and V. parahaemolyticus-mediated host-cell cytotoxicity during infection. These approaches should be valuable towards future detailed genetic and biochemical analyses of T3SS-1 in V. parahaemolyticus.

Dual temporal transcription activation mechanisms control cesT expression in enteropathogenic Escherichia coli.

The locus of enterocyte effacement (LEE) is a 35 kb pathogenicity island involved in attaching and effacing (A/E) Escherichia coli enteric infection. The LEE is organized into five large transcriptional operons (LEE1-LEE5) and a few bi- and monocistronic instances. The LEE5 operon co-transcribes three genes, tir-cesT-eae, although cesT can be transcribed in a separate mRNA from its own proximal promoter. The role of this separate promoter is not understood. In this study we characterized promoter activity for the type III secretion chaperone gene cesT. The cesT promoter, cesTp, has features consistent with σ(70) promoters that contain an extended -10 element. This was experimentally confirmed by mutations that altered cesTp activity. In stark contrast to LEE2-5 transcriptional operons, cesTp did not require the master regulator Ler for transcriptional activity. Moreover, cesTp activity was not dependent on the presence of GrlA or GrlR, two regulators associated with LEE gene expression. A cesTp-lux fusion was used in real-time assays and demonstrated initial cesTp activity that occurred before LEE5 promoter activity, which ensued after 120 min. cesTp was shown to be active during in vitro infection of HT-29 colonic epithelial cells. Inactivation of cesTp reduced CesT protein levels at early growth time points. These data indicate a Ler-, GrlA- and GrlR-independent activity for cesTp. We suggest that A/E pathogenic E. coli have evolved a mechanism to ready the cell for CesT protein expression in support of infection prior to Ler- and GrlA-related activities.

Expanded roles for multicargo and class 1B effector chaperones in type III secretion.

Bacterial type III secretion systems (T3SS) are complex protein assemblies that mediate the secretion of protein substrates outside the cell. Type III secretion chaperones (T3SC) are always found associated with T3SS, and they serve in multiple roles to ensure that protein substrates are efficiently targeted for secretion. Bacterial pathogens with T3SS express T3SC proteins that bind effectors, a process important for effector protein delivery into eukaryotic cells during infection. In this minireview, we focus on multicargo and class 1B T3SC that associate with effectors within significant pathogens of animals and plants. As a primary role, multicargo and class 1B T3SC form homodimers and specifically bind different effectors within the cytoplasm, maintaining the effectors in a secretion-competent state. This role makes T3SC initial and central contributors to effector-mediated pathogenesis. Recent findings have greatly expanded our understanding of cellular events linked to multicargo T3SC function. New binding interactions with T3SS components have been reported in different systems, thereby implicating multicargo T3SC in critical roles beyond effector binding. Three notable interactions with the YscN, YscV, and YscQ family members are well represented in the literature. Similar T3SC interactions are reported in the putative related flagellar T3SS, suggesting that secretion mechanisms may be more similar than previously thought. The evidence implicates multicargo and class 1B T3SC in effector binding and stabilization, in addition to T3SS recruitment and docking events.

Role of EscU auto-cleavage in promoting type III effector translocation into host cells by enteropathogenic Escherichia coli.

BACKGROUND: Type III secretion systems (T3SS) of bacterial pathogens coordinate effector protein injection into eukaryotic cells. The YscU/FlhB group of proteins comprises members associated with T3SS which undergo a specific auto-cleavage event at a conserved NPTH amino acid sequence. The crystal structure of the C-terminal portion of EscU from enteropathogenic Escherichia coli (EPEC) suggests this auto-cleaving protein provides an interface for substrate interactions involved in type III secretion events. RESULTS: We demonstrate EscU must be auto-cleaved for bacteria to efficiently deliver type III effectors into infected cells. A non-cleaving EscU(N262A) variant supported very low levels of in vitro effector secretion. These effector proteins were not able to support EPEC infection of cultured HeLa cells. In contrast, EscU(P263A) was demonstrated to be partially auto-cleaved and moderately restored effector translocation and functionality during EPEC infection, revealing an intermediate phenotype. EscU auto-cleavage was not required for inner membrane association of the T3SS ATPase EscN or the ring forming protein EscJ. In contrast, in the absence of EscU auto-cleavage, inner membrane association of the multicargo type III secretion chaperone CesT was altered suggesting that EscU auto-cleavage supports docking of chaperone-effector complexes at the inner membrane. In support of this interpretation, evidence of novel effector protein breakdown products in secretion assays were linked to the non-cleaved status of EscU(N262A). CONCLUSIONS: These data provide new insight into the role of EscU auto-cleavage in EPEC. The experimental data suggests that EscU auto-cleavage results in a suitable binding interface at the inner membrane that accommodates protein complexes during type III secretion events. The results also demonstrate that altered EPEC genetic backgrounds that display intermediate levels of effector secretion and translocation can be isolated and studied. These genetic backgrounds should be valuable in deciphering sequential and temporal events involved in EPEC type III secretion.

A comprehensive proteomic analysis of the type III secretome of Citrobacter rodentium.

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.

Regulation of expression and secretion of NleH, a new non-locus of enterocyte effacement-encoded effector in Citrobacter rodentium.

Together with enterohemorrhagic Escherichia coli and enteropathogenic Escherichia coli, Citrobacter rodentium is a member of the attaching-and-effacing (A/E) family of bacterial pathogens. A/E pathogens use a type III secretion system (T3SS) to translocate an assortment of effector proteins, encoded both within and outside the locus of enterocyte effacement (LEE), into the colonized host cell, leading to the formation of A/E lesions and disease. Here we report the identification and characterization of a new non-LEE encoded effector, NleH, in C. rodentium. NleH is conserved among A/E pathogens and shares identity with OspG, a type III secreted effector protein in Shigella flexneri. Downstream of nleH, genes encoding homologues of the non-LEE-encoded effectors EspJ and NleG/NleI are found. NleH secretion and translocation into Caco-2 cells requires a functional T3SS and signals located at its amino-terminal domain. Transcription of nleH is not significantly reduced in mutants lacking the LEE-encoded regulators Ler and GrlA; however, NleH protein levels are highly reduced in these strains, as well as in escN and cesT mutants. Inactivation of Lon, but not of ClpP, protease restores NleH levels even in the absence of CesT. Our results indicate that the efficient engagement of NleH in active secretion is needed for its stability, thus establishing a posttranslational regulatory mechanism that coregulates NleH levels with the expression of LEE-encoded proteins. A C. rodentium nleH mutant shows a moderate defect during the colonization of C57BL/6 mice at early stages of infection.

The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function.

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) maintain an extracellular lifestyle and use a type III secretion system to translocate effector proteins into the host cytosol. These effectors manipulate host pathways to favor bacterial replication and survival. NleA is an EHEC/EPEC- and related species-specific translocated effector protein that is essential for bacterial virulence. However, the mechanism by which NleA impacts virulence remains undetermined. Here we demonstrate that NleA compromises the Sec23/24 complex, a component of the mammalian COPII protein coat that shapes intracellular protein transport vesicles, by directly binding Sec24. Expression of an NleA-GFP fusion protein reduces the efficiency of cellular secretion by 50%, and secretion is inhibited in EPEC-infected cells. Direct biochemical experiments show that NleA inhibits COPII-dependent protein export from the endoplasmic reticulum. Collectively, these findings indicate that disruption of COPII function in host cells contributes to the virulence of EPEC and EHEC.