The type III secretion system effector EspO of enterohaemorrhagic Escherichia coli inhibits apoptosis through an interaction with HAX-1.

Many enteric pathogens employ a type III secretion system (T3SS) to translocate effector proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the intestinal epithelium. Here, we report that the anti-apoptotic regulator HS1-associated protein X1 (HAX-1) is an interaction partner of the T3SS effectors EspO of enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, OspE of Shigella flexneri and Osp1STYM of Salmonella enterica serovar Typhimurium. EspO, OspE and Osp1STYM have previously been reported to interact with the focal adhesions protein integrin linked kinase (ILK). We found that EspO localizes both to the focal adhesions (ILK localisation) and mitochondria (HAX-1 localisation), and that increased expression of HAX-1 leads to enhanced mitochondrial localisation of EspO. Ectopic expression of EspO, OspE and Osp1STYM protects cells from apoptosis induced by staurosporine and tunicamycin. Depleting cells of HAX-1 indicates that the anti-apoptotic activity of EspO is HAX-1 dependent. Both HAX-1 and ILK were further confirmed as EspO1-interacting proteins during infection using T3SS-delivered EspO1. Using cell detachment as a proxy for cell death we confirmed that T3SS-delivered EspO1 could inhibit cell death induced during EPEC infection, to a similar extent as the anti-apoptotic effector NleH, or treatment with the pan caspase inhibitor z-VAD. In contrast, in cells lacking HAX-1, EspO1 was no longer able to protect against cell detachment, while NleH1 and z-VAD maintained their protective activity. Therefore, during both infection and ectopic expression EspO protects cells from cell death by interacting with HAX-1. These results suggest that despite the differences between EHEC, C. rodentium, Shigella and S. typhimurium infections, hijacking HAX-1 anti-apoptotic signalling is a common strategy to maintain the viability of infected cells. TAKE AWAY: EspO homologues are found in EHEC, Shigella, S. typhimurium and some EPEC. EspO homologues interact with HAX-1. EspO protects infected cells from apoptosis. EspO joins a growing list of T3SS effectors that manipulate cell death pathways.

A One Health Approach to Child Stunting: Evidence and Research Agenda.

Stunting (low height for age) affects approximately one-quarter of children aged < 5 years worldwide. Given the limited impact of current interventions for stunting, new multisectoral evidence-based approaches are needed to decrease the burden of stunting in low- and middle-income countries (LMICs). Recognizing that the health of people, animals, and the environment are connected, we present the rationale and research agenda for considering a One Health approach to child stunting. We contend that a One Health strategy may uncover new approaches to tackling child stunting by addressing several interdependent factors that prevent children from thriving in LMICs, and that coordinated interventions among human health, animal health, and environmental health sectors may have a synergistic effect in stunting reduction.

The Citrobacter rodentium type III secretion system effector EspO affects mucosal damage repair and antimicrobial responses.

Infection with Citrobacter rodentium triggers robust tissue damage repair responses, manifested by secretion of IL-22, in the absence of which mice succumbed to the infection. Of the main hallmarks of C. rodentium infection are colonic crypt hyperplasia (CCH) and dysbiosis. In order to colonize the host and compete with the gut microbiota, C. rodentium employs a type III secretion system (T3SS) that injects effectors into colonic intestinal epithelial cells (IECs). Once injected, the effectors subvert processes involved in innate immune responses, cellular metabolism and oxygenation of the mucosa. Importantly, the identity of the effector/s triggering the tissue repair response is/are unknown. Here we report that the effector EspO ,an orthologue of OspE found in Shigella spp, affects proliferation of IECs 8 and 14 days post C. rodentium infection as well as secretion of IL-22 from colonic explants. While we observed no differences in the recruitment of group 3 innate lymphoid cells (ILC3s) and T cells, which are the main sources of IL-22 at the early and late stages of C. rodentium infection respectively, infection with ΔespO was characterized by diminished recruitment of sub-mucosal neutrophils, which coincided with lower abundance of Mmp9 and chemokines (e.g. S100a8/9) in IECs. Moreover, mice infected with ΔespO triggered significantly lesser nutritional immunity (e.g. calprotectin, Lcn2) and expression of antimicrobial peptides (Reg3ß, Reg3γ) compared to mice infected with WT C. rodentium. This overlapped with a decrease in STAT3 phosphorylation in IECs. Importantly, while the reduced CCH and abundance of antimicrobial proteins during ΔespO infection did not affect C. rodentium colonization or the composition of commensal Proteobacteria, they had a subtle consequence on Firmicutes subpopulations. EspO is the first bacterial virulence factor that affects neutrophil recruitment and secretion of IL-22, as well as expression of antimicrobial and nutritional immunity proteins in IECs.

Broad-Spectrum Regulation of Nonreceptor Tyrosine Kinases by the Bacterial ADP-Ribosyltransferase EspJ.

Tyrosine phosphorylation is key for signal transduction from exogenous stimuli, including the defense against pathogens. Conversely, pathogens can subvert protein phosphorylation to control host immune responses and facilitate invasion and dissemination. The bacterial effectors EspJ and SeoC are injected into host cells through a type III secretion system by enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), Citrobacter rodentium, and Salmonella enterica, where they inhibit Src kinase by coupled amidation and ADP-ribosylation. C. rodentium, which is used to model EPEC and EHEC infections in humans, is a mouse pathogen triggering colonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparison of the proteomes of intestinal epithelial cells isolated from mice infected with wild-type C. rodentium or C. rodentium encoding catalytically inactive EspJ revealed that EspJ-induced ADP-ribosylation regulates multiple nonreceptor tyrosine kinases in vivo Investigation of the substrate repertoire of EspJ revealed that in HeLa and A549 cells, Src and Csk were significantly targeted; in polarized Caco2 cells, EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 cells, EspJ modified Csk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, controls immune responses and the Src/Csk signaling axis.IMPORTANCE Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) strains cause significant mortality and morbidity worldwide. Citrobacter rodentium is a mouse pathogen used to model EPEC and EHEC pathogenesis in vivo Diarrheal disease is triggered following injection of bacterial effectors, via a type III secretion system (T3SS), into intestinal epithelial cells (IECs). While insights into the role of the effectors were historically obtained from pathological, immunologic, or cell culture phenotypes, subtle roles of individual effectors in vivo are often masked. The aim of this study was to elucidate the role and specificity of the ADP-ribosyltransferase effector EspJ. For the first time, we show that the in vivo processes affected by a T3SS effector can be studied by comparing the proteomes of IECs extracted from mice infected with wild-type C. rodentium or an espJ catalytic mutant. We show that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, regulates the host immune response in vivo.

Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors.

Enteropathogenic E. coli (EPEC) is a human pathogen that causes acute and chronic pediatric diarrhea. The hallmark of EPEC infection is the formation of attaching and effacing (A/E) lesions in the intestinal epithelium. Formation of A/E lesions is mediated by genes located on the pathogenicity island locus of enterocyte effacement (LEE), which encode the adhesin intimin, a type III secretion system (T3SS) and six effectors, including the essential translocated intimin receptor (Tir). Seventeen additional effectors are encoded by genes located outside the LEE, in insertion elements and prophages. Here, using a stepwise approach, we generated an EPEC mutant lacking the entire effector genes (EPEC0) and intermediate mutants. We show that EPEC0 contains a functional T3SS. An EPEC mutant expressing intimin but lacking all the LEE effectors but Tir (EPEC1) was able to trigger robust actin polymerization in HeLa cells and mucin-producing intestinal LS174T cells. However, EPEC1 was unable to form A/E lesions on human intestinal in vitro organ cultures (IVOC). Screening the intermediate mutants for genes involved in A/E lesion formation on IVOC revealed that strains lacking non-LEE effector/s have a marginal ability to form A/E lesions. Furthermore, we found that Efa1/LifA proteins are important for A/E lesion formation efficiency in EPEC strains lacking multiple effectors. Taken together, these results demonstrate the intricate relationships between T3SS effectors and the essential role non-LEE effectors play in A/E lesion formation on mucosal surfaces.

Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo.

The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem.

The Enterohemorrhagic Escherichia coli Effector EspW Triggers Actin Remodeling in a Rac1-Dependent Manner.

Enterohemorrhagic Escherichia coli (EHEC) is a diarrheagenic pathogen that colonizes the gut mucosa and induces attaching-and-effacing lesions. EHEC employs a type III secretion system (T3SS) to translocate 50 effector proteins that hijack and manipulate host cell signaling pathways, which allow bacterial colonization and subversion of immune responses and disease progression. The aim of this study was to characterize the T3SS effector EspW. We found espW in the sequenced O157:H7 and non-O157 EHEC strains as well as in Shigella boydii Furthermore, a truncated version of EspW, containing the first 206 residues, is present in EPEC strains belonging to serotype O55:H7. Screening a collection of clinical EPEC isolates revealed that espW is present in 52% of the tested strains. We report that EspW modulates actin dynamics in a Rac1-dependent manner. Ectopic expression of EspW results in formation of unique membrane protrusions. Infection of Swiss cells with an EHEC espW deletion mutant induces a cell shrinkage phenotype that could be rescued by Rac1 activation via expression of the bacterial guanine nucleotide exchange factor, EspT. Furthermore, using a yeast two-hybrid screen, we identified the motor protein Kif15 as a potential interacting partner of EspW. Kif15 and EspW colocalized in cotransfected cells, while ectopically expressed Kif15 localized to the actin pedestals following EHEC infection. The data suggest that Kif15 recruits EspW to the site of bacterial attachment, which in turn activates Rac1, resulting in modifications of the actin cytoskeleton that are essential to maintain cell shape during infection.

The Type III Secretion System Effector SptP of Salmonella enterica Serovar Typhi.

Strains of the various Salmonella enterica serovars cause gastroenteritis or typhoid fever in humans, with virulence depending on the action of two type III secretion systems (Salmonella pathogenicity island 1 [SPI-1] and SPI-2). SptP is a Salmonella SPI-1 effector, involved in mediating recovery of the host cytoskeleton postinfection. SptP requires a chaperone, SicP, for stability and secretion. SptP has 94% identity between S. enterica serovar Typhimurium and S Typhi; direct comparison of the protein sequences revealed that S Typhi SptP has numerous amino acid changes within its chaperone-binding domain. Subsequent comparison of ΔsptP S Typhi and S. Typhimurium strains demonstrated that, unlike SptP in S. Typhimurium, SptP in S Typhi was not involved in invasion or cytoskeletal recovery postinfection. Investigation of whether the observed amino acid changes within SptP of S Typhi affected its function revealed that S Typhi SptP was unable to complement S. Typhimurium ΔsptP due to an absence of secretion. We further demonstrated that while S. Typhimurium SptP is stable intracellularly within S Typhi, S Typhi SptP is unstable, although stability could be recovered following replacement of the chaperone-binding domain with that of S. Typhimurium. Direct assessment of the strength of the interaction between SptP and SicP of both serovars via bacterial two-hybrid analysis demonstrated that S Typhi SptP has a significantly weaker interaction with SicP than the equivalent proteins in S. Typhimurium. Taken together, our results suggest that changes within the chaperone-binding domain of SptP in S Typhi hinder binding to its chaperone, resulting in instability, preventing translocation, and therefore restricting the intracellular activity of this effector. IMPORTANCE: Studies investigating Salmonella pathogenesis typically rely on Salmonella Typhimurium, even though Salmonella Typhi causes the more severe disease in humans. As such, an understanding of S. Typhi pathogenesis is lacking. Differences within the type III secretion system effector SptP between typhoidal and nontyphoidal serovars led us to characterize this effector within S Typhi. Our results suggest that SptP is not translocated from typhoidal serovars, even though the loss of sptP results in virulence defects in S. Typhimurium. Although SptP is just one effector, our results exemplify that the behavior of these serovars is significantly different and genes identified to be important for S. Typhimurium virulence may not translate to S Typhi.

The Type III Secretion System Effector SeoC of Salmonella enterica subsp. salamae and S. enterica subsp. arizonae ADP-Ribosylates Src and Inhibits Opsonophagocytosis.

Salmonella species utilize type III secretion systems (T3SSs) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study, we compared a draft genome assembly of Salmonella enterica subsp. salamae strain 3588/07 against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and Salmonella bongori strain 12419. S. enterica subsp. salamae encodes the Salmonella pathogenicity island 1 (SPI-1), SPI-2, and the locus of enterocyte effacement (LEE) T3SSs. Though several key S Typhimurium effector genes are missing (e.g., avrA, sopB, and sseL), S. enterica subsp. salamae invades HeLa cells and contains homologues of S. bongori sboK and sboC, which we named seoC SboC and SeoC are homologues of EspJ from enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), which inhibit Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates, we identified EspJ homologues in S. bongori, S. enterica subsp. salamae, and Salmonella enterica subsp. arizonae The ß-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized beads into Cos-7 cells stably expressing green fluorescent protein (GFP)-FcγRIIa. Concurrently, S. enterica subsp. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC-dependent manner. These results show that S. bongori, S. enterica subsp. salamae, and S. enterica subsp. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and shed light on the complexities of the T3SS effector repertoires of Enterobacteriaceae.

Older leaves of lettuce (Lactuca spp.) support higher levels of Salmonella enterica ser. Senftenberg attachment and show greater variation between plant accessions than do younger leaves.

Salmonella can bind to the leaves of salad crops including lettuce and survive for commercially relevant periods. Previous studies have shown that younger leaves are more susceptible to colonization than older leaves and that colonization levels are dependent on both the bacterial serovar and the lettuce cultivar. In this study, we investigated the ability of two Lactuca sativa cultivars (Saladin and Iceberg) and an accession of wild lettuce (L. serriola) to support attachment of Salmonella enterica serovar Senftenberg, to the first and fifth to sixth true leaves and the associations between cultivar-dependent variation in plant leaf surface characteristics and bacterial attachment. Attachment levels were higher on older leaves than on the younger ones and these differences were associated with leaf vein and stomatal densities, leaf surface hydrophobicity and leaf surface soluble protein concentrations. Vein density and leaf surface hydrophobicity were also associated with cultivar-specific differences in Salmonella attachment, although the latter was only observed in the older leaves and was also associated with level of epicuticular wax.

The type III secretion effector NleF of enteropathogenic Escherichia coli activates NF-κB early during infection.

The enteric pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli employ a type 3 secretion system (T3SS) to manipulate the host inflammatory response during infection. Previously, it has been reported that EPEC, in a T3SS-dependent manner, induces an early proinflammatory response through activation of NF-κB via extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase Cζ (PKCζ). However, the activation of NF-κB during infection has not yet been attributed to an effector. At later time points postinfection, NF-κB signaling is inhibited through the translocation of multiple effectors, including NleE and NleC. Here we report that the highly conserved non-LEE (locus of enterocyte effacement)-encoded effector F (NleF) shows both diffuse and mitochondrial localization during ectopic expression. Moreover, NleF induces the nuclear translocation of NF-κB p65 and the expression of interleukin 8 (IL-8) following ectopic expression and during EPEC infection. Furthermore, the proinflammatory activity and localization of NleF were dependent on the C-terminal amino acids LQCG. While the C-terminal domain of NleF has previously been shown to be essential for interaction with caspase-4, caspase-8, and caspase-9, the proinflammatory activity of NleF was independent of interaction with caspase-4, -8, or -9. In conclusion, EPEC, through the T3SS-dependent translocation of NleF, induces a proinflammatory response in an NF-κB-dependent manner in the early stages of infection.

EspZ of enteropathogenic and enterohemorrhagic Escherichia coli regulates type III secretion system protein translocation.

UNLABELLED: Translocation of effector proteins via a type III secretion system (T3SS) is a widespread infection strategy among Gram-negative bacterial pathogens. Each pathogen translocates a particular set of effectors that subvert cell signaling in a way that suits its particular infection cycle. However, as effector unbalance might lead to cytotoxicity, the pathogens must employ mechanisms that regulate the intracellular effector concentration. We present evidence that the effector EspZ controls T3SS effector translocation from enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Consistently, an EPEC espZ mutant is highly cytotoxic. Following ectopic expression, we found that EspZ inhibited the formation of actin pedestals as it blocked the translocation of Tir, as well as other effectors, including Map and EspF. Moreover, during infection EspZ inhibited effector translocation following superinfection. Importantly, while EspZ of EHEC O157:H7 had a universal "translocation stop" activity, EspZ of EPEC inhibited effector translocation from typical EPEC strains but not from EHEC O157:H7 or its progenitor, atypical EPEC O55:H7. We found that the N and C termini of EspZ, which contains two transmembrane domains, face the cytosolic leaflet of the plasma membrane at the site of bacterial attachment, while the extracellular loop of EspZ is responsible for its strain-specific activity. These results show that EPEC and EHEC acquired a sophisticated mechanism to regulate the effector translocation. IMPORTANCE: Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important diarrheal pathogens responsible for significant morbidity and mortality in developing countries and the developed world, respectively. The virulence strategy of EPEC and EHEC revolves around a conserved type III secretion system (T3SS), which translocates bacterial proteins known as effectors directly into host cells. Previous studies have shown that when cells are infected in two waves with EPEC, the first wave inhibits effector translocation by the second wave in a T3SS-dependent manner, although the factor involved was not known. Importantly, we identified EspZ as the effector responsible for blocking protein translocation following a secondary EPEC infection. Interestingly, we found that while EspZ of EHEC can block protein translocation from both EPEC and EHEC strains, EPEC EspZ cannot block translocation from EHEC. These studies show that EPEC and EHEC employ a novel infection strategy to regulate T3SS translocation.

Origins of tumor-associated macrophages and neutrophils.

Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost all solid neoplasms. The cells are known to descend from immature monocytic and granulocytic cells, respectively, which are produced in the bone marrow. However, the spleen is also a recently identified reservoir of monocytes, which can play a significant role in the inflammatory response that follows acute injury. Here, we evaluated the role of the splenic reservoir in a genetic mouse model of lung adenocarcinoma driven by activation of oncogenic Kras and inactivation of p53. We found that high numbers of TAM and TAN precursors physically relocated from the spleen to the tumor stroma, and that recruitment of tumor-promoting spleen-derived TAMs required signaling of the chemokine receptor CCR2. Also, removal of the spleen, either before or after tumor initiation, reduced TAM and TAN responses significantly and delayed tumor growth. The mechanism by which the spleen was able to maintain its reservoir capacity throughout tumor progression involved, in part, local accumulation in the splenic red pulp of typically rare extramedullary hematopoietic stem and progenitor cells, notably granulocyte and macrophage progenitors, which produced CD11b(+) Ly-6C(hi) monocytic and CD11b(+) Ly-6G(hi) granulocytic cells locally. Splenic granulocyte and macrophage progenitors and their descendants were likewise identified in clinical specimens. The present study sheds light on the origins of TAMs and TANs, and positions the spleen as an important extramedullary site, which can continuously supply growing tumors with these cells.

Salmonella enterica strains belonging to O serogroup 1,3,19 induce chlorosis and wilting of Arabidopsis thaliana leaves.

The number of outbreaks and illness linked to the consumption of contaminated salad leaves have increased dramatically in the last decade. Escherichia coli and Salmonella enterica are the most common food-borne pathogens linked to consumption of fresh produce. Different serovars of S. enterica subspecies enterica have been shown to bind the surface of salad leaves, to exhibit tropism towards the stomata and to invade leaves and reach the underlying mesophyll. However the consequences of leaf invasion are not known. Here we show that following infiltration, serovars Typhimurium, Enteritidis, Heidelberg and Agona, as well as strains of S. enterica subspecies arizonae and diarizonae, survive in the mesophyll of Arabidopsis thaliana leaves but induce neither leaf chlorosis nor wilting. In contrast, S. Senftenberg induced strong leaf wilting 4 days post infiltration in A. thaliana accession Col-0 but not in accession Ws-0. Dead S. Senftenberg and bacterial lysates also induced leaf wilting. We found that mutations in the Arabidopsis pathogen associated molecular pattern (PAMP) recognition receptors (PRRs) FLS2, which recognizes flagellin, and EFR, which recognizes the bacterial elongation factor EF-Tu, had no effect on the wilting response of A. thaliana to S. Senftenberg. Infiltration of A. thaliana leaves with serovars Cannstatt, Krefeld and Liverpool, which like Senftenberg belong to Salmonella serogroup E(4) (O:1,3,19), also resulted in rapid leaf wilting, while all tested rough S. Senftenberg strains (lacking the O antigen) failed to elicit leaf wilting. These results suggest that the Salmonella O antigen 1,3,19 specifically triggers leaf chlorosis and wilting in A. thaliana.

Flagella mediate attachment of enterotoxigenic Escherichia coli to fresh salad leaves.

Enterotoxigenic Escherichia coli (ETEC) causes child and travelers' diarrhea and is presumed to be water- and food-borne. Sporadic outbreaks were traced to consumption of contaminated fresh produce, particularly salad leaves as lettuce and parsley. Importantly, the mechanism by which ETEC binds salad leaves is not known. In this study we investigated the ability of clinical ETEC isolates to adhere to Eruca vesicaria (commonly known as rocket). Towards this end we inoculated pieces of cut E. vesicaria leaves with clinical ETEC isolates grown in Luria broth at 20°C, conditions that are not permissive for expression of the plasmid-encoded colonization factors and hence mimic the actual transmission pathways of ETEC through intake of contaminated food. We found that ETEC strains bind E. vesicaria at various efficiencies. Examination of representative strains by scanning electron microscopy revealed that they adhere to the E. vesicaria surface in a diffuse pattern by extended filaments resembling flagella. Using the prototype ETEC strain H10407 we found that it also binds to lettuce, basil and spinach leaves. Binding of H10407 was dependent on flagella as a fliC mutant attached to leaves at a much lower efficiency. Interestingly, under the employed environmental conditions EtpA, which forms a flagellar tip structure, and colonization factor I are dispensable for leaf attachment. The results show that ETEC can bind specifically to salad leaves, which might represent an important, yet less recognized, source of infection.

Cellulose mediates attachment of Salmonella enterica Serovar Typhimurium to tomatoes.

Fresh fruit and vegetables are important components of a healthy and balanced diet. However, they are increasingly being recognized as important vehicles for transmission of human pathogens that were traditionally classified as zoonotic. There is a significant gap in our knowledge and understanding of the mechanisms by which human pathogens colonize and survive on or in fruits and vegetables. In this study we investigated the binding of Salmonella enterica to tomato fruits (Solanum lycopersicum), which is becoming a major source of human infection. We report that Salmonella enterica serovars Typhimurium and Senftenberg bound to the surface of unripe tomatoes in an aggregative pattern, while serovar Thompson adhered diffusely. We found that while flagella did not have a role in binding, bcsC S. Typhimurim mutants, deficient in cellulose production, exhibited significantly reduced level of attachment to tomatoes. Trans complementation of the mutation restored adhesion to the wild-type level.

Fresh fruit and vegetables as vehicles for the transmission of human pathogens.

Much research into food-borne human pathogens has focused on transmission from foods of animal origin. However, recent investigations have identified fruits and vegetables are the source of many disease outbreaks. Now believed to be a much larger contributor to produce-associated outbreaks than previously reported, norovirus outbreaks are commonly caused by contamination of foods from hands of infected workers. Although infections with Shiga toxin-producing E. coli O157 have been linked to beef more often than to any other food product, severe outbreaks have been traced to consumption of contaminated radish sprouts and pre-packaged spinach. Similarly, while infections with Salmonella have mainly been linked to consumption of foods of animal origin, many outbreaks have been traced to contaminated fresh produce. E. coli O157 binds to lettuce leaves by alternative mechanisms involving the filamentous type III secretions system, flagella and the pilus curli. Association of Salmonella with fresh produce appears to be serovar-specific involving flagella, curli, cellulose, and O antigen capsule. A better understanding of plant, microbiological, environmental, processing and food handling factors that facilitate contamination will allow development of evidence-based policies, procedures and technologies aimed at reducing the risk of contamination of fresh produce.

Binding to Na(+) /H(+) exchanger regulatory factor 2 (NHERF2) affects trafficking and function of the enteropathogenic Escherichia coli type III secretion system effectors Map, EspI and NleH.

Enteropathogenic Escherichia coli (EPEC) strains are diarrhoeal pathogens that use a type III secretion system to translocate effector proteins into host cells in order to colonize and multiply in the human gut. Map, EspI and NleH1 are conserved EPEC effectors that possess a C-terminal class I PSD-95/Disc Large/ZO-1 (PDZ)-binding motif. Using a PDZ array screen we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), a scaffold protein involved in tethering and recycling ion channels in polarized epithelia that contains two PDZ domains, as a common target of Map, EspI and NleH1. Using recombinant proteins and co-immunoprecipitation we confirmed that NHERF2 binds each of the effectors. We generated a HeLa cell line stably expressing HA-tagged NHERF2 and found that Map, EspI and NleH1 colocalize and interact with intracellular NHERF2 via their C-terminal PDZ-binding motif. Overexpression of NHERF2 enhanced the formation and persistence of Map-induced filopodia, accelerated the trafficking of EspI to the Golgi and diminished the anti-apoptotic activity of NleH1. The binding of multiple T3SS effectors to a single scaffold protein is unique. Our data suggest that NHERF2 may act as a plasma membrane sorting site, providing a novel regulatory mechanism to control the intracellular spatial and temporal effector protein activity.

The enteropathogenic Escherichia coli effector NleH inhibits apoptosis induced by Clostridium difficile toxin B.

Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coli (EPEC) type III secretion system effector NleH has a broad-range anti-apoptotic activity. In this study we examined the effects of NleH on cells challenged with TcdB. During infection with wild-type EPEC, NleH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NleH acts via a global anti-apoptotic pathway.