Cedric N. Berger

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.

pH-, Lactic Acid-, and Non-Lactic Acid-Dependent Activities of Probiotic Lactobacilli against Salmonella enterica Serovar Typhimurium

ABSTRACT The mechanism(s) underlying the antibacterial activity of probiotic Lactobacillus strains appears to be multifactorial and includes lowering of the pH and the production of lactic acid and of antibacterial compounds, including bacteriocins and nonbacteriocin, non-lactic acid molecules. Addition of Dulbeccos modified Eagles minimum essential medium to the incubating medium delays the killing activity of lactic acid. We found that the probiotic strains Lactobacillus johnsonii La1, Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029, L. casei DN-114 001, and L. rhamnosus GR1 induced a dramatic decrease in the viability of Salmonella enterica serovar Typhimurium SL1344 mainly attributable to non-lactic acid molecule(s) present in the cell-free culture supernatant (CFCS). These molecules were more active against serovar Typhimurium SL1344 in the exponential growth phase than in the stationary growth phase. We also showed that the production of the non-lactic acid substance(s) responsible for the killing activity was dependent on growth temperature and that both unstable and stable substances with killing activity were present in the CFCSs. We found that the complete inhibition of serovar Typhimurium SL1344 growth results from a pH-lowering effect.

NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic Escherichia coli infection

The human pathogens enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli and the related mouse pathogen Citrobacter rodentium subvert a variety of host cell signaling pathways via their plethora of type III secreted effectors, including triggering of an early apoptotic response. EPEC-infected cells do not develop late apoptotic symptoms, however. In this study we demonstrate that the NleH family effectors, homologs of the Shigella effector kinase OspG, blocks apoptosis. During EPEC infection, NleH effectors inhibit elevation of cytosolic Ca2+ concentrations, nuclear condensation, caspase-3 activation, and membrane blebbing and promote cell survival. NleH1 alone is sufficient to prevent procaspase-3 cleavage induced by the proapoptotic compounds staurosporine, brefeldin A, and tunicamycin. Using C. rodentium, we found that NleH inhibits procaspase-3 cleavage at the bacterial attachment sites in vivo. A yeast two-hybrid screen identified the endoplasmic reticulum six-transmembrane protein Bax inhibitor-1 (BI-1) as an NleH-interacting partner. We mapped the NleH-binding site to the N-terminal 40 amino acids of BI-1. Knockdown of BI-1 resulted in the loss of NleH’s antiapoptotic activity. These results indicate that NleH effectors are inhibitors of apoptosis that may act through BI-1 to carry out their cytoprotective function.

Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC)

Little is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins. These adhesins recognize as receptors the GPI‐anchored proteins CD55 (decay‐accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA). CD66e is a member of the CEA‐related cell adhesion molecules (CEACAM) family, comprising seven members. We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM‐expressing CHO and HeLa cells. The results demonstrate that only E. coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr‐I, including Dr, F1845 and AfaE‐III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6. Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr‐I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6. In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recruited around bacteria in HeLa cells. The recruited CEACAM1, CEA and CEACAM6 around adhering bacteria resist totally or in part a detergent extraction, whereas the recruited CEACAM3 does not. Finally, the results show that recognition of CEA and CEACAM6, but not CEACAM1, is accompanied by tight attachment to bacteria of cell surface microvilli‐like extensions, which are elongated. Moreover, recognition of CEA is accompanied by an activation of the Rho GTPase Cdc42 and by a phosphorylation of ERM, which in turn elicit the observed cell surface microvilli‐like extensions.

Enterohemorrhagic Escherichia coli Exploits EspA Filaments for Attachment to Salad Leaves

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens that use a filamentous type III secretion system (fT3SS) for colonization of the gut epithelium. In this study we have shown that EHEC O157 and O26 strains use the fT3SS apparatus for attachment to leaves. Leaf attachment was independent of effector protein translocation.

Interaction of Salmonella enterica with basil and other salad leaves

Contaminated salad leaves have emerged as important vehicles for the transmission of enteric pathogens to humans. A recent outbreak of Salmonella enterica serovar Senftenberg (S. Senftenberg) in the United Kingdom has been traced to the consumption of contaminated basil. Using the outbreak strain of S. Senftenberg, we found that it binds to basil, lettuce, rocket and spinach leaves showing a pattern of diffuse adhesion. Flagella were seen linking S. Senftenberg to the leaf epidermis, and the deletion of fliC (encoding phase-1 flagella) resulted in a significantly reduced level of adhesion. In contrast, although flagella linking S. enterica serovar Typhimurium to the basil leaf epidermis were widespread, deletion of fliC did not affect leaf attachment levels. These results implicate the role of flagella in Salmonella leaf attachment and suggest that different Salmonella serovars use strain-specific mechanisms to attach to salad leaves.

Bacterial Guanine Nucleotide Exchange Factors SopE-Like and WxxxE Effectors

ABSTRACT Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce “the trigger mechanism of cell entry.” Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection.

Subversion of actin dynamics by EspM effectors of attaching and effacing bacterial pathogens

Rho GTPases are common targets of bacterial toxins and type III secretion system effectors. IpgB1 and IpgB2 of Shigella and Map of enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli were recently grouped together on the basis that they share a conserved WxxxE motif. In this study, we characterized six WxxxE effectors from attaching and effacing pathogens: TrcA and EspM1 of EPEC strain B171, EspM1 and EspM2 of EHEC strain Sakai and EspM2 and EspM3 of Citrobacter rodentium. We show that EspM2 triggers formation of global parallel stress fibres, TrcA and EspM1 induce formation of localized parallel stress fibres and EspM3 triggers formation of localized radial stress fibres. Using EspM2 and EspM3 as model effectors, we report that while substituting the conserved Trp with Ala abolished activity, conservative Trp to Tyr or Glu to Asp substitutions did not affect stress‐fibre formation. We show, using dominant negative constructs and chemical inhibitors, that the activity of EspM2 and EspM3 is RhoA and ROCK‐dependent. Using Rhotekin pull‐downs, we have shown that EspM2 and EspM3 activate RhoA; translocation of EspM2 and EspM3 triggered phosphorylation of cofilin. These results suggest that the EspM effectors modulate actin dynamics by activating the RhoA signalling pathway.

EspM2 is a RhoA guanine nucleotide exchange factor.

We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotide‐free RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H‐Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the catalytic loop of EspM2, with alanine, greatly attenuated the RhoA GEF activity in vitro and the ability of EspM2 to induce stress fibres upon ectopic expression. These results suggest that binding of SifA to RhoA does not trigger nucleotide exchange while EspM2 is a unique Rho GTPase GEF.

The mechanisms used by enteropathogenic Escherichia coli to control filopodia dynamics

Enteropathogenic Escherichia coli (EPEC) subverts actin dynamics in eukaryotic cells by injecting effector proteins via a type III secretion system. First, WxxxE effector Map triggers transient formation of filopodia. Then, following recovery from the filopodial signals, EPEC triggers robust actin polymerization via a signalling complex comprising Tir and the adaptor proteins Nck. In this paper we show that Map triggers filopodia formation by activating Cdc42; expression of dominant‐negative Cdc42 or knock‐down of Cdc42 by siRNA impaired filopodia formation. In addition, Map binds PDZ1 of NHERF1. We show that Map–NHERF1 interaction is needed for filopodia stabilization in a process involving ezrin and the RhoA/ROCK cascade; expression of dominant‐negative ezrin and RhoA or siRNA knock‐down of RhoA lead to rapid elimination of filopodia. Moreover, we show that formation of the Tir‐Nck signalling complex leads to filopodia withdrawal. Recovery from the filopodial signals requires phosphorylation of a Tir tyrosine (Y474) residue and actin polymerization pathway as both infection of cells with EPEC expressing TirY474S or infection of Nck knockout cells with wild‐type EPEC resulted in persistence of filopodia. These results show that EPEC effectors modulate actin dynamics by temporal subverting the Rho GTPases and other actin polymerization pathways for the benefit of the adherent pathogen.