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Shiga toxin-producing Escherichia coli O157:H7 is a virulent strain causing severe gastrointestinal infection, hemolytic uremic syndrome and death. To date there are no specific therapies to reduce progression of disease. Here we investigated the effect of pooled immunoglobulins (IgG) on the course of disease in a mouse model of intragastric E. coli O157:H7 inoculation. Intraperitoneal administration of murine IgG on day 3, or both on day 3 and 6, post-inoculation improved survival and decreased intestinal and renal pathology. When given on both day 3 and 6 post-inoculation IgG treatment also improved kidney function in infected mice. Murine and human commercially available IgG preparations bound to proteins in culture filtrates from E. coli O157:H7. Bound proteins were extracted from membranes and peptide sequences were identified by mass spectrometry. The findings showed that murine and human IgG bound to E. coli extracellular serine protease P (EspP) in the culture filtrate, via the IgG Fc domain. These results were confirmed using purified recombinant EspP and comparing culture filtrates from the wild-type E. coli O157:H7 strain to a deletion mutant lacking espP. Culture filtrates from wild-type E. coli O157:H7 exhibited enzymatic activity, specifically associated with the presence of EspP and demonstrated as pepsin cleavage, which was reduced in the presence of murine and human IgG. EspP is a virulence factor previously shown to promote colonic cell injury and the uptake of Shiga toxin by intestinal cells. The results presented here suggest that IgG binds to EspP, blocks its enzymatic activity, and protects the host from E. coli O157:H7 infection, even when given post-inoculation. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli O157:H7 is a virulent strain causing severe gastrointestinal infection, hemolytic uremic syndrome and death. To date there are no specific therapies to reduce progression of disease. Here we investigated the effect of pooled immunoglobulins (IgG) on the course of disease in a mouse model of intragastric E. coli O157:H7 inoculation. Intraperitoneal administration of murine IgG on day 3, or both on day 3 and 6, post-inoculation improved survival and decreased intestinal and renal pathology. When given on both day 3 and 6 post-inoculation IgG treatment also improved kidney function in infected mice. Murine and human commercially available IgG preparations bound to proteins in culture filtrates from E. coli O157:H7. Bound proteins were extracted from membranes and peptide sequences were identified by mass spectrometry. The findings showed that murine and human IgG bound to E. coli extracellular serine protease P (EspP) in the culture filtrate, via the IgG Fc domain. These results were confirmed using purified recombinant EspP and comparing culture filtrates from the wild-type E. coli O157:H7 strain to a deletion mutant lacking espP. Culture filtrates from wild-type E. coli O157:H7 exhibited enzymatic activity, specifically associated with the presence of EspP and demonstrated as pepsin cleavage, which was reduced in the presence of murine and human IgG. EspP is a virulence factor previously shown to promote colonic cell injury and the uptake of Shiga toxin by intestinal cells. The results presented here suggest that IgG binds to EspP, blocks its enzymatic activity, and protects the host from E. coli O157:H7 infection, even when given post-inoculation.

ABSTRACT Enteric pathogens have complex interactions with the gut microbiota. Most of what is known about them has focused on microbiota-derived metabolites or small molecules that serve as nutrients and/or signals to aid in growth or transcriptionally regulate virulence gene expression. A common virulence strategy is to express a type III secretion system (T3SS), which is a molecular syringe deployed by many Gram-negative pathogens to hijack host cell function. Enterohemorrhagic Escherichia coli (EHEC) requires its T3SS to colonize the intestinal tract and cause disease. Here we report that a prominent member of the intestinal microbiota, Bacteroides thetaiotamicron (Bt), secretes proteases that cleave the translocon of the T3SS of EHEC to enhance effector translocation into host cells. This is in contrast from an endogenous protease from EHEC itself (namely, EspP) that cleaves the translocon protein EspB in a different site to limit effector translocation. The EspB protein forms the T3SS pore in mammalian cells, and pore proteins are conserved in the T3SSs from several pathogens. This is the first demonstration of a commensal species directly processing a pathogen’s T3SS, posing a new paradigm for how the microbiota can influence the severity of disease caused by bacterial pathogens. Because T3SSs are employed by many pathogens, this phenomenon has broad implications to commensal-pathogen relationships. IMPORTANCE The gut microbiota is usually regarded as providing colonization resistance against enteric pathogens. However, some pathogens evolved to thrive with the aid of certain members of the microbiota. Several Gram-negative bacteria employ type three secretion systems (T3SSs), which are molecular syringes that deliver effector proteins to host cells, hijacking host cell function. Here we show that the T3SS of enterohemorrhagic E. coli (EHEC) is cleaved by self and microbiota-derived proteases. Self-cleavage limits effector translocation, while cleavage by the microbiota member Bacteroides thetaiotamicron (Bt) exacerbates effector translocation and lesion formation on epithelial cells.

We previously developed attenuated rabbit enteropathogenic E. coli (rEPEC) strains which are effective oral vaccines against their parent pathogens by deleting le r, a global regulator of virulence genes. To use these strains as orally administered vectors to deliver other antigens we incorporated the B subunit of shiga-like toxin 1(Stx1) into the passenger domain of the autotransporter EspP expressed on a plasmid. Native EspP enters the periplasm where its passenger domain is exported to the bacterial surface through an outer membrane channel formed by its translocator domain, then cleaved and secreted. Since antigen localization may determine immunogenicity, we engineered derivatives of EspP expressing Stx1B- passenger domain fusions: 1. in cytoplasm 2. in periplasm, 3. surface-attached or 4. secreted. To determine which construct was most immunogenic, rabbits were immunized with attenuated O103 E. coli strain (E22 Δler) alone or expressing Stx1B in each of the above four cellular locations. IgG responses to Stx1B, and toxin-neutralizing antibodies were measured. Animals were challenged with a virulent rabbit Enterohemorrhagic E. coli (EHEC) strain of a different serogroup (O15) than the vaccine strain expressing Stx1 (RDEC-H19) and their clinical course observed. IgG responses to Stx1B subunit were induced in all animals vaccinated with the strain secreting Stx1B, in some vaccinated with surface-expressed Stx1B, but in not animals immunized with periplasmic or cytoplasmic Stx1B. Robust protection was observed only in the group immunized with the vaccine secreting Stx1B. Taken together, our data suggest that secretion of Stx1B, or other antigens, via an autotransporter, may maximize the protective response to live attenuated oral vaccine strains. [ABSTRACT FROM AUTHOR]

Enterotoxigenic Escherichia coli (ETEC), defined by the production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of diarrhea in animals, children in developing countries and to travelers. No broadly protective ETEC vaccine is available, largely because of the difficulty in inducing immunity to the small ST molecule. To take advantage of the demonstration (Liu et al., 2011; Zhang et al., 2013, 2010) that genetically produced fusions of mutant ST with LT subunits can induce effective immunity against both toxins, we engineered a live attenuated vaccine vector strain of E. coli (ZCR533), expressing the immunogenic LT-ST fusions. To present the LT-ST fusions to the mucosal immune system, we used restriction-free cloning to incorporate them into the passenger domain of the autotransporter protein (EspP) expressed on a medium copy number plasmid. This versatile system permits expression of incorporated antigens in either surface-bound or secreted forms by the ZCR533 vector, for delivery to the mucosal inductive sites. Incorporation of the fusions into EspP plasmids was confirmed by PCR and DNA sequencing. Protein expression was confirmed by Western blot of whole cell lysates and culture supernatents using polyclonal antisera to LT. Expression of the surface-targeted fusion on the surface of ZCR533 was confirmed by immuno-fluorescent staining. These studies show that antigenic LT-ST fusions can be surface-expressed or secreted, by our attenuated E. coli ZCR533 vaccine vector via the EspP autotransporter. These constructs could serve as broadly protective vaccine candidates to protect against both LT- and ST-producing ETEC. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic E. coli (EHEC) causes severe diseases in humans worldwide. One of its virulence factors is EspP, which belongs to the serine protease autotransporters of Enterobacteriaceae (SPATE) family. In this review we recapitulate the current data on prevalence, biogenesis, structural properties and functionality. EspP has been used to investigate mechanistic details of autotransport, and recent studies indicate that this transport mechanism is not autonomous but rather dependent on additional factors. Currently, five subtypes have been identified (EspPα-EspPε), with EspPα being associated with highly virulent EHEC serotypes and isolates from patients with severe disease. EspPα has been shown to degrade major proteins of the complement cascade, namely C3 and C5 and probably interferes with hemostasis by cleavage of coagulation factor V. Furthermore, EspPα is believed to contribute to biofilm formation perhaps by polymerization to rope-like structures. Together with the proteolytic activity, EspPα might ameliorate host colonization and interfere with host response.

Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins.

Using novel data on employee stock purchase plans (ESPPs), we show that aggregate purchases of company stock by lower-level employees predict future stock returns. Firms in the top quartile of ESPP purchases outperform those in the bottom quartile by 10% in the year after purchase. The relation between ESPP purchases and future stock returns is stronger for firms with high information asymmetry. Furthermore, we find that high ESPP purchases are associated with a lower likelihood of breaks in strings of consecutive earnings increases, as well as higher future sales growth and more innovation. These findings support the hypothesis that lower-level employees have information about future firm performance. We examine and reject a number of alternative explanations. Our results have implications for firms using employees as a source of capital, accounting issues related to expensing of equity-based compensation, and disclosure policy. Data, as supplemental material, are available at . This paper was accepted by Wei Jiang, finance. [ABSTRACT FROM AUTHOR]

Reports of renewed of interest in Employee Stock Purchase Plans have recently appeared, perhaps driven in part by the robust state of the stock market, that give a positive glow to these plans. But HR professionals should react with caution and carefully examine all aspects of these plans before enhancing or adopting one. Plans have recently been terminated and made less attractive to employees, and many companies have passed on the idea. Important data affecting plan design and employee participation have not been reported that contribute to a low level of interest. Involvement of major HR consultants is lacking and HR specialists appear to be in no hurry to champion these plans. Despite these factors, HR professionals should give these plans serious consideration because of their potential value for compensating employees in certain circumstances. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic E. coli (EHEC) causes severe diseases in humans worldwide. One of its virulence factors is EspP, which belongs to the serine protease autotransporters of Enterobacteriaceae (SPATE) family. In this review we recapitulate the current data on prevalence, biogenesis, structural properties and functionality. EspP has been used to investigate mechanistic details of autotransport, and recent studies indicate that this transport mechanism is not autonomous but rather dependent on additional factors. Currently, five subtypes have been identified (EspPα-EspPε), with EspPα being associated with highly virulent EHEC serotypes and isolates from patients with severe disease. EspPα has been shown to degrade major proteins of the complement cascade, namely C3 and C5 and probably interferes with hemostasis by cleavage of coagulation factor V. Furthermore, EspPα is believed to contribute to biofilm formation perhaps by polymerization to rope-like structures. Together with the proteolytic activity, EspPα might ameliorate host colonization and interfere with host response. [ABSTRACT FROM AUTHOR]

Abstract: Autotransporters are secreted proteins produced by pathogenic Gram-negative bacteria. They consist of a membrane-embedded β-domain and an extracellular passenger domain that is sometimes cleaved and released from the cell surface. We solved the structures of three noncleavable mutants of the autotransporter EspP to examine how it promotes asparagine cyclization to cleave its passenger. We found that cyclization is facilitated by multiple factors. The active-site asparagine is sterically constrained to conformations favorable for cyclization, while electrostatic interactions correctly orient the carboxamide group for nucleophilic attack. During molecular dynamics simulations, water molecules were observed to enter the active site and to form hydrogen bonds favorable for increasing the nucleophilicity of the active-site asparagine. When the activated asparagine attacks its main-chain carbonyl carbon, the resulting oxyanion is stabilized by a protonated glutamate. Upon cleavage, this proton could be transferred to the leaving amine group, helping overcome a significant energy barrier. Together, these findings provide insight into factors important for asparagine cyclization, a mechanism broadly used for protein cleavage. [Copyright &y& Elsevier]

Abstract: Besides Shiga toxins (Stx), Stx-producing Escherichia coli (STEC) harbour several other putative virulence factors, including the serine protease EspP. We have investigated 214 STEC strains from Austria belonging to 61 different serotypes from humans, animals, and food for the presence of this serine protease gene and have determined the espP subtypes and their association with clinical outcome. espP was detected in 121 (57%) out of 214 strains. Sixty-five of 68 strains (96%) of non-sorbitol-fermenting (NSF) O157:H7/NM (NM, non-motile) were positive for espP, while none of 8 SF E. coli O157:NM isolates contained this gene. All 9 strains of serotype O145:NM and 17 of 21 strains (81%) of serotype O26:H11/NM were positive for espP. Nineteen STEC serogroups including O103 and O111 serogroups – considered to be highly pathogenic – were completely negative for espP. Only 5 of 12 strains isolated from patients suffering from haemolytic uraemic syndrome (HUS) were espP-positive (all serogroup NSF O157) as well as 28 of 39 strains from patients with bloody diarrhoea, 40 of 63 strains from patients with non-bloody diarrhoea, and 15 of 19 strains from asymptomatic patients. In O157:H7/NM, O26:H11/NM, and O145:NM only espP subtype α was found, whereas in most of the other non-O157 serogroups, subtypes β and γ were found. Subtype δ was not detected in our strain collection. Regarding the espP subtypes, only subtype α, but not β and γ, were found in HUS patients. Moreover, we could demonstrate that espP, and in particular subtype α, is associated with highly pathogenic serogroups. [Copyright &y& Elsevier]

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of zoonotic diarrhoeal pathogens of worldwide importance. Cattle are a key reservoir; however the molecular mechanisms that promote persistent colonization of the bovine intestines by EHEC are ill-defined. The large plasmid of EHEC O157:H7 encodes several putative virulence factors. Here, it is reported that the pO157-encoded Type V-secreted serine protease EspP influences the intestinal colonization of calves. To dissect the basis of attenuation, a bovine primary rectal epithelial cell line was developed. Adherence of E. coli O157:H7 to such cells was significantly impaired by espP mutation but restored upon addition of highly purified exogenous EspP. Data of this study add to the growing body of evidence that cytotoxins facilitate intestinal colonization by EHEC. [ABSTRACT FROM AUTHOR]