Secreted Autotransporter Toxin (Sat) Mediates Innate Immune System Evasion.

Several strategies are used by Escherichia coli to evade the host innate immune system in the blood, such as the cleavage of complement system proteins by secreted proteases. Members of the Serine Proteases Autotransporters of Enterobacteriaceae (SPATE) family have been described as presenting proteolytic effects against complement proteins. Among the SPATE-encoding genes sat (secreted autotransporter toxin) has been detected in high frequencies among strains of E. coli isolated from bacteremia. Sat has been characterized for its cytotoxic action, but the possible immunomodulatory effects of Sat have not been investigated. Therefore, this study aimed to evaluate the proteolytic effects of Sat on complement proteins and the role in pathogenesis of BSI caused by extraintestinal E. coli (ExPEC). E. coli EC071 was selected as a Sat-producing ExPEC strain. Whole-genome sequencing showed that sat sequences of EC071 and uropathogenic E. coli CFT073 present 99% identity. EC071 was shown to be resistant to the bactericidal activity of normal human serum (NHS). Purified native Sat was used in proteolytic assays with proteins of the complement system and, except for C1q, all tested substrates were cleaved by Sat in a dose and time-dependent manner. Moreover, E. coli DH5α survived in NHS pre-incubated with Sat. EC071-derivative strains harboring sat knockout and in trans complementations producing either active or non-active Sat were tested in a murine sepsis model. Lethality was reduced by 50% when mice were inoculated with the sat mutant strain. The complemented strain producing active Sat partially restored the effect caused by the wild-type strain. The results presented in this study show that Sat presents immunomodulatory effects by cleaving several proteins of the three complement system pathways. Therefore, Sat plays an important role in the establishment of bloodstream infections and sepsis.

Secreted autotransporter toxin (Sat) mediates innate immune system evasion

Several strategies are used by Escherichia coli to evade the host innate immune system in the blood, such as the cleavage of complement system proteins by secreted proteases. Members of the Serine Proteases Autotransporters of Enterobacteriaceae (SPATE) family have been described as presenting proteolytic effects against complement proteins. Among the SPATE-encoding genes sat (secreted autotransporter toxin) has been detected in high frequencies among strains of E. coli isolated from bacteremia. Sat has been characterized for its cytotoxic action, but the possible immunomodulatory effects of Sat have not been investigated. Therefore, this study aimed to evaluate the proteolytic effects of Sat on complement proteins and the role in pathogenesis of BSI caused by extraintestinal E. coli (ExPEC). E. coli EC071 was selected as a Sat-producing ExPEC strain. Whole-genome sequencing showed that sat sequences of EC071 and uropathogenic E. coli CFT073 present 99% identity. EC071 was shown to be resistant to the bactericidal activity of normal human serum (NHS). Purified native Sat was used in proteolytic assays with proteins of the complement system and, except for C1q, all tested substrates were cleaved by Sat in a dose and time-dependent manner. Moreover, E. coli DH5α survived in NHS pre-incubated with Sat. EC071-derivative strains harboring sat knockout and in trans complementations producing either active or non-active Sat were tested in a murine sepsis model. Lethality was reduced by 50% when mice were inoculated with the sat mutant strain. The complemented strain producing active Sat partially restored the effect caused by the wild-type strain. The results presented in this study show that Sat presents immunomodulatory effects by cleaving several proteins of the three complement system pathways. Therefore, Sat plays an important role in the establishment of bloodstream infections and sepsis.

The Type III Secretion System (T3SS)-Translocon of Atypical Enteropathogenic Escherichia coli (aEPEC) Can Mediate Adherence.

The intimin protein is the major adhesin involved in the intimate adherence of atypical enteropathogenic Escherichia coli (aEPEC) strains to epithelial cells, but little is known about the structures involved in their early colonization process. A previous study demonstrated that the type III secretion system (T3SS) plays an additional role in the adherence of an Escherichia albertii strain. Therefore, we assumed that the T3SS could be related to the adherence efficiency of aEPEC during the first stages of contact with epithelial cells. To test this hypothesis, we examined the adherence of seven aEPEC strains and their eae (intimin) isogenic mutants in the standard HeLa adherence assay and observed that all wild-type strains were adherent while five isogenic eae mutants were not. The two eae mutant strains that remained adherent were then used to generate the eae/escN double mutants (encoding intimin and the T3SS ATPase, respectively) and after the adherence assay, we observed that one strain lost its adherence capacity. This suggested a role for the T3SS in the initial adherence steps of this strain. In addition, we demonstrated that this strain expressed the T3SS at significantly higher levels when compared to the other wild-type strains and that it produced longer translocon-filaments. Our findings reveal that the T3SS-translocon can play an additional role as an adhesin at the beginning of the colonization process of aEPEC.

The type III secretion system (T3SS)-Translocon of atypical enteropathogenic Escherichia coli (aEPEC) can mediate adherence

The intimin protein is the major adhesin involved in the intimate adherence of atypicalenteropathogenicEscherichia coli(aEPEC) strains to epithelial cells, but little is knownabout the structures involved in their early colonization process. A previous studydemonstrated that the type III secretion system (T3SS) plays an additional role in theadherence of anEscherichia albertiistrain. Therefore, we assumed that the T3SS couldbe related to the adherence efficiency of aEPEC during the first stages of contactwith epithelial cells. To test this hypothesis, we examined the adherence of sevenaEPEC strains and theireae(intimin) isogenic mutants in the standard HeLa adherenceassay and observed that all wild-type strains were adherent while five isogeniceaemutants were not. The twoeaemutant strains that remained adherent were then usedto generate theeae/escNdouble mutants (encoding intimin and the T3SS ATPase,respectively) and after the adherence assay, we observed that one strain lost itsadherence capacity. This suggested a role for the T3SS in the initial adherence stepsof this strain. In addition, we demonstrated that this strain expressed the T3SS atsignificantly higher levels when compared to the other wild-type strains and that itproduced longer translocon-filaments. Our findings reveal that the T3SS-transloconcan play an additional role as an adhesin at the beginning of the colonization processof aEPEC.

Draft Whole-Genome Sequences of 10 Atypical Enteropathogenic Escherichia coli Strains Isolated in Brazil.

The number of diarrhea cases caused by atypical enteropathogenic Escherichia coli (aEPEC) has been increasing worldwide. Here, we report the draft whole-genome sequences of 10 aEPEC strains isolated in Brazil. These sequences will provide an important source for future studies concerning aEPEC pathogenicity and genetic markers of potentially virulent strains.

Draft whole-genome sequences of 10 atypical enteropathogenic Escherichia coli strains isolated in Brazil

The number of diarrhea cases caused by atypical enteropathogenic Escherichia coli (aEPEC) has been increasing worldwide. Here, we report the draft whole-genome sequences of 10 aEPEC strains isolated in Brazil. These sequences will provide an important source for future studies concerning aEPEC pathogenicity and genetic markers of potentially virulent strains.

Natural and vaccine-mediated immunity to Salmonella Typhimurium is impaired by the helminth Nippostrongylus brasiliensis.

BACKGROUND: The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined. METHODOLOGY/PRINCIPAL FINDINGS: Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection. CONCLUSION: These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization.

Genotypic and phenotypic characterisation of enteroaggregative Escherichia coli from children in Rio de Janeiro, Brazil.

Enteroaggregative Escherichia coli (EAEC) is a significant cause of diarrhoeal illness in both children and adults. Genetic heterogeneity and recovery of EAEC strains from both healthy and diseased individuals complicates our understanding of EAEC pathogenesis. We wished to establish if genetic or phenotypic attributes could be used to distinguish between strains asymptomatically colonising healthy individuals and those which cause disease. Genotypic screening of a collection of twenty four EAEC isolates from children with and without diarrhoea revealed no significant differences in the repertoire of putative virulence factors present in either group of strains. In contrast, EAEC strains from phylogroup A were more strongly associated with asymptomatic groups whereas strains from phylogroup D were more associated with cases of diarrhoea. Phenotypic screening revealed no differences in the ability of strains from either cohort of children to form biofilms, to adhere to and invade cells in tissue culture or to cause disease in the Caenorhabditis elegans model of infection. However, the latter assay did reveal significant reduction in nematode killing rates when specific virulence factors were deleted from human pathogenic strains. Our results suggest that current models of infection are not useful for distinguishing avirulent from pathogenic strains of EAEC but can be useful in studying the effect of specific virulence factors.

SadA, a Trimeric Autotransporter from Salmonella enterica SerovarTyphimurium, Can Promote Biofilm Formation and Provides Limited Protection against Infection

Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromisedindividuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance becausethey are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA,a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. Wedemonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice.Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinalCaco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG responsewhich provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced byadministering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadAhaving pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection againstSalmonella.