Brucella abortus Choloylglycine Hydrolase Affects Cell Envelope Composition and Host Cell Internalization

Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization

Immunospecific Responses to Bacterial Elongation Factor Tu during Burkholderia Infection and Immunization

Burkholderia pseudomallei is the etiological agent of melioidosis, a disease endemic in parts of Southeast Asia and Northern Australia. Currently there is no licensed vaccine against infection with this biological threat agent. In this study, we employed an immunoproteomic approach and identified bacterial Elongation factor-Tu (EF-Tu) as a potential vaccine antigen. EF-Tu is membrane-associated, secreted in outer membrane vesicles (OMVs), and immunogenic during Burkholderia infection in the murine model of melioidosis. Active immunization with EF-Tu induced antigen-specific antibody and cell-mediated immune responses in mice. Mucosal immunization with EF-Tu also reduced lung bacterial loads in mice challenged with aerosolized B. thailandensis. Our data support the utility of EF-Tu as a novel vaccine immunogen against bacterial infection

Yersinia enterocolitica palearctica serobiotype O:3/4 - a successful group of emerging zoonotic pathogens

<p>Abstract</p> <p>Background</p> <p>High-pathogenic <it>Y. enterocolitica </it>ssp. <it>enterocolitica </it>caused several human outbreaks in Northern America. In contrast, low pathogenic <it>Y. enterocolitica </it>ssp. <it>palearctica </it>serobiotype O:3/4 is responsible for sporadic cases worldwide with asymptomatic pigs being the main source of infection. Genomes of three <it>Y. enterocolitica </it>ssp. <it>palearctica </it>serobiotype O:3/4 human isolates (including the completely sequenced Y11 German DSMZ type strain) were compared to the high-pathogenic <it>Y. enterocolitica </it>ssp. <it>enterocolitica </it>8081 O:8/1B to address the peculiarities of the O:3/4 group.</p> <p>Results</p> <p>Most high-pathogenicity-associated determinants of <it>Y. enterocolitica </it>ssp. <it>enterocolitica </it>(like the High-Pathogenicity Island, <it>yts1 </it>type 2 and <it>ysa </it>type 3 secretion systems) are absent in <it>Y. enterocolitica </it>ssp. <it>palearctica </it>serobiotype O:3/4 genomes. On the other hand they possess alternative putative virulence and fitness factors, such as a different <it>ysp </it>type 3 secretion system, an RtxA-like and insecticidal toxins, and a N-acetyl-galactosamine (GalNAc) PTS system (<it>aga</it>-operon). Horizontal acquisition of two prophages and a tRNA-Asn-associated GIYep-01 genomic island might also influence the <it>Y. enterocolitica </it>ssp. <it>palearctica </it>serobiotype O:3/4 pathoadaptation. We demonstrated recombination activity of the PhiYep-3 prophage and the GIYep-01 island and the ability of the <it>aga</it>-operon to support the growth of the <it>Y. enterocolitica </it>ssp. <it>enterocolitica </it>O:8/1B on GalNAc.</p> <p>Conclusions</p> <p><it>Y. enterocolitica </it>ssp. <it>palearctica </it>serobiotype O:3/4 experienced a shift to an alternative patchwork of virulence and fitness determinants that might play a significant role in its host pathoadaptation and successful worldwide dissemination.</p

An Oral Vaccine Based on U-Omp19 Induces Protection against B. abortus Mucosal Challenge by Inducing an Adaptive IL-17 Immune Response in Mice

As Brucella infections occur mainly through mucosal surfaces, the development of mucosal administered vaccines could be radical for the control of brucellosis. In this work we evaluated the potential of Brucella abortus 19 kDa outer membrane protein (U-Omp19) as an edible subunit vaccine against brucellosis. We investigated the protective immune response elicited against oral B. abortus infection after vaccination of mice with leaves from transgenic plants expressing U-Omp19; or with plant-made or E. coli-made purified U-Omp19. All tested U-Omp19 formulations induced protection against Brucella when orally administered without the need of adjuvants. U-Omp19 also induced protection against a systemic challenge when parenterally administered. This built-in adjuvant ability of U-Omp19 was independent of TLR4 and could be explained at least in part by its capability to activate dendritic cells in vivo. While unadjuvanted U-Omp19 intraperitoneally administered induced a specific Th1 response, following U-Omp19 oral delivery a mixed specific Th1-Th17 response was induced. Depletion of CD4+ T cells in mice orally vaccinated with U-Omp19 resulted in a loss of the elicited protection, indicating that this cell type mediates immune protection. The role of IL-17 against Brucella infection has never been explored. In this study, we determined that if IL-17A was neutralized in vivo during the challenge period, the mucosal U-Omp19 vaccine did not confer mucosal protection. On the contrary, IL-17A neutralization during the infection did not influence at all the subsistence and growth of this bacterium in PBS-immunized mice. All together, our results indicate that an oral unadjuvanted vaccine based on U-Omp19 induces protection against a mucosal challenge with Brucella abortus by inducing an adaptive IL-17 immune response. They also indicate different and important new aspects i) IL-17 does not contribute to reduce the bacterial burden in non vaccinated mice and ii) IL-17 plays a central role in vaccine mediated anti-Brucella mucosal immunity

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field