Bivalent copper ions presence triggers removal and homeostatic mechanisms in the metal-resistant microorganism Apiotrichum loubieri M12.

Microorganisms, especially those habiting mining environments, are of great importance for the retention of toxic metals in the environment. This work aimed to isolate a copper removing-microorganism from sediments of an Acid Mine Drainage-affected environment and to study the cellular responses trigger by metal presence. Apiotrichum loubieri M12 was able to tolerate and remove Cu(II) from liquid culture media, reaching a 30-35% removal capacity when it was exposed to 40 µg mL-1 Cu(II) after 48 h. Analysis of the biomass exposed to the metal through SEM-EDS showed copper presence on the cell surface and variations in the proportion of other biomass constituent elements. Proteomics revealed that the presence of Cu(II) induces differential expression of intracellular proteins involved in a wide variety of metabolic processes. Interestingly, a specific response to the metal was detected in cell-free supernatants, in which copper binding proteins were identified. A large number of proteins with metal ion binding sites were detected both at intra and extracellular levels. The microorganism responds not only by adjusting intracellular protein expression, but also by adjusting expression of proteins in the extracellular space.

Galectin-1 Cooperates with Yersinia Outer Protein (Yop) P to Thwart Protective Immunity by Repressing Nitric Oxide Production.

Yersinia enterocolitica (Ye) inserts outer proteins (Yops) into cytoplasm to infect host cells. However, in spite of considerable progress, the mechanisms implicated in this process, including the association of Yops with host proteins, remain unclear. Here, we evaluated the functional role of Galectin-1 (Gal1), an endogenous ß-galactoside-binding protein, in modulating Yop interactions with host cells. Our results showed that Gal1 binds to Yops in a carbohydrate-dependent manner. Interestingly, Gal1 binding to Yops protects these virulence factors from trypsin digestion. Given that early control of Ye infection involves activation of macrophages, we evaluated the role of Gal1 and YopP in the modulation of macrophage function. Although Gal1 and YopP did not influence production of superoxide anion and/or TNF by Ye-infected macrophages, they coordinately inhibited nitric oxide (NO) production. Notably, recombinant Gal1 (rGal1) did not rescue NO increase observed in Lgals1-/- macrophages infected with the YopP mutant Ye ∆yopP. Whereas NO induced apoptosis in macrophages, no significant differences in cell death were detected between Gal1-deficient macrophages infected with Ye ∆yopP, and WT macrophages infected with Ye wt. Strikingly, increased NO production was found in WT macrophages treated with MAPK inhibitors and infected with Ye wt. Finally, rGal1 administration did not reverse the protective effect in Peyer Patches (PPs) of Lgals1-/- mice infected with Ye ∆yopP. Our study reveals a cooperative role of YopP and endogenous Gal1 during Ye infection.

Characterization of copper stress response in Fusarium tricinctum M6: A metal-resistant microorganism isolated from an acid mine drainage-affected environment.

Acid mine drainage-affected environments are interesting microbial niches for the isolation of metal-resistant microorganisms. In this sense, the aim of the present work is to isolate and characterize metal-resistant microorganisms from sediments of an abandoned gold mine located in San Luis (Argentina). For these purposes, the metal removal capacity and the microelemental composition of the biomass exposed to metals were evaluated. Likewise, proteomic techniques were applied to understand the removal and resistance mechanisms. Fusarium tricinctum M6 was isolated and identified as tolerant to Cu(II), Fe(II) and Cr(VI). When faced with 40 µg mL-1 Cu(II), the growth was affected by 60% and the removal capacity was 30-35%. Copper was found uniformly distributed in the biomass (5.23% w/w) and variations in the proportion of other biomass constituent elements were detected. When exposed to Cu(II), F. tricinctum M6 showed differential expression of intra and extracellular proteins involved in different metabolic processes. A large number of proteins with metal ion binding sites were detected both at intra and extracellular levels. The results obtained in the present work indicated bioadsorption of the metal on the cell surface and an important readjustment of the protein expression to counteract the stress produced by Cu(II).

Identification of Clostridium difficile Immunoreactive Spore Proteins of the Epidemic Strain R20291.

PURPOSE: Clostridium difficile infections are the leading cause of diarrhea associated with the use of antibiotics. During infection, C. difficile initiates a sporulation cycle leading to the persistence of C. difficile spores in the host and disease dissemination. The development of vaccine and passive immunization therapies against C. difficile has focused on toxins A and B. In this study, an immunoproteome-based approach to identify immunogenic proteins located on the outer layers of C. difficile spores as potential candidates for the development of immunotherapy and/or diagnostic methods against this devastating infection is used. EXPERIMENTAL DESIGN: To identify potential immunogenic proteins on the surface of C. difficile R20291, spore coat/exosporium extracts are separated by 2D electrophoresis (2-DE) and analyzed for reactivity against C. difficile spore-specific goat sera. Finally, the selected spots are in-gel digested with chymotrypsin, peptides generated are separated by nanoUPLC followed by MS/MS using Quad-TOF-MS, corroborated by Ultimate 3000RS-nano-UHPLC coupled to Q-Exactive-Plus-Orbitrap MS. RESULTS: The analysis identify five immunoreactive proteins: spore coat proteins CotE, CotA, and CotCB; exosporium protein CdeC; and a cytosolic methyltransferase. CONCLUSION: This data provides a list of spore surface protein candidates as antigens for vaccine development against C. difficile infections.