No abstract

Cryptococcus neoformans, a pathogenic yeast, causes meningoencephalitis, especially in immunocompromised patients, leading in some cases to death. Microbes in biofilms can cause persistent infections, which are harder to treat. Cryptococcal biofilms are becoming common due to the growing use of brain valves and other medical devices. Using shotgun proteomics we determine the differences in protein abundance between biofilm and planktonic cells. Applying bioinformatic tools, we also evaluated the metabolic pathways involved in biofilm maintenance and protein interactions. Our proteomic data suggest general changes in metabolism, protein turnover, and global stress responses. Biofilm cells show an increase in proteins related to oxidation–reduction, proteolysis, and response to stress and a reduction in proteins related to metabolic process, transport, and translation. An increase in pyruvate-utilizing enzymes was detected, suggesting a shift from the TCA cycle to fermentation-derived energy acquisition. Additionally, we assign putative roles to 33 proteins previously categorized as hypothetical. Many changes in metabolic enzymes were identified in studies of bacterial biofilm, potentially revealing a conserved strategy in biofilm lifestyle.

Conidial contact with an arthropod surface is the first step of the fungal penetration and infection process. However, conidia of Metarhizium anisopliae have associated components, like enzymes that could be involved in triggering the penetration process and toxic effects that have not yet been well characterized. Fungi produce many enzymes that also are toxic components found in bacteria and animal venoms and thus may be considered as potential virulence factors. In this work, we report several enzymatic activities from spore surface protein extracts. The major proteolytic activities observed in spore surface proteins (SSP) were Pr1 and Pr2 activities, in that order. According to the zymograms obtained, SSP contain different proteases. SSP contain trehalase, exo- and endo-chitinase activities, and seven different chitinase bands which have been observed in zymograms. Activities involved in protection against reactive oxygen species (ROS) were also detected. Two lipolytic enzymes were also detected in lipase zymograms. Phospholipase C activity, closely related to microbial pathogenesis, was detected for the first time in M. anisopliae conidia. These activities described could be an initial step towards understanding the mechanisms involved in the first stage of M. anisopliae infection process and its toxic effects against arthropod hosts.

BackgroundMetarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins.ResultsWe determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity.ConclusionsThe data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-822) contains supplementary material, which is available to authorized users.

Both immunoreactive proteins and B-cell epitopes of C. gattii genotype VGII that were potentially targeted by a host humoral response were identified. Considering the evolutionary relevance of the identified proteins, we may speculate that they could be used as the initial targets for recombinant protein and peptide synthesis aimed at the development of immunodiagnostic tools for cryptococcosis.