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.

The pathogenic yeast Cryptococcus neoformans secretes numerous proteins, such as heat shock proteins, by unconventional mechanisms during its interaction with host cells. Hsp70 is a conserved chaperone that plays important roles in various cellular processes, including the interaction of fungi with host immune cells. Here, we report that sera from individuals with cryptococcosis infection recognize a recombinant C. neoformans Hsp70 (Cn_rHsp70). Moreover, immunofluorescence assays using antibodies against Cn_rHsp70 revealed the localization of this protein at the cell surface mainly in association with the capsular network. We found that the addition of Cn_rHsp70 positively modulated the interaction of C. neoformans with human alveolar epithelial cells and decreased fungal killing by mouse macrophages, without affecting phagocytosis rates. Immunofluorescence analysis showed that there was a competitive association among the receptor, GXM and Cn_rHsp70, indicating that the Hsp70-binding sites in host cells appear to be shared by glucuronoxylomannan (GXM), the major capsular antigen in C. neoformans. Our observations suggest additional mechanisms by which Hsp70 influences the interaction of C. neoformans with host cells.

SUMMARYCandida dubliniensis is an opportunistic yeast that has been recovered from several body sites in many populations; it is most often recovered from the oral cavities of human immunodeficiency virus-infected patients. Although extensive studies on epidemiology and phylogeny of C. dubliniensis have been performed, little is known about virulence factors such as exoenzymatic and hemolytic activities. In this study we compared proteinase, hyaluronidase, chondroitin sulphatase and hemolytic activities in 18 C. dubliniensis and 30 C. albicans strains isolated from AIDS patients. C. albicans isolates produced higher amounts of proteinase than C. dubliniensis (p < 0.05). All the tested C. dubliniensis strains expressed hyaluronidase and chondroitin sulphatase activities, but none of them were significantly different from those observed with C. albicans (p > 0.05). Hemolytic activity was affected by CaCl 2 ; when this component was absent, we did not notice any significant difference between C. albicans and C. dubliniensis hemolytic activities. On the contrary, when we added 2.5 g% CaCl 2 , the hemolytic activity was reduced on C. dubliniensis and stimulated on C. albicans tested strains (p < 0.05).

The in vitro susceptibility of 62 isolates of Sporothrix schenckii in its mycelial form, from LatinAmerican countries (Peru, Venezuela, Brazil and Uruguay) and Spain, to amphotericin B (AB), itraconazole (IZ), posaconazole (PZ) and terbinafine (TB) was determined by measuring the MICs and minimum fungicidal concentrations (MFCs) using a standardized Clinical and Laboratory Standards Institute method. In general, TB was the most active drug, with the lowest geometric mean (GM) MIC and MFC values amongst isolates from the five countries tested. IZ and PZ showed almost the same activity against all strains tested, except for isolates from Uruguay where IZ gave the highest GM MIC (10.68 mg l ). MFCs were 5 to 20 times higher than the MICs, but the lowest GM MFC and range values were found for TB. IZ and PZ gave the highest GM MFC. MFC may be a better predictor of therapeutic response than MIC, especially in immunosuppressed patients, making the use of IZ and PZ an inappropriate treatment. There were some differences in susceptibility according to the geographical source of the isolates, with the MIC being lower for TB in Venezuelan strains (P50.066) and the MFC higher for PZ in Peruvian strains (P50.02). Thus, geographical origin may be important for appropriate treatment, and may relate to the identification of species of the S. schenckii complex.

Cryptococcus neoformans and Cryptococcus gattii are encapsulated yeast agents of cryptococcosis and facultative intracellular pathogens. The interaction of these yeasts with macrophages is essential for containing the infection. However, Cryptococcus spp. overcome this initial host defense barrier using a unique pathogenic strategy involving intracellular replication and cytoplasmic accumulation of polysaccharide-containing vesicles. Here, we employed representational difference analysis (RDA) to identify C. neoformans and C. gattii genes differentially expressed during intracellular growth in rat peritoneal macrophages. The upregulated transcripts of C. neoformans during macrophage interaction were related to ATP-binding cassette (ABC) transporters, intra-golgi transport, chaperone activity, ribosomal maintenance, NAD metabolism, histone methylation, stress response, and monosaccharide metabolism. In contrast, with C. gattii, upregulated genes were associated with cell growth, aerobic respiration, protein binding, microtubule nucleation, monosaccharides and nitrogen metabolism, inositol or phosphatidylinositol phosphatase activity, cellular signaling, and stress response. Our findings reveal new genes that may be necessary for the intracellular parasitism of C. neoformans and C. gattii.

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Key message MdoDHN11 acts in the nucellus layer to protect the embryo and the endosperm from limited water availability during apple seed development. Abstract Dehydrins (DHNs) are protective proteins related to several plant developmental responses that involve dehydration such as seed desiccation and abiotic stresses. In apple (Malus × domestica Borkh.), the seed-specific MdoDHN11 was suggested to play important roles against dehydration during seed development. However, this hypothesis has not yet been evaluated. Within this context, several experiments were performed to functionally characterize MdoDHN11. In situ hybridization analysis during apple seed development showed that MdoDHN11 expression is confined to a maternal tissue called nucellus, a central mass of parenchyma between the endosperm and the testa. The MdoDHN11 protein was localized in the cytosol and nucleus. Finally, transgenic Arabidopsis plants expressing MdoDHN11 were generated and exposed to a severe water-deficit stress, aiming to mimic a situation that can occurs during seed development. All transgenic lines showed increased tolerance to water deficit in relation to wild-type plants. Taken together, our results provide evidences that MdoDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from limited water availability, and the mechanism of action probably involves the interaction of MdoDHN11 with proteins and other components in the cell.