Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensis Pb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways.

Background: Paracoccidioides brasiliensis is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi. Results: A cDNA (Pb sp) encoding a secreted serine protease (Pb SP), was isolated from a cDNA library constructed with RNAs of fungal yeast cells recovered from liver of infected mice. Recombinant Pb SP was produced in Escherichia coli, and used to develop polyclonal antibodies that were able to detect a 66 kDa protein in the P. brasiliensis proteome. In vitro deglycosylation assays with endoglycosidase H demonstrated that Pb SP is a N-glycosylated molecule. The Pb sp transcript and the protein were induced during nitrogen starvation. The Pb sp transcript was also induced in yeast cells infecting murine macrophages. Interactions of Pb SP with P. brasiliensis proteins were evaluated by two-hybrid assay in the yeast Saccharomyces cerevisiae. Pb SP interacts with a peptidyl prolyl cis-trans isomerase, calnexin, HSP70 and a cell wall protein PWP2. Conclusions: A secreted subtilisin induced during nitrogen starvation was characterized indicating the possible role of this protein in the nitrogen acquisition. Pb SP interactions with other P. brasiliensis proteins were reported. Proteins interacting with Pb SP are related to folding process, protein trafficking and cytoskeleton reorganization.

Staphylococcus saprophyticus is a Gram-positive and coagulase negative cocci that composes the skin microbiota and can act as an opportunistic agent causing urinary tract infections, being more frequent in sexually active young women. The ability of a pathogen to cause infection in the host is associated to its ability to adhere to host cells and to survive host immune defenses. In this work, we presented the comparative proteomic profile of three S. saprophyticus strains. It was possible to characterize differences in the proteome content, specially related to expression of virulence factors. We compiled this data and previous data and we detected one strain (9325) possessing higher production and secretion of proteins related to virulence. Our results show that phenotypic, genotypic, and proteomic differences reflect in the ability to survive during interaction with host cells, since the 9325 strain presented a higher survival rate after macrophage interaction. In counterpart, the 7108 strain that possesses lower content of proteins related to virulence presented higher ability to form biofilm suggesting that this strain can be better adapted to persist in the host and in the environment. Our work describes, for the first time, proteomic flexibility among S. saprophyticus strains, reflecting in virulence and persistence.

Background: The fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands. Results: The search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software. Conclusion: These observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell.