PCR-RLFP characterization of Leishmania spp. in domestic animals from the south-western border of Brazil.

The aim of this study was to characterize Leishmania spp. from canine and feline samples using Polymerase Chain Reaction (PCR)- Restriction Fragment Length Polymorphism (RFLP). It was conducted in the southern region of Brazil, located at border crossings to Argentina and Uruguay. Samples were collected from 116 dogs (Canis lupus familiaris) and 89 cats (Felis catus). The PCR was performed to screen for an LT1 fragment from kinetoplast DNA (kDNA) target gene, and positive samples were subjected to a second PCR for an internal transcribed spacers (ITS1) region from ribosomal DNA (rDNA) target. RFLP was performed using the Haemophilus aegyptius (HAE III) restriction endonuclease (Fermentas ®). Positive samples by PCR ITS1 were sequenced and deposited in NCBI GenBank, and a phylogenetic analysis was developed. We found that 12.9% (15/116) of the samples from dogs were positive. All the 89 cat samples were negative. Positive samples were tested against Leishmania reference strains presenting different patterns in PCR-RFLP, and these samples showed bands denoting similarity to the standard species of Leishmania infantum, proven through sequencing and phylogenetic analysis. The RFLP technique, alone, was shown to be feasible for practical application and confirmation of the involved Leishmania spp.

Identification of genes expressed by Cryptococcus gattii during iron deprivation.

Cryptococcus neoformans and C. gattii are pathogenic yeasts that cause life-threatening diseases in humans and animals. Iron is an essential nutrient for virtually every organism as it functions as a cofactor in numerous essential enzymatic reactions. In the literature, the competition for iron between microbes and mammalian hosts during infection is well documented. In this study, we used representational difference analysis (RDA) in order to gain a better understanding of how C. gattii responds to iron starvation. A total of 15 and 29 genes were identified as having altered expression levels due to iron depletion after 3 h and 12 h, respectively. Of these, eight genes were identified in both libraries. The transcripts were related to many biological processes, such as cell cycle, ergosterol metabolism, cell wall organization, transportation, translation, cell respiration and the stress response. These data suggest a remodeling of C. gattii metabolism during conditions of iron deprivation.

Cryptococcus neoformans and Cryptococcus gattii genes preferentially expressed during rat macrophage infection.

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.

Characterization and functional analysis of the beta-1,3-glucanosyltransferase 3 of the human pathogenic fungus Paracoccidioides brasiliensis.

The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in fungus cell wall assembly and morphogenesis, beta-1,3-glucanosyltransferase 3 (PbGel3p) is presented here. PbGel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The full-length cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis. The recombinant PbGel3p was overexpressed in Escherichia coli, and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of PbGel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1Delta mutant. The results indicated that PbGel3p is a cell wall-associated protein that probably works as a beta-1,3-glucan elongase capable of mediating fungal cell wall integrity.

Identification of novel temperature-regulated genes in the human pathogen Cryptococcus neoformans using representational difference analysis.

Cryptococcus neoformans is a basidiomycetous fungus and an opportunistic human pathogen that causes infections in both immunocompromised and immunocompetent hosts. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this microorganism. Representational difference analysis (RDA) was used to profile gene expression in C. neoformans grown at 37 degrees C or 25 degrees C. Contig assembly of 300 high-quality sequenced cDNAs and comparison analysis to the GenBank database led to the identification of transcripts that may be critical for both pathogen-host interactions and responses to either low or high temperature growth. Gene products involved in cell wall integrity, stress response, filamentation, oxidative metabolism, protein targeting and fatty acids metabolism were induced at 37 degrees C. In addition, genes related to chromatin silencing and phospholipid transport were upregulated at 25 degrees C. Therefore, our RDA analysis, comparing saprophytic and host temperature conditions, revealed new genes with potential involvement in C. neoformans virulence.

Molecular typing of clinical and environmental Cryptococcus neoformans isolates in the Brazilian state Rio Grande do Sul.

In Brazil, 4.5% of the AIDS-related opportunistic infections are caused by Cryptococcus neoformans. This pathogen is a ubiquitous environmental basidiomycetous encapsulated yeast, commonly found in soil and avian excreta. The present study investigates further the population structure of clinical and environmental C. neoformans isolates from south Brazil. One hundred five clinical and 19 environmental (pigeon excreta and Eucalyptus spp.) isolates from the Brazilian state Rio Grande do Sul were characterized based on morphological, biochemical, molecular and serological data. The majority of the clinical and environmental isolates analyzed belonged to C. neoformans var. grubii serotype A (89.5 and 52.6%, respectively), were mating type alpha (98.1 and 94.7%, respectively) and were phospholipase-positive (94.3 and 73.7%, respectively). PCR-fingerprinting with the microsatellite-specific primer M13 and the minisatellite-specific primer (GACA)(4) grouped the majority of the isolates into the molecular type VNI (89.5 of the clinical and 52.6% of the environmental isolates). Our results add considerable new information to the few available data on ecology, molecular biology and epidemiology of C. neoformans in the southern region of Brazil.