Characterization of a Cryptic and Intriguing Low Molecular Weight Plasmid.

The complete nucleotide sequence of cryptic plasmid pVCM04 isolated from Salmonella enterica serovar Enteritidis was determined and analyzed. pVCM04 contains 3853 bp with 53.6 % GC content and has twelve ORFs with more than 50 amino acids. Five of these sequences showed homology with replication and mobilization proteins. ORF1 and ORF2 showed homology with replication proteins, while ORFs 3-5 showed homology with mobilization proteins. The pVCM04 possesses a region associated with the theta-type replication mechanism. BLASTn search analysis revealed unexpectedly no similarity with sequences deposited in GenBank. The nucleotide sequence of pVCM04 can be divided into two arms: the region between nucleotides 552-1774 (encoding RepA and RepB) and the region between nucleotides 1775-3853 (encoding MobA, MobB and MobC). Codon bias pattern is distinct between mobA and repA, so the program Modeltest was used to select the best evolutionary model to study these genes. The result of ModelTest (model GTR+G for mobA and model HKY+G for repA) suggests that these genes would be subject to different selective pressures. Considering the differences in the codon usage, the selection of two different evolutionary models, and the absence of plasmids with homology to pVCM04 in GenBank, we believe that pVCM04 is a chimeric molecule and represents a new plasmid lineage.

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells.

BACKGROUND: Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. RESULTS: In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation - cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-beta-glucosidase) in mycelium cells; and ags (an alpha-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport - two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells - isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. CONCLUSION: Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.

A new Paracoccidioides brasiliensis 70-kDa heat shock protein reacts with sera from paracoccidioidomycosis patients.

A cDNA coding for a new member of the 70-kDa heat shock proteins (HSP70) family from the dimorphic and pathogenic fungus, Paracoccidioides brasiliensis, was cloned and characterized. The cDNA-deduced sequence coded for 655 amino acid residues and showed 95% identity to a previously described P. brasiliensis hsp70 gene. Cytoplasmic and typical nuclear localization signals, which indicate induction upon stress, were identified in the deduced peptide. The complete hsp70 cDNA coding region was cloned into a pGEX 4T-3 plasmid and expressed in Escherichia coli as a glutathione-S-transferase-tagged fusion protein. The recombinant protein reacted with a rabbit polyclonal antibody against HSP70. Western immunoblot experiments demonstrated that sera from paracoccidioidomycosis patients recognized the purified recombinant protein, suggesting an immunological role for this protein in the infectious process. The antigenicity analysis of rHSP70 detected three internal peptides that could act as activators of T-cell proliferation.

Overview and perspectives the transcriptome of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America that affects 10 million individuals. Pathogenicity is assumed to be a consequence of the dimorphic transition from mycelium to yeast cells during human infection. This review shows the results of the P. brasiliensis transcriptome project which generated 6,022 assembled groups from mycelium and yeast phases. Computer analysis using the tools of bioinformatics revealed several aspects from the transcriptome of this pathogen such as: general and differential metabolism in mycelium and yeast cells; cell cycle, DNA replication, repair and recombination; RNA biogenesis apparatus; translation and protein fate machineries; cell wall; hydrolytic enzymes; proteases; GPI-anchored proteins; molecular chaperones; insights into drug resistance and transporters; oxidative stress response and virulence. The present analysis has provided a more comprehensive view of some specific features considered relevant for the understanding of basic and applied knowledge of P. brasiliensis.

Regulation of N-acetyl-beta-D-glucosaminidase produced by Trichoderma harzianum: evidence that cAMP controls its expression.

Trichoderma harzianum is a filamentous fungus reported to be a producer of extracellular N-acetyl-beta-D-glucosaminidase (NAGase) when grown in chitin-containing medium. An approximately 64-kDa protein with NAGase activity was purified by gel filtration and ion exchange chromatography. The involvement of cyclic AMP (cAMP) in the synthesis of NAGase from T. harzianum in chitin-containing medium was also investigated. Molecules that increase the intracellular levels of cAMP, including caffeine, aluminium tetrafluoride and dinitrophenol, were used. Western blot analysis showed that NAGase synthesis was repressed by increasing the levels of intracellular cAMP. Using specific nag primers in a reverse transcription-polymerase chain reaction-based approach, NAGase synthesis was shown to be regulated at the level of gene transcription.