BINDING OF PARACOCCIDIOIDES-BRASILIENSIS TO LAMININ THROUGH SURFACE GLYCOPROTEIN GP43 LEADS TO ENHANCEMENT OF FUNGAL PATHOGENESIS

Extracellular matrix protein laminin binds specifically to yeast forms of Paracoccidioides brasiliensis and enhances adhesion of the fungus to the surface of epithelial Madin-Darby canine kidney cells in vitro. Immunoblotting of fungal extracts showed that the gp43 glycoprotein is responsible for adhesion. This was confirmed by binding assays using purified gp43, with a K-d of 3.7 nM. The coating of P. brasiliensis yeast forms with laminin before injection into hamster testicles enhanced the fungus virulence, resulting in a faster and more severe granulomatous disease. These results indicate that interaction of fungi with extracellular matrix elements may constitute a basis for the evolution of fungal infection toward regional spreading and dissemination.

Circadian rhythm of anti-fungal prenylated chromene in leaves of Piper aduncum

Leaves of Piper aduncum accumulate the anti-fungal chromenes methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate (2). The enzymatic formation of 2 from dimethylallyl diphosphate and I was investigated using cell-free extracts of the title plant. An HPLC assay for the prenylation reaction was developed and the enzyme activity measured in the protein extracts. The prenyltransferase that catalyses the transfer of the dimethylallyl group to C-2' of 1 was soluble and required dimethylallyl diphosphate as the prenyl donor. In the leaves, the biosynthesis of the prenylated chromene 2 was time-regulated and prenyltransferase activity depended upon circadian variation. Preliminary characterisation and purification experiments on the prenyltransferase from P. aduncum have been performed. Copyright (C) 2005 John Wiley & Sons, Ltd.

Comparative ''in vitro'' study on the susceptibility and emergence of mutants resistant to danofloxacin and ciprofloxacin among Staphylococcus aureus isolated from bovine mastitis

The antimicrobial susceptibility of danofloxacin was compared in vitro with ciprofloxacin against mastitogenic Staphylococcus aureus. Danofloxacin was more active than ciprofloxacin, showing minimal inhibitory concentrations (MIC90), minimal bactericidal concentrations (MBC90) and MBC/MIC ratio lower than those of ciprofloxacin. Ciprofloxacin-resistant mutants occurred at higher frequencies (greater than or equal to 10(-6)) than for danofloxacin (10(-9)).

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

COMPARATIVE INVITRO ACTIVITY OF 3 CEPHALOSPORINS AGAINST STAPHYLOCOCCUS-AUREUS ISOLATED FROM BOVINE MASTITIS

This report compares the in vitro activity of three cephalosporins (cephalothin, cefoxitin and ceftriaxone) against 57 Staphylococcus aureus strains isolated from cows with clinical mastitis on the basis of the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). The majority of the S aureus strains showed resistance to cefoxitin and ceftriaxone and sensitivity to cephalothin. The highest MICs and MBCs were found for cefoxitin and ceftriaxone. Antimicrobial tolerance (MBC/MIC greater-than-or-equal-to 32:1) was observed in relation to cephalothin and ceftriaxone. The data suggest that these cephalosporins may not be effective for the treatment of staphylococcal bovine mastitis. The precise definition of their antimicrobial efficacies requires more detailed in vitro and in vivo studies.

DECOMPOSITION RATES OF SPENT FUNGAL REFUSE OF ATTA-SEXDENS (HYMENOPTERA, FORMICIDAE) AND IMPLICATIONS FOR ESTIMATING FORAGE INTAKE BY LEAF-CUTTING ANTS

Using simulated ceramic refuse chambers, field decomposition studies were performed on the spent fungal refuse of the lead-cutting ant Atta sexdens rubropilosa. Refuse half life was estimated at 40 days, with complete decomposition at 100 days. These results suggest that the conversion-factor method used to estimate forage input into leaf-cutting ant colonies must be corrected for decomposition, or serious estimation errors will occur.

Monoclonal antibodies against the 43,000 da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis

The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin, Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction, Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells, Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity, Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.

Differential gene expression by Paracoccidioides brasiliensis in host interaction conditions: Representational difference analysis identifies candidate genes associated with fungal pathogenesis

Paracoccidioides brasiliensis causes infection by the host inhalation of airborne propagules of the mycelia phase of the fungus. These particles reach the lungs, and disseminate to virtually all organs. Here we describe the identification of differentially expressed genes in studies of host-fungus interaction. We analyzed two cDNA populations of P. brasiliensis, one obtained from infected animals and the other an admixture of fungus and human blood thus mimicking the hematologic events of the fungal dissemination. Our analysis identified transcripts differentially expressed. Genes related to iron acquisition, melanin synthesis and cell defense were specially upregulated in the mouse model of infection. The upregulated transcripts of yeast cells during incubation with human blood were those predominantly related to cell wall remodeling/synthesis. The expression pattern of genes was independently confirmed in host conditions, revealing their potential role in the infection process. This work can facilitate functional studies of novel regulated genes that may be important for the survival and growth strategies of P. brasiliensis in humans. (c) 2006 Elsevier Masson SAS. All rights reserved.

O-serogroups, eae gene and EAF plasmid in Escherichia coli isolates from cases of bovine mastitis in Brazil

Mastitis has been recognized for some time as the most costly disease in dairy herds. From March 1997 to August 1998. 2144 samples of bovine mastitic milk were collected, from which 182 Escherichia coli isolates were made, and from which 14 1 isolates had the somatic anti-en (serogroup) determined. Twelve different serogroups were isolated from mastitic milk, and among them were 026, 055, 0111 and 0 119, all of them classic enteropathogenic E. (oh (EPEC) serogroups. These represented 40.0% of the isolates. The 20 of 57 isolates tested had plasmids and in dot blot hybridization, nine isolates were positive for an EaeA probe and an EPEC adherence factor (EAF) probe while two isolates were negative for EaeA probe but positive for the EAF probe, the nine isolates were characterized as attaching and effacing (A/E) E, coli (AEEC) isolates. (C) 2002 Elsevier B.V. B.V. All rights reserved.