Proteomic analysis of the secretions of Pseudallescheria boydii, a human fungal pathogen with unknown genome.

Pseudallescheria boydii is a filamentous fungus that causes a wide array of infections that can affect practically all the organs of the human body. The treatment of pseudallescheriosis is difficult since P. boydii exhibits intrinsic resistance to the majority of antifungal drugs used in the clinic and the virulence attributes expressed by this fungus are unknown. The study of the secretion of molecules is an important approach for understanding the pathogenicity of fungi. With this task in mind, we have shown that mycelial cells of P. boydii were able to actively secrete proteins into the extracellular environment; some of them were recognized by antibodies present in the serum of a patient with pseudallescheriosis. Additionally, molecules secreted by P. boydii induced in vitro irreversible damage in pulmonary epithelial cells. Subsequently, two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of secreted proteins from P. boydii mycelial cells. The two-dimensional map showed that most of the proteins (around 100 spots) were focused at pH ranging from 4 to 7 with molecular masses ranging from 14 to >117 kDa. Fifty spots were randomly selected, of which 30 (60%) were consistently identified, while 20 (40%) spots generated peptides that showed no resemblance to any known protein from other fungi and/or MS with low quality. Notably, we identified proteins involved in metabolic pathways (energy/carbohydrate, nucleotide, and fatty acid), cell wall remodeling, RNA processing, signaling, protein degradation/nutrition, translation machinery, drug elimination and/or detoxification, protection against environmental stress, cytoskeleton/movement proteins, and immunogenic molecules. Since the genome of this fungus is not sequenced, we performed enzymatic and immunodetection assays in order to corroborate the presence of some released proteins. The identification of proteins actively secreted by P. boydii provides important new information for understanding immune modulation and provides important new perspectives on the biology of this intriguing fungus.

A new experimental culture medium for cultivation of Leishmania amazonensis: its efficacy for the continuous in vitro growth and differentiation of infective promastigote forms.

Parasites from the genus Leishmania cause a variety of disease states in humans and other mammals in tropical and subtropical regions, which include cutaneous, mucocutaneous and visceral leishmaniasis. The elaboration of a culture medium for the in vitro cultivation of Leishmania spp., which promotes the growth and differentiation of the parasites, is an important tool for diagnosis, biochemical, biological and immunological studies in the genus. Herein, we have reported the development of a rapid, inexpensive and reliable monophasic culture medium. The novel medium, designated PBHIL, promoted an excellent parasite growth, generating high quantities of promastigotes with long-term viability, and was able to induce cellular differentiation of L. amazonensis promastigotes to the amastigote-like forms (93%). Additionally, we reported the influence of this novel medium on the biochemical characteristics of L. amazonensis and on the interaction of this parasite parasites with mammalian macrophages.

Metallopeptidases produced by group B Streptococcus: influence of proteolytic inhibitors on growth and on interaction with human cell lineages.

Group B Streptococcus (GBS) is a major etiologic agent of neonatal bacterial infections and is the most common cause of sepsis and pneumonia in newborns. Surface and secreted molecules of GBS are often essential virulence factors which are involved in the adherence of the bacteria to host cells or are required to suppress the defense mechanisms of hosts. We analyzed the peptidase profiles of GBS by detection of proteolytic activities on SDS-PAGE containing copolymerized gelatin as substrate. Based on the inhibition by o-phenathroline and EGTA, three distinct peptidases of 220, 200 and 180 kDa were identified in the culture medium, besides one major cell-associated proteolytic activity, a 200-kDa metallopeptidase, suggesting that all were zinc-metallopeptidases. GBS culture supernatants, rich in metallotype peptidases, also cleaved fibronectin, laminin, type IV collagen, fibrinogen and albumin. Cleavage of the host extracellular matrix by GBS may be a relevant factor in the process of bacterial dissemination and/or invasion. Notably, metallopeptidase inhibitors strongly blocked GBS growth as well as its interaction with human cell lineages. Understanding the contribution of peptidases to the pathogenesis of GBS disease may broaden our perception of how this significant pathogen causes severe infections in newborn infants.

Extracellular peptidase in the fungal pathogen Pseudallescheria boydii.

Pseudallescheria boydii is a ubiquitous filamentous fungus capable of causing invasive disease in humans. In the present study, using sodium dodecyl sulfate-polyacrylamide gels containing bovine serum albumin as co-polymerized substrate, we identified a 28-kDa proteolytic activity released to the extracellular environment by mycelia of P. boydii. This peptidase was detected during the growth of P. boydii in Sabouraud-dextrose medium for 13 days and reached its maximal production on day 7. The 28-kDa peptidase was active in acidic pH (5.5) and had its activity completely blocked by 1,10-phenanthroline, a potent zinc-metallopeptidase inhibitor. Two other metallopeptidase inhibitors, EDTA and EGTA, were also tested and no alterations were observed in the activity of the 28-kDa extracellular peptidase. Likewise, E-64 (a cysteine peptidase inhibitor), phenylmethylsulphonyl fluoride (a serine peptidase inhibitor), and pepstatin A (an aspartyl peptidase inhibitor) did not significantly alter the enzymatic behavior. Collectively, we described for the first time the expression of an extracellular metallopeptidase in the human opportunistic fungal pathogen P. boydii.

Secretion of serine peptidase by a clinical strain of Candida albicans: influence of growth conditions and cleavage of human serum proteins and extracellular matrix components.

Candida albicans expresses a vast number of hydrolytic enzymes, playing roles in several phases of yeast-host interactions. Here, we identified two novel extracellular peptidase classes in C. albicans. Using gelatin-sodium dodecyl sulfate polyacrylamide gel electrophoresis two gelatinolytic activities were detected at physiological pH: a 60-kDa metallopeptidase, completely blocked by 1,10-phenanthroline, and a 50-kDa serine peptidase inhibited by phenylmethylsulfonyl fluoride. In an effort to establish a probable functional implication for these novel peptidase classes, we demonstrated that the 50-kDa secretory serine peptidase was active over a broad pH range (5.0-7.2) and was capable to hydrolyze some soluble human serum proteins and extracellular matrix components. Conversely, when this isolate was grown in yeast carbon base supplemented with bovine serum albumin, a secretory aspartyl peptidase activity was measured, instead of metallo- and serine peptidases, suggesting that distinct medium composition induces different expression of released peptidases in C. albicans. Additionally, we showed by quantitative proteolytic measurement, flow cytometry and immunoblotting assays that the brain heart infusion medium might repress the Sap1-3 production. Collectively, our results showed for the first time the capability of an extracellular proteolytic enzyme other than aspartic-type peptidases to cleave a broad spectrum of relevant host proteinaceous substrates by the human pathogen C. albicans.