Leishmania mexicana: promastigotes and amastigotes secrete protein phosphatases and this correlates with the production of inflammatory cytokines in macrophages.

Phosphatase activity of Leishmania spp. has been shown to deregulate the signalling pathways of the host cell. We here show that Leishmania mexicana promastigotes and amastigotes secrete proteins with phosphatase activity to the culture medium, which was higher in the Promastigote Secretion Medium (PSM) as compared with the Amastigote Secretion Medium (ASM) and was not due to cell lysis, since parasite viability was not affected by the secretion process. The biochemical characterization showed that the phosphatase activity present in PSM was higher in dephosphorylating the peptide END (pY) INASL as compared with the peptide RRA (pT)VA. In contrast, the phosphatase activity in ASM showed little dephosphorylating capacity for both peptides. Inhibition assays demonstrated that the phosphatase activity of both PSM and ASM was sensible only to protein tyrosine phosphatases inhibitors. An antibody against a protein phosphatase 2C (PP2C) of Leishmania major cross-reacted with a 44·9 kDa molecule in different cellular fractions of L. mexicana promastigotes and amastigotes, however, in PSM and ASM, the antibody recognized a protein about 70 kDa. By electron microscopy, the PP2C was localized in the flagellar pocket of amastigotes. PSM and ASM induced the production of tumor necrosis factor alpha, IL-1ß, IL-12p70 and IL-10 in human macrophages.

Ploidy study in sporothrix schenkii

Sporothrix schenckii is a dimorphic fungus that is pathogenic for humans. No sexual cycle has been reported for this fungus and little is known of its genetic constitution. To inquire into the ploidy state of Sporothrix schenckii, different approaches were followed: DNA content during transition from conidia to yeast, survival to ultraviolet irradiation, chemical mutagenesis, and induction of mitotic recombination. No change in ploidy was detected between the conidia and yeast phases of the fungi. Resistance to cell inactivation by UV irradiation was higher in S. schenckii and in Sacharomyces cerevisiae in its diploid state than in isogenic haploids a and alpha from S. cerevisiae that were inactivated at lower doses. Two mutant phenotypes, auxotrophy and albinism, were screened after chemical mutagenesis. One-step mutagenesis with either nitrous acid or ultraviolet light was unsuccessful in inducing auxotrophy but was sufficient to induce albino colonies. Two-step mutagenesis with nitrous acid in combination with UV light was necessary to attain two auxotrophic requirements: adenine and methionine. Prototrophic and pigmented revertants behaved as heterozygotes; after exposure to UV light they gave rise to derivatives which resemble the original mutation. The experiments presented in this work suggest that S. schenckii is a diploid, although aneuploidy cannot be excluded. Copyright 1999 Academic Press.

Chromatin organization in Entamoeba histolytica.

The chromatin structure of Entamoeba histolytica was investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particles 10 nm in diameter, but digestion of the chromatin with micrococcal nuclease did not render a regularly spaced DNA ladder in agarose gels. Southern blot analysis of the products of Entamoeba chromatin digestion using total amebic DNA and a non-transcribed repetitive sequence produced a banding pattern characteristic of eukaryotic chromatin with a repetitive size of approximately 130 bp. Conversely, hybridization with two active gene probes, actin and ribosomal RNA, showed that these sequences are not part of the chromatin organized in nucleosomes. It was also found that the basic nuclear proteins differ from histones of higher eukaryotes in electrophoretic mobility. Screening of an E. histolytica HM1-IMSS genomic library with Saccharomyces cerevisiae H3 and H4 genes and attempts to amplify E. histolytica sequences, homologous to these yeast histone genes, gave negative results suggesting that the Entamoeba proteins involved in chromatin organization are not typical histones.

Analysis of restriction profiles of mitochondrial DNA from Sporothrix schenckii.

Mitochondrial DNA (mtDNA) diversity was analyzed in 42 clinical isolates of Sporothrix schenckii from Mexico (n = 29), Guatemala (n = 4) and Colombia (n = 9). Based on HaeIII restriction digestion profiles, the isolates were classified into eight types. In addition to 24 mtDNA types previously reported in another study, 6 new types were found in this study. Most of the strains belong to type 14 and type 30, the former restricted to Mexico, whereas the latter was distributed in Mexico, Guatemala and Colombia. The new types (25-30) were identified in Mexico, Guatemala and Colombia. Restriction-fragment length polymorphism in mtDNA of S. schenckii revealed high levels of genetic variation attributable to differences in restriction sites as well as in mtDNA size. Based on genetic distances S. schenckii types were clustered into two main groups.

Biosynthesis and functions of melanin in Sporothrix schenckii.

Sporothrix schenckii is a human pathogen that causes sporotrichosis, an important cutaneous mycosis with a worldwide distribution. It produces dark-brown conidia, which infect the host. We found that S. schenckii synthesizes melanin via the 1,8-dihydroxynaphthalene pentaketide pathway. Melanin biosynthesis in the wild type was inhibited by tricyclazole, and colonies of the fungus were reddish brown instead of black on tricyclazole-amended medium. Two melanin-deficient mutant strains were analyzed in this study: an albino that produced normal-appearing melanin on scytalone-amended medium and a reddish brown mutant that accumulated and extruded melanin metabolites into its medium. Scytalone and flaviolin obtained from cultures of the reddish brown mutant were identified by thin-layer chromatography, high-performance liquid chromatography, and UV spectra. Transmission electron microscopy showed an electron-dense granular material believed to be melanin in wild-type conidial cell walls, and this was absent in conidial walls of the albino mutant unless the albino was grown on a scytalone-amended medium. Melanized cells of wild-type S. schenckii and the albino grown on scytalone-amended medium were less susceptible to killing by chemically generated oxygen- and nitrogen-derived radicals and by UV light than were conidia of the mutant strains. Melanized conidia of the wild type and the scytalone-treated albino were also more resistant to phagocytosis and killing by human monocytes and murine macrophages than were unmelanized conidia of the two mutants. These results demonstrate that melanin protects S. schenckii against certain oxidative antimicrobial compounds and against attack by macrophages.