RESUMO
Histoplasma capsulatum is a primary fungal pathogen of mammals responsible for histoplasmosis. During pathogenesis H. capsulatum yeast proliferate in phagosomes of macrophages. This extensive host/pathogen interaction involves a complex cascade of responses in both organisms. In the mammalian host, infection results in complex branched immunity that is initiated with an innate response and later induces an adaptive response but each response is difficult to resolve during fungal infection. Therefore, in an effort to identify less complex systems and to gain understanding of the host innate response to H. capsulatum, we constructed a mini-host survival assay. With this assay, we found ingestion of virulent Histoplasma capsulatum NAm 1 strain yeasts to be lethal to a Bristol-N2 Caenorhabditis elegans host. The virulent H. capsulatum NAm1 strain shows differential lethality under live/heat-killed infective conditions. Specifically, after ingestion of live yeast lethality is > or = 90% within 48 to 72 h, whereas worms ingesting heat-killed yeast reach equivalent mortality only after 10-14 days. On the other hand, ingestion of live H. capsulatum yeast of the nonvirulent NAm 1 (ura(-)) strain is no more lethal to the nematode than heat-killed yeast. Therefore, C. elegans provides an attractive model for further investigations of the ancient innate immune response during early host/pathogen (H. capsulatum/worm) interaction and pathogenesis.
Assuntos
Caenorhabditis elegans/imunologia , Histoplasma/imunologia , Histoplasmose/imunologia , Animais , Caenorhabditis elegans/microbiologia , Modelos Animais de Doenças , Histoplasma/patogenicidade , Histoplasmose/microbiologia , Interações Hospedeiro-Patógeno , Microscopia , VirulênciaRESUMO
Histoplasma capsulatum possesses a branched mitochondrial electron transport chain, with both cyanide-sensitive and -insensitive oxygen-consuming activities. The latter, carried out by a single subunit enzyme termed 'alternative oxidase', is the focus of this report. AOX1 cDNA clones were isolated and direct evidence that the cDNA ORF encodes functional alternative oxidase is reported. Also reported are the generation of an antiserum to the AOX1 protein product, and specific detection in vivo of the mRNA and protein products of the AOX1 gene. Finally, initial studies of regulation of H. capsulatum AOX1 gene expression demonstrated that RNA abundance was increased after hydrogen peroxide-mediated oxidative stress and after inhibition of mitochondrial electron transport enzymes with antimycin A or sodium cyanide. This pattern of regulation is consistent with the hypothesis that alternative oxidase contributes to survival of H. capsulatum after oxidative or metabolic stress and may be important for virulence of this pathogenic organism. The GenBank Accession Nos for the cDNA sequences reported in this paper are AF133236, AF133237 (AOX1).
Assuntos
Proteínas Fúngicas/metabolismo , Histoplasma/enzimologia , Oxirredutases/metabolismo , Sequência de Aminoácidos , Antimicina A/farmacologia , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , Cianetos/metabolismo , Cianetos/farmacologia , Transporte de Elétrons , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Regulação Fúngica da Expressão Gênica , Histoplasma/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Dados de Sequência Molecular , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Consumo de Oxigênio/fisiologia , Proteínas de Plantas , RNA Fúngico/química , RNA Fúngico/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Homologia de Sequência de AminoácidosRESUMO
Histoplasma capsulatum produces an extracellular catalase termed M antigen, which is similar to catalase B of Aspergillus and Emericella species. Evidence is presented here for two additional catalase isozymes in H. capsulatum. Catalase A is highly similar to a large-subunit catalase in Aspergillus and Emericella species, while catalase P is a small-subunit catalase protein with greatest similarity to known peroxisomal catalases of animals and Saccharomycotina yeasts. Complete cDNAs for the CATA and CATP genes (encoding catalases A and P, respectively) were isolated. The transcriptional expression of the H. capsulatum CATA, CATB (M antigen) and CATP genes was assessed by Northern blot hybridizations on total RNA. Results at the transcript levels for these genes are shown for three conditions: cell morphology (mycelial versus yeast phase cells), oxidative stress (in response to a challenge with H(2)O(2)) and carbon source (glucose vs glycerol). Collectively, these results demonstrated regulation of CATA by both cell morphology and oxidative stress, but not by carbon source, and regulation of CATB and CATP by carbon source but not cell morphology or oxidative stress. A phylogenetic analysis of presently available catalase sequences and intron residences was done. The results support a model for evolution of eukaryotic monofunctional catalase genes from prokaryotic genes.
