RESUMO
Candida albicans secreted aspartyl proteinases (Sap), products of the SAP genes, which are presumed to act as virulence factors. In the C. albicans strain WO-1, the ability to secrete Sap1 is regulated with switch phenotype, another putative virulence factor. KpnI restriction fragment length polymorphisms differentiate between several distinct SAP1 alleles in laboratory and clinical strains. Both SAP1 alleles from strain WO-1 along with their 5'- and 3'-flanking regions were cloned and sequenced, as were both alleles from another strain, SS. The 5'-flanking regions were remarkably similar in all four of the sequenced alleles over approximately 1,500 nucleotides. S1 analysis revealed that both alleles of WO-1 are transcribed. Characterization of the one allele from strain WO-1 identified a 284-nucleotide insertion flanked by 8-bp direct repeats that shows homology to the CARE2 repetitive element and that is not present in the other alleles. Characterization of the SAP1 alleles also identified a fourth SAP gene (SAP4) that includes an extended leader sequence. SAP4 is positioned upstream, in tandem to SAP1, in all strains tested and may encode another closely related secreted aspartyl proteinase.
Assuntos
Ácido Aspártico Endopeptidases/genética , Candida albicans/genética , Proteínas Fúngicas , Genes Fúngicos/genética , Sequências Repetitivas de Ácido Nucleico/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Candida albicans/patogenicidade , Elementos de DNA Transponíveis/genética , DNA Fúngico/genética , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Mapeamento por Restrição , Transcrição Gênica/genética , Virulência/genéticaRESUMO
The secreted aspartyl proteinases of Candida albicans (products of the SAP genes) are thought to contribute to virulence through their effects on Candida adherence, invasion, and pathogenicity. From a single strain of C. albicans (WO-1) which expresses a phenotypic switching system, three secreted aspartyl proteinases have been identified as determined by molecular weight and N-terminal sequence. Each of the three identified proteins represents the mature form of one of three distinct proteinase isoenzymes, two of which correspond to the recently cloned SAP1 and SAP2 genes (previously referred to as CAP, PEP, or PRA). A genomic library was screened under low-stringency hybridization conditions with a polymerase chain reaction fragment from SAP1. In addition to clones of SAP1 and SAP2, a clone containing SAP3, a novel third secreted proteinase gene, was identified and sequenced. The three aspartyl proteinase isoenzymes differ in primary sequence and pI, suggesting that they may play different roles in virulence and pathogenesis. All three of these proteinases are expressed in the same strain. However, the pattern of proteinase expression is correlated with the switch phenotype of the cell. Opaque cells of strain WO-1 express Sap1 and Sap3, while white cells of the same strain express Sap2. The differential expression of three Sap proteinases may contribute to virulence in C. albicans.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Candida albicans/enzimologia , Genes Fúngicos , Isoenzimas/metabolismo , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/isolamento & purificação , Sequência de Bases , Southern Blotting , Western Blotting , Candida albicans/crescimento & desenvolvimento , Clonagem Molecular , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Soros Imunes , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Cinética , Dados de Sequência Molecular , OligodesoxirribonucleotídeosRESUMO
The moderately repetitive Ca3 fragment of Candida albicans has been used as an effective DNA fingerprinting probe in epidemiological studies. EcoRI digestion of Ca3 DNA results in seven fragments of 4.2 kb (A), 2.98 kb (B), 2.85 kb (C), 0.77 kb (D1), 0.77 kb (D2), 0.38 kb (E), and 0.30 kb (F). Five of these EcoRI fragments have been mapped in the 5'-3' order C B D1 A D2. The intact Ca3 probe and the three largest EcoRI fragments, A, B, and C, were individually used to probe Southern blots of EcoRI-digested DNA of a set of test strains, transverse alternating field electrophoresis-separated chromosomes of strain 3153A, and Northern (RNA) blots of test strain 3153A. Fragments, A, B, and C each generate a different Southern blot hybridization pattern with EcoRI-digested whole-cell DNA; Ca3 sequences are present in at least five of seven separable chromosomes and a minichromosome of strain 3153A; fragments A, B, and C are distributed differently on chromosomes; and fragments A, B, and C do not cross-hybridize. Ca3 hybridizes to three major transcripts of 2.8, 2.3, and 1.5 kb. Fragment A hybridizes intensely to the 1.5-kb transcript, while fragments B and C both hybridize intensely to the 2.8- and 2.3-kb transcripts. The B fragment, which contains 2,980 bp and contributes to the major portion of the Ca3 pattern, was sequenced. Both direct and inverted repeat sequence motifs were identified. These results provide us with initial insights into the evolution of the Ca3 pattern and the nature of the probe.
Assuntos
Candida albicans/genética , Impressões Digitais de DNA , Sondas de DNA/genética , DNA Fúngico/genética , Sequência de Bases , Southern Blotting , Candidíase/epidemiologia , Candidíase/microbiologia , Desoxirribonuclease EcoRI , Desoxirribonucleases de Sítio Específico do Tipo II , Métodos Epidemiológicos , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sequências Repetitivas de Ácido NucleicoRESUMO
Restriction fragment polymorphism analysis was used to investigate the identity and genotypic relatedness of Candida albicans strains isolated from human immunodeficiency virus (HIV)-infected patients with or without oral candidiasis and from some of their sexual partners. Use of the species-specific DNA probe Ca3 revealed that most subjects carried a single distinct C. albicans strain throughout the course of the study, during both symptomatic and asymptomatic periods. Sexual partners were more likely to carry the same or similar C. albicans isolates than unrelated subjects, raising the possibility of transmission via intimate contact. One patient appeared to acquire his partner's isolate, which then became predominant in both partners in subsequent isolations. These findings indicate that recurrent oral candidiasis is usually caused by a single persistent strain unique to each patient, but that in some cases transmission via intimate contact may occur between sexual partners.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Candida albicans/isolamento & purificação , Soropositividade para HIV/microbiologia , Boca/microbiologia , Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Southern Blotting , Candida albicans/classificação , Candida albicans/genética , Candidíase/tratamento farmacológico , Candidíase/etiologia , Candidíase/microbiologia , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Feminino , Genoma Fúngico , Homossexualidade , Humanos , Cetoconazol/uso terapêutico , Masculino , Nistatina/uso terapêutico , Polimorfismo GenéticoRESUMO
The fungus Histoplasma capsulatum causes histoplasmosis, the most common endemic respiratory mycosis in the United States. Disseminated histoplasmosis in adults is often associated with immunosuppression, such as occurs in HIV infection. We report a case of oral histoplasmosis in an HIV-seropositive patient who presented with an ulceration on the left tip of the tongue, extending to the floor of the mouth, but was otherwise free of any active systemic disease. Histoplasma capsulatum was shown, by both histopathology and staining with a fluorescent antibody reagent specific for the organism, to be present in the lesion and was deduced to be the causative organism.
