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.

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.

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 Sapl is regulated with switch phenotype, another putative virulence factor. KpnI restriction fragment length polymorphisms differentiate between several distinct SAP] alleles in laboratory and clinical strains. Both SAPJ 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. Si 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 SAP] alleles also identified a fourth SAP gene (SAP4) that includes an extended leader sequence. SAP4 is positioned upstream, in tandem to SAP], in all strains tested and may encode another closely related secreted aspartyl proteinase.Candida albicans is one of the most common causes of opportunistic infections among immunocompromised patients. Factors which may contribute to the conversion of this normally commensal organism to a more virulent form include a morphological transformation between yeast and hyphal cells (6) and the ability of a Candida species to express alternative colony and cell shapes, a phenomenon which has been referred to as phenotypic switching (reviewed by Soll [31]). The phenotypic switching system of strain WO-1 has been characterized in detail (30, 32) and is easily manipulated, making it an important laboratory tool for examining the relationship between switch phenotype and the expression of virulence characteristics. In strain WO-1, the cells switch reversibly and at high frequency between predominantly spherical and smooth cells and cells which are larger, ovoid, and rough in appearance. Each of these cell types gives rise to a distinct colony phenotype, white (W) and opaque (0), respectively, on solid medium. The switch phenotype also affects properties associated with virulence, such as cell permeability, adhesiveness, drug susceptibility, and sensitivity to neutrophils and oxidants (32). The ability to switch between morphologic forms therefore seems to be a reflection of more pervasive changes in the spectrum of proteins expressed by each phenotype, a feature which is particularly interesting in the context of virulence, as each distinct cell type may be able to respond differently to the host.The secretion of proteolytic enzymes by C. albicans was first noted by Staib in 1965 (33), and since that time, a C. albicanssecreted aspartyl proteinase (Sap) activity has been linked with virulence through indirect observation. It has been reported that Sap activity is directly proportional to the virulence of * Corresponding au...

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.

Four proteins from Candida albicans extracts have been isolated by ATP affinity chromatography. These proteins were found to be at elevated levels in extracts of cells raised from 25 "C to 37 "C, but were present at low levels in cells grown at 25 "C. The molecular masses of the proteins (38-42 kDa, 66-68 kDa, 70-72 kDa and 74-76 kDa) correspond to the published sizes of C. albicans heat-shock proteins. Three of the four proteins were recognized by the sera of patients with oral and/or oesophageal C. albicans infections, with the 70-72 kDa protein reacting in all cases tested. Binding of antibodies to two of the other proteins (38-42 kDa and 74-76 kDa) differed from patient to patient. IgA antibodies were the dominant immunoglobulin class in these mucosal C. albicans infections. The IgA antibody titre may be of diagnostic value and seemed to be correlated to the severity of infections, with a higher level in oesophageal infections compared to oral infections. Antibody binding to these proteins was specific as the sera did not show the same enhanced recognition with bacterial or HeLa cell heat-shock proteins.