Secreted Aspartyl Proteinase Research Articles

Evidence for degradation of gastrointestinal mucin by Candida albicans secretory aspartyl proteinase.

A zone of extracellular digestion of the mucin layer around Candida albicans blastoconidia was observed by transmission electron microscopy in the jejunum of mice inoculated intragastrically (G. T. Cole, K. R. Seshan, L. M. Pope, and R. J. Yancey, J. Med. Vet. Mycol. 26:173-185, 1988). This observation prompted the hypothesis that a putative mucinolytic enzyme(s) may contribute to the virulence of C. albicans by facilitating penetration of the mucus barrier and subsequent adherence to and invasion of epithelial cells. Mucinolytic activity was observed as zones of clearing around colonies of C. albicans LAM-1 grown on agarose containing yeast nitrogen base, glucose, and hog gastric mucin. In addition, concentrated culture filtrate obtained after growth for 24 h in yeast nitrogen base, supplemented with glucose and mucin as the sole nitrogen source, contained proteolytic activity against biotin-labelled mucin which was inhibited by pepstatin A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the culture filtrate revealed two components of 42 and 45 kDa, with pIs of 4.1 and 5.3, respectively. A zymogram showed that mucin was degraded only by the 42-kDa component, which was also recognized by immunoblotting with an anti-secretory aspartyl proteinase (anti-Sap) 2p monoclonal antibody. The N-terminal sequence of the first 20 amino acids matched that reported for Sap2p. These results demonstrate that Sap2p is responsible for proteolysis of mucin by C. albicans in vitro and may be involved as a virulence factor in the breakdown of mucus and penetration of the mucin barrier by C. albicans.

Acid proteinase secreted by Candida tropicalis: functional analysis of preproregion cleavages in C. tropicalis and Saccharomyces cerevisiae.

The 40 kDa secreted aspartyl proteinase (Sapt1) of Candida tropicalis is a pepsin-like enzyme encoded by the SAPT1 gene. According to the deduced amino acid sequence. Sapt1 has a putative preproregion of 60 amino acids preceding the mature enzyme. Maturation and processing of Sapt1 was analysed in C. tropicalis and Saccharomyces cerevisiae strains expressing wild-type or mutated forms of SAPT1. In S. cerevisiae, the glycosylated 46 kDa proenzyme was converted to the mature 40 kDa form of Sapt1 by KEX2-dependent proteolytic cleavage following the Lys59-Arg60 sequence. The replacement of Lys59-Arg60 by Lys59-Gly60 revealed that the precursor can be processed by an autocatalytic cleavage. This alternative processing pathway to produce mature Sapt1 is less efficient than the Kex2-mediated pathway. Finally, it was shown that in C. tropicalis and S. cerevisiae the removal of the proregion was a prerequisite for the secretion of Sapt1.

Candida albicans: Morphogenesis and Pathogenesis.

Germ tube production of Candida albicans is favoured by, and is adapted to, conditions of nutrient deprivation, particularly low nitrogen. Hyphal development in media containing low serum concentrations is characterised by the formation of sparsely branched hyphae in which uninucleated, highly vacuolated intercalary compartments are arrested in the cell cycle, probably in G1. Re-entry into the cell cycle is accelerated by enrichment of the medium for nutrients and is preceded by reduction of the vacuolar space and concomitant increase in the size of the cytoplasmic volume. Hyphae exhibit contact guidance (thigmotropism) which may facilitate the invasion of tissues through local wounds, invaginations or sites of weakened surface integrity. Thus hyphal growth is adapted for nutrient scavenging and penetration of irregular surfaces.The genetics of dimorphism and virulence has been investigated through studies of differential gene expression, and by specific manipulation of gene expression by overexpression and the creation of null mutants. The expression of many genes is regulated during dimorphism however few of these play active roles in the regulation of morphogenesis. Disruption of genes encoding certain secreted aspartyl proteinases (SAP2) and a key step in the assembly of O-linked mannosylation (MNT1) led to attenuation of virulence. Manipulation of the expression of a hyphal specific gene HYR1 did not affect germ tube growth while overexpression of a constitutively expressed Candida gene HST7 stimulated pseudohyphal growth in Saccharomyces. Therefore, analysis and manipulation of the normal temporal pattern of gene expression is yielding valuable information regarding the regulation of morphogenesis and pathogenesis in this fungus.

Regulation of fungal extracellular proteases and their role in mammalian pathogenesis

Fungal infection in the immunocompromised host is a problem of increasing importance. The virulence determinants of Aspergillus fumigatus, the major agent of invasive aspergillosis, and of Candida albicans, causing candidiasis, are not well characterized. For both pathogens, the involvement of extracellular proteases in pathogenesis is discussed. The use of gene disruption techniques to inactivate the A. fumigatus alkaline protease and metalloprotease genes has led to the firm conclusion that neither of these enzymes has a significant role in virulence. The diploid nature of C. albicans (necessitating sequential inactivation of both alleles for gene disruption studies) and the presence of a multigene family encoding secreted aspartyl proteases has hampered progress in understanding the role of proteases in virulence. We discuss the involvement of wide-domain regulators in the control of protease production and give an example of how one of these regulators (encoded by the areA gene) has been used in virulence studies. Key words: Aspergillus, Candida, proteases, gene regulation.

Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors.

For the pathogenic yeast Candida albicans, secreted aspartyl proteinase (Sap) activity has been correlated with virulence. A family consisting of at least eight SAP genes can be drawn upon to produce Sap enzymatic activity. In this study, the levels of Sap1, Sap2, and Sap3 isoenzymes were monitored under a variety of growth conditions for several strains, including strain WO-1, which alternates between two switch phenotypes, white (W) and opaque (O). When cultured under proteinase-inducing conditions, most strains and W cells produce Sap2, while O cells produce Sap1, Sap2, and Sap3. Both W and O cells of strain WO-1 produce Saps in enriched and defined media that do not induce Saps from other strains. The specific Sap isoenzyme that is produced is determined by the cell type, while the level of Sap production is determined by environmental factors. The levels and temporal regulation of the SAP mRNAs as determined by Northern (RNA) analysis were consistent with Sap protein levels and with previous results. S1 analysis showed that SAP6 is the predominant SAP gene transcribed during hyphal induction at neutral pH. These studies define the culture conditions which control the levels of SAP mRNAs and Sap proteins, and they indicate that both the yeast/hyphal transition and phenotypic switching can determine which of the Sap isoenzymes is produced.

Rats clearing a vaginal infection by Candida albicans acquire specific, antibody-mediated resistance to vaginal reinfection

Oophorectomized, estrogen-treated rats were susceptible to experimental vaginal infection by Candida albicans. After spontaneous clearing of the primary infection, the animals were highly resistant to a second vaginal challenge with the fungus. The vaginal fluid of Candida-resistant rats contained antibodies directed against mannan constituents and secretory aspartyl proteinase(s) of C. albicans and was capable of transferring a degree of anti-Candida protection to naive, nonimmunized rats. This passive protection was mediated by the immunoglobulin fraction of the vaginal fluid and was substantially abolished by preabsorption of the vaginal fluid with C. albicans, but not with Saccharomyces cerevisiae, cells. Vaginal anti-mannan antibodies were also produced by active immunization with heat-killed cells of C. albicans or with a mannan extract when administered via the vaginal route. The protection conferred was comparable to that resulting from clearing of the primary infection. In summary, the data suggest that acquired anticandidal protection in this vaginitis model is mediated at least in part by antibodies, among which those directed against the mannan antigen(s) might play a dominant role.

The "universal" leucine codon CTG in the secreted aspartyl proteinase 1 (SAP1) gene of Candida albicans encodes a serine in vivo

A number of Candida species possess a tRNA(Ser)-like species that recognizes CTG codons that normally specify leucine (Leu) in the universal code of codon usage. Mass spectrometry and Edman sequencing of peptides from the secreted aspartyl proteinase isoenzyme (Sap1) demonstrate that positions specified by the CTG codon contain a nonmodified serine (Ser) in Candida albicans.

Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans

The opportunistic fungal pathogen Candida albicans produces secretory aspartyl proteinases, which are believed to be virulence factors in infection. We have studied the in vitro expression of seven known members of the SAP gene family in a range of strains and serotypes by Northern analysis. SAP1 and SAP3 were regulated during phenotypic switching between the white and opaque forms of the organism. The SAP2 mRNA, which was the dominant transcript in the yeast form, was found to be autoinduced by peptide products of Sap2 activity and to be repressed by amino acids. The expression of the closely related SAP4-SAP6 genes was observed only at neutral pH during serum-induced yeast to hyphal transition. No SAP7 mRNA was detected under any of the conditions or in any of the strains tested. Our data suggest that the various members of the SAP gene family may have distinct roles in the colonization and invasion of the host.

A fourth secreted aspartyl proteinase gene (SAP4) and a CARE2 repetitive element are located upstream of the SAP1 gene in Candida albicans.

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.

Three distinct secreted aspartyl proteinases in Candida albicans.

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 genes encoding the secreted aspartyl proteinases of Candida albicans constitute a family with at least three members

The secreted aspartyl proteinase activity from Candida albicans is thought to be a potential virulence factor. Four laboratories have cloned a gene from C. albicans encoding this enzyme. When two of these genes sharing 77% homology at the DNA level are hybridized under conditions of high stringency to contour-clamped homogeneous electric field chromosome separations of four different strains, they label different chromosomes: chromosome 6 for SAP1 and chromosome R for SAP2. The existence of different genes for the two sequences was confirmed by polymerase chain reaction. Genomic Southern blots probed with the genes and washed at low stringency revealed several cross-hybridizing bands. Contour-clamped homogeneous electric field chromosome separations probed at low stringency indicated that there was a cross-hybridizing sequence on chromosome 3 in addition to those on chromosomes R and 6. The genes for the secreted aspartyl proteinase activity in C. albicans thus constitute a gene family which we have called the SAP family.

Vaginopathic and proteolytic Candida species in outpatients attending a gynaecology clinic.

Non-pregnant, non-diabetic outpatients were examined for the presence of pathogenic vaginal yeasts to determine if a correlation existed between a specific yeast and clinical disease. Yeasts were isolated as single vaginal species from 186 of 228 subjects with clinically diagnosed candidal vaginitis, as well as from 122 out of 380 asymptomatic, age-matched controls. Apart from Candida albicans and C glabrata, other prevalent species were C krusei, C parapsilosis and Saccharomyces cerevisiae which accounted for 9.2%, 6.0% and 5.4%, and 9.0%, 2.4% and 19.7%, of yeasts from patients and carriers, respectively. Only C albicans and C parapsilosis were significantly more common in those with vaginitis. Only the isolates of these two species secreted aspartyl proteinase in vitro, and the amount of the enzymes secreted by the isolates from patients was significantly higher than that secreted by the isolates from carriers. These two species consistently produced vaginal infection in pseudoestrus rats, whereas none of the non-proteolytic species tested (C glabrata, C krusei, and S cerevisiae) colonised the vagina in these rats. Proteinase secretion correlated with experimental vaginal infection; it could also be a reliable factor for distinguishing clinically active infection from asymptomatic fungal carriage.

Molecular targets for the development of AIDS therapeutics : C. Debouck, Department of Molecular Genetics, SmitKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA

Candida virulence attributes include the ability to colonize and invade host tissues, and the secretion of hydrolytic enzymes. Although Candida albicans is regarded as the principal fungi causing infections in humans, other species, particularly Candida tropicalis, are increasingly being recognized as human pathogens. Relatively little is known, however, about the virulence attributes associated with C. tropicalis. The present study aimed to investigate epithelial infection by C. tropicalis using a reconstituted human oral epithelium (RHOE) together with confocal laser scanning microscopy and real-time PCR. A comparison of clinical strains was made in terms of tissue colonization, invasion and C. tropicalis secreted aspartyl proteinase (SAPT) gene expression. All C. tropicalis strains were able to colonize RHOE in a strain-dependent manner. After 12 h of infection, C. tropicalis was found to be highly invasive, with extensive tissue damage occurring after 24 h. Real-time PCR of C. tropicalis SAPT1-4 genes showed that expression was strain-dependent, with SAPT2-4 transcripts being frequently detected and SAPT1 rarely detected. Tissue invasion and damage was not inhibited by the presence of pepstatin A. Accordingly, and given that an increase in infection time was not accompanied with an increase in SAPT gene expression, it can be suggested that the proteinases are not involved in invasion and damage of RHOE by C. tropicalis. In summary, C. tropicalis can be considered as highly invasive with the ability to induce significant tissue damage. These features, however, do not appear to be related to specific SAPT gene expression.