Characterization of binding of Candida albicans to small intestinal mucin and its role in adherence to mucosal epithelial cells.

In order to approximate and adhere to mucosal epithelial cells, Candida must traverse the overlying mucus layer. Interactions of Candida species with mucin and human buccal epithelial cells (BECs) were thus investigated in vitro. Binding of the Candida species to purified small intestinal mucin showed a close correlation with their hierarchy of virulence. Significant differences (P < 0.05) were found among three categories of Candida species adhering highly (C. dubliniensis, C. tropicalis, and C. albicans), moderately (C. parapsilosis and C. lusitaniae) or weakly (C. krusei and C. glabrata) to mucin. Adherence of C. albicans to BECs was quantitatively inhibited by graded concentrations of mucin. However, inhibition of adherence was reversed by pretreatment of mucin with pronase or C. albicans secretory aspartyl proteinase Sap2p but not with sodium periodate. Saturable concentration- and time-dependent binding of mucin to C. albicans was abrogated by pronase or Sap2p treatment of mucin but was unaffected by beta-mercaptoethanol, sodium periodate, neuraminidase, lectins, or potentially inhibitory sugars. Probing of membrane blots of the mucin with C. albicans revealed binding of the yeast to the 66-kDa cleavage product of the 118-kDa C-terminal glycopeptide of mucin. Although no evidence was found for the participation of C. albicans cell surface mannoproteins in specific receptor-ligand binding to mucin, inhibition of binding by p-nitrophenol (1 mM) and tetramethylurea (0.36 M) revealed that hydrophobic interactions are involved in adherence of C. albicans to mucin. These results suggest that C. albicans may both adhere to and enzymatically degrade mucins by the action of Saps, and that both properties may act to modulate Candida populations in the oral cavity and gastrointestinal tract.

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.

Development of a method to detect secretory mucinolytic activity from Candida albicans.

Ultrastructural examinations of sites where Candida albicans invaded the bowel wall after oral intragastric inoculation of infant mice suggested that blastoconidia are capable of progressive extracellular digestion of the intestinal mucus barrier. Microplate assay methods, based on biotin or digoxigenin-labelling systems, were therefore devised for quantitation of protease and glycosidase activities against the glycoprotein mucin. Labelled mucin was adsorbed on microplate wells, incubated with sample to be assayed for enzyme activity, and the remaining labelled mucin was quantitated by spectrophotometry. Proteolytic activity against mucin was demonstrated using concentrated culture filtrate of C. albicans strain LAM-1, grown in yeast nitrogen base medium containing mucin as sole nitrogen source. The activity was inhibited by boiling for 10 min or by incubation with the aspartyl proteinase inhibitor pepstatin A.

Murine erythroleukemia cells contain two distinct GATA-binding proteins that have different patterns of expression during cellular differentiation.

GATA-1 is a major transcription factor of the erythroid lineage that has been implicated in the induced expression of a variety of red cell-specific genes during terminal differentiation of murine erythroleukemia cells. Although the GATA-1 protein is present at nearly equal levels before and after differentiation of murine erythroleukemia cells, in this study it was found that in the early commitment stages of the differentiation program there is a transient decrease in the GATA-1 mRNA and DNA binding activity levels due to a temporary block in transcription of the gene. Moreover, using a whole cell extraction procedure it was discovered that murine erythroleukemia cells contain a second GATA binding activity (denoted GATA-rel) which appears to be distinct from the GATA-1 factor based on its non-reactivity to two GATA-1 antisera. This protein has a limited tissue specificity, as it could not be detected in extracts from CHO, NIH 3T3, or COS cells. Similarly to the GATA-1 DNA-binding activity, the GATA-rel activity decreased during the early stages of differentiation. However, unlike GATA-1, GATA-rel activity did not return to pre-induced levels at later times. These results suggest that changes in gene expression during erythroid terminal differentiation may involve an interplay on levels of different GATA-binding factors.

Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66.

A new extracellular xylanase produced by Streptomyces lividans 66 was isolated from a genetically engineered clone of that strain. This enzyme, named xylanase B, has an Mr of 31,000 and acts specifically on xylan as an endo-type xylanase producing short-chain xylo-oligosaccharides. The activity is optimal at pH 6.5 and at a temperature of 55 degrees C, which is similar to that of the previously characterized xylanase A. Xylanase B is glycosylated and has a pI of 8.4; its Km and Vmax. values are 3.71 mg/ml and 1.96 mmol/mg of enzyme respectively. Specific antibodies raised against xylanase A show no cross-reaction with xylanase B; however, the anti-(xylanase B) antibodies react slightly with xylanase A. A comparison of the hydrolysis products obtained from oat-spelts xylan with both enzymes show that xylanase A preferentially degrades short-chain oligo-xylosides, whereas xylanase B acts on the longer, water-insoluble, molecules.