Ectophosphatase activity in Candida albicans influences fungal adhesion: study between HIV-positive and HIV-negative isolates.

OBJECTIVE: This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV-infected children (HIV+) and compares them with fifteen isolates from HIV-negative children (HIV-), as well as the fungal adhesion to epithelial cells and medical records. METHODS: The activities were measured in intact cells grown in BHI medium for 48 h at 37 degrees C. Phosphatase activity was assayed at pH 5.5 using 4-methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line. RESULTS: Mean values of ectophosphatase activity were 610.27 +/- 166.36 and 241.25 +/- 78.96 picomoles 4-methylumbelliferone/h/10(7) cells for HIV+ and HIV- group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV- (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor. CONCLUSION: These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.

Cell-associated and extracellular proteinases in Blastocrithidia culicis: influence of growth conditions.

The proteinase profile of Blastocrithidia culicis was analyzed, as well as how different growth conditions influenced its expression by gelatin-SDS-PAGE and the use of specific proteinase inhibitors. Multiple cell-associated proteinases with molecular masses corresponding to 33, 55, 60 kDa (cysteine proteinases) and 77, 80, 90, and 108 kDa (metalloproteinases) were detected using these methods. All the metalloproteinases identified were partitioned into the detergent phase after Triton X-114 extract, suggesting that they are membrane-bound, while all cysteine proteinases were partitioned into the aqueous phase. The proteolytic zymograms were similar when three different media were used for variable times of incubation. However, few quantitative and qualitative changes were observed. The secreted proteinase profile showed an heterogeneous pattern of enzymatic activities whose expression was dependent on time of growth and medium composition. However, the extracellular proteinase activities were abolished by 1,10-phenanthroline, suggesting that all of them are zinc-metalloproteinases.

Developmentally regulated protein expression mediated by dimethylsulfoxide in Herpetomonas samuelpessoai.

We have analyzed the total cell extract, cell surface, and secretory protein profiles related to cellular differentiation triggered by dimethylsulfoxide in the insect trypanosomatid Herpetomonas samuelpessoai. The flagellates were cultivated in chemically defined conditions in the absence or in the presence of 4% DMSO, and the resolved protein bands were detected by SDS-PAGE gels and avidin-Western blotting. The cell-associated proteins showed a complex pattern of around 40 silver-staining bands ranging from 15 to 150 kDa. There were generally minor quantitative differences in the protein profile between the non-treated and the DMSO-treated cells. The cell-surface protein profile revealed by the incubation of live parasites with biotin showed a decrease in the expression of the 120 kDa biotinylated polypeptide observed in the DMSO-treated cells when compared with untreated ones. However, control samples of both systems showed an endogenous biotinylated polypeptide of 63 kDa which also presented gelatinolytic activity. The trypanosomatids released at least 10 polypeptides to the culture medium. A low molecular mass exopolypeptide (35 kDa) was found exclusively in untreated cells, whereas a high-molecular-mass exopolypetide (250 kDa) was preferentially found in DMSO-treated cells.

Characterization of proteinases in Herpetomonas anglusteri and Herpetomonas roitmani.

We have analyzed the proteinase profile of two Herpetomonas species, H. anglusteri and H. roitmani (a symbiont-bearing trypanosomatid), by in situ detection of enzyme activities on SDS-PAGE gels containing copolymerized gelatin as substrate. Two major cell-associated proteolytic activities, a 60 kDa zinc-metalloproteinase and a 45 kDa cysteine proteinase could be detected based on the inhibition of their activities by 1, 10-phenathroline and E-64, respectively. The trypanosomatids released into the growth medium distinct proteinases. H. anglusteri expressed three digestion haloes in the gels of approximately 60, 50, and 40 kDa, whereas H. roitmani secreted only a 60 kDa enzyme. However, these activities were inhibited by 1,10-phenanthroline, suggesting that all of them are zinc-metalloproteinase.

Specific and reversible inhibition of Entamoeba histolytica cysteine-proteinase activities by Zn2+: implications for adhesion and cell damage.

BACKGROUND: Cysteine-proteinases are thought to play an important role in E. histolytica pathogenicity. Although effective blockage of proteolytic activities can be obtained with several known inhibitors, the high cellular toxicity of most of the inhibitors precludes experimentation with live cells or animal models. Specific cysteine-proteinase inhibitors that could be utilized in studies of virulence are of great need in the field of amebiasis. METHODS: Cysteine-proteinase activities were determined in trophozoite lysates by azocasein degradation and after PAGE and gelatin zymograms. Inhibition of the activities was assessed in the presence of 0.01-2.5 mM concentrations of divalent cations of the IIB and VIII series such as Zn, Cd, Hg, Ni, and Co. Reversibility was induced with 25 mM L-cysteine or 50 mM L-histidine and by metal chelation with 5 mM phenantroline. The inhibitory effect of ZnCl2 was tested with live cells in fibronectin-binding and cytotoxicity assays. RESULTS: ZnCl2 specifically inhibited cysteine-proteinase activities in trophozoite lysates in a concentration-dependent manner. Additionally, 1.0-2.5 mM ZnCl2 blocked proteolysis in more than 70%. This inhibition was completely reverted by L-cysteine, L-histidine, or phenantroline. Similar results were obtained by analyzing individual cysteine-proteinase activities separated in gelatin zymograms. At these concentrations, ZnCl2 significantly interfered with trophozoite adhesion, thus making amebas deficient in substrate degradation and cell damage. CONCLUSIONS: ZnCl2 is an effective inhibitor of amebic cysteine-proteinases. Its low toxicity at relatively high concentrations, high solubility, and low cost make it adequate for live cell experimentation and animal models of amebic virulence.