Biofilm of Candida albicans from oral cavity of an HIV-infected child: challenge on enamel microhardness.

OBJECTIVE: The aim of this study was to analyze the effect of C. albicans on enamel microhardness in vitro. STUDY DESIGN: Candida albicans was isolated from the oral mucosa (M) and dentin carious lesion (D) of an HIV+ child. Three groups of 12 enamel blocks each were placed in Petri plates (yeast carbon base agar/1% bovine serum albumin): G1, exposed to biofilm formed by C. albicans from M; G2, exposed to biofilm formed by C. albicans from D; G3, no biofilm. Three enamel blocks from each group were removed on days 3, 5, 8, and 10 after biofilm formation to measure the cross-sectional Knoop microhardness (CSMH) of the enamel areas, exposed and not exposed to biofilm. RESULTS: CSMH decreased in G1 and G2: in G1 on day 5, and in G2 on day 3 (analysis of variance: P < .05; Mann-Whitney test: P < .05), with a similar mean percentage reduction for both groups. CONCLUSIONS: Candida albicans can reduce enamel microhardness in vitro.

Differential collagenolytic activity of Candida albicans isolated from oral mucosa and dentinal carious lesions of HIV-infected children.

OBJECTIVE: The aim of this study was to compare type I collagen degradation by Candida albicans isolated from oral mucosa (M) and cavitated active dentinal caries (CAD) of HIV-infected children. STUDY DESIGN: To verify the proteolytic activity, the specimens were cultivated in brain-heart infusion medium and the supernatants were incubated in the presence or absence of type I collagen at 37°C for 12 hours and analyzed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Intensity of the bands on the gels was assessed by densitometric analysis using a scanner and images analyzed with software from Kodak Digital Science EDAS 120. RESULTS: Supernatants of all the C. albicans degraded type I collagen: that from M, on average, by 38.3% (SD 21.67) and that from CAD by 54% (SD 25.94; Wilcoxon test: P < .05). Predisposing factors had no association with the percentage of type I collagen degradation (Mann-Whitney test: P > .05). CONCLUSIONS: Candida albicans from different sites of the oral cavity of HIV-infected children has proteolytic activity for type I collagen.

Associations among the use of highly active antiretroviral therapy, oral candidiasis, oral Candida species and salivary immunoglobulin A in HIV-infected children.

OBJECTIVES: The aim was to examine the impact of antiretroviral therapy on the prevalence of oral candidiasis, recovery of oral Candida spp. , and salivary levels of total secretory immunoglobulin A (SIgA) and Candida-specific SIgA in human immunodeficiency virus (HIV)-infected children. STUDY DESIGN: Sixty-six HIV+ and 40 HIV- children were cross-sectionally examined for the presence of oral lesions. Whole stimulated saliva samples were collected for the identification of Candida spp. using culture and measurement of total and specific SIgA using enzyme-linked immunosorbent assay (ELISA). RESULTS: The HIV+ children had a higher prevalence of oral candidiasis (P < .05), higher frequency of detection of Candida spp. (P < .05), and higher levels of total (P < .05) and Candida-specific SIgA (P < .001) than the HIV- children. Among the HIV+ subjects, antiretroviral users had lower viral loads (P < .001) and lower levels of Candida spp. (P < .05) and total SIgA (P < .05) compared with antiretroviral nonusers. CONCLUSIONS: The use of antiretroviral therapy was associated with decreases in the prevalence of oral candidiasis. This diminished exposure to Candida spp. was accompanied by decreases in levels of total and Candida-specific SIgA.

Effect of suramin on the human pathogen Candida albicans: implications on the fungal development and virulence.

Candida albicans is an opportunistic pathogen that is of growing medical importance because it causes superficial, mucosal and systemic infections in susceptible individuals. Here, the effect of suramin, a polysulfonated naphthylurea derivative, on C. albicans development and virulence was evaluated. Firstly, it was demonstrated that suramin (500 microM) arrested its growth, showing a fungicidal action dependent on cell number. Suramin treatment caused profound changes in the yeast ultrastructure as shown by transmission electron microscopy. The more important changes were the enlargement of the fungi cytoplasmic vacuoles, the appearance of yeasts with an empty cytoplasm resembling ghost cells and a reduction in cell wall thickness. Suramin also blocked the transformation of yeast cells to the germ-tube and the interaction between C. albicans and epithelial cells. In order to ascertain that the action of suramin on C. albicans growth is a general feature instead of being strain-specific, the effects of suramin on 14 oral clinical strains isolated from healthy children and HIV-positive infants were analyzed. Interestingly, the strains of C. albicans isolated from HIV-positive patients were more resistant to suramin than strains isolated from healthy patients. Altogether, the results produced here show that suramin interfered with essential fungal processes, such as growth, differentiation and interaction with host cells.

Leishmania (Leishmania) amazonensis: differential expression of proteinases and cell-surface polypeptides in avirulent and virulent promastigotes.

A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania (Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE). The surface iodinated protein profiles showed two major polypeptides of 65-60 and 50-47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS-PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65-60, 52, and 50-47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65-60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. (L.) amazonensis.

Herpetomonas samuelpessoai: dimethylsulfoxide-induced differentiation is influenced by proteinase expression.

In this study, we analyzed the influence of proteinase expression on the cellular differentiation of Herpetomonas samuelpessoai. Along cellular differentiation, which was induced by dimethylsulfoxide (DMSO), the trypanosomatids secreted several molecules with variable proteolytic activity. All of them were inhibited by 10 m M 1,10-phenanthroline, suggesting that they are zinc-metalloproteinases. Analysis of parasite extracts revealed the occurrence of a 63-kDa metalloproteinase and a 45-kDa cysteine proteinase. After extraction with Triton X-114 followed by water-detergent partition, the 63-kDa component was present in both aqueous and detergent phases, which indicated that this enzyme may be distributed over different cellular compartments including membrane domains. The 45-kDa component, however, presented hydrophilic properties and was predominantly expressed by DMSO non-treated parasites, suggesting that proteinases may be involved in the process of cellular differentiation in H. samuelpessoai. This was confirmed by the fact that a cysteine proteinase inhibitor abrogated parasite differentiation. The role of proteinases and their relevance in the differentiation of H. samuelpessoai are discussed.

Changes of sialomolecules during the dimethylsulfoxide-induced differentiation of Herpetomonas samuelpessoai.

The expression of sialoglycoconjugates during the dimethylsulfoxide (DMSO)-induced differentiation of Herpetomonas samuelpessoai was analyzed by flow cytometry and Western blotting using sialic acid-specific lectins. Parasites reacted strongly with Limax flavus (LFA) and Sambucus nigra (SNA) agglutinins, and only weakly with Maackia amurensis (MAA) lectin. However, analysis of crude protein extracts by Western blotting revealed that bands with molecular masses corresponding to 15 and 40 kDa are recognized by MAA, and that treatment with DMSO induced the expression of two additional polypeptides with molecular masses of 65 and 90 kDa. Profiles of binding to LFA were indistinguishable when protein extracts from control or differentiated cells were analyzed. SNA recognized a major molecule with 25 kDa in extracts from non-differentiated forms and two low-molecular-weight bands from differentiated cells. These results indicate that molecules containing alpha2,6 and alpha2,3 sialyl-galactosyl sequences are present in H. samuelpessoai, and that their biosynthesis and expression are influenced by DMSO-induced differentiation.

Detection of sialoglycomolecules in five plant trypanosomatids and in an insect phytophagous isolate.

The sialoglycoprotein profiles of five plant trypanosomatids (Phytomonas spp.) and of one flagellate (Herpetomonas sp.) isolated from the salivary gland of a phytophagous insect (Phthia picta) were analyzed by Western blotting using three distinct lectins (LFA, SNA and MAA), which recognize specifically sialic acid residues in glycoconjugates. All six flagellates presented at least one polypeptide recognized by the lectins, with the exception of Phytomonas françai, which did not show any reactivity with SNA agglutinin. Phytomonas serpens and P. françai showed the most distinct pattern of sialoglycoproteins. Phytomonas mcgheei, Herpetomonas sp. and the two other Phytomonas spp., isolated from latex, displayed an identical sialomolecule profile. We discuss the possible role of the sialoglycoproteins in the physiology of these trypanosomatids.

Activation of the glycosylphosphatidylinositol-anchored membrane proteinase upon release from Herpetomonas samuelpessoai by phospholipase C.

We have analyzed the effects of exogenous phospholipase C (PLC) on the cell-surface polypeptides and proteinases of Herpetomonas samuelpessoai grown in chemically defined conditions by SDS-PAGE gels. Live parasites treated with PLC released into the extracellular medium a complex profile of glycosylphosphatidylinositol (GPI)-anchored polypeptides ranging from 15 to 100 kDa, some of them with proteolytic activity. Two major metalloproteinases with apparent molecular masses of 63 and 115 kDa were observed after PLC hydrolysis. Interestingly, only the PLC-released soluble form of the 115-kDa metalloenzyme, and not the membrane-anchored form, displayed proteolytic activity, demonstrating that cleavage of the GPI anchor can lead to enzymatic activation.