Niche-specific requirement for hyphal wall protein 1 in virulence of Candida albicans.

Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.

Characterization of Candida parapsilosis infection of an in vitro reconstituted human oral epithelium.

Oral candidosis is a common problem in immunocompromised patients, and whilst Candida albicans is regarded as the principal cause of infection, other non-Candida albicans Candida (NCAC) species are increasingly being recognized as human pathogens. Relatively little is known about the virulence factors associated with NCAC species, and the aim of this study was to use a reconstituted human oral epithelium (RHOE) to examine epithelial infection withCandida parapsilosis. Strains originating from the oral and vaginal mucosa and from the urinary tract were all shown to colonize RHOE in a strain-dependent manner. Strain differences were found in the colonizing morphology and in the extent of invasion of the RHOE. Low invasion of RHOE was detected for strains after 12 h, whereas extensive tissue damage was evident after 24 h when assessed using histological examination and lactate dehydrogenase activity determination. Tissue damage was reduced in the presence of pepstatin A, although C. parapsilosis invasion of the tissue was not inhibited. Real-time polymerase chain reaction of secreted aspartyl proteinase (SAP) genes (SAPP1-3) showed that expression was strain dependent, with an increased expression generally occurring for Candida infecting RHOE compared with planktonic equivalents. In summary, C. parapsilosis was not highly invasive of RHOE but did induce significant tissue damage, which could relate to specific SAPgene expression.

Genetic diversity and exoenzyme activities of Candida albicans and Candida dubliniensis isolated from the oral cavity of Brazilian periodontal patients.

OBJECTIVE: Mucosal surfaces are the primary oral reservoirs of Candida species, but these species can also be found in subgingival biofilm. The present study investigated the genetic diversity and production of exoenzymes of C. albicans and C. dubliniensis isolated from the oral cavity of systemically healthy patients with periodontitis. DESIGN: Fifty-three patients were analysed. Samples were collected from three oral cavity sites (periodontal pocket, gingival sulci and oral mucosa), plated and, after isolation, suspect strains of C. albicans and C. dubliniensis were identified by PCR. The genetic diversity of the isolates was evaluated by RAPD and the activities of the secreted aspartyl proteinases and phospholipases were evaluated by the agar plate method. RESULTS: Twenty-one patients showed positive results for Candida spp. There were no statistically significant differences between genders, or between sites. C. albicans was the most frequently found specie, while C. dubliniensis was isolated from the periodontal pocket of only one patient. Sixteen genotypes were detected among the C. albicans isolates, and one among the C. dubliniensis isolates. The similarity coefficient (S(SM)) values among the C. albicans genotypes ranged from 0.684 to 1.0 with an average of 0.905+/-0.074. All isolates produced high levels of Saps and most of them produced high levels of phospholipases. No relationship was found between the genotypes and the pattern of enzymatic production. There was no association between specific genotypes and their site of isolation. CONCLUSIONS: The results of the present study suggest that genetically homogeneous strains of C. albicans are present in the oral cavity of patients with periodontitis and that these strains are capable of producing high levels of exoenzyme.

Freqüência e atividade enzimática de Candida spp. na cavidade oral de pacientes diabéticos do serviço de endocrinologia de um hospital de Fortaleza-CE / Frequency and enzymatic activity of Candida spp. oral cavity of diabetic patients of the service of endocrinology of a hospital of Fortaleza-CE

O diabetes mellitus (DM), uma doença endócrino-metabólica de alta e crescente prevalência, é citada como responsável pela ocorrência das candidíases orais. A candidíase constitui um espectro de infecções causadas por fungos do gênero Candida, sendo o seu agente mais comum a Candida albicans, embora outras espécies tenham sido identificadas (Candida tropicalis, Candida guillermondii, Candida glabrata, Candida krusei). O objetivo deste trabalho foi avaliar a freqüência e a atividade enzimática de Candida spp. na cavidade oral de pacientes diabéticos atendidos no Serviço de Endocrinologia do Hospital Universitário Walter Cantídio da Universidade Federal do Ceará (HUWC/UFC). Foram coletadas amostras de 48 pacientes diabéticos, de ambos os sexos, com situações variáveis de controle glicêmico. Os materiais clínicos foram colhidos com ajuda de swabs e semeados em placas de Petri contendo ágar-Sabouraud dextrose com cloranfenicol e incubado a 37ºC. Os crescimentos foram identificados pelas provas clássicas usadas em micologia. Depois, essas cepas de Candida foram submetidas a provas de detecção de enzimas fosfolipase e proteinase. Destas, 15 amostras (31,25 por cento) apresentaram cultura positiva para o gênero Candida. A espécie mais freqüente foi a C. albicans, com 80 por cento, seguida de C. tropicalis (13,3 por cento) e C. guillermondii (6,7 por cento). Quanto à pesquisa da atividade enzimática de Candida spp., foi observado que 86,6 por cento delas apresentaram atividade de proteinase e 80 por cento, de fosfolipase. Conclui-se com tais resultados que a C. albicans é a mais freqüente e que as espécies de Candida isoladas possuem fortes atividades enzimáticas.

Diabetes mellitus, a endocrine-metabolic disease, of high and increasing prevalence, is cited as responsible by the occurrence of oral candidiasis. Candidiasis constitutes a specter of infections caused by fungi of genera Candida; the most common agent is Candida albicans, but other species have also been identified (Candida tropicalis, Candida guillermondii, Candida glabrata, Candida krusei). The objective of this work was to evaluate frequency and enzymatic activity of Candida spp. in the oral cavity of diabetic patients taken care in the service of endocrinology of the University Hospital Walter Cantídio of the Federal University of the Ceará. Samples had been collected of 48 diabetic patients, men and women, with various situations of glicemic control. Clinical materials had been collected with aid of swab and harvested in plates of Petri contend Sabouraud agar dextrose with cloranfenicol and incubated to 37ºC. The grown were identified by the used classic tests in mycology. In the following, these Candida strains were submitted to tests to detect phospholipase and proteinase enzymes. Of these, 15 samples (31,35 percent) presented positive culture for the genera Candida. The species more frequent was C. albicans with 80 percent, followed by C. tropicalis (13.3 percent) and C. guilliermondii (6.7 percent). Asfor the research on the enzymatic activity of Candida sp. it was observed that 86.6 percent presented activity of proteinase and 80 percent of phospholipase. It was concluded with these results that C. albicans is more frequent and that Candida spp. isolated species have strong enzymatic activity.

Oropharyngeal candidiasis in HIV-infected patients under treatment with protease inhibitors.

OBJECTIVE: Oropharyngeal candidiasis decreased when protease inhibitors were included with other antiretrovirals to treat HIV infection. We tested oral yeast isolates of Brazilian HIV-infected individuals receiving antiretroviral therapy for protease secretion and susceptibility to ritonavir and some antifungals. STUDY DESIGN: We collected oral samples and identified yeasts from 19 HIV-infected patients receiving highly active antiretroviral therapy (HAART) and suspected of having oral candidiasis. Ritonavir and its excipients' effects on the isolated yeasts were tested for protease secretion by Rüchel's technique. The yeasts' susceptibility to amphotericin B (AnB), fluorocitosine (5FC), fluconazole (FZL), ketoconazole (KZL), and itraconazole (IZL) was determined by E-test (AB Biodisk). Chi-squared test determined the statistical differences. RESULTS: Twenty-five different positive isolates were obtained. Sixty-eight percent were C. albicans. Other isolates included C. famata (16%), C. glabrata (4%), C. tropicalis (4%), T. capitatum (4%), and 1 isolate not identified. High protease secretion was observed for most of the isolates (20/25). Ritonavir only altered enzyme secretion in 6/20 of the protease-secreting isolates. All isolates were highly sensitive to both AnB and 5FC. Antifungal activity did not change when ritonavir was added to the culture media. Some isolates were highly resistant to studied antifungals (52.2% KZL, 30.4% FZL, and 26% IZL). Resistance significantly decreased when ritonavir was added to the medium with KZL and IZL (P <.5 by chi-squared). A trend to decreased resistance was also observed with FZL but the results were not statistically significant. CONCLUSION: Candida continues to be the most prevalent fungus in the oral cavity. Although oral candidal isolates secrete protease, ritonavir does not inhibit all protease-secreting oral yeast isolates. There seems to be a synergistic effect between ritonavir and oral antifungals against fungal resistance.

In vitro secreted aspartyl proteinase activity of Candida albicans isolated from oral diseases and healthy oral cavities.

The in vitro secreted aspartyl proteinase (SAP) activity of Candida albicans isolated from a variety of oral conditions, including healthy oral cavities, was determined. SAP activity (units/10(6) cells/ml, +/-SD) was 0.28 +/- 0.33 for pseudomembranous candidosis isolates (n = 18), 0.35 +/- 0.46 for chronic erythematous candidosis isolates (n = 21) and 0.30 +/- 0.32 for chronic hyperplastic candidosis isolates (n = 50). SAP activity of 0.19 +/- 0.22 was recorded for isolates from squamous cell carcinoma (n = 18), 0.26 +/- 0.37 for burning mouth syndrome isolates (n = 29), 0.25 +/- 0.38 for isolates from xerostomia (n = 15) and 0.39 +/- 0.50 for isolates from lichen planus (n = 13). The SAP activity of isolates from oral disease states was significantly (P < 0.05) higher than that recorded for 28 isolates from healthy mouths (activity of 0.04 +/- 0.03). However, there was no significant difference in the SAP activity between the three forms of clinical oral candidosis (P > 0.05). SAP activity was inhibited in control samples containing the SAP inhibitor, pepstatin A. These results indicate that C. albicans strains associated with oral disease have inherently higher SAP activity.

Different isoforms of secreted aspartyl proteinases (Sap) are expressed by Candida albicans during oral and cutaneous candidosis in vivo.

Distinct isoforms of secreted aspartyl proteinases (Sap) of Candida albicans are important virulence factors for different types of candidosis. Predominant expression of Sap1-3 has been shown to be crucial for superficial infections in experimental mucosal and cutaneous candidosis, whereas Sap4-6 might be important for systemic disease. This in-vivo study investigated Sap expression in two samples from patients with oral candidosis and from cutaneous infection. Two different polyclonal antibodies directed against Sap1-3 and Sap4-6 were used for ultrastructural characterisation of protein localisation and expression. Post-embedding immuno-electron microscopy revealed Sap1-3 and Sap4-6 immunoreactivity in all samples. All C. albicans cells expressed predominantly the proteinases Sap1-3 which were evenly distributed within the cell wall and cytoplasmic membrane. In contrast, Sap4-6 labelling was only evident in a few fungal cells. In particular it was localised at the tips of hyphal cells during invasion. These data suggest a different pathogenetic role for Sap1-3 and Sap4-6 during host-fungal interaction.

Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity.

Candida albicans, an opportunistic pathogen in humans, secretes secretory aspartyl proteinases (Saps), which have been correlated with virulence. We examined the temporal regulation of the mRNA expression of seven known members of the SAP gene family by reverse transcription polymerase chain reaction (RT-PCR) in (i) an in vitro model of oral candidosis based on reconstituted human epithelium (RHE); and (ii) clinical samples from patients with oral candidosis. SAP1 and SAP3 transcripts were first detected 42 h after inoculation of RHE, while at the same time, slight morphological alterations in the epithelium were documented by light microscopy. SAP6 expression occurred 6 h later concomitantly with germ tube formation of some infecting Candida cells and severe lesions of the epithelial tissue. SAP2 and SAP8 RT-PCR products were first detected 60 h after infection, while SAP4 and SAP5 transcripts were never discovered. Thus, a temporal progression of SAP expression in the order SAP1 and SAP3 > SAP6 > SAP2 and SAP8 was observed at the same time as increasing RHE damage occurred. At the protein level, Sap antigen was found within the C. albicans yeast cells and the epithelial cells by immunoelectron microscopy using an anti-Sap murine monoclonal antibody directed against the gene products Sap1-3. Expression of SAP1-3 and 6 was also detected by RT-PCR in samples from patients suffering from oral candidosis. Our results suggest that the pathogenesis of experimental and clinical oral candidosis is associated with the differential and temporal regulation of SAP gene expression.

Increased expression of Candida albicans secretory proteinase, a putative virulence factor, in isolates from human immunodeficiency virus-positive patients.

The increased prevalence and the severity of oropharyngeal candidiasis in human immunodeficiency virus (HIV)-positive patients are attributed exclusively to the virus-induced immune deficiency of the host. The present study was aimed at answering the question of whether Candida albicans secretory proteinase, a putative virulence factor of the opportunistic C. albicans yeast, has any potential influence on the clinical manifestation of oropharyngeal candidiasis in HIV-positive patients. We measured the secretory proteinase activities of clinical C. albicans isolates from the oropharynges of either HIV-positive individuals (n = 100) or a control group (n = 122). The mean secretory proteinase activity of C. albicans isolates from the HIV-positive group (4,255 +/- 2,372 U/liter) was significantly higher compared with that of isolates from the control group (2,324 +/- 1,487 U/liter) (P < 0.05). The higher level of secretory proteinase activity in the culture supernatants of individual C. albicans isolates correlated with the increased level of proteinase expression on the cell surface, as revealed by cytofluorometry, and with higher levels of secretion of the immunodetectable protein, as shown by Western blotting (immunoblotting). Proteinase activity within the population of C. albicans isolates from HIV-positive individuals was independent of the patient's clinical disease stage and the CD4+/CD8+ cell numbers. Furthermore, no correlation of the proteinase activities with the C. albicans serotype was found, although C. albicans serotype B was significantly more frequent in the HIV-positive group (40%) compared with that in the control group (12%). However, a positive correlation of proteinase activity to antifungal susceptibility was evident. The C. albicans isolates from the HIV-positive group that were characterized by higher levels of proteinase activity were also less susceptible to the widely used azole antifungal ketoconazole and fluconazole. Collectively, the present data are consistent with a concept of early preferential selection of a subpopulation of C. albicans in HIV-infected patients.

Immunocytochemical detection of ornithine decarboxylase (ODC) in paraffin-embedded tissues as a possible prognostic indicator for oral lesions.

Intracellular levels of ornithine decarboxylase (ODC) are raised following mitogenic stimulus and in neoplasia. Because lesions of the oral cavity are often difficult to assess histologically, we have determined the value of immunocytochemical detection of ODC as a prognostic indicator in 74 routinely fixed and paraffin-embedded oral biopsies using peroxidase-antiperoxidase and immunogold-silver staining. The latter proved more sensitive, yielding positive reactions in 32 of 43 oral carcinomas (11/14 well differentiated, 16/21 moderately differentiated and 3/5 poorly differentiated) and 7/11 potentially malignant lesions, compared with 19/45 carcinomas and 1/15 potentially malignant lesions, by peroxidase anti-peroxidase. Hyperplastic lesions (n = 7) and normal non-keratinized buccal mucosa (n = 7) were all negative. Follow-up was possible in 13 of the carcinoma patients. Of 7 positive ODC reactions but clinically node-free at biopsy, 2 died and 2 had recurrences within 3 years, whereas all of 6 with no immunoreactivity were symptom-free after 3-5 years. Immunostaining for ODC may be helpful for the prognostic assessment of routinely processed oral lesions and in choosing treatment.