Louis de Repentigny

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

ABSTRACT 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 β-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 Candidapopulations in the oral cavity and gastrointestinal tract.

Evidence for Differential Expression of Candida albicans Virulence Genes during Oral Infection in Intact and Human Immunodeficiency Virus Type 1-Transgenic Mice

To comprehensively assess the in vivo expression of Candida albicans hydrolytic enzyme genes during oropharyngeal candidiasis (OPC), a controlled sequential analysis of the temporal expression of individual members of the SAP (secretory aspartyl proteinase) gene family and PLB1 (phospholipase B) in a murine model of OPC was conducted. Acute infections in intact C3H and DBA/2 mice were terminated by clearance of C. albicans within 7 days after oral inoculation, but transgenic (Tg) mice expressing human immunodeficiency virus type 1 were persistently colonized until a final outgrowth before death. In contrast to the sustained expression of other SAP genes and PLB1, SAP7 and SAP8 were conspicuously distinguished by their transient expression in both intact and Tg mice. SAP5 and SAP9 were most strongly expressed throughout the course of infection in the Tg mice. These findings indicate that expression of individual members of the C. albicans SAP gene family is differentially regulated during experimental OPC.

Mucosal Candidiasis in Transgenic Mice Expressing Human Immunodeficiency Virus Type 1

The availability of CD4C/HIV(MutA) transgenic (Tg) mice expressing human immunodeficiency virus type 1 in immune cells and developing an AIDS-like disease has provided the opportunity to devise a model of mucosal candidiasis that closely mimics the clinical and pathologic features of candidal infection in human AIDS. After intraoral infection with Candida albicans, oral burdens were strikingly elevated in the Tg mice, compared with non-Tg littermates (P<.05), during primary infection, a 6-10-week carrier state, and a marked terminal outgrowth preceding death. The chronic carrier state was absent in the non-Tg mice because of clearing of C. albicans. Candida hyphae penetrated the epithelium of the oral cavity, esophagus, and cardial-atrium fold of the stomach, accompanied by a mononuclear cell infiltrate. Immunohistochemical analysis suggested that decreased frequencies of major histocompatibility complex class II-expressing cells, combined with reduced CD4+ cells, may underlie the susceptibility to mucosal candidiasis in these Tg mice.

Characteristics and outcome of infants with candiduria in neonatal intensive care - a Paediatric Investigators Collaborative Network on Infections in Canada (PICNIC) study

BackgroundThere is limited information in the literature on the presentation and prognosis of candidal urinary tract infection (UTI) in infants in the neonatal intensive care unit (NICU).MethodsThis was a prospective cohort study performed in 13 Canadian NICUs. Infants with candidal UTI without extra-renal candidal infection at presentation were enrolled.ResultsThirty infants fit the study criteria. Median birth weight and gestational age were 2595 grams (range 575-4255) and 35 weeks (range 24-41) with 10 infants being < 30 weeks gestation. The most common primary underlying diagnosis was congenital heart disease (n = 10). The median age at initial diagnosis was 16 days (range 6-84 days). Renal ultrasonography findings were compatible with possible fungal disease in 15 of the 26 infants (58%) in whom it was performed. Treatment was variable, but fluconazole and either amphotericin B deoxycholate or lipid-based amphotericin B in combination or sequentially were used most frequently. Extra-renal candidiasis subsequently developed in 4 infants. In 2 of these 4 infants, dissemination happened during prolonged courses of anti-fungal therapy. Three of 9 deaths were considered to be related to candidal infection. No recurrences of candiduria or episodes of invasive candidiasis following treatment were documented.ConclusionCandidal UTI in the NICU population occurs both in term infants with congenital abnormalities and in preterm infants, and is associated with renal parenchymal disease and extra-renal dissemination. A wide variation in clinical approach was documented in this multicenter study. The overall mortality rate in these infants was significant (30%). In one third of the deaths, Candida infection was deemed to be a contributing factor, suggesting the need for antifungal therapy with repeat evaluation for dissemination in infants who are slow to respond to therapy.

CD8+ T cells but not polymorphonuclear leukocytes are required to limit chronic oral carriage of Candida albicans in transgenic mice expressing human immunodeficiency virus type 1

ABSTRACT Candida albicans causes oropharyngeal candidiasis (OPC) but rarely disseminates to deep organs in human immunodeficiency virus (HIV) infection. Here, we used a model of OPC in CD4C/HIVMut transgenic (Tg) mice to investigate the role of polymorphonuclear leukocytes (PMNs) and CD8+ T cells in limiting candidiasis to the mucosa. Numbers of circulating PMNs and their oxidative burst were both augmented in CD4C/HIVMutA Tg mice expressing rev, env, and nef of HIV type 1 (HIV-1), while phagocytosis and killing of C. albicans were largely unimpaired compared to those in non-Tg mice. Depletion of PMNs in these Tg mice did not alter oral or gastrointestinal burdens of C. albicans or cause systemic dissemination. However, oral burdens of C. albicans were increased in CD4C/HIVMutG Tg mice expressing only the nef gene of HIV-1 and bred on a CD8 gene-deficient background (CD8−/−), compared to control or heterozygous CD8+/− CD4C/HIVMutG Tg mice. Thus, CD8+ T cells contribute to the host defense against oral candidiasis in vivo, specifically in the context of nef expression in a subset of immune cells.

Comparison of Itraconazole and Ketoconazole in HIV-Positive Patients with Oropharyngeal or Esophageal Candidiasis

The efficacy of oral itraconazole and ketoconazole in the treatment of oropharyngeal and/or esophageal candidiasis, and the rate of post-treatment relapse, were compared in a multicenter, prospective, double-blind, double-dummy, randomized, parallel-group trial. A total of 143 adult HIV-positive patients with oropharyngeal and/or esophageal candidiasis were assigned to receive either itraconazole or ketoconazole (200 mg/day). Patients with oropharyngeal and esophageal candidiasis were treated for 2 and 4 weeks, respectively. Patients were evaluated clinically and mycologically after 1, 2 and 4 (for esophageal patients) weeks of therapy, and relapses were compared in a 6-week post-treatment follow-up period. Of 129 evaluable patients, 98 had oropharyngeal candidiasis and 31 esophageal infection. CDC classification, CD4+ cell counts, and number of previous episodes of oropharyngeal or esophageal candidiasis were comparable in both groups. Oropharyngeal infection was cleared clinically at 21 days in 71% of patients receiving itraconazole and 60% receiving ketoconazole, and esophageal candidiasis was cleared at 41 days in 100% of patients receiving itraconazole and 91% receiving ketoconazole. Marginally significant differences were found between itraconazole and ketoconazole in rates of clearing of infection clinically in patients with oropharyngeal and esophageal candidiasis (p = 0.0614 and 0.0781, respectively). Mean rates of infection relapse were not statistically different in the two treatment groups. Adverse events were generally mild and not considered drug related. Itraconazole is marginally more efficacious than ketoconazole in the treatment of oropharyngeal and esophageal candidiasis in HIV-positive patients and both drugs appear safe and well tolerated.

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

ABSTRACT We previously reported that CD4C/human immunodeficiency virus (HIV)Nef transgenic (Tg) mice, expressing Nef in CD4+ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4+ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4+ T cells, DCs, and macrophages (CD4E/HIVNef); in CD4+ T cells and DCs (mCD4/HIVNef and CD4F/HIVNef); in macrophages and DCs (CD68/HIVNef); or mainly in DCs (CD11c/HIVNef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIVNef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4+ T cells showed CD4+ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4+ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.

Effect of the Echinocandin Caspofungin on Expression of Candida albicans Secretory Aspartyl Proteinases and Phospholipase In Vitro

ABSTRACT Although the echinocandin caspofungin primarily inhibits the synthesis of cell wall 1,3-β-d-glucan, its fungicidal activity could also potentially perturb the expression of virulence factors involved in the ability of Candida albicans to cause infection. Expression of the C. albicans secretory aspartyl proteinase (SAP) and phospholipase B (PLB) virulence genes was determined by reverse transcription-PCR after the addition of caspofungin to cells grown for 15 h in Sabouraud dextrose broth. In cells that remained viable, expression of SAP1 to SAP3, SAP7 to SAP9, and PLB1 was unaltered after exposure to fungicidal concentrations (4 to 16 μg/ml) of caspofungin over a period of 7 h. However, expression of SAP5 increased steadily beginning 1 h after exposure to caspofungin. These results indicate that caspofungin is rapidly fungicidal against C. albicans, before any suppression of SAP or PLB1 gene expression can occur.

Isolation and characterization of the Candida albicans SEC4 Gene

The SEC4 gene product is a major component of the protein secretion machinery. More specifically, it is believed to play a pivotal role in targeting and fusion of secretory vesicles to the plasma membrane. Its recently described implication with the Saccharomyces cerevisiae Rho3p, which is required for directing growing points during bud formation, has prompted us to investigate the role and function of Sec4p in the morphological changes of the yeast pathogen Candida albicans. We have therefore cloned the C. albicans SEC4 gene. It encodes a 210 amino acids long protein sharing up to 75% homology to the S. cerevisiae homolog, when conserved changes are allowed. Its RNA is constitutively expressed in C. albicans grown under various physiological conditions. We also show that it can functionally complement a S. cerevisiae sec4 thermosensitive mutant. The sequence of the C. albicans SEC4 gene has been deposited in GenBank under Accession Number AF017183.

A defect in ATP‐citrate lyase links acetyl‐CoA production, virulence factor elaboration and virulence in Cryptococcus neoformans

The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment. We focused on the ACL1 gene for ATP‐citrate lyase, which converts citrate to acetyl‐CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl‐CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl‐CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unable to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl‐CoA production (e.g. an acetyl‐CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modelling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen‐specific inhibitors.