Toll-like receptor 2 is dispensable for acquired host immune resistance to Candida albicans in a murine model of disseminated candidiasis

Previous work by our group showed that Toll-like receptor 2 (TLR2) is essential for activation of innate immunity, playing a major role in the response of macrophages to Candida albicans, triggering cytokine and chemokine expression, and therefore TLR2 -/- mice are more susceptible to systemic primary candidiasis. In this work, we used a murine model of systemic C. albicans infection, in which resistance to reinfection with virulent wild-type cells is induced by prior exposure of mice to a low-virulence agerminative strain of C. albicans (primary sublethal infection), to study the influence of TLR2 gene deletion on (i) the ability to develop an acquired resistance upon vaccination; (ii) the…

The Candida albicans UBI3 gene encoding a hybrid ubiquitin fusion protein involved in ribosome biogenesis is essential for growth.

We have constructed a conditional null mutant Candida albicans strain for the UBI3 gene which encodes a ubiquitin fusion protein involved in ribosome biogenesis. A one-step gene disruption procedure, using the plasmid pCaDis, was designed to place the second copy of the UBI3 gene under the control of the tightly regulated MET3 promoter in a C. albicans heterozygous strain (UBI3/Deltaubi3::hisG), previously isolated in the first step of the ura-blaster protocol. Analysis of the conditional null mutant in repressing and inducing conditions indicates that UBI3 is an essential gene whose expression is required for growth of C. albicans.

Toll-like receptor 2 mediates prostaglandin E2 production in murine peritoneal macrophages and splenocytes in response to Candida albicans

The involvement of Toll-like receptor 2 (TLR2) and TLR4 in triggering signal transduction pathways leading to prostaglandin E(2) (PGE(2)) production in response to Candida albicans has been studied in cells from wild-type, TLR2-/- and TLR4-/- knockout mice. In vitro PGE(2) production by macrophages challenged with zymosan, yeast or hypha cells was strongly inhibited in TLR2-deficient cells, but not in TLR4-/- cells, as compared to macrophages from wild-type mice. PGE(2) production was dependent on de novo cyclooxygenase-2 (Cox2) synthesis, since unchallenged cells failed to produce PGE(2) and specific Cox2 inhibition during challenge totally blocked PGE(2) production. Similar results were o…

Candida and Candidiasis: The Cell Wall as a Potential Molecular Target for Antifungal Therapy

The fungal species Candida albicans is an opportunistic pathogen, which causes serious infections in humans, particularly in immunocompromised patients. Depending on the underlying host defect, C. albicans causes a variety of infections, ranging from superficial mucocutaneous candidiasis to life-threatening disseminated infections. Both the limited spectrum of antifungal drugs currently in clinical use and the emergence of resistances make necessary the development of new effective antifungal drugs with minimal side effects; however, such a research is limited by the small number of specific target sites identified to date. The cell wall is a fungal specific dynamic structure essential to a…

Evaluation of the Ability of Polyphenol Extracts of Cocoa and Red Grape to Promote the Antioxidant Response in Yeast Using a Rapid Multiwell Assay

Saccharomyces cerevisiae has been used as a model organism to study the capacity of cocoa and red grape extracts to trigger an antioxidant response. A methodology adapted to microtiter plates has been developed to monitor yeast growth after culture preincubation with food ingredients and exposure to oxidative stress by hydrogen peroxide and menadione. This methodology proved effective in measuring the ability of cocoa and red grape extracts to promote an antioxidant response in yeast, and also the prospect of conducting dose-response studies. Additionally, the method has proven useful to perform studies with mutant strains lacking genes that may be related to the mechanism of action underly…

The Yeast External Invertase as a Reporter to Study Regulation ofCandida Albicans Promoter Sequences inSaccharomyces Cerevisiae

Depletion of polyubiquitin encoded by the UBI4 gene confers pleiotropic phenotype to Candida albicans cells.

We have studied the roles of polyubiquitin in Candida albicans physiology. Heterologous expression of the C. albicans polyubiquitin (UBI4) gene in a ubi4 Saccharomyces cerevisiae strain suppressed the mutant phenotype (hypersensitivity to heat shock). A heterozygous strain UBI4/Deltaubi4::hisG, obtained following the ura-blaster procedure, was used to construct a conditional mutant using a pCaDis derivative plasmid. By serendipity we isolated the UBI4 conditional mutant as well as a UBI4 mutant containing a non-functional MET3 promoter. Depletion of polyubiquitin conferred pleiotropic effects to mutant cells: (i) a limited increased sensitivity to mild heat shock; (ii) increased formation o…

Cloning of a cDNA fragment encoding part of the protein moiety of the 58-kDa fibrinogen-binding mannoprotein of Candida albicans

Immunoscreening of a Candida albicans expression library with antibodies against the 58 kDa fibrinogen-binding mannoprotein (mp58) of the fungus resulted in the isolation of clones encoding the protein moiety of this molecule. Sequence of the 0.9 kb cDNA of one of the clones selected for further analysis, revealed an open reading frame coding for 292 amino acids, which displays sequence similarity to proteins belonging to a family of immunodominant antigens of Aspergillus spp. The gene corresponding to this cDNA was named FBP1 (fibrinogen-binding protein). These results represent the first report on the identification of C. albicans genes encoding surface receptors for host proteins.

Proteomic Analysis of Saccharomyces cerevisiae Response to Oxidative Stress Mediated by Cocoa Polyphenols Extract

The present study addressed the protective effects against oxidative stress (OS) of a cocoa powder extract (CPEX) on the protein expression profile of S. cerevisiae. A proteomic analysis was performed after culture preincubation with CPEX either without stress (&minus

Toll-like receptor-2 is essential in murine defenses against Candida albicans infections

In this work, we studied the role of toll-like receptor-2 (TLR2) in murine defenses against Candida albicans. TLR2-deficient mice experimentally infected intraperitoneally (i.p.) or intravenously (i.v.) in vivo had very significant impaired survival compared with that of control mice. In vitro production of TNF-alpha and macrophage inhibitory protein-2 (MIP-2) by macrophages from TLR2-/- mice in response to yeasts and hyphae of C. albicans were significantly lower (80% and 40%, respectively; P <0.05) than production by macrophages from wild-type mice. This impaired production of TNF-alpha and MIP-2 probably contributed to the 41% decreased recruitment of neutrophils to the peritoneal cavity…

Toll-like receptor 4 defective mice carrying point or null mutations do not show increased susceptibility toCandida albicansin a model of hematogenously disseminated infection

We have studied the role of TLR4 in murine defenses against Candida albicans in two TLR4-defective mouse strains: C3H/HeJ mice which have defective TLR4 signaling, and TLR4-/- knockout mice. Both TLR4-defective mice strains experimentally infected with virulent C. albicans cells showed no significant difference in survival as compared with their respective controls. Recruitment of neutrophils to the peritoneal cavity of i.p. infected mice was not affected in TLR4-/-animals, but significantly enhanced in C3H/HeJ mice, compared with their control mice. In vitro production of TNF-alpha by macrophages from both types of TLR4-defective mice, in response to yeasts and hyphae of C. albicans, was n…

Complementation of Saccharomyces cerevisiae mutationsin genes involved in translation and protein folding (EFB1 and SSB1)with Candida albicans cloned genes

We have demonstrated that the expression of Candida albicans genes involved in translation and protein folding (EFB1 and SSB1) complements the phenotype of Saccharomyces cerevisiae mutants. The elongation factor 1beta (EF-1beta) is essential for growth and efb1 S. cerevisiae null mutant cells are not viable; however, viable haploid cells, carrying the disrupted chromosomal allele of the S. cerevisiae EFB1 gene and pEFB1, were isolated upon sporulation of a diploid strain which was heterozygous at the EFB1 locus and transformed with pEFB1 (a pEMBLYe23 derivative plasmid containing an 8-kb DNA fragment from the C. albicans genome which contains the EFB1 gene). This indicates that the C. albic…

Molecular cloning and characterization of theCandida albicansUBI3 gene coding for a ubiquitin-hybrid protein

Using a polyubiquitin cDNA as a probe, we have isolated a clone (pPR3, a pEMBLYe23 derivative plasmid) containing the Candida albicans UBI3 gene coding for a fusion protein. This protein is formed by one ubiquitin subunit fused, at its C-terminus, to an unrelated peptide which is similar to the ribosomal protein encoded by the 3' tail of the Saccharomyces cerevisiae UBI3 gene. Southern blot analysis of chromosomal DNA probed with the 3' non-ubiquitin tail of UBI3 indicated that only one homologous gene is present in the C. albicans genome. Heterelogous expression of pPR3 in a S. cerevisiae ubi3 mutant strain complements the mutant phenotype (slow growth) conferred by the ubi3 defect; this p…