Reduced CX3CL1 Secretion Contributes to the Susceptibility of Oral Leukoplakia-Associated Fibroblasts to Candida albicans.
Ontology highlight
ABSTRACT: Candida leukoplakia (OLK) is a kind of oral leukoplakia combined with chronic candidal infection, which plays an important role in the malignant transformation of OLK. However, little is known about the etiology, including susceptibility of leukoplakia to candidal adhesion, invasion and infection. Some antimicrobial peptides secreted by oral epithelial cells or fibroblasts potentially have antifungal activities against Candida albicans (C. albicans). In this study, we established three co-culture models to simulate different C. albicans-fibroblasts interactions during progression of candida leukoplakia. The susceptibility of oral leukoplakia-associated fibroblasts (LKAFs) to C. albicans and its underlying mechanism were determined. Samples of 14 LKAFs and 10 normal fibroblasts (NFs) were collected. The co-culture models showed that LKAFs had promoted the adhesion, invasion, and survival of C. albicans compared with NFs. CX3CL1, a chemokine with antifungal activity, was less abundant in LKAFs than NFs. Overexpression of CX3CL1 via transfection in LKAFs could partly restore the resistance to C. albicans. We also showed that inhibition of ERK could suppress CX3CL1 secretion. While phosphor-ERK was inhibited in LKAFs compared with NFs. Besides, the mRNA expression of a shedding enzyme for CX3CL1, disintegrin and metalloproteinase domain (ADAM) 17 was decreased in LKAFs than NFs. In conclusion, LKAFs produced and secreted less CX3CL1 by inhibiting the ERK signaling pathway, thereby contributing to impaired cell resistance to C. albicans.
Project description:A recognized hotspot for mutations conferring reduced echinocandin susceptibility (RES) is residue S645 of Candida albicans Gsc1(Fks1). We report that the mutation F641Y is associated with RES in a C. albicans isolate. The analogous Fks2 residue is mutated F to V in a Candida glabrata RES isolate; the introduction of this mutation into susceptible C. glabrata confirmed its role in RES. Y641-equivalent Fks residues were identified in intrinsically RES Fusarium species and Candida guilliermondii.
Project description:To investigate the pre-vacuolar secretory pathway in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS1. C. albicans VPS1 encodes a predicted 694-aa dynamin-like GTPase that is 73.3% similar to S. cerevisiae Vps1p. Plasmids bearing C. albicans VPS1 complemented the temperature-sensitive growth, abnormal class F vacuolar morphology, and carboxypeptidase missorting of a S. cerevisiae vps1 null mutant. To study VPS1 function in C. albicans, a conditional mutant strain (tetR-VPS1) was generated by deleting the first allele of VPS1 and placing the second allele under control of a tetracycline-regulatable promoter. With doxycycline, the tetR-VPS1 mutant was hyper-susceptible to sub-inhibitory concentrations of fluconazole, but not amphotericin B, 5-fluorocytosine, or non-specific osmotic stresses. The repressed tetR-VPS1 mutant was defective in filamentation and secreted less extracellular protease activity. Biofilm production and filamentation within the biofilm were markedly reduced. These results suggest that C. albicans VPS1 has a key role in several important virulence-related phenotypes.
Project description:Candida albicans SUR7 has been shown to be required for plasma membrane organization and cell wall synthesis, but its role in virulence is not known. Using a bioinformatics strategy, we previously identified several novel putative secretion pathway proteins potentially involved in virulence, including the C. albicans homolog of the Saccharomyces cerevisiae endocytosis-related protein Sur7p. We therefore generated a C. albicans sur7Delta null mutant and examined its contribution to key virulence attributes.Structurally, the C. albicans sur7Delta mutant was impaired in response to filamentation-inducing conditions, and formed aberrant hyphae with extensive accumulation of plasma membrane-derived structures within the cell. Absence of SUR7 resulted in a temperature-sensitive growth defect at high temperatures (42 degrees C), which was partially rescued by addition of NaCl. We next examined the role of the SUR7 paralog C. albicans FMP45 in this temperature-sensitive phenotype. Analysis of C. albicans Fmp45p-GFP demonstrated co-localization of Fmp45p with Sur7p and increased fluorescence in the plasma membrane in the presence of high salt. We next focused on key virulence-related phenotypes. The C. albicans sur7Delta null mutant exhibited secretory defects: reduced lipase secretion, and increased levels of secreted Sap2p. The null mutant was hyper-susceptible to sub-inhibitory concentrations of caspofungin, but not amphotericin B and 5-fluorocytosine. Functionally, the sur7Delta mutant demonstrated increased adhesion to polystyrene and of note, was markedly defective in biofilm formation. In an in vitro macrophage model of virulence, the sur7Delta mutant was impaired in macrophage killing.Plasma membrane and cell wall organization are important for cell morphology, and alterations of these structures contributed to impairment of several key virulence-associated phenotypes in the C. albicans sur7Delta mutant.
Project description:Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.
Project description:The vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems in Saccharomyces cerevisiae. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogen Candida albicans, we created a conditional mutant in which VMA3 was placed under the control of a tetracycline-regulated promoter (tetR-VMA3 strain). Repression of VMA3 in the tetR-VMA3 strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss of C. albicans V-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-VMA3 strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-VMA3 strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in an in vitro macrophage killing model. Repression of VMA3 suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression of RIM8, MDS3, EFG1, CST20, or UME6, which encode positive regulators of filamentation. Specific chemical inhibition of Vma3p function also results in defective filamentation. These findings suggest either that V-ATPase functions downstream of these transcriptional regulators or that V-ATPase function during filamentation involves independent mechanisms and alternative signaling pathways. Taken together, these data indicate that V-ATPase activity is a fundamental requirement for several key virulence-associated traits in C. albicans.
Project description:ObjectiveThe objectives of this study were to investigate the antifungal susceptibility and genetic diversity of Candida albicans isolated from HIV+ patients with oropharyngeal candidiasis. A total of 50 C. albicans isolates were cultured on Sabouraud glucose agar containing chloramophenicol. The antifungal susceptibility of the isolates against fluconazole, clotrimazole, nystatin, amphotericin B, ketoconazole and flucytosine was assessed using disc diffusion method. The genetic diversity of C. albicans isolates was determined using random amplified polymorphic DNA marker.ResultsThe inhibition zones ranged from 4?±?1.8 to 40?±?3.8 mm for fluconazole, 7?±?1.0 to 37?±?1.8 mm for ketoconazole, 14?±?0.8 to24?±?0.8 mm for amphotericin B, 25?±?0.0 to 33?±?0.0 mm for nystatin and 7?±?4.2 to 40?±?0.0 mm for clotrimazole. At 90% similarity, three distinct groups were observed. The smallest cluster composed of 3 isolates, whereas the largest one composed of 17 isolates. 32% (16/50), 28% (14/50) and 14% (7/50) were resistant to fluconazole, ketoconazole and clotrimazole, respectively.
Project description:C5-deficiency in the A/J and recombinant congenic strain, BcA17, is linked to a dysregulated inflammatory response to Candida albicans infection and high morbidity compared to C5-sufficient C57Bl/6J (B6) mice. The recombinant congenic BcA70 strain thus provides an ideal model to study the effect of C5-deficiency on a B6 genetic background. We have used the A/J and BcA70 strains to carry out a detailed analysis of the pathophysiological effects of the uncontrolled inflammatory activity as a result of Candida infection. We present several lines of evidence to indicate that the heart of C5-deficient mice is severely affected by infection, although the kidney is the site of highest fungal replication. Histological sections of Candida-infected cardiac and kidney tissues from A/J mice reveal cellular infiltration in the vicinity of hyphal foci in the heart, leaving more dense fungal colonies in the kidney unaffected. As well as high-level inflammatory activity in the infected heart tissue, gene expression profiles revealed a striking lack of cardio-protective stress response linked to C5-deficiency. An inability of the A/J and BcA17 hearts to make the appropriate adaptive changes resulted in lower levels of both fatty acid and glucose metabolism, leading to cardiac failure. Keywords: differential response to infection; genetic variation
Project description:Real-time quantitative PCR was used to measure expression levels of genes encoding efflux pumps, ERG11 and two control genes, ACT1 and PMA1, in a collection of 14 fluconazole-susceptible Candida albicans isolates. For each gene, average expression levels and variations within the population were determined. These values were then used as reference points to make predictions about the molecular basis of resistance in 38 clinical isolates (the majority of which were resistant to fluconazole) obtained from 18 patients treated with posaconazole for refractory oropharyngeal candidiasis. For each of the 38 isolates, the expression levels of genes encoding efflux pumps, ERG11 and the control genes, were measured as above. Comparison of the two data sets revealed that expression of ACT1 and PMA1 did not vary significantly between the two sets of isolates. In contrast, MDR1, ERG11, CDR1, and CDR2 were overexpressed in 3, 4, 14, and 35, respectively, of the isolates from patients treated with azoles. In addition to these changes, the patient isolates all had at least one and often multiple missense mutations in ERG11. Select ERG11 alleles were expressed in Saccharomyces cerevisiae; all of the alleles tested conferred reduced susceptibility to fluconazole. Despite both the increases in pump expression and the ERG11 mutations, only one of the patient isolates exhibited a large decrease in posaconazole susceptibility.
Project description:The rise in the use of biomedical devices and implants has seen a concomitant surge in the advent of device-related nosocomial (hospital-acquired) infections of bacterial and fungal origins. The most common nosocomial fungal infection is candidiasis caused mainly by Candida albicans biofilms. Candidiasis is associated with an unacceptably high mortality rate, and there is an urgent need for the discovery of new antifungal drugs that prevent or control biofilm formation. To this end, we recently developed an ultra-high-throughput microarray platform consisting of nano-scale biofilms of C. albicans encapsulated in collagen or alginate hydrogel matrices for antifungal drug screening. Here, we report that the choice of matrix influences the apparent susceptibility of C. albicans to the common antifungal drugs, amphotericin B, and caspofungin. While amphotericin B is equally effective against biofilms grown in collagen and alginate matrices, caspofungin is effective only against biofilms grown only in alginate, but not in collagen. We demonstrate differences in the distribution of the drugs in the two matrices may contribute to the susceptibility of C. albicans nano-biofilms. In a larger context, our results highlight the importance of the choice of matrix as a parameter in 3D cell encapsulation, and suggest a screening strategy to predict drug performance in vivo.
Project description:The diploid heterozygous yeast Candida albicans is the most common cause of fungal infection. Here, we report the genome sequence assembly of the clinical oral isolate 529L. As this isolate grows as a commensal, this genome will serve as a reference for experimental and genetic studies of mucosal colonization.