Effects of efflux pumps on antifungal activity of chitosan against Candida albicans.

Background: Antifungal resistance is a major problem, commonly caused by drug-efflux pump overexpression. To evaluate if chitosan could be effective in drug-resistant Candida infections, we investigated the effects of efflux pumps on antifungal activity of chitosan. Materials and Methods: The minimal fungicidal concentration (MFC) of oligomer (7-9 kD) and polymer (900-1,000 kD) chitosan against Saccharomyces cerevisiae and Candida albicans were evaluated by broth and agar dilution methods. The MFCs of S. cerevisiae with single deletion of efflux pump genes, with deletion of seven efflux pumps (AD∆), and AD∆ overexpressing C. albicans efflux pump genes (CDR1, CDR2 and MDR1) were determined. C. albicans with homozygous deletions of CDR1 and of CDR2 were generated using CRISPR-Cas9 system and tested for chitosan susceptibility. Results: While deleting any individual efflux pump genes had no effect on chitosan susceptibility, simultaneous deletion of multiple pumps (in AD∆) increased sensitivity to both types of chitosan. Interestingly, the overexpression of CDR1, CDR2 or MDR1 in AD∆ barely affected its sensitivity. Moreover, C. albicans with homozygous deletions of CDR1 and/or CDR2 showed similar sensitivity to wildtype. Conclusion: Thus, C. albicans susceptibility to chitosan was not affected by drug-efflux pumps. Chitosan may be a promising antifungal agent against pump-overexpressing azole-resistant C. albicans.

1. Neither deletion of efflux pump genes, nor overexpression of major C. albicans efflux pumps in pump-deficient S. cerevisiae, nor deletion of major efflux pumps in C. albicans affects yeast susceptibility to chitosan. 2. Chitosan may be an effective antifungal agent against drug-resistant C. albicans.

Efficacy of low-molecular weight chitosan against Candida albicans biofilm on polymethyl methacrylate resin.

BACKGROUND: Candida biofilm is a major cause of denture stomatitis. We aimed to compare the efficacy of low-molecular-weight chitosan solutions against Candida albicans biofilm on polymethyl methacrylate (PMMA) resin. METHODS: Various types of chitosan were tested for anti-Candida activity by broth dilution. Two types were selected for further testing on 24-hour C.albicans biofilm formed on PMMA specimens. Specimens were randomly distributed among experimental groups, including 0.1% and 0.2% acetic acid, 3 and 6 mg/mL of oligomer chitosan and 30 kDa chitosan solutions, effervescent tablet (Polident), and 0.2% chlorhexidine, and immersed for 5 min to 12 h. The viability of C. albicans after cleansing were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Remaining viability was calculated into percentage relative to respective controls and analyzed using ANOVA with Tukey post-hoc tests. Live/dead fluorescence microscopy was also performed. RESULTS: Chitosan solutions had high efficacy against C. albicans biofilm on PMMA. The mean relative viability compared to control after 12-h immersion was 6.60 ± 4.75% and 12.72 ± 6.96% for 3 and 6 mg/mL oligomer, respectively, and 11.68 ± 4.81% and 18.08 ± 6.20% for 3 and 6 mg/mL 30 kDa chitosan, respectively. CONCLUSIONS: Low-molecular-weight chitosan solution is an effective antifungal denture cleanser that can significantly reduce C. albicans viability in biofilm on PMMA.

Activity of chitosan antifungal denture adhesive against common Candida species and Candida albicans adherence on denture base acrylic resin.

STATEMENT OF PROBLEM: Candida adherence to the denture base is an important cause of denture stomatitis. In addition, infections with drug-resistant Candida have become more prevalent, especially among elderly and immunocompromised patients. Thus, alternative safe antifungal agents for oral applications are needed. PURPOSE: The purpose of this in vitro study was to investigate the activity of chitosan, a natural biopolymer, against common oral Candida species and its efficacy in inhibiting C albicans adherence to denture-base acrylic resin. MATERIAL AND METHODS: The minimum fungicidal concentrations (MFCs) of 5 types of chitosan against 6 species of Candida and 10 C albicans clinical isolates were determined by broth and agar dilution, respectively. N-succinyl chitosan (NSC), low- and high-molecular-weight water-soluble chitosan (LMWC and HMWC), and oligomer and polymer shrimp-chitosan were examined. NSC and HMWC, as pure gel and as a mixture with carboxymethylcellulose (CMC), were applied to acrylic resin disks, incubated with C albicans for 24 hours, and washed, and adherent cells were collected for colony count. The effects of HMWC on human gingival fibroblasts after 1 and 24 hours of treatment were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The retention force of HMWC gel was measured by using a universal testing machine. The Kruskal-Wallis and Mann-Whitney U tests were used to compare the antiadherence activity (α=.05). RESULTS: HMWC had the highest antifungal activity against most Candida species tested and C albicans clinical isolates. HMWC gel completely inhibited C albicans adherence to denture base acrylic resin (P<.001). CMC denture adhesive significantly increased C albicans adherence (P<.001), but adding 2×MFC HMWC into CMC reduced the adherence, although this was not statistically significant (P=.06). HMWC at 1×MFC and 2×MFC showed no toxic effect on gingival fibroblast viability and proliferation. Moreover, the retention force provided by HMWC gel was sufficient for use as a denture adhesive (>5000 Pa). CONCLUSIONS: High-molecular-weight, water-soluble chitosan is a biocompatible biopolymer that could inhibit C albicans adherence and that showed properties suitable for development into an antifungal denture adhesive.

Multiple E-boxes in the distal promoter of the rat pyruvate carboxylase gene function as a glucose-responsive element.

Pyruvate carboxylase (PC) is an anaplerotic enzyme that regulates glucose-induced insulin secretion in pancreatic islets. Dysregulation of its expression is associated with type 2 diabetes. Herein we describe the molecular mechanism underlying the glucose-mediated transcriptional regulation of the PC gene. Incubation of the rat insulin cell line INS-1 832/13 with glucose resulted in a 2-fold increase in PC mRNA expression. Transient transfections of the rat PC promoter-luciferase reporter construct in the above cell line combined with mutational analysis indicated that the rat PC gene promoter contains the glucose-responsive element (GRE), comprising three canonical E-boxes (E1, E3 and E4) and one E-box-like element (E2) clustering between nucleotides -546 and -399, upstream of the transcription start site. Mutation of any of these E-boxes resulted in a marked reduction of glucose-mediated transcriptional induction of the reporter gene. Electrophoretic mobility shift assays revealed that the upstream stimulatory factors 1 and 2 (USF1 and USF2) bind to E1, the Specificity Protein-1 (Sp1) binds to E2, USF2 and the carbohydrate responsive element binding protein (ChREBP) binds to E4, while unknown factors binds to E3. High glucose promotes the recruitment of Sp1 to E2 and, USF2 and ChREBP to E4. Silencing the expression of Sp1, USF2 and ChREBP by their respective siRNAs in INS-1 832/13 cells blunted glucose-induced expression of endogenous PC. We conclude that the glucose-mediated transcriptional activation of the rat PC gene is regulated by at least these three transcription factors.

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter.

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (-386/-374), non-perfect DR1 (-118/-106) and HNF4α-specific binding motif (H4-SBM) (-26/-14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.

Functional characterization of the proximal promoter of the murine pyruvate carboxylase gene in hepatocytes: role of multiple gc boxes.

Pyruvate carboxylase (PC) catalyzes the first committed step in gluconeogenesis in the liver. The murine PC gene possesses two promoters, the proximal (P1) and the distal (P2) which mediate production of distinct tissue-specific mRNA isoforms. By comparing the luciferase activities of 5'-nested deletions of the P1-promoter in the AML12 mouse hepatocyte cell line, the critical cis-acting elements required for maintaining basal transcription were located within the 166 nucleotides proximal to the transcription start site. Three GC boxes were identified within this region and shown by gel shift and ChIP assays to bind Sp1/Sp3. Over-expression of Sp1/Sp3 in AML12 and NIH3T3 cells increased P1-promoter activity, with Sp1 being a stronger activator than Sp3. Mutation of any one of the three GC boxes dramatically reduced basal promoter activity by 60-80% suggesting that all three boxes are equally strong regulatory elements. In AML12 cells, over-expression of Sp1/Sp3 restored the transcriptional activity of GC1 and GC2 but not GC3 mutants to levels similar to that of the WT construct, suggesting that GC3 is particularly critical for Sp1/Sp3-mediated induction. In NIH3T3 cells, however, the three boxes were equally important, indicating that the GC boxes differentially contribute to transcriptional regulation of the P1-promoter in the two cell lines. Mutants harboring two disrupted GC boxes showed a further decrease in promoter activity similar to the triple GC box mutant. Neither Sp1 nor Sp3 was able to fully restore the promoter activities of these mutants to that the WT level.