Chitosan Nanogel Design on Gymnema sylvestre Essential Oils to Inhibit Growth of Candida albicans Biofilm and Investigation of Gene Expression ALS1, ALS3

Chitosan (CS) is a polycation with a pka of about 6.3 with a charge density dependent on pH and the %DA-value which can interact with polyanions to form complex and gels. Nanoparticles (CNPs) in addition can increase the antifungal potential of bioactive compounds like essential oils by increasing cellular interactions between them and the fungal as a result of the very small size that enhances cellular uptake. In this study, was set to investigate the encapsulation of the Gymnema sylvestre essential oils (G.EOs) using Chitosan and Myrestic acid made Nanogel in order to enhance its antifungal activity and stability to the oil against C. albicans strain (ATCC 10231). To procedure this, the self-assembled process of Chitosan and Myrestic acid Nanogel (CS-MA) through the 1- ethyl - 3- (3 dimethyl aminoprophyl) carbodiimide (EDC) was designed. Its physicochemical properties were determined by Fourier Transforms Infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and microscopic methods by Atomic force microscopy (AFM), Transmission Electron Microscopy (TEM), and Scanning Electron Microscopy (SEM). Minimum inhibitory concentration (MIC) at 18.7 to 37.5 µg/ml and 2.3 to 4.6 µg/ml and minimal fungicidal concentration (MFC) at 75 µg/ml and 5.38 µg/ml using by broth micro dilution (BMD) method for G. sylvestre oils (G.EOs) and oil-loaded Nanogels (G.OLNPs) were measurement. The susceptibility of C. albicans biofilm to fractions was examined by 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[8phenylamino) carbonyl] - 2H - tetrazolium hydroxide (XTT) quantitatively.The concentration of G.OLNPs required to inhibit 50 % biofilm formation was 4.68 µg/ml, while that to remove 90 % biofilm growth was 18.07 µg/ml. In addition, it was observed that cell uptake of G.OLNPs was much higher compared with free G.EOs. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to determine the effect of sub-MIC concentrations of G.EOs and G.OLNPs on expression of the biofilm–related gene ALS1 / ALS3, and indicated the G.OLNPs down-regulated the expression of hypha-specific gene ALS3. Furthermore, the data strongly suggested that G.OLNPs more effective suppressed C. albicans planktonic cells and reduction biofilm biomass

International Journal of Molecular and Clinical Microbiology Hwp1 gene Expression of Candida albicans and Study its role in adherence

Candida albicans is an opportunistic fungal pathogen found as mycoflora in the human body surfaces. Sevral genes play a crucial rule in its virulence including Hwp1 (hyphal wall protein 1), BCR1 and ALS gene family. Hwp1 gene is a responsible for coding a cell surface protein, which mediates biofilm formation in candida albicans. Here we investigated the presence of the HWP1gene was characterised among Candida albicans isolates in women with recurrent vaginal canididasis and further we studies its role in cell adherence. We used 50 Candida albicans clinical isolates resistant to Fluconazole. RNA (of samples were extracted using glass bead and lysis buffer and cDNA was synthesized using reverse transcriptase enzyme. Expression of (HWP1) gene was analysed using reverse transcriptase-plolymerase chain reaction (RT-PCR). The ability of adherence of the isolates with or without the expression of HWP1 were characterized using Hela cells. Statistical analysis were performed using t-test and two-way ANOVA SPSS software. Our results showed that the HWP1 gene were present in 47 samples (94%) out of 50 isolates, 27 samples (57%) had expression of HWP1. The result of adherence assay revealed that the isolates with the expression of HWP1 gene and control isolates was statistically different (p<0.05). In conclusion, the isolates with the expression of HWP1 gene has the higher ability to adhere the epithelial mucosal cell surface. Our data support a positive correlation between the expression of HWP1 gene and the ability of adherence to epithelial cells

ALS1 and ALS3 gene expression and biofilm formation in Candida albicans isolated from vulvovaginal candidiasis

Background: A cluster of genes are involved in the pathogenesis and adhesion of Candida albicans to mucosa and epithelial cells in the vagina, the important of which is agglutinin-like sequence (ALS) genes. As well as vaginitis is a significant health problem among women, the antifungal resistance of Candida species is continually increasing. This cross-sectional study investigates the expression of ALS1 and ALS3 genes and biofilm formation in C. albicans isolate isolated from vaginitis. Materials and Methods: Fifty-three recognized isolates of C. albicans were collected from women with recurrent vulvovaginal candidiasis in Iran, cultured on sabouraud dextrose agar, and then examined for gene expression. Total messenger RNA (mRNA) extracted from C. albicans isolates and complementary DNA synthesized using reverse transcriptase enzyme. Reverse transcription-polymerase chain reaction (RT-PCR) using specific primer was used to evaluate the expression of ALS1 and ALS3 through housekeeping (ACT1) genes. 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide assay was performed to assess adherence capacity and biofilm formation in the isolated. Results: Forty isolates (75.8%) expressed ALS1 and 41 isolates (77.7%) expressed ALS3 gene. Moreover, 39 isolates (74%) were positive for both ALS1 and ALS3 mRNA by the RT-PCR. Adherence capability in isolates with ALS1 or ALS3 genes expression was greater than the control group (with any gene expression), besides, it was significantly for the most in the isolates that expressed both ALS1 and ALS3 genes simultaneously. Conclusion: The results attained indicated that there is an association between the expression of ALS1 and ALS3 genes and fluconazole resistance in C. albicans. A considerable percent of the isolates expressing the ALS1 and ALS3 genes may have contributed to their adherence to vagina and biofilm formation