The Discrepancy between Clove and Non-Clove Cigarette Smoke-Promoted Candida albicans Biofilm Formation with Precoating RNA-aptamer

Abstract

This study explores the influence of precoating aptamer (Ca-apt1) on C. albicans viability while the fungus was growing in the presence of exposing condensed cigarette smoke (CSC), prepared from clove (CCSC) and non-clove (NCSC) cigarettes, for 48 h. Using qPCR, we found that mRNA expression of adhesion-associated genes ( ALS3 and HWP1) was impaired by precoating C. albicans yeast cells with the aptamer. Conversely, the gene transcription was upregulated when aptamer-uncoated yeast was pre-treated with either CSC. In addition, by analysing the result of MTT ([3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay, we found that the presence of added CCSC or NCSC in growth medium for 48 h was significantly enhanced C. albicans biofilm development. However, the presence of precoated aptamer was significantly impaired biofilm development accelerated by the NCSC. The inhibitory effect of the Ca-apt1 was not dependent on the precoated aptamer (1ng/μL and 10 ng/μL). Interestingly, we noted that the enhancer effect of treated CCSC was no longer effective when the yeast had been precoated with 10 ng/μL aptamer tested. Additionally, light microscopy analysis revealed that precoating aptamer alleviates morphological changes of C. albicans (from yeast to hypha formation) that are enhanced by adding CCSC or NCSC in the growth medium. In conclusion, these results suggest that administration on Ca-ap1 exhibits a significant protective effect on CSC-induced biofilm formation by C. albicans.

Keywords: ALS3 and HWP1.; Biofilm; Candida albicans; Cigarette Smoke; RNA-aptamer.

Figure 1.. Cigarettes used to prepare condensate cigarette smoke.

A and B are Clove and non-clove cigarette, respectively.

Figure 2.. Induction of selected biofilm-associated genes by C. albicans following precoating with different concentrations of Ca-apt1.

The upper panel shows the effect of precoating aptamer on ALS3 and HWP1 expressed by C. albicans in preformed biofilm (90 min incubation time) were analysed by qPCR. The mRNA expression levels were normalized relative to the control (yeast cells without aptamer precoating), which was set to one for each gene to determine the fold change in expression of genes in C. albicans clinical isolate ( A) and the ATCC 10231 ( B). The results are expressed as the mean and standard deviation (SD) of triplicate experiments and repeated two times independently. *Significantly higher downregulation in the expression of mRNA (P < 0.05) in the presence of precoating aptamer. The lower panel is representative of micrograph of C. albicans yeast cell (that only shown in clinical isolate) without precoating aptamer ( C), and the reduced germinated cells after incubating for 90 min with precoating aptamer 1 ng/μL ( D) and 10 ng/μL ( E). All images were captured by using light microscopic at X 200 magnification.

Figure 3.. Effect of cigarette smoke condensate on the transcription level of selected C. albicans biofilm-associated genes.

The qPCR analysis of ALS3 and HWP1 genes is shown after the preformed biofilm (90 min incubation) was treated by growth medium supplemented with different CSC (CCSC or NCSC). The mRNA level of each gene was normalized to that of 18S rRNA, while the expression level of the control (untreated biofilm) was set to one for each gene to determine the fold change in the expression of each targeted gene in clinical isolate ( A) and the ATCC strain ( B) of C. albicans. *Significantly higher upregulation of mRNA expression of HWP1 induced by CCSC than NCSC (P < 0.05). Data are expressed as the mean and standard deviation (SD) of triplicates from two separate experiments. The CCSC and NCSC are clove and non-clove cigarettes smoke condensate, respectively. The lower panel shows the 48-h biofilm formation that only shown in clinical isolate; the control ( C) and those biofilms treated with CCSC ( D) and NCSC ( E), respectively. All the biofilms are visualized by using light microscopic images at X 200 magnification.

Figure 4.. Effect of condensate smoke on preformed biofilm cells.

The effect of CCSC ( A) and NCSC ( B) exposure on biofilm formation of C. albicans, which had been precoated with different concentration of Ca-apt1, evaluated after incubation for 48-h. The relative biofilm formation compared to biofilm without the precoating aptamer (control) was calculated. Data represent the mean and standard deviation (SD) of three biofilms grown on two separate occasions. Asterisks denote statistically significant differences between the effect of cigarette smoke condensate determined by MTT assay, p < 0.05. The CCSC and NCSC are clove and non-clove cigarette smoke condensate, respectively.

Figure 5.. Light microscopy photomicrograph illustrating the different morphology of C. albicans with and without precoating aptamer (Ca-apt1) and grown for 48-h in the growth medium supplemented with cigarette smoke condensate (CSC).

The pictures show biofilm of C. albicans derived from clinical isolate, without either precoating aptamer or added CSC ( A), C. albicans with precoated aptamer but untreated with either CSC/ CCSC or NCSC ( B), C. albicans precoated with 1 ng/μL aptamer, CCSC ( C) and NCSC ( E), and 10% aptamer with CCSC ( D) and NCSC ( F). All microscopic image show at X 200 magnification.