ANTIBIOFILM ACTIVITIES AND PROTEOMIC ANALYSIS OF CANDIDA TROPICALIS IN RESPONSE TO TERPENES

Abstract

Candida albicans and the emerging threats caused by non-albicans Candida species have started to show resistance which calls for identification of new alternatives or modification of the existing antifungal agents to defy the dominant tendency of infection. Presently, available anti-Candida drugs exhibit a high frequency of resistance, low specificity and toxicity at a higher dosage. In addition, the discovery of natural or synthetic anti-Candida drugs is slow-paced and often does not pass clinical trials. Among non-albicans Candida species, Candida tropicalis is frequently emerging fungal pathogen in the Asia-Pacific region, causing a high mortality rate due to candidiasis. The preponderance of C. tropicalis in clinically isolated strains is recorded as higher as 42.1%. Also, the resistance in isolated C. tropicalis strains towards fluconazole is observed to be 38.5%. This increased rate of fluconazole resistance and frequent isolation of C. tropicalis makes it a major concern. C. tropicalis is an opportunistic human pathogen with an ability to cause superficial as well as systemic infections in immunocompromised patients. The formation of biofilm by C. tropicalis can cause dreadful and persistent infections which are difficult to treat due to acquired resistance. Azole drugs including fluconazole are enormously used in several antifungal treatments. However, C. tropicalis isolates showing significant azole drug resistance attributed to the point mutations in cytochrome P-450 lanosterol 14-α-demethylase (Erg11p) protein. Therefore, alternative drugs against Erg11p are immensely required to combat the acquired resistance. Owing to the potential of essential oils in terms of their antimicrobial activities as well as traditional usage has emphasized their applicability in various fields ranging from agriculture to food technology and as natural alternatives alongside synthetic drugs. However, more clinical studies are required to confirm and verify the true potency of these natural substances for their safe applications in human health and the environment. Essential oils are found to be rich in terpenes which are known to have good antimicrobial properties along with their explicit aroma. Various terpenes have shown multiple antifungal activities against planktonic cells in Candida species. However, their effects on the biofilm formation and its eradication are still in infancy. Previous studies have revealed antifungal activities of these terpenes at multiple levels in different organisms. Therefore, the effect of antifungal agents could not be specified by studying it over one organism or even on established targets. More studies are required to be done in respect to finding probable genes, proteins and metabolites responsible for resistance against antifungal agents. Furthermore, many terpenes have been tried as antifungal agents against Candida species. These are found as a potent anti-Candida compound with a broad ii range of antimicrobial properties. Thus, the exploitation of antifungal and antibiofilm properties could serve as a bridge between traditional uses and rational utilization of essential oils and citral. In the present thesis entitled “Antibiofilm activities and proteomic analysis of Candida tropicalis in response to terpenes”, an extensive study has been done to elaborate on the effects of terpenes on C. tropicalis biofilm. Firstly, in vitro growth and development of C. tropicalis biofilm were recognized and antifungal activities of the seven terpenes, namely, geraniol, menthol, citral, cinnamaldehyde, carvacrol, eugenol and thymol were screened. They were further checked for the abilities of biofilm inhibition and eradication. The calculated MIC50, BIC50 and BEC50 values for citral and thymol were lesser than other terpenes, therefore, observed to be more effective against C. tropicalis. The morphological changes have also shown damaged surface in the presence of these terpenes. These results clearly indicated that among these terpenes; citral and thymol were significantly effective against both planktonic and biofilm forms of C. tropicalis, compared to others. Secondly, the comparative potentials of citral and thymol against C. tropicalis were explored. The administration of citral and thymol on C. tropicalis biofilm leads to a fungicidal effect. The relative fold change in the expression of certain key genes which are involved in major pathways followed by C. tropicalis, to reduce the effect of well-known drugs, has also been investigated to get the insights in the plausible mechanism for its survival in the presence of these two components. Citral and thymol have formed indentations in the cells depicting distorted surface and decreased viability. Also, the cell membrane and DNA damage were determined in C. tropicalis biofilm cells when treated with citral and thymol as antifungal agents. The cells membrane integrity was compromised and DNA damage was observed. Quantitative real-time PCR analysis showed augmented expression of the cell membrane biosynthesis genes including ERG11/ CYT450 against citral and the cell wall-related tolerance genes involving CNB1 against thymol thus, depicting their differential mode of actions. Next, the homology model of well-known azole drug target Erg11p of C. tropicalis was employed to unravel the interaction between citral and lanosterol-14-α-demethylase (Erg11p). The molecular interactions of two isomers of citral namely neral and geranial, with CtErg11p, were evaluated. The three-dimensional structure of CtErg11p was prepared through in silico structural modelling approach. The best-generated model was validated and further used for the molecular docking studies with citral. The molecular docking studies provide insights into citral-CtErg11p interactions, which could be helpful for further optimization and development of other inhibitory analogues of citral asserting the expansion of proficient broad-spectrum antifungals. The molecular interactions between citral, heme group and participating amino iii acid residues of CtErg11p protein have been identified that could lead to understanding the protein-ligand interaction. Furthermore, insights into the changes in the biofilm cell proteome and the extracellular matrix of C. tropicalis in the presence of citral were obtained. One-dimensional polyacrylamide gel electrophoresis (1D-PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF-MS) were employed to identify the changes in the protein expression of C. tropicalis in response to the sub-lethal concentration of citral. The results revealed citral-induced proteins of C. tropicalis biofilm. In 1D-PAGE, a total of six differential proteins, involved in oxidative stress, amino acid biosynthesis, heme biosynthesis and glucose metabolism pathways were detected. However, in 2D-PAGE, the differential expression of proteome has revealed a total of twenty-five proteins in C. tropicalis biofilm which were induced in the presence of citral. Among these, amino-acid biosynthesis (Met6p, Gln1p, Pha2p); nucleotide biosynthesis (Xpt1p); carbohydrate metabolism (Eno1p, Fba1p, Gpm1p); sterol biosynthesis (Mvd1p/ Erg19p, Hem13p); energy metabolism (Dnm1p, Coa1p, Ndk1p, Atp2p, Atp4p, Hts1p); oxidative-stress (Hda2p, Gre22p, Tsa1p, Pst2p, Sod2p); and biofilm specific (Adh1p, Ape1p, Gsp1p) proteins were identified. The overexpression of oxidative-stress related proteins indicates the response of biofilm cell in combating oxidative-stress during citral treatment. Moreover, the upregulation of Adh1 is of particular interest because it subsidises the biofilm inhibition through ethanol production as a cellular response. The augmented expression of Mvd1/ Erg19 signifies the effect of citral on ergosterol biosynthesis. The overall sugar and protein moieties were diminished in the treated extracellular matrix; however, ergosterol and cell wall content e.g. hexosamine were relatively increased. Therefore, it is clearly indicated that the cellular response towards citral acts through multifactorial processes.