Candida virulence attributes: exploring a link to oral mucosal carcinogenesis

© 2014 Dr. Ali Daoud Al NuaimiThe fungus Candida has been implicated in the aetiology of oral cancer; however, the exact carcinogenic mechanisms are still under investigation. A persuasive body of evidence suggests a direct causal role involving metabolism and production of carcinogenic compounds such as acetaldehyde. Recently, the hypothesis of microbial induced inflammation is gaining an increased interest and thought to play an important role in the multi-step process of carcinogenesis. Chronic inflammatory mediators, such as cytokines and growth factors, may act as a link between infection, innate immunity, inflammation and cancer through their tumour growth stimulatory effect. Inadequate pathogen elimination, prolonged inflammatory signalling, and defects in anti-inflammatory mechanisms can lead to chronic inflammation and subsequent release of cytokines and growth factors that benefit tumour development. Previous research has found a significant association between the virulence potential of pathogenic fungi, such as secreted aspartyl proteinases and the expression of inflammatory cytokines and chemokines. In addition, differences have been observed in Candida strains/biotypes colonizing premalignant/malignant and healthy oral mucosa. The present research had four major aims: (1) To study the major virulence attributes and biofilm antifungal susceptibility of medically important Candida species and strains (2) To develop a rapid, accurate and economic genetic method for identification and genotyping of medically relevant fungal pathogens using real time polymerase chain reaction-high resolution melting curve analysis (HRMA) (3) To assess the prevalence of Candida carriage and level of colonization in oral cancer and matched non-oral cancer patients (4) To investigate the major virulence attributes and ethanol-derived acetaldehyde production of Candida isolated from oral cancer patients and compare these with oral yeasts isolated from patients with no evidence of oral cancer. Results showed a significant variation in biofilm forming ability and hydrolytic enzyme production between different Candida species and strains, with high antifungal resistance observed for C. albicans bioflms in comparison to planktonic Candida cells. Furthermore, correlation analysis revealed a significant positive correlation between biofilm forming ability and hydrolytic enzymes production. The HRMA-based genetic identification method developed during this study was rapid, cheap and highly accurate in identifying all unknown Candida isolates and C. albicans genotypes. The melting curve profiles of internal transcribed spacer regions and transposable intron in 25S of ribosomal DNA were highly discriminative and showed consistency over replicates and repeated assays. It had the advantages of using a single primer set to identify all unknown isolates with high potential to distinguish clinical isolates not included in the reference strain list. In the present study, the frequencies of oral yeasts’ presence and high oral colonization were significantly higher in oral cancer than non-oral cancer patients (p=001; p=0.033, respectively). No significant difference in the isolation profile of Candida species was found between the two groups, except C. parapsilosis was more frequent in the non-oral cancer group. Differences were noticed in the incidence of C. albicans strains where significantly more C. albicans genotype-A was isolated from cancer patients and significantly more C. albicans genotype-B isolated from non-cancer patients. Multiple regression analyses showed significant association with cancer observed for alcohol drinking (OR=4.253; 95% CI=1.351, 13.386), Candida presence (OR=3.242; 95% CI=1.505, 6.984) and high oral colonization (OR=3.587; 95% CI=1.153, 11.162). Finally, statistical analysis demonstrated significantly enhanced biofilm forming ability, phosphoplipase, proteinase and ethanol-derived acetaldehyde production by Candida isolated from oral cancer patients compared with isolates from matched non-oral cancer patients. A significant association was found between Candida species and certain virulence properties with higher frequency of high biofilm mass, high biofilm growth activity, high phospholipase and esterase activity was observed in C. albicans species as compared with the non-albicans Candida species. Acetaldehyde production was higher in C. albicans than non-albicans Candida isolates, but did not reach the level of statistical significance. A significantly higher frequency of high proteinase activity was seen in the non-albicans Candida compared with C. albicans isolates. No significant association was found between the genotype of C. albicans and any of the investigated virulence factors other than significantly higher biofilm mass was obtained in C. albicans genotype A compared with genotype B. Correlation analysis revealed a significant positive correlation between both biofilm mass and growth activity and phospholipase enzyme production