IMPACT OF ENVIRONMENTAL FACTORS ON CANDIDA ALBICANS BIOFILM

Abstract

Biofilms are complex, interdependent community of surface associated microbial cells enclosed in an extracellular matrix. They can adhere to any surface ranging from industrial and aquatic water systems to medical devices and host tissues. The unique feature of biofilm structure is its increased resistance to conventional antimicrobial agents and immune system which made them difficult to treat from clinical point of view. Biofilms formed by Candida sp., Staphylococcus sp., Streptococcus sp., and Escherichia coli, Pseudomonas aeruginosa are responsible for majority of nosocomial infections. Among these, Candida albicans is the predominant fungal species associated with biofilm related infections. C. albicans, dimorphic yeast of human microbiota colonizing oral, gastrointestinal and urogenital tracts of healthy individuals. Nevertheless, C. albicans is the most common fungal pathogen causing superficial and systemic infections. During colonisation, C. albicans is exposed to a variety of environmental factors at diverse host niches such as nutrient resources, oxidative and osmotic stresses, innate immune secretory factors and other co-infecting microorganisms. The environments encountered by C. albicans within a host depend on the niche it occupies. Adaptation to these different environments is crucial for C. albicans virulence as it increases the survival of this pathogen. In adaptation process, these environmental factors can influence the cell physiology, morphology, adherence and architecture of biofilms which results in coordinated changes in expression of virulence factors and cell wall composition. The altered virulence traits in the biofilm can influence the pathogenicity of C. albicans. Keeping the significance of above facts in view, the main objective of the work carried out in this thesis was to investigate the effect of environmental factors on C. albicans biofilm and how this in turn affects the virulence factors and cell wall composition of this fungal pathogen during biofilm growth. C. albicans needs to assimilate locally available or alternative nutrients for their survival and multiplication in the dynamic environments. During infection, carbon sources play a central role in metabolism and critical for colonization in C. albicans. The role of different carbon sources such as glucose, lactate, sucrose, and arabinose on C. albicans biofilm development and virulence factors was investigated. Quantitative analysis of biofilm formation was analyzed by XTT (2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5- carboxanilide) reduction assay. Qualitative analysis of biofilm development was determined by confocal laser scanning, scanning electron and atomic force microscopy. Glucose grown cells exhibited the highest ii metabolic activity during adhesion among all carbon sources tested. However, cells exposed to sucrose exhibited highest biofilm formation and matrix polysaccharides secretion. Exposure to lactate induced hyphal structures with the highest proteinase activity while arabinose grown cells formed pseudohyphal structures possessing the highest phospholipase activity. β-glucans are the major structural components of the cell wall of C. albicans as well as most important fungal pathogen associated molecular patterns (PAMPs). Therefore, structural changes in β- glucan was characterised by Fourier transform infrared (FT-IR) spectroscopy. Curve fitting analysis of the spectrum revealed a significant changes in β(1→6) to β(1→3) glucan ratio in the carbon sources tested. These results signify that carbon sources influence C. albicans biofilm development, modulate virulence factors and structural organization of cell wall component β- glucan. During colonisation, C. albicans encounters environmental stresses namely osmotic and oxidative stresses. Therefore, the study was focused on the impact of these stress factors on C. albicans biofilm formation and virulence factors. C. albicans cells were incubated in the presence of 5 mM H2O2 and 2 M NaCl to induce oxidative and osmotic stresses. Oxidative stress enhanced extracellular DNA secretion into the biofilm matrix via reactive oxygen species mediated cell lysis, increased chitin levels, and reduced the extracellular phospholipase as well as the proteinase activity. While osmotic stress notably stimulated biofilm formation with increased proteinase and decreased phospholipase activity. FT-IR and nuclear magnetic resonance (NMR) spectroscopy analysis of the cell wall component mannan revealed a decrease in mannan content and reduced β-linked mannose moieties under stress conditions. The ability of C. albicans to survive in the presence of sub-lethal concentrations of oxidative and osmotic stress inducing agents can be attributed to the highly resistant biofilm mode of growth response by C. albicans. The constant flow of saliva in the oral milieu containing innate immune secretory factors acts as a chemical barrier to restrict pathogens. The response of C. albicans to the innate immune defense factors like mucin, lactoferrin, and lysozyme was tested. In addition, the effect of extracellular adenosine tri-phosphate (eATP) released by damaged cells to signal ‘danger’ to immune system were also studied. Experiments were conducted by incubating C. albicans in the presence of different test compounds mentioned above and biofilm formation was evaluated using XTT assay. Biofilm formation was suppressed by lysozyme and lactoferrin in a dose dependent manner. However, lactoferrin and lysozyme were more effective at higher concentrations (≥2 mg mL-1) in disrupting preformed C. albicans biofilm. Mucin is another host derived secretory compound that suppressed biofilm formation and virulence traits in C. iii albicans by inhibiting cell attachment to polystyrene surface. On the other hand, danger signalling molecule extracellular dATP/ATP (500 μM) stimulated biofilm formation and the release of eDNA into C. albicans biofilm matrix. Treatment of C. albicans with dATP activated ROS mediated cell lysis thereby releasing DNA extracellularly into matrix. Overall, results indicated that modulation of C. albicans biofilm formation by these host innate immune factors can alter the dynamic interplay between host immune system and the clearance of the pathogen. Biofilm communities are far more complex than recognised. Microorganisms present in the surrounding environment can have a major impact largely on the growth of other microbes. The influence of pathogenic bacteria namely Pseudomonas aeruginosa Staphylococcus aureus and Escherichia coli on C. albicans biofilm formation was studied. Different responses were observed when different pathogenic bacteria were co-incubated with C. albicans. Both P. aeruginosa and E. coli suppressed C. albicans biofilm formation, conversely S. aureus did not exert any inhibitory effect on C. albicans biofilm growth. The specific activity of phospholipase and proteinase was also reduced in mixed species biofilms as compared to C. albicans biofilm. Along with the above studies, attempts were made to identify the agents that interfere with the adhesion and biofilm formation by C. albicans. Several phytocompounds were selected from the chemical library available in the literature and screened for their antifungal activity. Antifungal activity of certain plant extracts and phytochemicals is due to the presence of biologically active compounds like terpenes and polyphenolic substances. Hence, in this investigation the role of terpenes namely eugenol, menthol and thymol and fluconazole (FLA) on C. albicans biofilm inhibition were studied. The minimum inhibitory concentration evaluated by broth micro-dilution method showed antifungal activity against C. albicans at a concentration of 0.12 % (v/v) for both thymol and eugenol as compared to 0.25 % (v/v) for menthol. Thymol and eugenol were more effective in inhibition of preformed biofilm than menthol. Synergistic studies using checkerboard micro-dilution assay showed fractional inhibitory concentration index between thymol/FLA effectively against pre-formed C. albicans biofilms. Thymol with fluconazole showed highest synergy in reduction of biofilm formation than eugenol and menthol which was not observed when their activities were observed independently. Adherence studies and microscopic analysis showed reduction in cell number and alteration in morphology of C. albicans. The distinct ability of C. albicans to grow at diverse host niches is attributed to the adaptive response generated with respect to the environmental conditions it encounters in a specific iv niche. Significant findings from these studies indicated that host derived environmental factors like nutrient resources and stresses promote biofilm formation. It also affects virulence factors and cell wall composition in C. albicans biofilm. On the other hand, microbe derived environments can inhibit biofilm formation and virulence factors in C. albicans. Thus, biofilm formation in response to different host environmental factors is integral part of C. albicans pathogenicity. The cell phenotypes of C. albicans within biofilms are highly resistant to antifungal agents and innate immune factors. To counteract this, several natural phytochemicals in form of combinatorial therapy either by using conventional antifungal agents or by innate immune secretory factors like mucin, lactoferrin or lysozyme could be a promising medication in the treatment of biofilm infections.