BIOCHEMICAL AND METABOLOMICS PROFILING OF Candida glabrata CLINICAL ISOLATES FOR PHARMACOLOGICAL TARGETING

Abstract

Fungal infections are very common across the world due to the natural abundance of many fungi in the environment. Although many fungal species are known to mankind, around 600 are infectious to humans, particularly in patients with compromised immunity. These commensal fungi undergo colonization and cause infection associated with inflammation of the affected area. These infections range from mild to moderate surface infections to severe invasive fungal infections. Candida species are among the most prevalent human-associated fungus, with skin and mucous membranes as the top sites of their occurrence. The skin, oral and vaginal mucosa are among the leading sites of Candida infections in humans, commonly called candidiasis. According to recent consensus, vulvovaginal candidiasis (VVC) affects millions of women each year and is the second most common cause of vaginitis. Candida glabrata secure the second position among the leading causes of VVC. Although less common than Candida albicans, C. glabrata-associated VVC is the primary cause of concern due to its unique virulence attributes. C. glabrata, like other fungal species, adapt to the adverse host defense mechanisms. The metabolic flexibility is one of the critical factors that help C. glabrata growth and survival inside the host. This metabolic flexibility is associated with alterations at a transcriptomic level to cope with the surrounding environment. Such metabolically and genomically altered strains obtained from the body of infected patients are known as clinical isolates. These clinical strains are different from the wild-type strains as they become more virulent while struggling for their existence in the host niche. Hence, it becomes imperative to understand the genotypic and phenotypic traits acquired by the clinical isolates that transform them from commensals to infectious agents. Besides studying the characteristics of the species, the elucidation about its growth requirements and the surrounding environment is also crucial in the understanding the pathogenicity of the pathogen. C. glabrata is a part of vulvovaginal mycobiota, as it survives there along with other fungal and bacterial species in harmony, without causing any illness in healthy individuals. The environment in the vaginal mucosa is unique, as it is composed of lactic and acetic acid with relatively less glucose. Thus, C. glabrata needs to depend on other carbon sources instead of glucose. Thus, while shifting from glucose to lactic acid as a carbon source, C. glabrata may acquire some metabolic and biochemical changes. Furthermore, the vaginal pH is acidic. This distinct niche makes the vaginal mucosa more challenging for the C. glabrata, and it has to modify itself to become competent enough to survive and grow in these conditions. Therefore, the study of lactic acid conditioning of C. glabrata may help explore the morphological, biochemical, and metabolic changes that might be responsible for acquiring virulence in this species. Clinically, there are three primary classes of drugs that are currently used for the treatment of candidiasis: azoles, polyenes, and echinocandins. These drugs offer excellent coverage for most fungal infections. However, these drugs are associated with many risks limiting their affordability in the clinical setting. These drugs pose the problem of bioavailability, toxicity, side effects, and drug-drug interactions. Moreover, C. glabrata is inherently less sensitive to azoles, which is the only class of antifungal agents with acceptable oral bioavailability. Also, the incidences of resistance to other antifungal drugs by C. glabrata have been reported recently. These factors collectively demand the search for alternative treatment options for successful therapeutic outcomes against C. glabrata infections. Several natural compounds have been claimed to exhibit antifungal effects and act by distinct mechanisms uncommon to classical drugs. When used concomitantly, many natural compounds have shown synergism with classical drugs. These compounds belong to different chemical classes like phenols, flavonoids, coumarins, quinones, saponins, xanthones, alkaloids, lectins, peptides, terpenoids, essential oils, and many more. The link between biochemical changes and metabolic alterations induced by these novel antifungal agents and their antifungal effect is yet to be explored. In the present thesis, four clinical isolates of C. glabrata (C1-C4), previously collected from the patients of VVC in the Dehradun district of Uttarakhand, were investigated and the differences were marked in reference to wild type strain. These strains of C. glabrata had different susceptibility to fluconazole. All the strains were characterized along with wild-type (C0) by morphological, physiological, biochemical, metabolomics, and transcriptional analyses. These studies were further continued to observe the effect of lactic acid conditioning on selected clinical isolates to track the differences at the metabolic level when the carbon source changed from glucose to lactic acid. Finally, some natural antifungal agents were screened for their potency against the clinical isolates of C. glabrata. Out of these, alkaloid berberine was selected for further studies to explore its mechanism of antifungal action through the biochemical analysis, NMR based metabolomics approach and RT-PCR assays. The entire study is systematically organized in the chapters (Chapters 1 to 4) in the thesis. These chapters are discussed below. Chapter 1 gives an overview of the human fungal infections (mycoses) with their classification on the basis of body site affected. Three major categories of mycoses, superficial, mucosal, and invasive, are described with emphasis on the most common causative fungal species. The pathogenic traits and epidemiology of these fungal species are briefly summarized. Many fungal species that are commensals to humans live at different body sites that are exposed to the outer environment directly or indirectly, forming mycobiota. A snapshot of the distribution of these fungal species in a human host is given to understand the general occurrence of different fungal species that have the potential to cause opportunistic infection in the immune-compromised host. Among the most common fungal infections in humans, Candidiasis is discussed in detail with reference to common species, epidemiology, risk factors, and pathogenesis. A summary of the major species of NAC group, mainly C. glabrata is provided. Further, the differences between C. albicans and C. glabrata, the two top causes of VVC across the globe, and the virulence attributes of C. glabrata that make it a species of concern are described. Finally, the techniques used for metabolomics with particular emphasis on NMR, and a summary of the successful applications of fungal metabolomics are also appraised. Chapter 2 is dedicated to the characterization of C. glabrata clinical isolates selected for the study. The clinical isolates (C1-C4) and wild-type (C0) C. glabrata strains were systematically investigated at the molecular level by biochemical, morphological, and chemical profiling techniques. Biochemical analysis revealed significant differences in azole susceptibility, membrane ergosterol content, cell surface hydrophobicity (CSH), and reactive oxygen species (ROS) generation among the clinical isolates compared to C0. Chemical profiling studies suggested more robust cell membrane and ergosterol biosynthesis pathways in clinical isolates than C0. The intracellular metabolites were analyzed by one dimensional proton NMR, where a total of 83 compounds were annotated, including amino acids (24), nitrogen bases (15), organic acids (22), sugars and derivatives (13), vitamins and coenzymes (4), choline derivatives (2) and alcohols (2), and acetamide. Further, the multivariate analysis of metabolomics data revealed 18 metabolites responsible for group discrimination among the five strains studied. Metabolomics data and transcriptional analysis of certain regulatory proteins (enzymes) advocated significant alterations in cell membrane stress, carbohydrate metabolism and trehalose biosynthesis, amino acid biosynthesis, ergosterol pathway, and turnover of clinical isolates (C3 and C4) as compared to C0. Thus, this study delineated the principal cellular metabolic pathways and associated biochemical and morphological characteristics altered in clinical isolates that may be responsible for adaptation and survival in susceptible hosts as these pathways can be targeted in formulating new antifungal agents. Chapter 3 is based on the investigation of the effect of a simulated host vulvovaginal microenvironment on the metabolomics and physiology of clinical isolates. For this, two clinical isolates and a wild-type strain of C. glabrata were grown in minimal media yeast nitrogen base (YNB) supplemented with either dextrose or lactic acid (LA) as a carbon source throughout all the experiments. The growth patterns, metabolic alterations, and cellular pathways were investigated by employing biochemical assays, NMR-based metabolomics, and transcriptional studies. C. glabrata showed considerable variations in its topological and biochemical features without compromising growth in LA media. Chemical profiling data highlighted involvement of cell wall/membrane, ergosterol, and oxidative stress related pathways in mediating adaptative response of C. glabrata towards LA. Further, the metabolomics analysis by 1D 1H NMR spectroscopy revealed 59 common intracellular metabolites categorized by their chemical nature as sugars and their derivatives (12), amino acids and their derivatives (22), organic acids (9), nitrogen bases (8), amines and amides (4), alcohols (3), and choline. A significant alteration was observed in 19 metabolites of C. glabrata cells upon growth in LA. Interestingly myo-inositol, xylose, putrescine and betaine which are key metabolites for cell growth and viability, were found to be differentially expressed by clinical isolates. These observations were supported by the transcriptional expression study of selected genes evidencing cell wall/membrane re-organization, altered oxidative stress, and reprogramming of carbon metabolic pathways. Collectively, the study advanced our understanding of the adaptative response of C. glabrata in the vaginal microenvironment to lactic acid for survival and virulence. Chapter 4 deals with screening some natural antifungal agents for their potency against selected clinical isolates of C. glabrata. Of the nine compounds screened, berberine, an isoquinoline alkaloid, was selected due to its low MIC against fluconazole resistant clinical isolates. The checkerboard analysis revealed that berberine showed synergism with the classical antifungal drugs of class azole and polyene. To explore the mechanisms behind selective action of berberine over clinical isolates of C. glabrata, the effect of subinhibitory concentration of berberine on their biochemical and metabolic properties was studied. Significant changes were observed in CSH, membrane ergosterol, ROS generation and antioxidant systems, surface charge, and membrane efflux activity, which are essential factors contributing to virulence. Further, the effect of berberine on metabolic profiles of strains under study was determined to understand the link between modulation of metabolism and antifungal effect. Through metabolomics analysis, 46 metabolites were annotated, including amino acids and peptides (17), organic acids (10), sugars (8), pyrimidines (3), choline derivatives (2), purines (2), betaine, glutamine, propylene glycol, and NADP+. Multivariate analysis of the metabolomics data identified 18 metabolites responsible for group discrimination among all the berberine treated and non-treated groups, and the fold change analysis identified twenty metabolites to be differentially regulated by berberine. Thus, the biochemical and metabolomics analysis suggested that the cell wall attributes, antioxidant systems, and central carbon and amino acid metabolism were differentially affected by berberine. Further, the synergistic studies of berberine with antifungal drugs suggested for synergistic effect, thus making the natural product as a potent combination for designing future antifungal formulations. In summary, the present thesis focused on elucidating the metabolic and biochemical characteristics of clinical isolates of C. glabrata evolved with time upon exposure to host micro-environment and drug therapy. Through NMR metabolomics approach, the distinct pathways associated with development of virulence in clinical isolates were identified. Among these metabolic pathways, particularly the trehalose pathway, can be targeted to treat resistant clinical isolates. Lactic acid influenced the metabolism of C. glabrata strains, suggesting obvious changes in their pathogenicity evidenced by alterations in biochemical and metabolic traits and stronger stress response. Finally, NMR-based metabolomics and biochemical analysis revealed key mechanisms behind the higher potency of berberine against clinical isolates of C. glabrata than the wild-type strain. As berberine showed synergism with other classically used antifungal drugs, it can be used as an additive to antifungal treatment to reduce the toxicity of currently used drugs and to improve the therapeutic outcome.