Molecular Mechanisms of Pathogenesis in Candidiasis
Research in the Sundstrom laboratory focuses on elucidating the molecular mechanisms of fungal pathogenesis in candidiasis, the most common fungal infection in humans. Factors that promote disease from both the pathogen and the host are prioritized. The results of this research will be translated into the development of new strategies for inhibiting candidiasis.
Candidiasis is most frequently caused by Candida albicans, a yeast that rapidly adapts to environmental changes by altering its growth form to true hyphae. Hyphae are pro-adherent and pro-invasive and are required for virulence in animal models. Interactions between human hosts and C. albicans are initiated at birth when material from the mother comes in contact with the skin and mucous membranes of the infant. Thrush and skin infections such as diaper rash frequently stimulate host innate immune defenses as C. albicans activates its natural adhesive and invasive properties targeting the stratified squamous epithelium. Following treatment with antifungal drugs and with the subsequent development of adaptive immunity, the early battle between fungus and host settles into a quiescent state in which C. albicans persists in low numbers in the gastrointestinal tract as a member of the normal flora, inhibited by adaptive host immunity. Since C. albicans is not completely eliminated from the host, its pathogenic potential for invading diverse mucosal and epidermal surfaces frequently emerges in clinical settings involving loss of immunity such as in the presence of HIV, leukemia, malignancy, radiation therapy for head and neck cancer or other risk factors such as treatment with antibacterial drugs that reduce the normal bacterial flora. The protean pathogenic potential of C. albicans includes the capacity to disseminate via the blood stream to internal organs, potentially causing system failure in cases of severe immunosuppression. Perilously few antifungal drugs are available that selectively inhibit this eukaryotic pathogen without causing harm to the host. More information is needed to understand pathogenic mechanisms that can be targeted for development of antifungal strategies.
A major discovery made in the Sundstrom laboratory important for the pro-adhesive properties of C. albicans is that hyphal growth forms are abundantly coated with an adhesin denoted Hwp1 (Hyphal Wall Protein 1). Unlike other microbial adhesins which adhere through hydrobic or lectin-like interactions, Hwp1 forms covalent attachments to proteins on human buccal epithelial cells in host tissue. Covalent bond formation is catalyzed by mammalian epithelial cell transglutaminases that lead to the formation of cross-links between Hwp1 and host proteins. Epithelial transglutaminases are essential for creating the primary host defense barrier and are constitutively expressed on epithelial cells lining the oral cavity. Hwp1 is required for oroesophageal candidiasis in immunodeficient mice, suggesting that Hwp1 plays an important role in allowing C. albicans to maintain a tight foothold on the oral mucosa, contributing to microabscess formation, which is destructive and impairs the function to the oral mucosa.
To elucidate host factors that contribute to attachment, the properties of epithelial cells that become cross-linked to Hwp1 are being investigated. Using differentiation markers to evaluate epithelial cells, it was shown that the differentiation status of oral epithelial cells is critical for Hwp1-mediated attachment by C. albicans. Epithelial cells with late differentiation markers bind more Hwp1 than cells expressing early differentiation markers. The enhanced binding of Hwp1 to terminally differentiated epithelial cells correlates well with a clinical feature of thrush; the presence of hyperkeratinization that involves increased differentiation of the epithelium. Hyperkeratinization is a general keratinocyte response to an impairment in the permeability barrier of the epithelium. In the specific case of candidiasis the hyperkeratinzed region provides an environment that supports heavy growth of C. albicans. Further experiments investigating the interactions of C. albicans with the epithelium led to the discovery that soluble factors produced by hyphal forms of C. albicans stimulate a keratinocyte response involving actin cytoskeletal reorganization, motility and ultimately up-regulation of differentiation markers. Secreted aspartyl proteases of C. albicans are implicated in triggering a response in the epithelial cells that leads to their differentiation. Thus hyphal growth forms can influence the differentiation program of epithelial cells in a manner that promotes fungal growth.
One of the most important topics for fungal pathogenesis is how fungi couple changes in gene expression to fungal morphogenesis. To study this question, C. albicans strains with the upstream region of Hwp1 coupled to a GFP reporter gene have been created. These strains have been used to map the region of Hwp1 that activates gene expression in hyphal growth. In other experiments, it was found that changes in actin dynamics that occur during the transition to germ tube growth promote Hwp1 gene expression. Using cAMP hypomorphic mutants, we discovered that changes in actin dynamics are transmitted through the cAMP signaling pathway.
Ongoing work in the Sundstrom laboratory continues to advance the understanding of host pathogen interactions as well as to investigate the mechanisms that couple gene expression to morphogenesis. For the first time, the ability of C. albicans to attach to differentiated keratinocytes and to manipulate the keratinocyte differentiation program has been elucidated. Many questions remain unanswered. Experiments to identify epithelial proteins that bind to Hwp1 and to map the exact sites of attachment of Hwp1 to host proteins are in progress. The mechanisms that trigger keratinocytes to differentiate and become less toxic to C. albicans are also being investigated. These results will lead to translational studies that will evaluate the potential for therapies that inhibit covalent attachment and resist candidal influences on differentiation to inhibit candidiasis. Other research involves investigating the mechanisms that regulate Hwp1 expression by identifying DNA binding proteins that influence Hwp1 gene expression and by investigating the epigenetic mechanisms important for expression. An important goal is to extend the studies on Hwp1 to other hypha-specifically expressed genes to unravel general mechanisms that lead to hypha-specific gene expression. These avenues of investigation will contribute to both the general understanding of fungal strategies for adaptation to different environments through morphogenesis and changes in gene expression as well as to provide strategies for advancing the treatment of candidiasis for future generations.