Virulence of Candida albicans: The goal of our research is to understand the mechanisms that control the pathogenicity of the yeast C. albicans. C. albicans is the most common and arguably the most important causative agent of human fungal infections. It is a major opportunistic pathogen of immunocompromised hosts, including AIDS patients and those undergoing chemotherapy, tissue transplants or with central venous catheters. Studies indicate that up to 90% of AIDS patients suffer from oropharyngeal and esophageal candidiasis, in which C. albicans is the major causative agent. C. albicans can exist in different morphological states including budded or yeast-like cells and hyphal forms (Fig. 1), and there is strong evidence that the ability to switch between these morphological states is essential for its virulence. Understanding the input signals and output responses that lead to the budded-to-hyphal transition (BHT) will undoubtedly lead to tremendous insight into virulence mechanisms and may ultimately lead to new anti-fungal therapeutic targets and drugs. This point is especially critical since there are serious side effects due to renal and liver dysfunction associated with the polyenes (i.e., amphotericin B, nystatin) that are usually used to treat C. albicans infections. In addition, a significant increase in resistance to the less toxic azole drugs (i.e., fluconoazole) has occurred within the patient population, especially HIV-positive patients.
C. albicans pathogenicity can be attributed to its ability to survive and thrive in multiple microenvironments within the host, including multiple organs, the mucosa, and the bloodstream, and to virulence factors that aid in the adherence and invasion of multiple cell types. One of the major virulence factors for C. albicans is its ability to form biofilms on indwelling medical devices such as catheters. Systemic candidiasis can arise from C. albicans cells being released from these biofilms, followed by colonization of mucosal surfaces, penetration of epithelial and endothelial cell barriers, and dissemination throughout the body. Both budded and hyphal forms of C. albicans have been identified in infections and are important for virulence, and the BHT is critical for systemic infections, a premise that was reinforced in non-virulent C. albicans mutants defective in hyphal formation.
The BHT occurs in response to environmental stress signals such as temperature above 35oC, pH above 6.5, nitrogen and/or carbon starvation, low oxygen concentration, and changes in cell density, growth in serum or other chemicals such as N-acetylglucosamine, proline and other amino acids, or alcohols, and the presence of host macrophages. Given that C. albicans cells respond to a variety of different growth and environmental signals to induce the budded-to-hyphal transition, it is not surprising that multiple signaling pathways function in hyphal development. However, the molecular mechanisms that underlie these different signaling pathways and their potential cross-talk are still unclear.
Use of small molecules to study cell
biological process: There is no question that the use of
organic molecules in deciphering complex biological processes has been
tremendously fruitful. For example, much of what is known about
processes within the cell comes from studies using specific inhibitors
structure or function such as the cytochalasins, latrunculins, and
phalloidins. These types of small molecules have proven invaluable
interact with their target proteins as agonists or antagonists in a
specific manner, which allows definitive conclusions to be drawn
protein¹s function in a particular process. This type of approach
analogous to a reverse genetic approach in that molecules are used to
known protein targets that are presumed to be involved in a cellular
process. Small molecules can also be used in a forward genetic approach
in which no
presumptions are made about what proteins are involved in the process.
occurs through the screening of large, diverse sets of individual
a phenotype (or reversal of a phenotype) associated with a cellular
process. This approach has been successful in studying a wide variety
processes. It should be noted that
this type of phenotype-based screening is particularly useful in
where classical forward genetic approaches are not possible (i.e.,
recessive mutations in a diploid species), and is another reason why we
decided to use this approach to study biofilm formation and the BHT in
constitutive diploid C. albicans.
We have identified 21 molecules that inhibit the BHT without affecting budded growth: 5 are novel molecules (Toenjes et al., 2005) and 16 molecules affect known cellular targets or signaling pathways in the cell (Toenjes et al., submitted). Our studies bring to bear an arsenal of genetic, molecular genetic, biochemical, and cell biological approaches. Specifically, we are elucidating the mechanisms by which environmental signals that induce hyphal growth and virulence also regulate biofilm formation. In addition, we are circumventing the inherent complexities associated with classical genetic screens with the diploid C. albicans by using a new high-throughput small molecule screen to identify inhibitors and enhancers of biofilm formation. The bioactive molecules identified in this screen are being used to identify and characterize components of the pathways regulating the BHT and the molecular mechanisms by which these components function. Information on the effects of these bioactive molecules, or their derivatives, on C. albicans biofilm formation and virulence will be critical in acquiring the ultimate goal of identifying new anti-fungal molecules or targets for anti-fungal therapeutics.