Differential Roles of a Family of Flavodoxin-Like Proteins That Promote Resistance to Quinone-Mediated Oxidative Stress in Candida albicans.

Survival of the fungal pathogen Candida albicans within a mammalian host relies on its ability to resist oxidative stress. The four flavodoxin-like proteins (Pst1, Pst2, Pst3, and Ycp4) that reside on the inner surface of the C. albicans plasma membrane represent a recently discovered antioxidant mechanism that is essential for virulence. Flavodoxin-like proteins combat oxidative stress by promoting a two-electron reduction of quinone molecules, which prevents the formation of toxic semiquinone radicals. Previous studies indicated that Pst3 played a major role in promoting resistance to the small quinone molecules p-benzoquinone and menadione. Analysis of additional quinones confirmed this role for Pst3. To better define their function, antibodies were raised against each of the four flavodoxin-like proteins and used to quantify protein levels. Interestingly, the basal level of flavodoxin-like proteins differed, with Pst3 and Ycp4 being the most abundant. However, after induction with p-benzoquinone, Pst1 and Pst3 were the most highly induced, resulting in Pst3 becoming the most abundant. Constitutive expression of the flavodoxin-like protein genes from a TDH3 promoter resulted in similar protein levels and showed that Pst1 and Pst3 were better at protecting C. albicans against p-benzoquinone than Pst2 or Ycp4. In contrast, Pst1 and Ycp4 provided better protection against oxidative damage induced by tert-butyl hydroperoxide. Thus, both the functional properties and the relative abundance contribute to the distinct roles of the flavodoxin-like proteins in resisting oxidative stress. These results further define how C. albicans combats the host immune response and survives in an environment rich in oxidative stress.

Plasma Membrane MCC/Eisosome Domains Promote Stress Resistance in Fungi.

There is growing appreciation that the plasma membrane orchestrates a diverse array of functions by segregating different activities into specialized domains that vary in size, stability, and composition. Studies with the budding yeast Saccharomyces cerevisiae have identified a novel type of plasma membrane domain known as the MCC (membrane compartment of Can1)/eisosomes that correspond to stable furrows in the plasma membrane. MCC/eisosomes maintain proteins at the cell surface, such as nutrient transporters like the Can1 arginine symporter, by protecting them from endocytosis and degradation. Recent studies from several fungal species are now revealing new functional roles for MCC/eisosomes that enable cells to respond to a wide range of stressors, including changes in membrane tension, nutrition, cell wall integrity, oxidation, and copper toxicity. The different MCC/eisosome functions are often intertwined through the roles of these domains in lipid homeostasis, which is important for proper plasma membrane architecture and cell signaling. Therefore, this review will emphasize the emerging models that explain how MCC/eisosomes act as hubs to coordinate cellular responses to stress. The importance of MCC/eisosomes is underscored by their roles in virulence for fungal pathogens of plants, animals, and humans, which also highlights the potential of these domains to act as novel therapeutic targets.

MCC/Eisosomes Regulate Cell Wall Synthesis and Stress Responses in Fungi.

The fungal plasma membrane is critical for cell wall synthesis and other important processes including nutrient uptake, secretion, endocytosis, morphogenesis, and response to stress. To coordinate these diverse functions, the plasma membrane is organized into specialized compartments that vary in size, stability, and composition. One recently identified domain known as the Membrane Compartment of Can1 (MCC)/eisosome is distinctive in that it corresponds to a furrow-like invagination in the plasma membrane. MCC/eisosomes have been shown to be formed by the Bin/Amphiphysin/Rvs (BAR) domain proteins Lsp1 and Pil1 in a range of fungi. MCC/eisosome domains influence multiple cellular functions; but a very pronounced defect in cell wall synthesis has been observed for mutants with defects in MCC/eisosomes in some yeast species. For example, Candida albicans MCC/eisosome mutants display abnormal spatial regulation of cell wall synthesis, including large invaginations and altered chemical composition of the walls. Recent studies indicate that MCC/eisosomes affect cell wall synthesis in part by regulating the levels of the key regulatory lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P2) in the plasma membrane. One general way MCC/eisosomes function is by acting as protected islands in the plasma membrane, since these domains are very stable. They also act as scaffolds to recruit >20 proteins. Genetic studies aimed at defining the function of the MCC/eisosome proteins have identified important roles in resistance to stress, such as resistance to oxidative stress mediated by the flavodoxin-like proteins Pst1, Pst2, Pst3 and Ycp4. Thus, MCC/eisosomes play multiple roles in plasma membrane organization that protect fungal cells from the environment.

Yeast three-hybrid screen identifies TgBRADIN/GRA24 as a negative regulator of Toxoplasma gondii bradyzoite differentiation.

Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.

Identification of Cryptosporidium parvum active chemical series by Repurposing the open access malaria box.

The apicomplexan parasites Cryptosporidium parvum and Cryptosporidium hominis are major etiologic agents of human cryptosporidiosis. The infection is typically self-limited in immunocompetent adults, but it can cause chronic fulminant diarrhea in immunocompromised patients and malnutrition and stunting in children. Nitazoxanide, the current standard of care for cryptosporidiosis, is only partially efficacious for children and is no more effective than a placebo for AIDS patients. Unfortunately, financial obstacles to drug discovery for diseases that disproportionately affect low-income countries and technical limitations associated with studies of Cryptosporidium biology impede the development of better drugs for treating cryptosporidiosis. Using a cell-based high-throughput screen, we queried the Medicines for Malaria Venture (MMV) Open Access Malaria Box for activity against C. parvum. We identified 3 novel chemical series derived from the quinolin-8-ol, allopurinol-based, and 2,4-diamino-quinazoline chemical scaffolds that exhibited submicromolar potency against C. parvum. Potency was conserved in a subset of compounds from each scaffold with varied physicochemical properties, and two of the scaffolds identified exhibit more rapid inhibition of C. parvum growth than nitazoxanide, making them excellent candidates for further development. The 2,4-diamino-quinazoline and allopurinol-based compounds were also potent growth inhibitors of the related apicomplexan parasite Toxoplasma gondii, and a good correlation was observed in the relative activities of the compounds in the allopurinol-based series against T. gondii and C. parvum. Taken together, these data illustrate the utility of the Open Access Malaria Box as a source of both potential leads for drug development and chemical probes to elucidate basic biological processes in C. parvum and other apicomplexan parasites.