Grf10 and Bas1 Regulate Transcription of Adenylate and One-Carbon Biosynthesis Genes and Affect Virulence in the Human Fungal Pathogen Candida albicans.

Candida albicans is an opportunistic human fungal pathogen that causes superficial fungal infections and lethal systemic infections. To colonize and establish infections, C. albicans coordinates the expression of virulence and metabolic genes. Previous work showed that the homeodomain transcription factor Grf10 is required for formation of hyphae, a virulence factor. Here we report global gene expression analysis of a grf10Δ strain using a DNA microarray and identify genes for de novo adenylate biosynthesis (ADE genes), one-carbon metabolism, and a nucleoside permease (NUP). Upregulation of these genes in response to adenine limitation required both Grf10 and the myb protein Bas1, as shown by quantitative real-time PCR (qRT-PCR). Phenotypic analysis showed that both mutants exhibited growth defects when grown in the absence of adenine, and the doubling time was slower for the bas1Δ mutant. Bas1 is required for basal expression of these genes, whereas NUP expression is more dependent upon Grf10. Disruption of BAS1 led to only modest defects in hypha formation and weak attenuation of virulence in a systemic mouse model of infection, as opposed to the previously reported strong effects found in the grf10Δ mutant. Our data are consistent with a model in which Grf10 coordinates metabolic effects on nucleotide metabolism by interaction with Bas1 and indicate that AMP biosynthesis and its regulation are important for C. albicans growth and virulence. IMPORTANCECandida albicans is a commensal and a common constituent of the human microbiota; however, it can become pathogenic and cause infections in both immunocompetent and immunocompromised people. C. albicans exhibits remarkable metabolic versatility as it can colonize multiple body sites as a commensal or pathogen. Understanding how C. albicans adapts metabolically to each ecological niche is essential for developing novel therapeutic approaches. Purine metabolism has been targeted pharmaceutically in several diseases; however, the regulation of this pathway has not been fully elucidated in C. albicans. Here, we report how C. albicans controls the AMP de novo biosynthesis pathway in response to purine availability. We show that the lack of the transcription factors Grf10 and Bas1 leads to purine metabolic dysfunction, and this dysfunction affects the ability of C. albicans to establish infections.

Flow Cytometry-Based Assessment of Antibody Function Against Malaria Pre-erythrocytic Infection.

The development of new interventional strategies against pre-erythrocytic malaria is hampered by the lack of standardized approaches to assess inhibition of sporozoite infection of hepatocytes. The following methodology, based on flow cytometry, can be used to quantitatively assess P. falciparum sporozoite infection in vitro in medium throughput. In addition to assessing the efficacy of antibodies, this assay has a wide variety of applications for investigating basic science questions about the malaria liver stage. This approach is easily applied in a variety of laboratory settings, assesses the functionality of antibody responses against malaria sporozoites, and can be adapted for the limited quantities of sample which are typically available from clinical investigations.

Suppression of host p53 is critical for Plasmodium liver-stage infection.

Plasmodium parasites infect the liver and replicate inside hepatocytes before they invade erythrocytes and trigger clinical malaria. Analysis of host signaling pathways affected by liver-stage infection could provide critical insights into host-pathogen interactions and reveal targets for intervention. Using protein lysate microarrays, we found that Plasmodium yoelii rodent malaria parasites perturb hepatocyte regulatory pathways involved in cell survival, proliferation, and autophagy. Notably, the prodeath protein p53 was substantially decreased in infected hepatocytes, suggesting that it could be targeted by the parasite to foster survival. Indeed, mice that express increased levels of p53 showed reduced liver-stage parasite burden, whereas p53 knockout mice suffered increased liver-stage burden. Furthermore, boosting p53 levels with the use of the small molecule Nutlin-3 dramatically reduced liver-stage burden in vitro and in vivo. We conclude that perturbation of the hepatocyte p53 pathway critically impacts parasite survival. Thus, host pathways might constitute potential targets for host-based antimalarial prophylaxis.