Audrey R. Odom

Calcineurin is required for virulence of Cryptococcus neoformans

Cyclosporin A (CsA) and FK506 are antimicrobial, immunosuppressive natural products that inhibit signal transduction. In T cells and Saccharomyces cerevisiae, CsA and FK506 bind to the immunophilins cyclophilin A and FKBP12 and the resulting complexes inhibit the Ca2+‐regulated protein phosphatase calcineurin. We find that growth of the opportunistic fungal pathogen Cryptococcus neoformans is sensitive to CsA and FK506 at 37°C but not at 24°C, suggesting that CsA and FK506 inhibit a protein required for C.neoformans growth at elevated temperature. Genetic evidence supports a model in which immunophilin–drug complexes inhibit calcineurin to prevent growth at 37°C. The gene encoding the C.neoformans calcineurin A catalytic subunit was cloned and disrupted by homologous recombination. Calcineurin mutant strains are viable but do not survive in vitro conditions that mimic the host environment (elevated temperature, 5% CO2 or alkaline pH) and are no longer pathogenic in an animal model of cryptococcal meningitis. Introduction of the wild‐type calcineurin A gene complemented these growth defects and restored virulence. Our findings demonstrate that calcineurin is required for C.neoformans virulence and may define signal transduction elements required for fungal pathogenesis that could be targets for therapeutic intervention.

A Second Target of the Antimalarial and Antibacterial Agent Fosmidomycin Revealed by Cellular Metabolic Profiling

Antimicrobial drug resistance is an urgent problem in the control and treatment of many of the worlds most serious infections, including Plasmodium falciparum malaria, tuberculosis, and healthcare-associated infections with Gram-negative bacteria. Because the non-mevalonate pathway of isoprenoid biosynthesis is essential in eubacteria and P. falciparum and this pathway is not present in humans, there is great interest in targeting the enzymes of non-mevalonate metabolism for antibacterial and antiparasitic drug development. Fosmidomycin is a broad-spectrum antimicrobial agent currently in clinical trials of combination therapies for the treatment of malaria. In vitro, fosmidomycin is known to inhibit the deoxyxylulose phosphate reductoisomerase (DXR) enzyme of isoprenoid biosynthesis from multiple pathogenic organisms. To define the in vivo metabolic response to fosmidomycin, we developed a novel mass spectrometry method to quantitate six metabolites of non-mevalonate isoprenoid metabolism from complex biological samples. Using this technique, we validate that the biological effects of fosmidomycin are mediated through blockade of de novo isoprenoid biosynthesis in both P. falciparum malaria parasites and Escherichia coli bacteria: in both organisms, metabolic profiling demonstrated a block of isoprenoid metabolism following fosmidomycin treatment, and growth inhibition due to fosmidomycin was rescued by media supplemented with isoprenoid metabolites. Isoprenoid metabolism proceeded through DXR even in the presence of fosmidomycin but was inhibited at the level of the downstream enzyme, methylerythritol phosphate cytidyltransferase (IspD). Overexpression of IspD in E. coli conferred fosmidomycin resistance, and fosmidomycin was found to inhibit IspD in vitro. This work has validated fosmidomycin as a biological reagent for blocking non-mevalonate isoprenoid metabolism and suggests a second in vivo target for fosmidomycin within isoprenoid biosynthesis, in two evolutionarily diverse pathogens.

Characterization of the MFα pheromone of the human fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is an important human pathogenic fungus with a defined sexual cycle and well‐developed molecular and genetic approaches. C. neoformans is predominantly haploid and has two mating types, MATa and MATα. Mating is known to be regulated by nutritional limitation and thought also to be regulated by pheromones. Previously, a portion of the MATα locus was cloned, and a presumptive pheromone gene, MFα1, was identified by its ability to induce conjugation tube‐like filaments when introduced by transformation into MATa cells. Here, the ability of the MFα1 gene to induce these morphological changes in MATa cells was used as a phenotypic assay to perform a structure–function analysis of the gene. We show that the MFα1 open reading frame is required for the morphological response of MATa cells. We also find that the cysteine residue of the C‐terminal CAAX motif is required for activity of the MFα1 pheromone. In addition, we use a reporter system to measure the expression levels of the MFα1 pheromone gene and find that two signals, nutrient starvation and the presence of factors secreted by mating partner cells, impinge on this promoter and regulate MFα1 expression. We identify a second pheromone gene, MFα2, and show phenotypically that this gene is also expressed. Finally, we have synthesized the MFα1 pheromone and show that only the predicted mature modified form of the α‐factor peptide triggers morphological responses in MATa cells.

Functional genetic analysis of the Plasmodium falciparum deoxyxylulose 5-phosphate reductoisomerase gene

Novel antimalarial drugs are urgently needed to treat severe malaria caused by Plasmodium falciparum. Isoprenoid biosynthesis is a promising target pathway, since the biosynthetic route in Plasmodia is biochemically distinct from the mevalonate pathway in humans. The small molecule fosmidomycin is an inhibitor of the enzyme responsible for the first dedicated step in isoprenoid biosynthesis, deoxyxylulose 5-phosphate reductoisomerase (DXR). However, the antimalarial effects of fosmidomycin might not be specific to DXR inhibition and further validation of DXR is warranted. We present the first functional genetic validation of P. falciparum DXR (PF14_0641). Using a single cross-over strategy, we show that plasmid integration occurs at the DXR locus but only when DXR gene function is preserved, but not when integration disrupts gene function. These data indicate that DXR is required for intraerythrocytic development of P. falciparum.

A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites

Isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway generates commercially important products and is a target for antimicrobial drug development. MEP pathway regulation is poorly understood in microorganisms. We employ a forward genetics approach to understand MEP pathway regulation in the malaria parasite, Plasmodium falciparum. The antimalarial fosmidomycin inhibits the MEP pathway enzyme deoxyxylulose 5-phosphate reductoisomerase (DXR). Fosmidomycin-resistant P. falciparum are enriched for changes in the PF3D7_1033400 locus (hereafter referred to as PfHAD1), encoding a homologue of haloacid dehalogenase (HAD)-like sugar phosphatases. We describe the structural basis for loss-of-function PfHAD1 alleles and find that PfHAD1 dephosphorylates a variety of sugar phosphates, including glycolytic intermediates. Loss of PfHAD1 is required for fosmidomycin resistance. Parasites lacking PfHAD1 have increased MEP pathway metabolites, particularly the DXR substrate, deoxyxylulose 5-phosphate. PfHAD1 therefore controls substrate availability to the MEP pathway. Because PfHAD1 has homologs in plants and bacteria, other HAD proteins may be MEP pathway regulators.

The MEP pathway and the development of inhibitors as potential anti-infective agents

The non-mevalonate (or MEP) pathway represents an essential biosynthetic route used by plants, algae, and eubacteria to generate isoprenoid precursors. The MEP pathway has also been genetically validated in pathogenic organisms such as P. falciparum and M. tuberculosis. As this pathway is absent in mammalian systems, the enzymes of the MEP pathway represent attractive targets for the development of novel herbicides and antimicrobial chemotherapeutics. This review examines the enzymes in the MEP pathway from a detailed medicinal chemistry and structural biology perspective. The binding modes of substrates and inhibitors are discussed, identifying key interactions that maybe exploitable in small molecule inhibitor design.

Five questions about non-mevalonate isoprenoid biosynthesis.

The author has declared that no competing interests exist. The authors studies are supported by NIH K08 AI079010, Doris Duke Charitable Foundation Clinical Scientist Development Award, and the Childrens Discovery Institute of St. Louis, MO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Malaria Parasites Produce Volatile Mosquito Attractants

ABSTRACT The malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid organelle that possesses plant-like metabolic pathways. Plants use the plastidial isoprenoid biosynthesis pathway to produce volatile odorants, known as terpenes. In this work, we describe the volatile chemical profile of cultured malaria parasites. Among the identified compounds are several plant-like terpenes and terpene derivatives, including known mosquito attractants. We establish the molecular identity of the odorant receptors of the malaria mosquito vector Anopheles gambiae, which responds to these compounds. The malaria parasite produces volatile signals that are recognized by mosquitoes and may thereby mediate host attraction and facilitate transmission. IMPORTANCE Malaria is a key global health concern. Mosquitoes that transmit malaria are more attracted to malaria parasite-infected mammalian hosts. These studies aimed to understand the chemical signals produced by malaria parasites; such an understanding may lead to new transmission-blocking strategies or noninvasive malaria diagnostics. Malaria is a key global health concern. Mosquitoes that transmit malaria are more attracted to malaria parasite-infected mammalian hosts. These studies aimed to understand the chemical signals produced by malaria parasites; such an understanding may lead to new transmission-blocking strategies or noninvasive malaria diagnostics.

Isoprenoid Biosynthesis Inhibition Disrupts Rab5 Localization and Food Vacuolar Integrity in Plasmodium falciparum

ABSTRACT The antimalarial agent fosmidomycin is a validated inhibitor of the nonmevalonate isoprenoid biosynthesis (methylerythritol 4-phosphate [MEP]) pathway in the malaria parasite, Plasmodium falciparum. Since multiple classes of prenyltransferase inhibitors kill P. falciparum, we hypothesized that protein prenylation was one of the essential functions of this pathway. We found that MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking. We have found that Rab5 proteins dramatically mislocalize upon fosmidomycin treatment, consistent with a loss of protein prenylation. Fosmidomycin treatment caused marked defects in food vacuolar morphology and integrity, consistent with a defect in Rab-mediated vesicular trafficking. These results provide insights to the biological functions of isoprenoids in malaria parasites and may assist the rational selection of secondary agents that will be useful in combination therapy with new isoprenoid biosynthesis inhibitors.

Structural Studies and Protein Engineering of Inositol Phosphate Multikinase

Background: IPMK is a key enzyme in signaling required for cellular adaptation and organismal development. Results: We report the structure of IPMK, use structure-based enzyme design and perform complementation analysis in model systems. Conclusion: We define a basis for substrate selectivity and report that 6-kinase activity is important for signaling. Significance: Determination of the IPMK structure enabled rewiring of signaling in organisms. Inositol phosphates (IPs) regulate vital processes in eukaryotes, and their production downstream of phospholipase C activation is controlled through a network of evolutionarily conserved kinases and phosphatases. Inositol phosphate multikinase (IPMK, also called Ipk2 and Arg82) accounts for phosphorylation of IP3 to IP5, as well as production of several other IP molecules. Here, we report the structure of Arabidopsis thaliana IPMKα at 2.9 Å and find it is similar to the yeast homolog Ipk2, despite 17% sequence identity, as well as the active site architecture of human IP3 3-kinase. Structural comparison and substrate modeling were used to identify a putative basis for IPMK selectivity. To test this model, we re-engineered binding site residues predicted to have restricted substrate specificity. Using steady-state kinetics and in vivo metabolic labeling studies in modified yeast strains, we observed that K117W and K117W:K121W mutants exhibited nearly normal 6-kinase function but harbored significantly reduced 3-kinase activity. These mutants complemented conditional nutritional growth defects observed in ipmk null yeast and, remarkably, suppressed lethality observed in ipmk null flies. Our data are consistent with the hypothesis that IPMK 6-kinase activity and production of Ins(1,4,5,6)P4 are critical for cellular signaling. Overall, our studies provide new insights into the structure and function of IPMK and utilize a synthetic biological approach to redesign inositol phosphate signaling pathways.