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Evidence has accumulated to demonstrate that the intestinal microbiota enhances mammalian enteric virus infections1. For example, we and others previously reported that commensal bacteria stimulate acute and persistent murine norovirus infections2-4. However, in apparent contradiction of these results, the virulence of murine norovirus infection was unaffected by antibiotic treatment. This prompted us to perform a detailed investigation of murine norovirus infection in microbially deplete mice, revealing a more complex picture in which commensal bacteria inhibit viral infection of the proximal small intestine while simultaneously stimulating the infection of distal regions of the gut. Thus, commensal bacteria can regulate viral regionalization along the intestinal tract. We further show that the mechanism underlying bacteria-dependent inhibition of norovirus infection in the proximal gut involves bile acid priming of type III interferon. Finally, the regional effects of the microbiota on norovirus infection may result from distinct regional expression profiles of key bile acid receptors that regulate the type III interferon response. Overall, these findings reveal that the biotransformation of host metabolites by the intestinal microbiota directly and regionally impacts infection by a pathogenic enteric virus.

The malaria parasite, Plasmodium falciparum, proliferates rapidly in human erythrocytes by actively scavenging multiple carbon sources and essential nutrients from its host cell. However, a global overview of the metabolic capacity of intraerythrocytic stages is missing. Using multiplex 13C‐labelling coupled with untargeted mass spectrometry and unsupervised isotopologue grouping, we have generated a draft metabolome of P. falciparum and its host erythrocyte consisting of 911 and 577 metabolites, respectively, corresponding to 41% of metabolites and over 70% of the metabolic reaction predicted from the parasite genome. An additional 89 metabolites and 92 reactions were identified that were not predicted from genomic reconstructions, with the largest group being associated with metabolite damage‐repair systems. Validation of the draft metabolome revealed four previously uncharacterised enzymes which impact isoprenoid biosynthesis, lipid homeostasis and mitochondrial metabolism and are necessary for parasite development and proliferation. This study defines the metabolic fate of multiple carbon sources in P. falciparum, and highlights the activity of metabolite repair pathways in these rapidly growing parasite stages, opening new avenues for drug discovery., Multiplex stable‐isotope labelling defines the observable metabolome of red blood cell stages of the malaria parasite Plasmodium falciparum and indicates potential roles for metabolic enzymes of unknown function.

The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of parasite GPI anchor biosynthesis, the putative phosphomannomutase (PMM) (EC 5.4.2.8), HAD5 (PF3D7_1017400). We confirmed the PMM and phosphoglucomutase activities of purified recombinant HAD5 by developing novel linked enzyme biochemical assays. By regulating the expression of HAD5 in transgenic parasites with a TetR-DOZI-inducible knockdown system, we demonstrated that HAD5 is required for malaria parasite egress and erythrocyte reinvasion, and we assessed the role of HAD5 in GPI anchor synthesis by autoradiography of radiolabeled glucosamine and thin layer chromatography. Finally, we determined the three-dimensional X-ray crystal structure of HAD5 and identified a substrate analog that specifically inhibits HAD5 compared to orthologous human PMMs in a time-dependent manner. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition in parasites and that HAD5 has potential as a specific, multistage antimalarial target.

Widespread antimalarial drug resistance has prompted the need for new therapeutics and greater understanding of malaria parasite biology. To this end, the isoprenoid biosynthesis inhibitor fosmidomycin has been used to probe the metabolic regulation in the malaria parasite, Plasmodium falciparum . Genetic changes in the haloacid dehalogenase (HAD) superfamily member HAD2 conferred resistance to fosmidomycin, at the cost of decreased fitness. In the absence of fosmidomycin, parasites gained mutations to phosphofructokinase that restored growth and fosmidomycin sensitivity, thus revealing an intriguing example of plasticity in a core glycolytic process. Moreover, this study marks a second report of a HAD superfamily protein-modulating metabolic homeostasis in P falciparum parasites. Haloacid dehalogenase enzymes are distributed across all domains of life and have increasingly been found to influence central carbon metabolism and drug sensitivity in P falciparum . Investigating the mechanisms by which HAD superfamily members modulate metabolism may shed light on how metabolic networks are connected in apicomplexan parasites and other organisms and may guide future therapeutic endeavors.

Background Malaria continues to pose an enormous economic and global health threat, killing over 200,000 people annually, primarily children under the age of 5. With the constant barrier of antimalarial resistance and the rise of delayed clearance by artemisinin, it is especially important to identify drug/target pairs that rapidly kill parasites. We study targetable metabolic pathways in the malaria parasite, Plasmodium falciparum, to guide such future drug development against this disease. In recent years, we have discovered that a large family of hydrolases, the Haloacid Dehalogenase (HAD) Superfamily of proteins, are implicated in regulating a variety of P. falciparum metabolic pathways, which can lead to dramatic changes in central carbon metabolism and drug resistance. We now turn our attention to a related HAD protein, the putative phosphomannomutase in these parasites, HAD5, responsible for the interconversion of mannose-6-phosphate and mannose-1-phosphate. This is an essential process for all stages of the parasite, and thus has potential as a broad antimalarial target. We examined the role of HAD5 in these parasites, and its potential to be chemically inhibited. Methods Recombinant protein was generated and purified for enzymatic assays to determine HAD5 activity and test inhibitor potency against HAD5 compared to recombinant human orthologs, PMM1 and PMM2. In parallel, CRISPR/Cas9 was used to generate inducible knockdown parasite strains to demonstrate this gene’s essentiality and its role in parasite biology. Parasite growth was measured by flow cytometry and light microscopy. Immunofluorescence analysis (IFA) was used to track the parasite development on a molecular scale. Results Inhibition of HAD5 was achieved in biochemical assays, with an IC50 of 68µM in our most potent compound, representing roughly 10-fold increased potency against the parasite protein compared to human orthologs. In culture, knockdown of HAD5 leads to interrupted egress from and reinvasion into red blood cells, culminating in parasite death. In IFA-visualized parasites, reinvasion-facilitating proteins were no longer anchored to parasite surfaces, accounting for the inhibition of the parasite life cycle. Conclusion In the search for new antimalarial targets, identifying proteins that are essential across multiple parasite life-stages while being distinct from human orthologs is necessary to block parasite transmission, cure symptomatic infection, and minimize off-target effects. HAD5 is an essential protein in malaria parasites that is expressed throughout the parasite’s life cycle, and can be specifically targeted by inhibitors, giving it promise as a future drug target.

Unique and essential aspects of parasite metabolism are excellent targets for development of new antimalarials. An improved understanding of parasite metabolism and drug resistance mechanisms is urgently needed. The antibiotic fosmidomycin targets the synthesis of essential isoprenoid compounds from glucose and is a candidate for antimalarial development. Our report identifies a novel mechanism of drug resistance and further describes a family of metabolic regulators in the parasite. Using a novel forward genetic approach, we also uncovered mutations that suppress drug resistance in the glycolytic enzyme PFK9. Thus, we identify an unexpected genetic mechanism of adaptation to metabolic insult that influences parasite fitness and tolerance of antimalarials., In the malaria parasite Plasmodium falciparum, synthesis of isoprenoids from glycolytic intermediates is essential for survival. The antimalarial fosmidomycin (FSM) inhibits isoprenoid synthesis. In P. falciparum, we identified a loss-of-function mutation in HAD2 (P. falciparum 3D7_1226300 [PF3D7_1226300]) as necessary for FSM resistance. Enzymatic characterization revealed that HAD2, a member of the haloacid dehalogenase-like hydrolase (HAD) superfamily, is a phosphatase. Harnessing a growth defect in resistant parasites, we selected for suppression of HAD2-mediated FSM resistance and uncovered hypomorphic suppressor mutations in the locus encoding the glycolytic enzyme phosphofructokinase 9 (PFK9). Metabolic profiling demonstrated that FSM resistance is achieved via increased steady-state levels of methylerythritol phosphate (MEP) pathway and glycolytic intermediates and confirmed reduced PFK9 function in the suppressed strains. We identified HAD2 as a novel regulator of malaria parasite metabolism and drug sensitivity and uncovered PFK9 as a novel site of genetic metabolic plasticity in the parasite. Our report informs the biological functions of an evolutionarily conserved family of metabolic regulators and reveals a previously undescribed strategy by which malaria parasites adapt to cellular metabolic dysregulation.

Widespread antimalarial drug resistance has prompted the need for new therapeutics and greater understanding of malaria parasite biology. To this end, the isoprenoid biosynthesis inhibitor fosmidomycin has been used to probe the metabolic regulation in the malaria parasite, Plasmodium falciparum. Genetic changes in the haloacid dehalogenase (HAD) superfamily member HAD2 conferred resistance to fosmidomycin, at the cost of decreased fitness. In the absence of fosmidomycin, parasites gained mutations to phosphofructokinase that restored growth and fosmidomycin sensitivity, thus revealing an intriguing example of plasticity in a core glycolytic process. Moreover, this study marks a second report of a HAD superfamily protein-modulating metabolic homeostasis in P falciparum parasites. Haloacid dehalogenase enzymes are distributed across all domains of life and have increasingly been found to influence central carbon metabolism and drug sensitivity in P falciparum. Investigating the mechanisms by which HAD superfamily members modulate metabolism may shed light on how metabolic networks are connected in apicomplexan parasites and other organisms and may guide future therapeutic endeavors.