Accumulating evidence demonstrates that the intestinal microbiota enhance mammalian enteric virus infections. For example, we and others have previously reported that commensal bacteria stimulate acute and persistent murine norovirus infections. In apparent contradiction to these results however, 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 whereby commensal bacteria inhibit viral infection of the proximal small intestine while simultaneously stimulating 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 is 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 which regulate the type III interferon response. Overall, these findings reveal that biotransformation of host metabolites by the intestinal microbiota directly and regionally impacts infection by a pathogenic enteric virus.

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.

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 13 C‐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.

29In the malaria parasite Plasmodium falciparum, isoprenoid synthesis from glycolytic 30 intermediates is essential for survival. The antibiotic and antimalarial fosmidomycin (FSM) 31 inhibits isoprenoid synthesis. In FSM-resistant P. falciparum, we identify a loss-of-function 32 mutation in HAD2 as causative for resistance. Enzymatic characterization shows that HAD2, a 33 member of the haloacid dehalogenase-like hydrolase (HAD) superfamily, functions as a 34 nucleotidase. Harnessing a growth defect in HAD2-mutant parasites, we select for suppression of 35 HAD2-mediated FSM resistance and uncover hypomorphic suppressor mutations in the locus 36 encoding the glycolytic enzyme phosphofructokinase. Metabolic profiling demonstrates that 37 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/155523 doi: bioRxiv preprint first posted online 2! FSM resistance is achieved via increased steady-state levels of MEP pathway and glycolytic 38 intermediates and confirms reduced PFK9 function in the suppressed strains. We identify HAD2 39 as a novel regulator of malaria glycolytic metabolism and drug sensitivity. Our study informs the 40 biological functions of an evolutionarily conserved family of metabolic regulators and reveal a 41 previously undescribed strategy for cellular glycolytic regulation. 42 43

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.