Clostridium difficile ( C. difficile ) incidence has tripled over the past 15 years and is attributed to the emergence of hypervirulent strains. While it is clear that C. difficile toxins cause damaging colonic inflammation, the immune mechanisms protecting from tissue damage require further investigation. Through a transcriptome analysis, we identify IL-33 as an immune target upregulated in response to hypervirulent C. difficile . We demonstrate that IL-33 prevents C. difficile -associated mortality and epithelial disruption independently of bacterial burden or toxin expression. IL-33 drives colonic group 2 innate lymphoid cell (ILC2) activation during infection and IL-33 activated ILC2s are sufficient to prevent disease. Furthermore, intestinal IL-33 expression is regulated by the microbiota as fecal microbiota transplantation (FMT) rescues antibiotic-associated depletion of IL-33. Lastly, dysregulated IL-33 signaling via the decoy receptor, sST2, predicts C. difficile -associated mortality in human patients. Thus, IL-33 signaling to ILC2s is an important mechanism of defense from C. difficile colitis.

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Malaria continues to be one of the deadliest diseases worldwide, and the emergence of drug resistance parasites is a constant threat. Plasmodium parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are essential for parasite growth. Previously, we and others identified that the Malaria Box compound MMV008138 targets the apicoplast and that parasite growth inhibition by this compound can be reversed by supplementation of IPP. Further work has revealed that MMV008138 targets the enzyme 2- C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD) in the MEP pathway, which converts MEP and cytidine triphosphate (CTP) to cytidinediphosphate methylerythritol (CDP-ME) and pyrophosphate. In this work, we sought to gain insight into the structure-activity relationships by probing the ability of MMV008138 analogs to inhibit PfIspD recombinant enzyme. Here, we report PfIspD inhibition data for fosmidomycin (FOS) and 19 previously disclosed analogs and report parasite growth and PfIspD inhibition data for 27 new analogs of MMV008138. In addition, we show that MMV008138 does not target the recently characterized human IspD, reinforcing MMV008138 as a prototype of a new class of species-selective IspD-targeting antimalarial agents.

Compounds that target isoprenoid biosynthesis in Plasmodium falciparum could be a welcome addition to malaria chemotherapy, since the methylerythritol phosphate (MEP) pathway used by the parasite is not present in humans. We previously reported that MMV008138 targets the apicoplast of P. falciparum and that its target in the MEP pathway differs from that of Fosmidomycin. In this article, we determine that the active stereoisomer of MMV008138 is 4a, which is (1R,3S)-configured. 2′,4′-Disubstitution of the D ring was also found to be crucial for inhibition of the parasite growth. Limited variation of the C3-carboxylic acid substitutent was carried out, and methylamide derivative 8a was found to be more potent than 4a; other amides, acylhydrazines, and esters were less potent. Finally, lead compounds 4a, 4e, 4f, 4h, 8a, and 8e did not inhibit growth of Escherichia coli, suggesting that protozoan-selective inhibition of the MEP pathway of P. falciparum can be achieved.

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C . scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E . histolytica . Introduction of C . scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C . scindens –colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E . histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C . scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C . scindens –colonized specific pathogen–free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C . scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E . histolytica .