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Clostridioides difficile infection (CDI) represents a significant burden on the health care system, one that is exacerbated by the emergence of binary toxin (CDT)-producing hypervirulent C. difficile strains. Previous work from our laboratory has shown that Toll-like receptor 2 (TLR2) recognizes CDT to induce inflammation. Here we explore the interactions of CDT with TLR2 and the impact on host immunity during CDI. We found that the TLR2/6 heterodimer, not TLR2/1, is responsible for CDT recognition, and that gene pathways including nuclear factor-κB and MAPK downstream of TLR2/6 are upregulated in mice with intact TLR2/6 signaling during CDI.

Clostridioides difficile is the leading cause of hospital-acquired gastrointestinal infection, in part due to the existence of binary toxin (CDT)-expressing hypervirulent strains. We have previously shown that CDT interacts with the TLR2/6 heterodimer to induce inflammation, and in this study we further explore this interaction as well as the contribution of the separate components of CDT, CDTa and CDTb. We found that the binding component, CDTb, is capable of inducing inflammation. Additionally, complementation of a CDT-deficient C. difficile strain with CDTb alone restored virulence in a hamster model of C. difficile infection. Overall, this study demonstrates that the binding component of C. difficile binary toxin contributes to virulence during infection.

Clostridioides difficile infection (CDI) represents a significant burden on the health care system, one that is exacerbated by the emergence of binary toxin (CDT)-producing hypervirulent C. difficile strains. Previous work from our lab has shown that TLR2 recognizes CDT to induce inflammation. Here we explore the interactions of CDT with TLR2 and the impact on host immunity during CDI. We found that the TLR2/6 heterodimer, not TLR2/1, is responsible for CDT recognition, and that gene pathways including NF-κB and MAPK downstream of TLR2/6 are upregulated in mice with intact TLR2/6 signaling during CDI.

The gut microbiome provides resistance to infection. However, the mechanisms for this are poorly understood. Colonization with the intestinal bacteriumClostridium scindensprovided protection from the parasiteEntamoeba histolyticavia innate immunity. Introduction ofC. scindensinto the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte-progenitors (GMPs) and provided neutrophil-mediated protection against subsequent challenge withE. histolytica. Adoptive transfer of bone-marrow fromC. scindenscolonized-mice into naïve-mice protected against ameba infection and increased intestinal neutrophils. Because of the known ability ofC. scindensto metabolize the bile salt cholate, we measured deoxycholate and discovered that it was increased in the sera ofC. scindenscolonized mice, as well as in children protected from amebiasis. Administration of deoxycholate alone (in the absence ofC. scindens) increased the epigenetic mediator JMJD3 and GMPs and provided protection from amebiasis. In conclusion the microbiota was shown to communicate to the bone marrow via microbially-metabolized bile salts to train innate immune memory to provide antigen-nonspecific protection from subsequent infection. This represents a novel mechanism by which the microbiome protects from disease.One Sentence SummaryIntroduction of the human commensal bacteriaClostridium scindensinto the intestinal microbiota epigenetically alters bone marrow and protects from future parasite infection.

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 Letter, we determine that the active stereoisomer of MMV008138 is 4a , which is (1 R ,3 S )-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 substituent 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.

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 Pf IspD recombinant enzyme. Here, we report Pf IspD inhibition data for fosmidomycin (FOS) and 19 previously disclosed analogs and report parasite growth and Pf IspD 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.