The innate immune response of Drosophila melanogaster is governed by a complex set of signaling pathways that trigger antimicrobial peptide (AMP) production, phagocytosis, melanization, and encapsulation. Although immune responses against both bacteria and fungi have been demonstrated in Drosophila, identification of an antiviral response has yet to be found. To investigate what responses Drosophila mounts against a viral infection, we have developed an in vivo Drosophila X virus (DXV)-based screening system that identifies altered sensitivity to viral infection by using DXV's anoxia-induced death pathology. Using this system to screen flies with mutations in genes with known or suggested immune activity, we identified the Toll pathway as a vital part of the Drosophila antiviral response. Inactivation of this pathway instigated a rapid onset of anoxia induced death in infected flies and increases in viral titers compared to those in WT flies. Although constitutive activation of the pathway resulted in similar rapid onset of anoxia sensitivity, it also resulted in decreased viral titer. Additionally, AMP genes were induced in response to viral infection similar to levels observed during Escherichia coli infection. However, enhanced expression of single AMPs did not alter resistance to viral infection or viral titer levels, suggesting that the main antiviral response is cellular rather than humoral. Our results show that the Toll pathway is required for efficient inhibition of DXV replication in Drosophila. Additionally, our results demonstrate the validity of using a genetic approach to identify genes and pathways used in viral innate immune responses in Drosophila.Drosophila X virus ͉ innate immunity ͉ virus ͉ Dif

Mycobacterium tuberculosis (Mtb) is a persistent intracellular pathogen intrinsically tolerant to most antibiotics. However, the specific factors that mediate this tolerance remain incompletely defined. Here we apply metabolomic profiling to discover a common set of metabolic changes associated with the activities of three clinically used tuberculosis drugs, isoniazid, rifampicin and streptomycin. Despite targeting diverse cellular processes, all three drugs trigger activation of Mtb's isocitrate lyases (ICLs), metabolic enzymes commonly assumed to be involved in replenishing of tricarboxylic acid (TCA) cycle intermediates. We further show that ICL-deficient Mtb strains are significantly more susceptible than wild-type Mtb to all three antibiotics, and that this susceptibility can be chemically rescued when Mtb is co-incubated with an antioxidant. These results identify a previously undescribed role for Mtb's ICLs in antioxidant defense as a mechanism of antibiotic tolerance.

Background Recurrent Clostridioides difficile infection (rCDI) is associated with loss of microbial diversity and microbe-derived secondary bile acids, which inhibit C. difficile germination and growth. SER-109, an investigational microbiome drug of donor-derived, purified spores, reduced recurrence in a dose-ranging, phase (P) 1 study in subjects with multiple rCDIs. Methods In a P2 double-blind trial, subjects with clinical resolution on standard-of-care antibiotics were stratified by age (< or ≥65 years) and randomized 2:1 to single-dose SER-109 or placebo. Subjects were diagnosed at study entry by PCR or toxin testing. Safety, C. difficile–positive diarrhea through week 8, SER-109 engraftment, and bile acid changes were assessed. Results 89 subjects enrolled (67% female; 80.9% diagnosed by PCR). rCDI rates were lower in the SER-109 arm than placebo (44.1% vs 53.3%) but did not meet statistical significance. In a preplanned analysis, rates were reduced among subjects ≥65 years (45.2% vs 80%, respectively; RR, 1.77; 95% CI, 1.11–2.81), while the <65 group showed no benefit. Early engraftment of SER-109 was associated with nonrecurrence (P < .05) and increased secondary bile acid concentrations (P < .0001). Whole-metagenomic sequencing from this study and the P1 study revealed previously unappreciated dose-dependent engraftment kinetics and confirmed an association between early engraftment and nonrecurrence. Engraftment kinetics suggest that P2 dosing was suboptimal. Adverse events were generally mild to moderate in severity. Conclusions Early SER-109 engraftment was associated with reduced CDI recurrence and favorable safety was observed. A higher dose of SER-109 and requirements for toxin testing were implemented in the current P3 trial. Clinical Trials Registration NCT02437487, https://clinicaltrials.gov/ct2/show/NCT02437487?term=SER-109&draw= 2&rank=4.

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-β-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.