Summary Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram-positive and Gram-negative bacteria through simultaneous induction of oxidative, thiol, and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP-induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP-Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidoreductases to oxygen. CpxRA two-component system was a negative regulator of PGRP-induced oxidative stress. By contrast, PGRP-induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn2+ through influx of extracellular Zn2+) were mostly independent of oxidative stress. Thus, manipulating pathways that induce oxidative, thiol, and metal stress in bacteria could be a useful strategy to design new approaches to antibacterial therapy.

Mammalian peptidoglycan Recognition proteins (pGRps) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. pGRps induce oxidative stress in bacteria through a block in the respiratory chain, which results in decreased respiration and incomplete reduction of oxygen (o 2) to hydrogen peroxide (H 2 o 2). in this study we identify the site of pGRp-induced generation of H 2 o 2 in Escherichia coli. tn-seq screening of E. coli Tn10 insertion library revealed that mutants in formate dehydrogenase (fDH) genes had the highest survival following pGRp treatment. Mutants lacking functional fDH-o had abolished pGRp-induced H 2 o 2 production and the highest resistance to pGRpinduced killing, and formate enhanced pGRp-induced killing and H 2 o 2 production in an fDH-dependent manner. Mutants in ubiquinone synthesis (but not menaquinone and demethylmenaquinone) and cytochrome bd-i (but not cytochromes bo 3 and bd-ii) also had completely abolished pGRp-induced H 2 o 2 production and high resistance to PGRP-induced killing. Because electrons in the respiratory chain flow from dehydrogenases' substrates through quinones and then cytochromes to o 2 , these results imply that the site of pGRp-induced incomplete reduction of o 2 to H 2 o 2 is downstream from dehydrogenases and ubiquinone at the level of cytochrome bd-i, which results in oxidative stress. these results reveal several essential steps in pGRp-induced bacterial killing. Peptidoglycan Recognition Proteins (PGRPs) are evolutionarily conserved and function in antibacterial innate immunity 1,2. Mammals have four PGRPs coded by PGLYRP1-4 genes. PGLYRP1, PGLYRP3, and PGLYRP4 are directly bactericidal for both Gram-positive and Gram-negative bacteria 3-6 , whereas PGLYRP2 is an enzyme, peptidoglycan amidohydrolase 7,8. All PGRPs have one or two conserved PGRP domains, which bind muramyl-peptide fragments of bacterial peptidoglycan 1,2. Mammalian PGRPs also bind bacterial lipopolysaccharide (LPS) with a binding site located outside the peptidoglycan-binding groove 5,9. Bacterial killing by PGRPs requires binding of PGRP to peptidoglycan in Gram-positive bacteria or to the outer membrane in Gram-negative bacteria 10. However, PGRPs do not enter the cytoplasm and exert bacterial killing from this extracellular site by simultaneously inducing three severe stress responses in bacteria: oxidative stress, thiol stress, and metal stress 10,11. Simultaneous induction of all three stress responses is required for efficient PGRP-induced bacterial killing, because: (i) each stress response is required for PGRP-induced killing but individually each stress response is only bacteriostatic, but not bactericidal; and (ii) bacterial killing can be recapitulated by the simultaneous treatment of bacteria with paraquat (which induces H 2 O 2 production), diamide (which depletes thiols), and metals (which increase intracellular metal concentrations) 11 .

LL-37, the only member of the mammalian cathelicidin in humans, plays an essential role in innate immunity by killing pathogens and regulating the inflammatory response. However, at an inflammatory focus, arginine residues in LL-37 can be converted to citrulline via a reaction catalyzed by peptidyl-arginine deiminases (PAD2 and PAD4), which are expressed in neutrophils and are highly active during the formation of neutrophil extracellular traps (NETs). Citrullination impairs the bactericidal activity of LL-37 and abrogates its immunomodulatory functions. Therefore, we hypothesized that citrullination-resistant LL-37 variants would retain the functionality of the native peptide in the presence of PADs. To test this hypothesis, we synthetized LL-37 in which arginine residues were substituted by homoarginine (hArg-LL-37). Bactericidal activity of hArg-LL-37 was comparable with that of native LL-37, but neither treatment with PAD4 nor exposure to NETs affected the antibacterial and immunomodulatory activities of hArg-LL-37. Importantly, the susceptibilities of LL-37 and hArg-LL-37 to degradation by proteases did not significantly differ. Collectively, we demonstrated that citrullination-resistant hArg-LL-37 is an attractive lead compound for the generation of new agents to treat bacterial infections and other inflammatory diseases associated with enhanced PAD activity. Moreover, our results provide a proof-of-concept for synthesis of therapeutic peptides using homoarginine.

In this paper, we study AI approaches to successfully play a 2-4 players, full information, Bomberman variant published on the CodinGame platform. We compare the behavior of three search algorithms: Monte Carlo Tree Search, Rolling Horizon Evolution, and Beam Search. We present various enhancements leading to improve the agents' strength that concern search, opponent prediction, game state evaluation, and game engine encoding. Our top agent variant is based on a Beam Search with low-level bit-based state representation and evaluation function heavy relying on pruning unpromising states based on simulation-based estimation of survival. It reached the top one position among the 2,300 AI agents submitted on the CodinGame arena.

Mammalian peptidoglycan recognition proteins (PGRPs or PGLYRPs) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. Tn-seq screening of Bacillus subtilis transposon insertion library revealed that mutants in the shikimate pathway of chorismate synthesis had high survival following PGLYRP4 treatment. Deletion mutants for these genes had decreased amounts of menaquinone (MK), increased resistance to killing, and attenuated depletion of thiols following PGLYRP4 treatment. These effects were reversed by MK or reproduced by inhibiting MK synthesis. Deletion of cytochrome aa3-600 or NADH dehydrogenase (NDH) genes also increased B. subtilis resistance to PGLYRP4-induced killing and attenuated thiol depletion. PGLYRP4 treatment also inhibited B. subtilis respiration. Similarly in Escherichia coli, deletion of ubiquinone (UQ) synthesis, formate dehydrogenases (FDH), NDH-1, or cytochrome bd-I genes attenuated PGLYRP4-induced thiol depletion. PGLYRP4-induced low level of cytoplasmic membrane depolarization in B. subtilis and E. coli was likely not responsible for thiol depletion. Thus, our results show that the respiratory electron transport chain components, cytochrome aa3-600, MK, and NDH in B. subtilis, and cytochrome bd-I, UQ, FDH-O, and NDH-1 in E. coli, are required for both PGLYRP4-induced killing and thiol depletion and indicate conservation of the PGLYRP4-induced thiol depletion and killing mechanisms in Gram-positive and Gram-negative bacteria.