Abstract
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 (O2) to hydrogen peroxide (H2O2). In this study we identify the site of PGRP-induced generation of H2O2 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 H2O2 production and the highest resistance to PGRP-induced killing, and formate enhanced PGRP-induced killing and H2O2 production in an FDH-dependent manner. Mutants in ubiquinone synthesis (but not menaquinone and demethylmenaquinone) and cytochrome bd-I (but not cytochromes bo3 and bd-II) also had completely abolished PGRP-induced H2O2 production and high resistance to PGRP-induced killing. Because electrons in the respiratory chain flow from dehydrogenases’ substrates through quinones and then cytochromes to O2, these results imply that the site of PGRP-induced incomplete reduction of O2 to H2O2 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.
Highlights
Peptidoglycan Recognition Proteins (PGRPs) are evolutionarily conserved and function in antibacterial innate immunity[1,2]
Our results further show that ubiquinone and cytochrome bd-I are required for PGRP-induced killing, and that the site of PGRP-induced H2O2 production in the respiratory chain is downstream from formate dehydrogenase (FDH)-O, NDH-1, NDH-2, and ubiquinone at the level of cytochrome bd-I
We used human recombinant PGLYRP4 as a representative bactericidal PGRP, as we have previously shown that all human bactericidal PGRPs had similar activity and mechanism of bacterial killing[4,6,10,11]
Summary
Peptidoglycan Recognition Proteins (PGRPs) are evolutionarily conserved and function in antibacterial innate immunity[1,2]. This conclusion was based on: (i) increased resistance to PGRP-induced killing and inability of PGRP to induce increased production of H2O2 in several deletion mutants for the components of this pathway, including Δnuo (NDH-1 deficient), Δndh (NDH-2 deficient), several TCA-cycle enzymes (ΔsucB, ΔsucD, Δicd, ΔsdhD, and ΔlpdA), and Δcrp and ΔcyaA (deficient in the cAMP-Crp regulator of TCA cycle and central carbon catabolism); (ii) correlated PGRP-induced increase in the expression of cAMP-Crp-controlled genes for central carbon catabolism and respiratory oxidoreductases; and (iii) correlated PGRP-induced increases in NADH, phosphoenolpyruvate, and cAMP in wild-type cells but not in the above PGRP-resistant mutants[12] These results indicated that the PGRP-induced block in the respiratory chain and the site of generation of H2O2 occurred at or down-stream from NDH-1 and NDH-2. The exact location of this block and the site of production of H2O2 remained unknown
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