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Staphylococcus aureus is a leading cause of chronic and recurrent infections of the skin, bones, joints and bloodstream. During infection, S. aureus faces the twin threats of the host immune system and therapeutic antibiotics. Since both host immune cells and antibiotics generate reactive oxygen species, we hypothesised that S. aureus may employ a common mechanism to repair damage caused by either threat. We found that staphylococcal DNA is damaged during exposure to both human neutrophils and most bactericidal antibiotics. To understand the nature of this damage and how S. aureus repairs it, we screened a panel of transposon mutants defective for various DNA repair processes. This revealed that loss of the rexBA operon significantly reduced staphylococcal survival in human blood, during incubation with purified neutrophils, in the peritoneal cavity of mice and during exposure to a large panel of bactericidal antibiotics. We then used biochemical assays to demonstrate that RexAB is a member of the AddAB family of ATP-dependent helicase/nucleases that are required for the repair of DNA double strand breaks. Finally, we found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted survival of these pathogens in human blood, suggesting that DNA repair constitutes a broadly conserved defence against neutrophils. Together, these data demonstrate that DNA is a target of host immune cells and several antibiotics, leading to double-strand breaks, and that repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.

Local structural frustration, the existence of mutually exclusive competing interactions, may explain why some proteins are dynamic when others are rigid. More specifically, frustration is thought to play a key role in biomolecular recognition while it can also underpin the flexibility of binding sites. Here we show how a seemingly small chemical modification, the oxidation of two cysteine thiols to form a disulfide bond, during the biological function of the N-terminal domain of the bacterial oxidoreductase DsbD (nDsbD), introduces frustration. In oxidised nDsbD, local frustration disrupts the packing of the protective cap loop region against the active site of the protein allowing loop opening By contrast, in reduced nDsbD, lacking a disulfide bond, the cap loop is rigid, always shielding the active-site cysteines and protecting them from the otherwise oxidising environment of the bacterial periplasm. Our results point towards an intricate coupling between the dynamics of the active-site cysteines and those of the cap loop, which shapes the protein-protein association reactions of nDsbD resulting in optimised protein function.

Background Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming. Objectives To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances. Methods We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in Results Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains. Conclusions The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.

Nitrate and nitrite transport across biological membranes is often facilitated by protein transporters that are members of the major facilitator superfamily. Paracoccus denitrificans contains an unusual arrangement whereby two of these transporters, NarK1 and NarK2, are fused into a single protein, NarK, which delivers nitrate to the respiratory nitrate reductase and transfers the product, nitrite, to the periplasm. Our complementation studies, using a mutant lacking the nitrate/proton symporter NasA from the assimilatory nitrate reductase pathway, support that NarK1 functions as a nitrate/proton symporter while NarK2 is a nitrate/nitrite antiporter. Through the same experimental system, we find that Escherichia coli NarK and NarU can complement deletions in both narKand nasAin P. denitrificans, suggesting that, while these proteins are most likely nitrate/nitrite antiporters, they can also act in the net uptake of nitrate. Finally, we argue that primary sequence analysis and structural modelling do not readily explain why NasA, NarK1 and NarK2, as well as othertransporters from this protein family, have such different functions, ranging fromnet nitrate uptake to nitrate/nitrite exchange.

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion-site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing-based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria., L.M.N. was supported by Medical Research Council (MRC) Grant MR/N023250/1 and a Marie Curie Fellowship (PIIF-GA-2013-625318). A.F. was supported by MRC Grants MR/K001930/1 and MR/N023250/1 and Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/N002539/1. R.C.D.F. and D.A.I.M. were supported by the MRC Career Development Award MR/M009505/1. D.A.-J. acknowledges support by the MINECO Contract CTQ2016-76941-R, Fundación Biofísica Bizkaia, the Basque Excellence Research Centre (BERC) program and IT709-13 of the Basque Government, and Fundación BBVA. M.A.S.-P. was supported by the MINECO under the “Juan de la Cierva Postdoctoral program” (position FJCI-2015-25725).

Extensive use of antibiotics in medicine and agriculture has led to increasing emergence of antimicrobial resistance in bacterial populations. Dwindling resources in the discovery of novel active compound leads and the increasing demands for safety and efficacy of new drugs mean that we are now faced with treatment failures due to multi-drug resistant pathogens. In the quest for new targets that will enable us to counter antibiotic resistance, it is often ignored that many resistance mechanisms precede the clinical use of antibiotics. Instead, the ability to adapt, survive and bypass the toxicity of many chemical compounds is wired within the bacterial genome. Continuous inter-strain and inter-species competition have given microorganisms tools to thrive under conditions of chemical warfare. Recognising this is important when characterising mechanisms underpinning bacterial antimicrobial resistance, as it can lead to novel strategies that can help us bypass it. The work described here explores the connection between the disulfide bond formation system, a key oxidative protein folding pathway in the cell envelope of Gram-negative bacteria, and two widespread antimicrobial resistance mechanisms, b-lactamase catalysed hydrolysis of b-lactam antibiotics and efflux-mediated drug expulsion. It is demonstrated that oxidative-protein-folding-mediated proteostasis is crucial for both resistance mechanisms, and its inhibition can sensitise multidrug-resistant pathogens to existing antibiotics. Preliminary results from an experimental evolution approach, set the scene for future exploration of the importance of disulfide linkages for the capacity of b-lactamase enzymes to evolve under selective pressure. Together, these findings aim to address the mechanistic basis of a new avenue for antibiotic adjuvant therapy, whereby targeting a non-essential process would allow us to potentiate existing antibiotics towards previously resistant bacterial strains. With novel essential targets against bacteria being scarce, adjuvant approaches like this one could prolong the use and efficacy of existing drugs against some of the most resistant Gram-negative pathogens. Open Access