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Abstract
Exposure to brake abrasion dust (BAD) reduces the phagocytic capacity of macrophages, possibly contributing to increased risk of airway infections in polluted regions. The mechanisms that underlie this toxicity remain to be characterised and, considering that pre-exposure of respiratory bacteria to alternative traffic-related particulates enhances their dissemination into the lungs of mice, may include changes in the virulence of pathogens themselves. Dual proteomic screening (TMT-labelled UPLC-MS) was performed for RAW 264.7 monocyte-derived macrophages and S.aureus (USA300) following 24h exposures to BAD (0-16 ug/ml). In S.aureus, expression of agr-regulated, anti-leukocytic virulence factors (lipases, staphopains, hemolysins) increased in a dose-dependent manner as well as proteins required for epithelial invasion (serine proteases) and antibiotic resistance. RAW 264.7 exhibited a predominantly adaptive response following exposures, enhancing expression of proteins required for metal homeostasis, NFkB-mediated inflammation and endosomal maturation. However, disrupted expression of proteins required for electron transport chain activity and Fe-S cluster synthesis accompanied significant dose-dependent reductions in ATP production (60-82%, p< 0.001) while transition electron microscopy (TEM) detected disrupted mitochondrial membrane morphology. TEM also identified reductions in pseudopodia length in exposed macrophages (54% shorter than control, p< 0.05), highlighting a mechanism by which BAD exposure may decrease the incidence of host-pathogen interactions. These data highlight dual mechanisms by which BAD emissions could impair bacterial phagocytosis and enhance susceptibility to airway infections. Through avoidance and suppression of host anti-microbial mechanisms by the bacteria and, as actin polymerisation depends on ATP availability, loss of mitochondrial function and downstream pseudopodia projection in macrophages.

M.Z. and J.A.G. were supported by funding from the European Union's Horizon 2020 research and innovation programme under grant agreement 634588. K.J.W. was supported by a Sir Henry Dale Fellowship funded by the Wellcome Trust and the Royal Society (098375/Z/12/Z). G.P.R. was funded by a CAPES Science Without Borders scholarship (BEX 2445/13-1). M.T.G.H. is funded by the Chief Scientist Office through the Scottish Infection Research Network, a part of the SHAIPI consortium (grant SIRN/10). ; Pathogens are exposed to toxic levels of copper during infection, and copper tolerance may be a general virulence mechanism used by bacteria to resist host defenses. In support of this, inactivation of copper exporter genes has been found to reduce the virulence of bacterial pathogens in vivo. Here we investigate the role of copper hypertolerance in methicillin-resistant Staphylococcus aureus (MRSA). We show that a copper hypertolerance operon (copB-mco), carried on a mobile genetic element (MGE), is prevalent in a collection of invasive S. aureus strains and more widely among clonal complex 22, 30, and 398 strains. The copB and mco genes encode a copper efflux pump and a multicopper oxidase, respectively. Isogenic mutants lacking copB or mco had impaired growth in subinhibitory concentrations of copper. Transfer of a copB-mco-carrying plasmid to a naive clinical isolate resulted in a gain of copper hypertolerance and enhanced bacterial survival inside primed macrophages. The copB and mco genes were upregulated within infected macrophages, and their expression was dependent on the copper-sensitive operon repressor CsoR. Isogenic copB and mco mutants were impaired in their ability to persist intracellularly in macrophages and were less resistant to phagocytic killing in human blood than the parent strain. The importance of copper-regulated genes in resistance to phagocytic killing was further elaborated using mutants expressing a copper-insensitive variant of CsoR. Our findings suggest that the gain of mobile genetic elements carrying copper hypertolerance genes contributes to the evolution of virulent strains of S. aureus that are better equipped to resist killing by host immune cells. ; Publisher PDF ; Peer reviewed