Bacterial membrane vesicle research has so far focused mainly on Gram-negative bacteria. Only recently have Gram-positive bacteria been demonstrated to produce and release extracellular membrane vesicles (MVs) that contribute to bacterial virulence. Although treatment of bacteria with antibiotics is a well-established trigger of bacterial MV formation, the underlying mechanisms are poorly understood. In this study, we show that antibiotics can induce MVs through different routes in the important human pathogen Staphylococcus aureus. DNA-damaging agents and antibiotics inducing the SOS response triggered vesicle formation in lysogenic strains of S. aureus but not in their phage-devoid counterparts. The β-lactam antibiotics flucloxacillin and ceftaroline increased vesicle formation in a prophage-independent manner by weakening the peptidoglycan layer. We present evidence that the amount of DNA associated with MVs formed by phage lysis is greater than that for MVs formed by β-lactam antibiotic-induced blebbing. The purified MVs derived from S. aureus protected the bacteria from challenge with daptomycin, a membrane-targeting antibiotic, both in vitro and ex vivo in whole blood. In addition, the MVs protected S. aureus from killing in whole blood, indicating that antibiotic-induced MVs function as a decoy and thereby contribute to the survival of the bacterium.
Staphylococcus aureus is the leading cause of infective endocarditis (IE). While the role of S. aureus cell-wall associated protein clumping factor A (ClfA) in promoting IE has been already demonstrated, that of the secreted plasma-clotting factors staphylocoagulase (Coa) and von Willebrand factor-binding protein (vWbp) has not yet been elucidated. We investigated the role of Coa and vWbp in IE initiation in rats with catheter-induced aortic vegetations, using Lactococcus lactis expressing coa, vWbp, clfA or vWbp/clfA, and S. aureus Newman Δcoa, ΔvWbp, ΔclfA or Δcoa/ ΔvWbp/ΔclfA mutants. vWbp-expression increased L. lactis valve infection compared to parent and coa-expressing strains (incidence: 62%, versus 0% and 13%, respectively; P < 0.01). Likewise, expression of clfA increased L. lactis infectivity (incidence: 80%), which was not further affected by co-expression of vWbp. In symmetry, deletion of the coa or vWbp genes in S. aureus did not decrease infectivity (incidence: 68 and 64%, respectively) whereas deletion of clfA did decrease valve infection (incidence: 45%; P = 0.03 versus parent), which was not further affected by the triple deletion Δcoa/ΔvWbp/ΔclfA (incidence: 36%; P > 0.05 versus ΔclfA mutant). Coa does not support the initial colonization of IE (in L. lactis) without other key virulence factors and vWbp contributes to initiation of IE (in L. lactis) but is marginal in the present of ClfA.
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Microbiomes are an essential contributor to the metabolic activity in the human gastrointestinal tract. The fermentation of otherwise indigestible nutritional components like dietary fibers relies on a complex interplay of metabolic pathways that are distributed across the individual bacteria. Yet, which of the bacteria are responsible for which parts of the distributed metabolism and how they should be grouped together is insufficiently understood. Here, we present the NicheMap(TM), an approach to map the different bacterial taxa that make up the gut microbiome onto the different functional niches of microbial carbohydrate fermentation. Our approach uses in vitro measurements of bacterial growth and metabolic activity to identify which bacterial taxa are responsible for which metabolic function in the relevant complex context of whole human fecal microbiomes. We identified 'characteristic taxa' selected for by a panel growth substrates representative of dietary components that are resistant to digestion by host enzymes. These characteristic taxa offer predictions of which bacteria are stimulated by the various components of human diet. We validated these predictions using microbiome data from a human nutritional supplementation study. We suggest a template of how bacterial taxonomic diversity is organized along the trophic cascade of intestinal carbohydrate fermentation. We anticipate that our results and our approach will provide a key contribution towards building a structure-function map for gut microbiomes. Having such a map on hand is an important step in moving the microbiome from a descriptive science to an interventional one.
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