The effect of imipenem and diffusible signaling factors on the secretion of outer membrane vesicles and associated Ax21 proteins in Stenotrophomonas maltophilia.

Simon Devos,Bart Devreese,Stephan Stremersch,Gonzalez Van Driessche,Jolien Vitse,Riet De Rycke,Koen Raemdonck,Wouter Van Putte,Laurence Van Oudenhove

doi:10.3389/fmicb.2015.00298

Abstract

Outer membrane vesicles (OMVs) are small nanoscale structures that are secreted by bacteria and that can carry nucleic acids, proteins, and small metabolites. They can mediate intracellular communication and play a role in virulence. In this study, we show that treatment with the β-lactam antibiotic imipenem leads to a dramatic increase in the secretion of outer membrane vesicles in the nosocomial pathogen Stenotrophomonas maltophilia. Proteomic analysis of their protein content demonstrated that the OMVs contain the chromosomal encoded L1 metallo-β-lactamase and L2 serine-β-lactamase. Moreover, the secreted OMVs contain large amounts of two Ax21 homologs, i.e., outer membrane proteins known to be involved in virulence and biofilm formation. We show that OMV secretion and the levels of Ax21 in the OMVs are dependent on the quorum sensing diffusible signal system (DSF). More specific, we demonstrate that the S. maltophilia DSF cis-Δ2-11-methyl-dodecenoic acid and, to a lesser extent, the Burkholderia cenocepacia DSF cis-Δ2-dodecenoic acid, stimulate OMV secretion. By a targeted proteomic analysis, we confirmed that DSF-induced OMVs contain large amounts of the Ax21 homologs, but not the β-lactamases. This work illustrates that both quorum sensing and disturbance of the peptidoglycan biosynthesis provoke the release of OMVs and that OMV content is context dependent.

Highlights

Stenotrophomonas maltophilia is the most frequently isolated unusual non-fermenting Gramnegative bacterium in hospitalized patients (Fihman et al, 2012)
Analyzing the protein sequences of the S. maltophilia Ax21 homologs Smlt0387 and Smlt0184 with Pfam (Finn et al, 2014) showed that they belong to the outer membrane protein β-barrel domain family, with e-values 1.5 e-09 and 5.7 e-11, respectively
The genome sequence of the pathogenic Stenotrophomonas maltophilia K279a strain revealed an organism that is well adjusted for living in an environment with antibiotics (Crossman et al, 2008)

Summary

Introduction

Stenotrophomonas maltophilia is the most frequently isolated unusual non-fermenting Gramnegative bacterium in hospitalized patients (Fihman et al, 2012) It is associated with an expanding range of clinical syndromes like bacteraemia, pneumonia and soft-tissue infections. Chromosomal encoded β-lactamases can be secreted in outer membrane vesicles (OMVs), enabling extracellular β-lactam degradation (Ciofu et al, 2000). Such OMVs are secreted by all Gram-negative bacteria and have different biological functions including protection of the secreted cargo, long-distance transport of toxins and virulence factors, cell-cell communication, pathogenesis, antibiotic resistance and aiding in biofilm formation (Deatherage et al, 2009; Bonnington and Kuehn, 2013; Tiwari, 2014). Xanthomonas Ax21, recently renamed to omp1X, is an outer membrane β-barrel protein that is secreted by the general secretion (Sec) system, and it is associated with outer membrane vesicles (OMVs) as well (Bahar et al, 2014)

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