RND-4 efflux transporter gene deletion in Burkholderia cenocepacia J2315: a proteomic analysis

Abstract

Background: The Burkholderia cenocepacia J2315 is Gram-negative bacterium that is a pathogen for cystic fibrosis (CF) patients. It displays a high-level of resistance to most antimicrobial drugs. In Gram-negative bacteria, the ResistanceNodulation-Cell Division (RND) transporter family has a poorly understood role in multidrug resistance. In a previous publication we analysed the RND-4 and RND-9 transporters by microarray analysis. The obtained results suggested that only RND-4 contributes to the antibiotic resistance. The aim of this study was to investigate the role of this efflux transporter from a proteomic point of view.

Efflux pump-mediated drug resistance in Burkholderia

“…A proteomic analysis of the effects of RND-4 gene deletion revealed about 70 differentially expressed proteins, most of which were associated with cellular functions other than drug resistance. This suggests that RND-4 plays a more general role in B. cenocepacia ’s biology ( Gamberi et al, 2013 ). Aside from the key role that efflux, especially RND-mediated efflux, plays in adaptation to antibiotic exposure ( Bazzini et al, 2011b ; Sass et al, 2011 ; Tseng et al, 2014 ), survival of Burkholderia species in various niche environments and accompanying conditions, e.g., the cystic fibrosis airways ( Mira et al, 2011 ), marine habitats ( Maravic et al, 2012 ), oxygen levels ( Hemsley et al, 2014 ), exposure to noxious chemicals ( Rushton et al, 2013 ), and other ecological niches ( Liu et al, 2015 ), involves to various degrees changes in efflux pump expression.…”

Subfunctionalization influences the expansion of bacterial multidrug antibiotic resistance

“…The search for RND 2 and RND 4 operons in 797 Burkholderia genomes revealed that the RND 4 operon is highly conserved, being present in almost all the genomes examined. The high conservation of RND 4 is consistent with the critical role of this operon in multiple antibiotic resistance, virulence, and other cellular processes [ 23 – 26 , 28 ]. Further, a gene encoding a TetR family transcriptional regulator located upstream of RND 4 is highly conserved, suggesting that regulatory elements controlling the RND 4 operon expression are also common to all Burkholderia species.…”

“…This pump is also associated with the modulation of some virulence factors (motility, biofilm formation, chemotaxis and quorum sensing ) [ 24 ]. A proteomic analysis comparing the parental strain and the deletion mutant suggests a more general role for RND 4 in the physiology of B. cenocepacia cells [ 28 ]. Conversely, the Burkholderia RND 2 protein is not expressed during growth on LB medium, and its role in antibiotic resistance can only be revealed when overexpressed in E. coli [ 29 ] .…”

Cellular response to empty and palladium‐conjugated amino‐polystyrene nanospheres uptake: A proteomic study

“…IEF was performed on IPG strips (BioRad) having pH 3–10 nonlinear and 18 cm length by using an Ettan IPGphor™ system with Ettan IPGphor Manifold™ (GE Healthcare). Briefly, IPG strips were rehydrated overnight with 350 μL of rehydration solution (8 M urea, 2% w/v CHAPS, 0.5% w/v DTE) supplemented with 0.5% v/v carrier ampholyte (Biorad) and a trace of bromophenol blue by using IPGbox . Then the strips were transferred to Ettan IPGphor Manifold, covered with inert oil.…”

“…A total of 600 μg of protein sample was loaded into the sample cups placed near the anode. The strips were focused at 16°C according to the following electrical conditions: 200 V for 1 h, 300 V for 1 h, from 300 to 3500 V in 30 min, 3500 V for 4 h, 5000 for 2 h, from 5000 to 8000 V in 30 min, and 8000 V until a total of 90 000 V/h was reached, with a limiting current of 50 μA/strip . After IEF, the equilibration steps were performed in 6 M urea, 2% w/v SDS, 2% w/v DTE, 30% v/v glycerol, and 0.05 M Tris‐HCL pH 6.8 for 10 min, then in the same buffer containing 2.5% w/v iodoacetamide (IA) in place of DTE, for other 10 min .…”