Abstract
Among the different mechanisms used by bacteria to resist antibiotics, active efflux plays a major role. In Gram-negative bacteria, active efflux is carried out by tripartite efflux pumps that form a macromolecular assembly spanning both membranes of the cellular wall. At the outer membrane level, a well-conserved outer membrane factor (OMF) protein acts as an exit duct, but its sequence varies greatly among different species. The OMFs share a similar tri-dimensional structure that includes a beta-barrel pore domain that stabilizes the channel within the membrane. In addition, OMFs are often subjected to different N-terminal post-translational modifications (PTMs), such as an acylation with a lipid. The role of additional N-terminal anchors is all the more intriguing since it is not always required among the OMFs family. Understanding this optional PTM could open new research lines in the field of antibiotics resistance. In Escherichia coli, it has been shown that CusC is modified with a tri-acylated lipid, whereas TolC does not show any modification. In the case of OprM from Pseudomonas aeruginosa, the N-terminal modification remains a matter of debate, therefore, we used several approaches to investigate this issue. As definitive evidence, we present a new X-ray structure at 3.8 Å resolution that was solved in a new space group, making it possible to model the N-terminal residue as a palmitoylated cysteine.
Highlights
After several decades of continuous antibiotic therapy success, we are facing the appearance of multi-drug resistant strains and the near absence of new antibiotic family development for more than 10 years (Fischbach and Walsh, 2009; Hede, 2014)
Chemical Analysis of the Lipoyl Position Among the different posttranslational modification (PTM) that can occur on an N-terminal cysteine (Chalker et al, 2009) N- or S-palmitoylation or acetylation are readily observed (Resh, 1999; Tooley and Schaner Tooley, 2014)
As these different modifications are regulated by specific transferases, it is important to characterize the exact nature of OprM PTM
Summary
After several decades of continuous antibiotic therapy success, we are facing the appearance of multi-drug resistant strains and the near absence of new antibiotic family development for more than 10 years (Fischbach and Walsh, 2009; Hede, 2014) These facts highlight the need for new anti-infection strategies (Walsh, 2003; Olivares et al, 2013), a promising compound isolated from natural soil bacteria that is able to kill Gram-positive pathogens, was recently reported (Ling et al, 2015). Among the most virulent nosocomial pathogens are Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococci, and Acinetobacter baumannii (Poole, 2004; Lister et al, 2009; Bereket et al, 2012; Bayram et al, 2013) These strains have developed several resistance strategies including active efflux pumps (Cattoir, 2004; Li and Nikaido, 2009; Nikaido, 2009; Nikaido and Pages, 2012). This study will focus on the versatile membrane channel OprM, which has the ability to work with at least four different pumps, including OprM-MexAB, OprMMexXY (Aires et al, 1999; Morita et al, 2012), OprM-MexJK (Chuanchuen et al, 2002), and OprM-MexMN (Mima et al, 2005)
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