Critical Role of 3'-Downstream Region of pmrB in Polymyxin Resistance in Escherichia coli BL21(DE3).

Fuzhou Xu,Atsushi Hinenoya,Ziqiang Guan,Ximin Zeng,Xing-Ping Li,Jun Lin

doi:10.3390/microorganisms9030655

Abstract

Polymyxins, such as colistin and polymyxin B, are the drugs used as a last resort to treat multidrug-resistant Gram-negative bacterial infections in humans. Increasing colistin resistance has posed a serious threat to human health, warranting in-depth mechanistic research. In this study, using a functional cloning approach, we examined the molecular basis of colistin resistance in Escherichia coli BL21(DE3). Five transformants with inserts ranging from 3.8 to 10.7 kb displayed significantly increased colistin resistance, three of which containing pmrB locus and two containing pmrD locus. Stepwise subcloning indicated that both the pmrB with a single G361A mutation and at least a 103 bp downstream region of pmrB are essential for conferring colistin resistance. Analysis of the mRNA level and stability showed that the length of the downstream region drastically affected the pmrB mRNA level but not its half-life. Lipid A analysis, by mass spectrometry, revealed that the constructs containing pmrB with a longer downstream region (103 or 126 bp) have charge-altering l-4-aminoarabinose (Ara4N) and phosphoethanolamine (pEtN) modifications in lipid A, which were not observed in both vector control and the construct containing pmrB with an 86 bp downstream region. Together, the findings from this study indicate that the 3′-downstream region of pmrB is critical for the PmrB-mediated lipid A modifications and colistin resistance in E. coli BL21(DE3), suggesting a novel regulatory mechanism of PmrB-mediated colistin resistance in E. coli.

Highlights

IntroductionPolymyxins, such as colistin ( known as “polymyxin E”) and polymyxin B, are polycationic peptide antibiotics with broad-spectrum activity against Gram-negative bacterial pathogens (such as Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae)
Polymyxins, such as colistin and polymyxin B, are polycationic peptide antibiotics with broad-spectrum activity against Gram-negative bacterial pathogens
We examined a panel of genes potentially involved in polymyxin resistance by using the same functional rescuing approach previously described [21]

Summary

Introduction

Polymyxins, such as colistin ( known as “polymyxin E”) and polymyxin B, are polycationic peptide antibiotics with broad-spectrum activity against Gram-negative bacterial pathogens (such as Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae). Colistin was introduced into clinical usage in the 1960s but was replaced by other antibiotics due to concerns of nephrotoxicity and neurotoxicity [1]. Since the 1990s, with the advent of multidrug-resistant (MDR) Gram-negative bacteria, colistin has been re-introduced and considered as one of the drugs used as a last resort to treat. Colistin resistance has been arising in various significant Gram-negative bacteria, posing a serious threat to the clinical treatment of MDR pathogens [3]. Cationic colistin can bind negatively charged lipopolysaccharide (LPS), which is anchored at the outer leaflet of the outer membrane through lipid

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Conclusion