Abstract
Rapid dissemination of the plasmid-born polymyxin resistance gene mcr-1 poses a critical medical challenge. MCR-1 expression is tightly controlled and imposes a fitness cost on the bacteria. We used growth studies and metabolomics to examine growth and metabolic changes within E. coli TOP10 at 8 and 24 h in response to different levels of expression of mcr-1. Induction of mcr-1 greatly increased expression at 8 h and markedly reduced bacterial growth; membrane disruption and cell lysis were evident at this time. At 24 h, the expression of mcr-1 dramatically declined with restored growth and membrane integrity, indicating regulation of mcr-1 expression in bacteria to maintain membrane homeostasis. Intermediates of peptide and lipid biosynthesis were the most commonly affected metabolites when mcr-1 was overexpressed in E. coli. Cell wall biosynthesis was dramatically affected with the accumulation of lipids including fatty acids, glycerophospholipids and lysophosphatidylethanolamines, especially at 8 h. In contrast, levels of intermediate metabolites of peptides, amino sugars, carbohydrates and nucleotide metabolism and secondary metabolites significantly decreased. Moreover, the over-expression of mcr-1 resulted in a prolonged reduction in intermediates associated with pentose phosphate pathway and pantothenate and CoA biosynthesis. These findings indicate that over-expression of mcr-1 results in global metabolic perturbations that mainly involve disruption to the bacterial membrane, pentose phosphate pathway as well as pantothenate and CoA biosynthesis.
Highlights
The emergence and spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria represents an increasing threat to global public health (McEwen and Collignon, 2018; Lerminiaux and Cameron, 2019)
The expression vector pBAD-hisA (Thermo Fisher Scientific, United States) was used to construct recombinant plasmids. pBAD-hisA contains a promoter for the araBAD operon that controls the expression of genes, which can be regulated with L-Arabinose (Guzman et al, 1995). mcr-1 was cloned from pHNSHP45 (GenBank: KP347127.1) using primer pair to pBADhisA, yielding pBAD-mcr-1 (Supplementary Tables S1, S2)
It is clear that mcr-1 encodes a phosphoethanolamine transferase, an inner membrane protein which attaches phosphoethanolamine to the lipid A, reducing susceptibility to polymyxins (Liu et al, 2017)
Summary
The emergence and spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria represents an increasing threat to global public health (McEwen and Collignon, 2018; Lerminiaux and Cameron, 2019). Intrinsic resistance involves sensing the presence of polymyxins in the environment via two-component regulatory systems (TCSs, e.g., PmrAB and PhoPQ). These TCSs upregulate the expression of pmrC and/or arnBCADTEF-ugd leading to the modification of the phosphate groups of lipid A via the addition of cationic 4-amino-4-deoxyL-arabinose (L-Ara4N) and/or phosphoethanolamine (pEtN) moieties, thereby attenuating the disorganization of the OM (Falagas and Kasiakou, 2005; Olaitan et al, 2014). The mechanisms of polymyxin resistance in these organisms include mutations in pmrAB and phoPQ and the negative regulator mgrB (in Klebsiella pneumoniae) that lead to constitutive expression of lipid A modification genes, and mutations in lipid A biosynthesis genes (e.g., lpxACD) that cause the loss of LPS and prevent polymyxin binding (Cannatelli et al, 2013; Olaitan et al, 2014)
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