Exogenous and Endogenous Phosphoethanolamine Transferases Differently Affect Colistin Resistance and Fitness in Pseudomonas aeruginosa

Abstract

Colistin represents a last-line treatment option for infections caused by multidrug resistant Gram-negative pathogens, including Pseudomonas aeruginosa. Colistin resistance generally involves the modification of the lipid A moiety of lipopolysaccharide (LPS) with positively charged molecules, namely phosphoethanolamine (PEtN) or 4-amino-4-deoxy-L-arabinose (Ara4N), that reduce colistin affinity for its target. Several lines of evidence highlighted lipid A aminoarabinosylation as the primary colistin resistance mechanism in P. aeruginosa, while the contribution of phosphoethanolamination remains elusive. PEtN modification can be due to either endogenous (chromosomally encoded) PEtN transferase(s) (e.g., EptA in P. aeruginosa) or plasmid borne MCR enzymes, commonly found in enterobacteria. By individually cloning eptA and mcr-1 into a plasmid for inducible gene expression, we demonstrated that MCR-1 and EptA have comparable PEtN transferase activity in P. aeruginosa and confer colistin resistance levels similar to those provided by lipid A aminoarabinosylation. Notably, EptA, but not MCR-1, negatively affects P. aeruginosa growth and, to a lesser extent, cell envelope integrity when expressed at high levels. Mutagenesis experiments revealed that PEtN transferase activity does not account for the noxious effects of EptA overexpression, that instead requires a C-terminal tail unique to P. aeruginosa EptA, whose function remains unknown. Overall, this study shows that both endogenous and exogenous PEtN transferases can promote colistin resistance in P. aeruginosa, and that PEtN and MCR-1 mediated resistance has no impact on growth and cell envelope homeostasis, suggesting that there may be no fitness barriers to the spread of mcr-1 in P. aeruginosa.

An Update of Mobile Colistin Resistance in Non-Fermentative Gram-Negative Bacilli

“…Cervoni et al also demonstrated that MCR-1 increases colistin resistance in recipient cells. Moreover, the expression of MCR-1 in Pseudomonas does not affect bacterial growth or cell envelope homeostasis (Cervoni et al, 2021).…”

Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa

“…Except for pMEimuBC, the overexpression of all DNA polymerases inhibited the growth of HolD-depleted cells with respect to the empty plasmid controls, and none of them rescued the growth of HolD-depleted RecA-deficient cells (Figure S4). The inhibitory effect of polymerase-expressing constructs was also observed in HolD-depleted cells cultured without IPTG (Figure S4), plausibly because of leaky gene expression from the pME6032 plasmid in the absence of the inducer [57]. This effect appears to be dependent on HolD depletion, as the overexpression of the DNA polymerases marginally affected the growth of wild-type and ∆recA cells (Figure S4).…”

More than mcr: Canonical Plasmid- and Transposon-Encoded Mobilized Colistin Resistance (mcr) Genes Represent a Subset of Phosphoethanolamine Transferases

“…However, these approaches are time-consuming and require a hard-to-achieve standardization process to ensure similar gene expression and active protein levels. Moreover, it has been shown that heterologous overexpression of P. aeruginosa eptA (Cervoni et al, 2021) or dysregulation of S. Typhimurium eptA transcription (Vaara et al, 1981;Olaitan et al, 2014) lead to colistin resistance. Hence, the identification of other bioinformatic criteria that can be used to discriminate between mcr and eptA can improve the ability to detect colistin resistance genes in bacterial WGS data and provide an important basis for selecting genes for phenotypic characterization.…”

“…However, these approaches are time-consuming and require a hard-to-achieve standardization process to ensure similar gene expression and active protein levels. Moreover, it has been shown that heterologous overexpression of P. aeruginosa eptA (Cervoni et al, 2021) or dysregulation of S . Typhimurium eptA transcription (Vaara et al, 1981; Olaitan et al, 2014) lead to colistin resistance.…”