Spermidine and spermine are the natural substrates of theAcinetobacter baumanniiAmvA multidrug efflux pump

Abstract

Multidrug efflux pumps are important drivers of antibiotic resistance in Acinetobacter baumannii and other pathogens, however their ‘natural’ roles beyond transport of clinical antimicrobials are poorly described. Polyamines are an ancient class of molecules with broad roles in all three kingdoms of life, and are the likely natural substrate of at least one efflux pump family. We have defined the transcriptome of A. baumannii following treatment with high levels of the polyamines putrescine, cadaverine, spermidine and spermine. These molecules influenced expression of multiple gene classes in A. baumannii including those associated with virulence, and the four polyamines induced distinct but overlapping transcriptional responses. Polyamine shock also induced expression of the MFS-family efflux pump gene amvA and its repressor gene amvR. Loss of amvA dramatically reduced tolerance to the long-chain triaamine spermidine, but caused only modest changes in resistance to known AmvA substrates such as acriflavine. We confirmed reduced accumulation of spermidine in amvA-deficient A. baumannii, and showed that its expression is induced by long-chain polyamines through its cognate regulator AmvR. Our findings suggest that the conserved A. baumannii efflux pump AmvA has evolved to export spermidine from the cell, but that its substrate recognition promiscuity also allows activity against clinically-important biocides and antibiotics.ImportanceAMR genes, including multidrug efflux pumps, evolved long before the ubiquitous use of antimicrobials in medicine and infection control. Multidrug efflux pumps often transport metabolites, signals and host-derived molecules in addition to antibiotics or biocides. Understanding the ancestral physiological roles of multidrug efflux pumps could help to inform the development of strategies to subvert their activity. In this study, we investigated the response of Acinetobacter baumannii to polyamines, a widespread, abundant class of amino acid-derived metabolites, which led us to identify long-chain polyamines as natural substrates of the disinfectant efflux pump AmvA. A second clinically-important efflux pump, AdeABC, also contributed to polyamine tolerance. Our results suggest that the disinfectant resistance capability that allows A. baumannii to survive in hospitals may have evolutionary origins in the transport of polyamine metabolites.

Physiological Functions of Bacterial “Multidrug” Efflux Pumps

“…A. baumannii cells were grown in the absence and presence of 5 mg/mL cadaverine, putrescine, spermidine, or 1.25 mg/mL spermine, mRNA was extracted and examined to test expression of the aceI gene using qRT-PCR 412 and RNA-Seq transcriptomics. 413 Cadaverine and putrescine produced an 18−22-fold induction of expression of this gene, whereas spermidine was less effective and spermine ineffective for induction (Figure 30A). or putrescine was mildly toxic toward A. baumannii wild-type cells, but this was extremely severe in cells from which the aceI gene had been deleted (Figure 30B).…”

“…Following on from experiments on AceI, a more extensive analysis of polyamine regulated gene expression was performed in A. baumannii. 413 Here, the full transcriptomic response of cells exposed to polyamines (Figure 19) was determined. These experiments showed aceI to be the most highly induced gene under putrescine and cadaverine stress, consistent with it having a physiological function in diamine efflux.…”

“…These experiments showed aceI to be the most highly induced gene under putrescine and cadaverine stress, consistent with it having a physiological function in diamine efflux. 413 Additionally, exposure of A. baumannii to spermidine and spermine induced expression of the gene encoding AmvA, a DHA2 family transport protein. AmvA has been characterized as conferring resistance to a diverse range of antibiotics and biocides.…”

“…These results are consistent with AdeABC functioning as a polyamine efflux pump, that may cooperate with AceI and AmvA to promote the export of a diverse array of polyamines across the inner and outer membranes of A. baumannii. 413 Studies examining random mutants of the E. coli SMR family transporter EmrE for novel drug resistance phenotypes identified one mutant, W63G, whose expression promoted resistance to erythromycin in E. coli but additionally led to significantly reduced fitness on media buffered with bis-trispropane. 264 It was found that the polyamine chain in bis-trispropane was responsible for the toxic effect and that putrescine (23; Figure 19) mimicked the phenomenon of toxicity.…”

Efflux Pump Mediated Antimicrobial Resistance by Staphylococci in Health-Related Environments: Challenges and the Quest for Inhibition

“…In addition to antimicrobial resistance, efflux pumps in bacteria play numerous vital physiological roles including extrusion of endogenous noxious metabolites, host-derived antimicrobials and virulence factors, suggesting that the synthetic antimicrobials could be “accidental substrates” of these membrane transport proteins [ 86 , 87 , 88 ]. There is growing evidence from many studies that highlight the contribution of efflux pumps towards bacterial biofilm formation [ 89 ], particularly in several important pathogenic species of bacteria such as S. aureus , Acinetobacter baumannii , Escherichia coli , and Pseudomonas aeruginosa .…”