2.A.117 The Proteobacterial Antimicrobial Compound Efflux (PACE) Family

Chlorhexidine, an antiseptic or disinfectant, is a membrane-active biocide. Hassan et al. 2013 examined the transcriptomic response of a representative nosocomial human pathogen, Acinetobacter baumannii, to chlorhexidine, to identify the primary chlorhexidine resistance elements. The most highly up-regulated genes encoded components of a major multidrug efflux system, AdeABC (TC# 2.A.6.2.40). The next most highly overexpressed gene under chlorhexidine stress was designated AceI. Orthologs of the aceI gene are conserved within the genomes of a broad range of proteobacterial species, but are also found in other bacterial phyla. Expression of aceI or its orthologs from several other γ- or β-proteobacterial species in Escherichia coli resulted in significant increases in resistance to chlorhexidine. Additionally, disruption of the aceI ortholog in Acinetobacter baylyi rendered it more susceptible to chlorhexidine. The AceI protein was localized to the membrane after overexpression in E. coli. This protein was purified, and binding assays demonstrated direct and specific interactions between AceI and chlorhexidine. Transport assays using [14C]-chlorhexidine showed that AceI mediates the energy-dependent efflux of chlorhexidine.  It was later found to  transport other drugs including benzalkonium, dequalinium, proflavine, and acriflavine. The physiological substrates are polyamines (diamines) such as cadaverine and putrescine (Hassan et al. 2019). An E15Q AceI mutant with a mutation in a conserved acidic residue, although unable to mediate chlorhexidine resistance and transport, was still able to bind chlorhexidine. AceI forms a dimeric exporter under the control of AceR (Liu et al. 2018). These data are consistent with AceI being an active chlorhexidine efflux protein and the founding member of a family of bacterial polyamine and drug efflux transporters, structurally similar to the DMT superfamily (2.A.7).

The generalized transport reaction catalyzed by AceI is:

Chlorhexidine (in)  →  chlorhexidine (out)


 

References:

Bolla, J.R., A.C. Howes, F. Fiorentino, and C.V. Robinson. (2020). Assembly and regulation of the chlorhexidine-specific efflux pump AceI. Proc. Natl. Acad. Sci. USA 117: 17011-17018.
Hassan, K.A., L.D. Elbourne, L. Li, H.K. Gamage, Q. Liu, S.M. Jackson, D. Sharples, A.B. Kolstø, P.J. Henderson, and I.T. Paulsen. (2015). An ace up their sleeve: a transcriptomic approach exposes the AceI efflux protein of Acinetobacter baumannii and reveals the drug efflux potential hidden in many microbial pathogens. Front Microbiol 6: 333.
Hassan, K.A., Q. Liu, L.D.H. Elbourne, I. Ahmad, D. Sharples, V. Naidu, C.L. Chan, L. Li, S.P.D. Harborne, A. Pokhrel, V.L.G. Postis, A. Goldman, P.J.F. Henderson, and I.T. Paulsen. (2018). Pacing across the membrane: the novel PACE family of efflux pumps is widespread in Gram-negative pathogens. Res. Microbiol. [Epub: Ahead of Print]
Hassan, K.A., Q. Liu, P.J. Henderson, and I.T. Paulsen. (2015). Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems. mBio 6:.
Hassan, K.A., S.M. Jackson, A. Penesyan, S.G. Patching, S.G. Tetu, B.A. Eijkelkamp, M.H. Brown, P.J. Henderson, and I.T. Paulsen. (2013). Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins. Proc. Natl. Acad. Sci. USA 110: 20254-20259.
Hassan, K.A., V. Naidu, J.R. Edgerton, K.A. Mettrick, Q. Liu, L. Fahmy, L. Li, S.M. Jackson, I. Ahmad, D. Sharples, P.J.F. Henderson, and I.T. Paulsen. (2019). Short-chain diamines are the physiological substrates of PACE family efflux pumps. Proc. Natl. Acad. Sci. USA 116: 18015-18020.
Liu, Q., K.A. Hassan, H.E. Ashwood, H.K.A.H. Gamage, L. Li, B.C. Mabbutt, and I.T. Paulsen. (2018). Regulation of the aceI multidrug efflux pump gene in Acinetobacter baumannii. J Antimicrob Chemother 73: 1492-1500.


2.A.117.1 The (mainly) Proteobacterial Chlorhexidine Efflux (PCE) family


Examples:

TC#NameOrganismal TypeExample
2.A.117.1.1

The Chlorhexidine drug-resistance exporter, AceI of 179 aas and 4 TMSs (Hassan et al. 2013) .  It is capable of exporting multiple drugs such as benzalkonium, dequalinium, proflavine, and acriflavine. (Hassan et al. 2015). The aceI gene is induced in A. baumannii by the short-chain diamines, cadaverine and putrescine. Membrane transport experiments conducted in whole cells of A. baumannii and Escherichia coli and also in proteoliposomes showed that AceI mediates the efflux of these short-chain diamines (polyamines) such as  when energized by an electrochemical gradient (Hassan et al. 2019), suggesting that they are the phsiological substrates of AceI. AceI can form dimers and is regulated at the transcriptional level by AceR (Bolla et al. 2020).

Proteobacteria

AceI of Acinetobacter baumannii

 
2.A.117.1.10

Uncharacterized putative drug exporter of 148 aas and 4 TMSs.

Proteobacteria

Transmembrane poir domain-protein of Azospira oryzae (Dechlorosoma suillum)

 
2.A.117.1.11

Uncharacterized putative drug exporter of 140 aas and 4 TMSs.

Proteobacteria

UP of Shewanella hoihica

 
2.A.117.1.12

The Unknown Bacterial Transmembrane Pair (UBTP) family member of 146 aas and 4 TMSs.  Chlorhexidine-unresponsive (Hassan et al. 2013).

Proteobacteria

UBTP family member of Burkholderia cenocepacia

 
2.A.117.1.13

Member of the Proteobacterial Antimicrobial Compound Efflux (PACE) family.  This protein is of 140 aas with 4 TMSs.  It has been shown to be an active drug exporter, conferring resistance to both proflavine and acriflavine, mediated by an active efflux mechanism (Hassan et al. 2018).

Proteobacteria

UBTP2 family member of Vibrio parahaemolyticus

 
2.A.117.1.2

Chlorhexidine-responsive chlorhexadine exporter of 171 aas and 4 TMSs.

Proteobacteria

AceI of Pseudomonas aeruginosa

 
2.A.117.1.3

Chlorhexidine-responsive putative chlorhxidine exporter of 160 aas and 4 TMSs, AceI (Hassan et al. 2013). It confers resistance to both proflavine and acriflavine by an active efflux mechanism (Hassan et al. 2015). AceR is an activator of aceI gene expression when challenged with chlorhexidine (Liu et al. 2018). This system also exports polyamines (organic diamines) such as cadaverine and putrescine (and possibly spermidine with low affinity).  It is induced preferentially by cadaverine and putrescine, and to a much lesser extent by spermidine. An AceI-E15Q mutant is inactive (Hassan et al. 2019).

Proteobacteria

AceI of Salmonella typhi

 
2.A.117.1.4

Chlorhexidine-unresponsive putative drug exporter of 147 aas and 4 TMSs.

Proteobacteria

Drug exporter of Pseudomonas aeruginosa

 
2.A.117.1.5

Chlorhexidine-unresponsive putative drug exporter of 144 aas and 4 TMSs.

Proteobacteria

Drug exporter of Desulfovibrio vulgaris

 
2.A.117.1.6

Uncharacterized putative drug exporter of 134 aas and 4 TMSs.

Firmicutes

UP of Veillonella parvula

 
2.A.117.1.7

Uncharacterized putative drug exporter of 147 aas and 4 TMSs.

Proteobacteria

UP of Methylobacterium populi

 
2.A.117.1.8

Uncharacterized putative drug exporter of 179 aas and 4 TMSs.

Actinobacteria

UP of Micrococcus luteus

 
2.A.117.1.9

Uncharacterized putative drug exporter of 346 aas and 4 N-terminal TMSs with a long C-terminal hydrophilic domain.

Proteobacteria

UP of Pseudomonas fluorescens

 
Examples:

TC#NameOrganismal TypeExample
2.A.117.2.1

Uncharacterized putative drug exporter of 142 aas and 4 TMSs.

Proteobacteria

UP of Rhodobacter spheroides

 
2.A.117.2.2

Uncharacterized protein of 148 aas and 4 TMSs in a 2 + 2 TMS arrangement.

UP of Hyphomicrobium sp. (freshwater metagenome)

 
2.A.117.2.3

PACE efflux transporter protein of 153 aas and 4 TMSs in a 2 + 2 TMS arrangement.

PACE exporter of Planctomycetes bacterium (activated sludge metagenome)

 
Examples:

TC#NameOrganismal TypeExample
2.A.117.3.1

Uncharacterized protein of 88 aas and 2 TMSs. Its sequence correspondes to the N-terminus of full length homologues and may therefore be a truncated version of a 4 TMS proteins.

UP of Yangia sp. PrR003