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Review
. 2018 Sep-Oct;169(7-8):450-454.
doi: 10.1016/j.resmic.2018.01.001. Epub 2018 Feb 2.

Pacing across the membrane: the novel PACE family of efflux pumps is widespread in Gram-negative pathogens

Karl A Hassan  1 , Qi Liu  2 , Liam D H Elbourne  2 , Irshad Ahmad  3 , David Sharples  3 , Varsha Naidu  2 , Chak Lam Chan  3 , Liping Li  2 , Steven P D Harborne  3 , Alaska Pokhrel  2 , Vincent L G Postis  4 , Adrian Goldman  5 , Peter J F Henderson  3 , Ian T Paulsen  2
Affiliations
Review

Pacing across the membrane: the novel PACE family of efflux pumps is widespread in Gram-negative pathogens

Karl A Hassan et al. Res Microbiol. 2018 Sep-Oct.

Abstract

The proteobacterial antimicrobial compound efflux (PACE) family of transport proteins was only recently described. PACE family transport proteins can confer resistance to a range of biocides used as disinfectants and antiseptics, and are encoded by many important Gram-negative human pathogens. However, we are only just beginning to appreciate the range of functions and the mechanism(s) of transport operating in these proteins. Genes encoding PACE family proteins are typically conserved in the core genomes of bacterial species rather than on recently acquired mobile genetic elements, suggesting that they confer important core functions in addition to biocide resistance. Three-dimensional structural information is not yet available for PACE family proteins. However, PACE proteins have several very highly conserved amino acid sequence motifs that are likely to be important for substrate transport. PACE proteins also display strong amino acid sequence conservation between their N and C-terminal halves, suggesting that they evolved by duplication of an ancestral protein comprised of two transmembrane helices. In light of their drug resistance functions in Gram-negative pathogens, PACE proteins should be the subject of detailed future investigation.

Keywords: Antimicrobial resistance; Bacterial transmembrane pair domain; Efflux; Gram-negative pathogen; Membrane transport; PACE.

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Figures

Fig. 1
Fig. 1
Predicted transmembrane topology and conserved amino acid sequence motifs present in PACE family proteins. An amino acid sequence alignment of 47 diverse PACE family proteins (Supplemental Fig. S1), encoded by a broad range of hosts, was used to identify amino acid sequence motifs that are conserved across the family (A) Predicted topology of PACE family proteins. Upper case characters are conserved in greater than 90% of protein sequences and characters in lower case are conserved in greater than 65% but fewer than 90% of the aligned protein sequences. Conserved residues are coloured (red: negatively charged, blue: positively charged, orange: aromatic, black: others). The similar amino acid sequence motifs in helices 1 and 3 (1A and 1B, respectively) are surrounded by purple lines, and the similar sequence motifs in helices 2 and 4 (2A and 2B, respectively) are surrounded by green lines (B–E) Sequence logos, made using WebLogo 3 , showing conservation of amino acid residues in the four sequence motifs identified in PACE proteins; error bars show an approximate Bayesian 95% confidence interval . (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2
Fig. 2
Acriflavine transport mediated by PACE family proteins encoded by the human pathogen Burkholderia cenocepacia HI2424. A) Transport experiments performed using E. coli OmniMax cells expressing the proteins of interest, essentially as described previously . The cells were preloaded with 5 μM acriflavine in the presence of 10 μM CCCP. The cells were washed and re-energised using glucose at the point marked with an arrow and transport monitored fluorimetrically. Acriflavine fluorescence is quenched when it is intercalated into nucleic acids. Therefore, its transport out of the cell is associated with an increase in fluorescence. B) Western blot performed on the cells used in the assay, probed using His-Probe-HRP. The B. cenocepacia HI2424 PACE family proteins Bcen2424_2356 and Bcen2424_5347 were expressed at easily detectable levels after the 1-h induction used for this assay. Bcen2424_2356 promoted the rapid efflux of acriflavine, whereas Bcen2424_5347 did not promote acriflavine transport above background. The third PACE family protein encoded by B. cenocepacia HI2424, Bcen2424_5167 was not expressed and was excluded from the figure. Bcen2424_5167 and Bcen2424_5347 are phylogenetically distinct from Bcen2424_2356. Error bars show the standard deviation of three replicate experiments.
Fig. s1
Fig. s1
Solubilisation and purification of the Ferrimonas balearica DSM 9799 PACE family protein, Fbal_3166 , using styrene maleic acid co-polymer and Ni-affinity purification. Fbal_3166 protein was overexpressed in E. coli BL21 cells grown in a 30 L fermenter using the pTTQ18 expression system , . Styrene maleic acid co-polymer preparation, membrane solubilisation and Ni-affinity purification were performed as previously described . Samples consisting of solubilised membrane proteins (lane A), proteins that did not bind to the Ni-affinity column (lane B) and purified Fbal_3166 (lane C) were run on a 15% SDS-PAGE gel and stained with Coomassie brilliant blue R-250. The size (kDa) of co-migrated soluble molecular weight markers is shown on the left side of the gel.
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