Functionally Cloned pdrM from Streptococcus pneumoniae Encodes a Na+ Coupled Multidrug Efflux Pump

Abstract

Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na+- or Li+-dependent manner. Moreover, Na+ efflux via PdrM was observed when acriflavine was added to Na+-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na+/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.

Multidrug Transport Protein NorM from Vibrio cholerae Simultaneously Couples to Sodium- and Proton-Motive Force

“…Hence, carboxylates can be part of Na ϩ binding sites but can also act as proton-binding sites. As the relevance of carboxylates in Na ϩ -dependent MATE transporters has been discussed only in the context of Na ϩ and cationic substrates (13)(14)(15)(16)20), our novel observations on the SMF and PMF dependence of NorM-VC-mediated transport lead us to re-examine the functional roles of the catalytically important carboxylate Asp-36 (in TM1) in the N-terminal half, and Glu-255 (in TM7) and Asp-371 (in TM10) in a network of aromatic residues in the C-terminal half (Fig. 3).…”

“…Depending on the effect of Na ϩ on substrate efflux in intact cells, earlier studies on bacterial MATE transporters grouped these proteins into two classes, those that are Na ϩ dependent, including NorM from Vibrio parahaemeolyticus (13), Vibrio cholerae (14), Neisseria gonorrhoeae (15), and PdrM from Streptococcus pneumonia (16), and those that are not Na ϩ dependent, including PmpM from Pseudomonas aeruginosa (17), PfMATE from Pyrococcus furiosus (18), and DinF from Bacillus halodurans (19), but that are H ϩ dependent instead. Evidence for this H ϩ dependence came from substrate-induced proton transport by PmpM and DinF in inside-out membrane vesicles (17,19) and PfMATE in spheroplasts (18).…”

Broadly conserved Na + -binding site in the N-lobe of prokaryotic multidrug MATE transporters

“…However, other prokaryotic MATEs have been reported to be coupled to H + instead [e.g., PfMATE (13), DinF-BH (12), and NorM-PS (21)] or, intriguingly, to both Na + and H + acting additively [e.g., NorM-VC (22)]. Other monovalent cations such as K + , Rb + , and Li + have also been reported to influence substrate efflux by some MATEs (23)(24)(25)(26). A caveat, however, is that the specificity of these transporters is often probed through cell-based resistance assays, and therefore it is not always completely clear that the effects observed reflect directly or exclusively on MATE activity.…”

Characterization of MATE-Type Multidrug Efflux Pumps from Klebsiella pneumoniae MGH78578

“…cholera non-O1 [ 8 , 9 ]. PdrM, which we recently reported, also displayed sodium-driven drug efflux ability and was the first multidrug efflux pump coupled with Na + to be identified in Gram-positive bacteria [ 10 ]. The efflux activity of substrates coupled with protons has been reported in AbeM from Acinetobacter baumannii [ 11 ], EmmdR from Enterobacter cloacae [ 12 ], PmpM from Pseudomonas aeruginosa [ 13 ], and PfMATE from Pyrococcus furiosus [ 14 ], which belong to cluster 1 in the MATE family.…”

“…However, DinF from E . coli and most DinF homologues that we have investigated did not significantly change resistance levels to antimicrobial agents (our unpublished data and reference 10) [ 10 ]. DinF from E .…”