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A noncanonical chaperone interacts with drug efflux pumps during their assembly into bacterial outer membranes

['Christopher J. Stubenrauch', 'Infection', 'Immunity Program', 'Biomedicine Discovery Institute', 'Department Of Microbiology', 'Monash University', 'Clayton', 'Victoria', 'Centre To Impact Amr', 'Rebecca S. Bamert']

Date: 2022-01 

Bacteria have membrane-spanning efflux pumps to secrete toxic compounds ranging from heavy metal ions to organic chemicals, including antibiotic drugs. The overall architecture of these efflux pumps is highly conserved: with an inner membrane energy-transducing subunit coupled via an adaptor protein to an outer membrane conduit subunit that enables toxic compounds to be expelled into the environment. Here, we map the distribution of efflux pumps across bacterial lineages to show these proteins are more widespread than previously recognised. Complex phylogenetics support the concept that gene cassettes encoding the subunits for these pumps are commonly acquired by horizontal gene transfer. Using TolC as a model protein, we demonstrate that assembly of conduit subunits into the outer membrane uses the chaperone TAM to physically organise the membrane-embedded staves of the conduit subunit of the efflux pump. The characteristics of this assembly pathway have impact for the acquisition of efflux pumps across bacterial species and for the development of new antimicrobial compounds that inhibit efflux pump function.

Here, we provide direct evidence of the interaction between TamA and TolC, where each β-strand of the TolC stave domain was found to interact with the lateral gate of TamA. Building on previous observations of mutant phenotypes [ 35 , 36 , 38 ], and incorporating new findings from in situ cross-linking and pulse-chase assays, we suggest a promiscuous assembly process for TolC-like proteins such that they can use either the BAM complex or the TAM for assembly. We discuss how this promiscuity would be highly relevant in enhancing the success rates with which HGT could establish functional efflux pumps in diverse lineages of bacteria. This study underscores the bottlenecks in acquisition, expression, and activity of drug efflux pumps and informs on the interplay between the BamA and TamA chaperones mediating outer membrane protein assembly.

TolC-like proteins are topologically unlike any other substrate handled by the BAM complex or the TAM, because their β-barrel fold is not evolutionarily related to other outer membrane proteins, but instead evolved from the periplasmic component of the efflux pump, with convergent evolution bringing it to resemble the β-barrel fold [ 30 ]. As such, the assembly pathway for TolC-like proteins is far from simple. The membrane-embedded β-barrel of TolC is composed of 3 identical stave domains. Each stave is constructed through assembling an antiparallel array of 4 beta-strands into a TolC monomer that alone is not ideally suited to either the aqueous environment of the periplasm, nor the hydrophobic environment of the outer membrane. Each of these stave domains then needs to be brought together to construct the final β-barrel domain of the trimeric TolC protein ( Fig 1B and 1C ). While the BAM complex impacts on TolC assembly in E. coli [ 31 , 32 ], the BAM complex is paradoxically not involved in assembly of the TolC-like proteins OprM in P. aeruginosa [ 33 ] nor HgdD in the cyanobacterium Anabaena sp. PCC 7120 [ 34 ]. Mutant forms of the BAM complex (BamAΔR64 and BamAΔR36-K89), which impact the assembly of 14 well-characterised β-barrel proteins, do not affect TolC assembly [ 35 , 36 ], yet deletion of the BAM complex accessory lipoprotein BamB significantly increases TolC levels in the outer membrane [ 37 ] and also confers an increased TolC trimerisation rate [ 38 ]. Furthermore, the assembly kinetics of TolC trimerisation are, at least partially, reduced in the absence of the TAM chaperone, suggesting it to be important for TolC assembly but with no clear indication of the mechanism underlying the assembly process [ 38 ]. Together, these phenotypic data highlight the unusual nature of TolC assembly.

The major, membrane-embedded chaperone catalysing outer membrane protein assembly is the β-barrel assembly machinery (BAM) complex, which works together with the translocation and assembly machinery (TAM) [ 17 ]. Architecturally, the BAM complex is composed from a protein of the Omp85 superfamily, BamA, and a set of accessory proteins the identity of which vary across the lineages of gram-negative bacteria [ 18 – 24 ]. The TAM is a second module of the BAM composed of an inner membrane protein, TamB, and a protein of the Omp85 superfamily, TamA, in the outer membrane [ 20 , 25 – 27 ]. The TAM has a more limited phylogenetic distribution than the BAM complex [ 28 ] and has only been characterised functionally in 3 bacterial species: E. coli, Citrobacter rodentium, and Klebsiella pneumoniae [ 25 – 27 , 29 ].

TolC is the archetypal conduit of tripartite efflux pumps and contains a characteristic αβ-barrel architecture ( Fig 1B ). To achieve this structure, the TolC polypeptide needs to be folded into staves, 3 of which are assembled together to form a transmembrane β-barrel embedded in the outer membrane ( Fig 1C ) as well as an α-barrel that extends 100 Å into the periplasm [ 7 ]. In E. coli, this trimeric TolC conduit subunit is then engaged either onto AcrAB or onto one of 8 other distinct efflux pumps [ 9 ]. In other bacterial species, multiple proteins with structural homology to TolC exist, such as in Pseudomonas species, where at least 18 of these TolC-like proteins have been identified as purpose-evolved conduits [ 10 ]. Through horizontal gene transfer (HGT), the genes encoding TolC-like proteins, and their cognate partner proteins, can be acquired across diverse bacterial lineages promoting the spread of AMR phenotypes [ 11 – 15 ]. The acquisition of these genes is the first committed step to having a functional pump, but the phenotype can only be enacted if the complex folding pathway for the conduit subunit can be mediated in the host species acquiring those genes. Catalysing the assembly pathway for TolC-like proteins would depend on the host having the necessary chaperones to drive the protein folding and assembly and might be limited by sequence diversity in the acquired polypeptide, which did not coevolve with those chaperones [ 16 ].

(A) Schematic of an efflux pump removing harmful substances. The pump comprises an IMC protein from the RND, ABC, or MFS superfamilies, a PAP, and an OMF family component, like TolC [ 1 ]. (B) Ribbon diagram of TolC (PDB: 1EK9). Sizes of each structural domain are indicated to the right [ 7 ]. One of the 3 monomers is coloured pink for clarity. (C) Structural map of one TolC monomer (without its signal peptide), based on its crystal structure (PDB: 1EK9) over residues 1–428 and as predicted using PSIPRED 4.0 [ 8 ] over residues 429–471. The characteristic OEP regions are indicated. Cylinders represent α-helices; arrows represent β-strands. IMC, inner membrane channel; OEP, outer membrane efflux protein; OMF, outer membrane factor; PAP, periplasmic adaptor protein; PGN, peptidoglycan.

One of the most commonly deployed mechanisms of antimicrobial resistance (AMR) is mediated by bacterial efflux pumps, protein complexes that have evolved to rid cells of toxic compounds [ 1 ]. The fundamental mechanism by which these efflux pumps function in gram-negative bacteria depends on a tripartite architecture, comprised of 3 components that combine to span the inner membrane, periplasm, and outer membrane ( Fig 1A ), as understood from several landmark structural studies on these tripartite complexes [ 2 – 5 ]. Most of the structural and functional work has been focussed on the prototypical pumps AcrAB-TolC and MexAB-OprM. These pumps are constitutively expressed in Escherichia coli and Pseudomonas aeruginosa, respectively, and are the workhorses that confer low levels of resistance to clinically relevant drugs before more specialised resistance mechanisms can evolve [ 6 ].

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001523

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