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

Abstract

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.

Role of Efflux Pumps on Antimicrobial Resistance in Pseudomonas aeruginosa

“…Its emergence earlier than the extensive exposure to antimicrobial agents suggested that their evolution as a resistance mechanism occurred independently of the use of antimicrobials [ 44 ]. The efflux system allows the bacteria to survive at low levels of drugs, conferring time to develop a more specialized mechanism of resistance [ 116 ]. When combined with the outer membrane barrier, the efflux system guarantees cellular protection from a range of compounds [ 106 ], favoring the dissemination of gene coding for such systems.…”

“…Some isolates have genes for different efflux pumps in their genome, acting as genetic reservoirs [ 48 , 52 ]. This organization can also occur from gene duplication events [ 117 ] and gene cassettes [ 116 ].…”

The TAM, a Translocation and Assembly Module for protein assembly and potential conduit for phospholipid transfer

“…1D , inset), with this feature interacting with TamA (Selkrig et al, 2015 ; Selkrig et al, 2012 ). Consistent with the TamB-TamA interaction, comparative genomic analysis has revealed that most, but not all, bacterial lineages that encode a TamB, also encode a TamA (Heinz et al, 2015 ; Stubenrauch et al, 2022 ). In species such as Borrelia burgdorferi , a bacterium where TamB is present but TamA is not, co-immunoprecipitation analysis suggests that TamB is a partner protein of BamA (Iqbal et al, 2016 ), contributing its function to β-barrel protein assembly through the BAM complex (Heinz et al, 2015 ; Iqbal et al, 2016 ; Stubenrauch et al, 2016b ).…”

“…A third assay system uses disulfide-mediated cross-linking (Kuszak et al, 2015 ) to address whether the TAM participates in folding of TolC, a β-barrel protein where the BAM complex provides for efficient folding (Stubenrauch et al, 2016a ; Werner and Misra, 2005 ). Loss of either TamA or TamB, did not significantly affect the rate of assembly of 35 S-TolC (Stubenrauch et al, 2016a ), and there was no functional defect in antibiotic efflux either (Stubenrauch et al, 2022 ). To test the hypothesis that nascent TolC substrate molecules engage the lateral gate (Bamert et al, 2017 ) in TamA, a series of cysteine residues were incorporated into the gate and used to assess substrate engagement via disulphide cross-linking experiments.…”

“…To test the hypothesis that nascent TolC substrate molecules engage the lateral gate (Bamert et al, 2017 ) in TamA, a series of cysteine residues were incorporated into the gate and used to assess substrate engagement via disulphide cross-linking experiments. TolC engages the lateral gate of TamA in transit into the bacterial outer membrane, contributing to a process where it is the BAM complex activity that is rate-limiting (Stubenrauch et al, 2022 ).…”