Biology subjects

Blair, J. M. A.

Publications and source records attributed to Blair, J. M. A..

4 recordsLinked to original sources

bioRxiv2021

The membrane-proximal domain of the periplasmic adapter protein plays a role in vetting substrates utilising channels 1 and 2 of RND efflux transporters

Active efflux by resistance-nodulation-division (RND) efflux pumps is a major contributor to antibiotic resistance in clinically relevant Gram-negative bacteria. Tripartite RND pumps, such as AcrAB-TolC of Salmonella enterica serovar Typhimurium, comprise of an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor. Previously, we elucidated binding sites within the PAP AcrA (termed binding boxes) that were important for AcrB-transporter recognition. Here, we have refined the binding box model by identifying the most critical residues involved in PAP-RND binding and show that the corresponding RND-binding residues in the closely related PAP AcrE are also important for AcrB interactions. In addition, our analysis identified a membrane-proximal domain (MPD)-residue in AcrA (K366), that when mutated, differentially affects transport of substrates utilising different AcrB efflux-channels, namely channels 1 and 2, supporting a potential role for the PAP in sensing the substrate-occupied state of the proximal binding pocket (PBP) of the transporter and substrate vetting. Our model predicts that there is a close interplay between the MPD of the PAP and the RND transporter in the productive export of substrates utilising the PBP. ImportanceAntibiotic resistance greatly threatens our ability to treat infectious diseases. In Gram-negative bacteria, overexpression of tripartite efflux pumps, such as AcrAB-TolC, contributes to multidrug resistance because they export many different classes of antibiotics. The AcrAB-TolC pump is made up of three components: the periplasmic adaptor protein (PAP) AcrA, the RND-transporter AcrB, and the outer-membrane factor TolC. Here, we identified critical residues of AcrA that are important for its function with AcrB in Salmonella enterica serovar Typhimurium. Also, we show that AcrA shares these critical residues with AcrE, a closely related PAP, explaining their interoperability with AcrB. Importantly, we identified a residue in the membrane-proximal domain of AcrA that when mutated affected how different substrates access AcrB and impacted downstream efflux via TolC channel. Understanding the role that PAPs play in the assembly and function of tripartite RND pumps can guide novel ways to inhibit their function to combat antibiotic resistance.

microbiology↗

bioRxiv2021

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse {beta}-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug- resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers. IMPACT STATEMENTDisruption of disulfide bond formation sensitizes resistant Gram- negative bacteria expressing {beta}-lactamases and mobile colistin resistance enzymes to currently available antibiotics.

microbiology↗

bioRxiv2021

Efflux only impacts drug accumulation in actively growing cells

For antibiotics with intracellular targets, effective treatment of bacterial infections requires the drug to accumulate to a high concentration inside cells. Bacteria produce a complex cell envelope and possess drug-export efflux pumps to limit drug accumulation inside cells. Decreasing cell envelope permeability and increasing efflux pump activity can reduce intracellular accumulation of antibiotics, and are commonly seen in antibiotic resistant strains. Here, we show that the balance between influx and efflux differs depending on bacterial growth phase in Gramnegative bacteria. Accumulation of the model fluorescent drug, ethidium bromide (EtBr) was measured in S. Typhimurium SL1344 (wild-type) and efflux deficient ({Delta}acrB) strains during growth. In SL1344, EtBr accumulation remained low, regardless of growth phase and did not correlate with acrAB transcription. EtBr accumulation in {Delta}acrB was high in exponential phase but dropped sharply later in growth, with no significant difference to SL1344 in stationary phase. Low EtBr accumulation in stationary phase was not due to the upregulation of other efflux pumps, but instead, due to decreased permeability of the envelope in stationary phase. RNAseq identified changes in expression of several pathways that remodel the envelope in stationary phase, leading to lower permeability. This study shows that efflux is only important for maintaining low drug accumulation in actively growing cells, and that envelope permeability is the predominant factor dictating the rate of drug entry in stationary phase cells. This conclusion means that (i) antibiotics with intracellular targets may be less effective in complex non-growing or slow-growing bacterial infections where intracellular accumulation may be low, (ii) efflux inhibitors may be successful in potentiating the activity of existing antibiotics, but potentially only for bacterial infections where cells are actively growing and (iii) the remodelling of the cell envelope prior to stationary phase could provide novel drug targets.

microbiology↗

bioRxiv2021

Interchangeability of Periplasmic Adaptor Proteins AcrA and AcrE in forming functional efflux pumps with AcrD in Salmonella Typhimurium

BackgroundRND efflux pumps are important mediators of antibiotic resistance. RND pumps including the principal multidrug-efflux pump AcrAB-TolC in Salmonella, are tripartite systems, with an inner membrane RND-transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). We previously identified the residues required for binding between the PAP AcrA and the RND-transporter AcrB and have demonstrated that PAPs can function with non-cognate transporters. AcrE and AcrD/AcrF are homologues of AcrA and AcrB, respectively. Here, we show that AcrE can interact with AcrD, which does not possess its own PAP, and establish that the residues previously identified in AcrB-binding are also involved in AcrD-binding. MethodsThe acrD and acrE genes were expressed into a strain lacking acrABDEF ({Delta}3RND). PAP residues involved in promiscuous interactions were predicted based on previously defined PAP-RND interactions and corresponding mutations generated in acrA and acrE. Antimicrobial susceptibility of the mutant strains was determined. ResultsCo-expression of acrD and acrE significantly decreased susceptibility of the {Delta}3RND strain to AcrD substrates showing that AcrE can form a functional complex with AcrD. The substrate profile of Salmonella AcrD differed from that of E. coli AcrD. Mutations targeting the previously defined PAP-RND interaction sites in AcrA/AcrE impaired efflux of AcrD-dependent substrates. ConclusionsThese data indicate that AcrE forms an efflux-competent pump with AcrD and thus presents an alternative PAP for this pump. Mutagenesis of the conserved RND-binding sites validates the interchangeability of AcrA and AcrE, highlighting them as potential drug targets for efflux inhibition.

microbiology↗