Diversity and conservation of RND efflux pumps across Escherichia coli

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Pugh, Hannah ORCID: https://orcid.org/0000-0002-5163-3196 (2022). Diversity and conservation of RND efflux pumps across Escherichia coli. University of Birmingham. Ph.D.

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Abstract

RND efflux pumps are located in the inner membrane of Gram-negative bacteria and form tripartite complexes that span the inner membrane, periplasm and outer membrane. It is widely accepted that Escherichia coli isolates possess six RND pumps; AcrB, AcrD, AcrF, MdtBC, MdtF and CusA. These pumps are involved in a wide range of physiological functions but are best characterised for their role in antimicrobial efflux. Furthermore, overexpression of RND pumps in clinical isolates confers an MDR phenotype. Though once a clinical isolate, E. coli K-12 and derivatives such as K-12 MG1655 have lost virulence factors and contain significant mutations as a result of years of laboratory passage. Despite this most work determining the structure and function of E. coli RND pumps has been carried out using E. coli K-12 MG1655.
Work in this study found that RND efflux pumps were highly conserved across E. coli although level of conservation varied between systems. In addition, when using the K-12 protein sequences as a reference, amino acid substitutions were present and highly conserved across 20 E. coli lineages. Moreover AcrA, AcrB and TolC which form the main RND efflux pump in E. coli each possessed an amino acid substitution which was present in almost 100% of assemblies belonging to phylogroups B2, D and F. These phylogroups are most commonly associated with extraintestinal E. coli infections and AMR carriage. Furthermore, ST11 which is commonly referred to as E. coli O157:H7 was found to lack to a functional AcrF protein due to a highly conserved 45 nucleotide insertion half way through the gene which encodes two stop codons.
An additional seventh RND protein, EefB, was also identified in the work and found to be highly prevalent across phylogroups B2, D, E, F and G but completely absent from phylogroups A, B1 and C which are more commonly associated with commensal living. This RND pump was found encoded on a five gene operon that encoded a TetR regulator (EefR), PAP (EefA), RND pump (EefB), OMP (EefC), and an MFS transporter (EefD). In E. coli which encode the operon, the genes were found to be highly conserved suggesting strong positive selection for an important biological function.
Taken together, this study demonstrates that RND pumps are highly conserved across E. coli but the extent to which systems are conserved and also the number of systems present varies across E. coli phylogroups. In addition it shows that the use of a model strain not only removes strain specific diversity but prevents the identification and subsequent characterisation of RND systems that are highly prevalent across a species.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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