A gene in dis-surA: Determining the true identity of JW0052 Escherichia coli and assessing the role of frmB and BrkA expression on outer membrane stability

Abstract

Bordetella pertussis resistance to killing A (BrkA) protein is a virulence factor found in the outer membrane (OM) of the human pathogen Bordetella pertussis, the causative agent of whooping cough. BrkA functions as an autotransporter and has been implicated in mediating adherence and conferring serum resistance by inhibiting the classical complement pathway. SurA is a periplasmic chaperone protein that prevents aggregation of outer membrane proteins (OMPs) such as BrkA, delivering them to the β-barrel assembly machinery (BAM) complex for proper integration into the OM. Previous studies have reported conflicting phenotypes regarding BrkA expression in the absence of SurA. JW0052 (ΔsurA) Escherichia coli transformed with the BrkA expression vector pPALMC1 have exhibited inconsistent growth rates when compared to wildtype (WT) strain BW25113 E. coli expressing the same vector. This study initially set out to clarify these conflicting findings by further exploring the impact of surA deletion and BrkA expression on OM stability. However, PCR and sequencing confirmation of the strain indicated that JW0052 was not a true surA knockout, despite showing kanamycin resistance. Whole genome sequencing revealed that the JW0052 strain was a knockout of frmB, encoding a serine hydrolase involved in formaldehyde detoxification. This study found that the gene frmB is not required for BrkA expression in E. coli. Further, ΔfrmB E. coli expressing BrkA exhibited reduced growth compared to WT E. coli expressing BrkA. A spot plate assay demonstrated greater sensitivity in both WT and ΔfrmB E. coli expressing BrkA to vancomycin, sodium dodecyl sulfate (SDS), and ethylenediaminetetraacetic acid (EDTA) compared to their untransformed counterparts, suggesting that the expression of BrkA compromises OM stability. These findings confirm the true identity of the JW0052 E. coli knockout and provide further insight into potential therapeutics targeting essential proteins in the virulence of Bordetella pertussis.