Abstract
The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to its clinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightly regulated by a transcriptional activator, Caf1R, of the AraC/XylS family, proteins notoriously prone to aggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression from the native and synthetic codon-optimised caf1R cloned in three different expression plasmids was examined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstrated in vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R were produced from codon optimised caf1R. Transcriptional-lacZ reporter fusions defined the PM promoter and Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identified Caf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correct folding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availability of functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 and Caf1R-regulated pathophysiology of Y. pestis.
Highlights
Due to its surface location, high level of expression and the fact that the F1 polymer is unique to Y. pestis, F1 remains a primary target for plague diagnostics [11,12,13,14] and a key component of anti-plague vaccines [6,15,16,17]
With so little known about regulation of the caf locus, in contrast to the wealth of data available on structure, assembly and vaccine potential of the F1 capsular antigen itself, this study has focused on assessing different expression systems for recombinant production of functional Caf1R in E. coli
Bacterial proteins with a PROSITE scan score of 12.52–30.74 are considered a member of DNA-binding transcriptional regulators of the extensively dispersed AraC/Xyls (A/X)
Summary
Yersinia pestis is a deadly zoonotic bacterial pathogen. It has killed around 200 million people in three major global plague pandemics and continues to be a threat to this day with endemic outbreaks and sporadic cases [1,2]. Production of F1 is induced during infection following transmission from the flea vector to humans or to a rodent reservoir [9,10]. This surface capsule-like structure helps bacteria neutralise a robust immune response by conferring antiphagocytic ability [10]. Due to its surface location, high level of expression and the fact that the F1 polymer is unique to Y. pestis, F1 remains a primary target for plague diagnostics [11,12,13,14] and a key component of anti-plague vaccines [6,15,16,17]
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