Rifampicin exerts anti-mucoviscous activity against hypervirulent Klebsiella pneumoniae via binding to the RNA polymerase β subunit

Abstract

In hypervirulent Klebsiella pneumoniae (hvKP), the hypermucoviscous capsule is known to be a major virulence determinant. We previously discovered that rifampicin (RFP), a bactericidal drug that binds to and inhibits the β subunit of RNA polymerase (RpoB), elicits anti-mucoviscous activity against hvKP by suppressing rmpA, a regulator of capsule production. Here, we aimed to determine whether RFP exerts this effect at sub-growth-inhibitory concentrations via its binding to RpoB. Five spontaneous RFP-resistant mutants (R1-R5) were prepared from an hvKP clinical isolate and subjected to whole genome sequencing and mucoviscosity analyses. Subsequently, a two-step allelic exchange procedure was used to create a rpoB mutant R6 and revertants with wild-type rpoB from R1-R5 (named R1'-R5'). Transcription levels of rmpA and the capsular polysaccharide polymerase gene magA and capsule thickness of R1-R5 and R1'-R5' grown without or with RFP were evaluated by quantitative reverse transcription polymerase chain reaction and microscopic observation using India ink staining. R1-R5 all had non-synonymous point mutations in rpoB and were highly resistant to the bactericidal effects and anti-mucoviscous activity of RFP. While the properties of R6 were similar to those of R1-R5, the responses of R1'-R5' to RFP were identical to those of the wild type. rmpA and magA transcription levels and capsule thickness correlated well with the mucoviscosity levels. RFP exerts anti-mucoviscous activity by binding to RpoB. The mechanism of how this causes rmpA suppression remains to be explored.