A functional menadione biosynthesis pathway is required for capsule production by Staphylococcus aureus

Abstract

Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus, we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV, we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione is involved in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.

Triclosan-resistant small-colony variants of Staphylococcus aureus produce less capsule, less phenol-soluble modulins, and are attenuated in a Galleria mellonella model of infection

“…In previous work we analysed capsule production by both hemB and menD SCVs of S. aureus , where we found that those that were auxotrophic for menadione ( menD ) produced little capsule, whereas those that were auxotrophic for hemin were unaffected [20]. In this study we demonstrate that SCV associated with triclosan resistance and auxotrophic for fatty acids also produce less capsule, an effect that appears to be mediated at the transcriptional level.…”

“…In recent work we identified an association between genes associated with the SCV phenotype and capsule production [20]. We functionally verified this association and demonstrated that a menadione auxotrophic SCV did not produce capsule, whereas a hemin autotropic SCV did.…”

“…In previous work we identified mutations in genes involved in the triclosan-resistant SCV (tr-SCV) form of S. aureus as being associated with capsule production [20]. To examine this association, we isolated a tr-SCV by growing S. aureus strain Newman in presence of 32 µg ml −1 of triclosan for 3 days.…”

“…The production of the capsule for each strain was determined using a previously described dot immunoblot assay [20]. Each strain was grown on TSA and incubated at 37 °C for 18 h. Colonies were scraped from the plate surface and resuspended in TSB.…”

Targeted control of pneumolysin production by a mobile genetic element in Streptococcus pneumoniae

“…For this reason we utilized in this study three different approaches, which allowed us to consider a wider range of loci with a focus on loci identified by more than one of the GWAS approaches. Our usual approach of functionally validating identified associations [63–67] has proven to be challenging in S. pneumoniae , as our clinical strains are highly resistant to transformation and the more genetically amenable laboratory strains do not always contain the associated gene, as we found in the case of zomB .…”