Staphylococcus aureus is a highly successful human pathogen capable of colonising and spreading easily between humans while causing a wide range of infections, including life threatening bacteraemias, endocarditis and pneumonia. Virulence gene expression is regulated primarily by the staphylococcal accessory gene regulator (agr) in a cell density-dependent manner termed quorum sensing. Strains of S. aureus, particularly community-associated methicillin resistant S. aureus (CA-MRSA), carrying the genes for a pore forming exotoxin, the Panton Valentine Leukocidin (PVL), have been spreading worldwide over the last 10 to 15 years. As high level production of exotoxins has been implicated in the success of the CA-MRSA clone USA300, a collection of PVL producing clinical isolates from Nottingham were studied with the aim of understanding why PVL production might vary between strains. Sequence typing of the Nottingham strain collection revealed that high PVL producing ST22 agr group 1 and low PVL producing ST30 agr group 3 strains were the most common types. PVL production was stimulated by addition of the type specific exogenous autoinducing peptide (AIP), which activates the agr sensor AgrC, and inhibited by the AgrC antagonist (ala5)AIP-1 if added before a critical cell population density was reached. Analysis of the pvl promoter identified predicted binding sites for RNA polymerase and the SarA protein family of regulators. Promoter pull down experiments confirmed the binding of several staphylococcal regulators to both the agr and pvl promoters including SarA, SarS, Rot and MgrA indicating that these are likely to play a role in the regulation of PVL production. Analysis of the agr locus of high and low PVL producing strains found that a low PVL producing ST22 agr group 1 strain TS13 had a single nucleotide polymorphism (SNP) conferring an N267I substitution in the cytoplasmic domain region of agrC not present in a high PVL producing ST22 strain TS14. Whole genome sequencing of these strains revealed them to be closely related to each other, differing by less than 200 SNPs across their core genomes. While not possessing an SCCmec element, they carried phages encoding pvl and the immune evasion complex (IEC) comprising staphylokinase, the chemotaxis inhibitory protein (CHIPS) and the staphylococcal complement inhibitor (SCIN). AgrC N267I reduced sensitivity to AIP in a bioluminescent reporter for agr which responds to but does not produce AIP. Introducing AgrC N267I into an agr reporter which both produces and senses AIP resulted in delayed autoinduction. This appeared to explain the delay in AIP autoinduction observed in TS13 as compared with TS14 with resulting low PVL production. Other naturally occurring agrC cytoplasmic mutations including T247I and I311T/A343T reduced AIP sensitivity and were associated with delayed autoinduction and reduced exotoxin production. PVL production is closely regulated by agr with expression mediated by several staphylococcal regulators in a cell population density dependent manner. Mutations in agrC occur naturally, delay autoinduction, can have a marked impact on exotoxin production and may be a form of adaptation to niches where high level agr expression is not required.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:632447 |
| Date | January 2014 |
| Creators | Sloan, Tim J. |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/14075/ |
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