Analysis of the association between polymorphisms in intergenic regions of Staphylococcus aureus genes involved in biofilm formation and periprosthetic joint infections

Abstract

In this thesis, we have focused on studying variants found in IGRs adjacent to the most important genes involved in S. aureus biofilm formation; the icaADBCR locus, and the genes encoding the family of surface adhesins. For this purpose, we sequenced the whole genome of a collection of 71 S. aureus isolates from periprosthetic joint infections (PJI) and wound infections stored at the Clinical Bacteriological Laboratory of the Sahlgrenska University Hospital and at the Culture Collection University of Gothenburg (CCUG), respectively. In the first chapter, we explored the regulatory regions of the icaADBCR locus to identify patterns that might be associated with an increased capacity of the isolates to produce PIA/PNAG and form a biofilm. This study compared the regulatory regions of the icaADBCR locus in the genomes of PJI and wound isolates with those in the genome of the reference strain MW2. From these analyses, strains were grouped based on the SNPs found in the IGRs of the operon and also within the coding region of the transcriptional regulator IcaR. These regions showed high conservation rates, and no pattern associated with the origin of the isolates, either PJI or wounds, was detected. On the other hand, using transcriptional fusions between the regulatory region of the icaADBCR locus and the green fluorescent protein gene (gfp), we demonstrated that the expression of icaADBC genes was not affected by the presence of variations in IGRs. Notably, a SNP within the coding region of icaR, which results in an amino acid change in the transcriptional repressor IcaR V176E, led to a significant increase in the transcription of the icaADBC operon and the production of PIA/PNAG. Using a Galleria mellonella infection model, we were able to demonstrate a significant reduction in S. aureus virulence associated with the increase in PIA/PNAG production. In the second chapter, we focused on analyzing the association between SNPs in the promoter regions of genes encoding adhesion-related proteins with adhesins expression levels and therefore, the ability of the strain to adhere to medical devices. Genome analyses of PJI and wound isolates showed different profiles in the content of adhesin-encoding genes. Some of these, such as sasG and cna, were lineage-associated, and fifteen genes were present in the whole collection of strains. When the variability in the SNPs contained in regulatory regions that control the expression of each adhesin was investigated, different variation rates were found among the isolates. Following the same approach as in chapter I, based on transcriptional fusions between regulatory regions and the gfp gene, results showed that each genetic lineage contained a specific profile of adhesins expression under the same environmental condition. Moreover, we developed a biomaterial-associated murine infection model together with a metagenomic analysis to simultaneously compare the capacity of different S. aureus isolates to colonize medical implants. In summary, our results evidenced that SNPs in the IGRs flanking the genes encoding factors important for biofilm development may contribute to the generation of variability in the capacity of S. aureus to colonize medical implants. In particular, our results revealed that IGRs controlling the expression of the icaADBC locus and production of the PIA/PNAG exopolysaccharide are highly conserved and that very few silent SNPs can be detected between strains. On the contrary, SNPs in the IGRs of genes encoding surface adhesins provide a profile of proteins expression that is specific for each S. aureus clonal complex (CC). Altogether, these studies emphasize the importance of investigating the potential impact of SNPs inside IGRs on gene expression and specific bacterial traits, such as pathogen colonization success.