Elucidation of the role of icaR and rob regulation for mucoid Staphylococcus aureus isolates, which do not carry the 5 bp-deletion in the intergenic region of the ica operon

• Applicant: Dr. Bianca Schwartbeck
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 521973082

Recently, we described that during the persistence of Staphylococcus aureus in the airways of people with cystic fibrosis (pwCF), mucoid isolates emerged from normal ones and that they carried a 5bp-deletion (TATTT; 5bp-del) within the intergenic region (IGR) of the intercellular adhesion operon (icaADBC). This operon synthesizes polysaccharide intercellular adhesin (PIA) when not repressed by icaR by binding to a 42nt-sequence upstream of the start codon of icaA. The start codons of icaR and icaA are separated by a 164nt-long intergenic region (IGR). While Jefferson et al. revealed that IcaR does not bind to the TATTT sequence, Yu et al. reported that the new identified “repressor of biofilm” (ROB) binds to an approximately 25nt-long sequence within the IGR that contains this TATTT motif, and thus, also represses the expression of icaADBC. In our DFG funded prospective pilot study, we identified the 5bp-del within the IGR of the ica operon in mucoid isolates from 6 of 7 individuals, while in one patient the 5bp-del was not detected. Just recently we finished a multicenter study, in which we analyzed isolates from pwCF from 13 centers (12 in Germany, 1 in Austria). In specimens of 41 of 435 pwCF (9.43%), mucoid isolates were identified. In 3 individuals with mucoid isolates, no 5bp-del could be identified and also in mucoid isolates of 2 pwCF with long-term S. aureus infection from the CF cohort in Münster (4 and 18 years). The entire ica operon of these isolates was investigated by Sanger sequencing. First results show that these mucoid isolates without the 5bp-del carried various mutations in icaR. All mutations lead to a frame shift and to a premature stop codon and a much shorter protein. Furthermore, we identified mucoid isolates with mutations in rob. The understanding of how the ica locus and PIA biosynthesis are regulated on the molecular level by ROB and IcaR is still far from complete. With our project, we plan: 1. to investigate the physical interaction between IcaR and ROB as well as with the IGR. Based on the results of mutations found in icaR and rob of our clinical mucoid S. aureus isolates, we hypothesize that (i) both regulators interact with each other, (ii) both proteins are needed for repression and (iii) that the supposed binding sites of the two repressors are longer than suggested. 2. to use different environmental set-ups to find different conditions that might trigger the emergence of mutations leading to mucoidy. 3. to investigate the host-pathogen interaction of mucoid S. aureus in contact with host cells to reveal the overall virulence of mucoid isolates in comparison to the wild-type normal S. aureus. 4. to provide a systematic pheno- and genotypic analyses of mucoid S. aureus on Congo Red agar to facilitate identification of mucoid variants in the routine microbial diagnostics and for research.

• DFG Programme: Research Grants