RpiRc regulates RsbU to modulate eDNA-dependent biofilm formation andin vivovirulence ofStaphylococcus aureusin a mouse model of catheter infection

Abstract

15Staphylococcus aureus is a major human pathogen. Despite high incidence and morbidity, molecular 16 mechanisms occurring during infection remain largely unknown. Under defined conditions, biofilm 17 formation contributes to the severity of S. aureus related infections. Extracellular DNA (eDNA), a 18 component of biofilm matrix released from apoptotic bacteria, is involved in biofilm structure and 19 stability. In many bacterial biofilms, eDNA originates from cell lysis although eDNA can also be 20 actively secreted or exported by bacterial membrane vesicles. By screening the Nebraska transposon 21 library, we identified rpiRc as a biofilm regulator involved in eDNA regulation. RpiRc is a transcription 22 factor from the pentose phosphate pathway (PPP) whose product is a polysaccharide intercellular 23 adhesin (PIA) precursor. However, rpiRc mutant strain showed neither susceptibility to DispersinB® (a 24 commercially available enzyme disrupting PIA biofilms) nor alteration of ica transcription (the operon 25 regulating PIA production). Decreased biofilm formation was linked to Sln, an extracellular compound 26 degrading eDNA in an autolysis independent pathway. Biofilm susceptibility to antibiotics in wt and 27 mutant strains was tested using a similar protocol as the Calgary biofilm device. Involvement of RpiRc 28 in S. aureus virulence was assessed ex vivo by internalization experiments into HEK293 cells and in 29 vivo in a mouse model of subcutaneous catheter infection. While minimum inhibitory concentrations 30 (MICs) of planktonic cells were not affected in the mutant strain, we observed increased biofilm 31 susceptibility to almost all tested antibiotics, regardless of their mode of action. More importantly, 32 the rpiRc mutant showed reduced virulence in both ex vivo and in vivo experiments related to 33 decreased fnbpA-B transcription and eDNA production. RpiRc is an important regulator involved in 34 eDNA degradation inside the matrix of mature PIA independent biofilms. These results illustrate that 35 RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also to S. aureus 36 cell adhesion and virulence during subcutaneous infection. 37 38 3 39 Author summary 40 Biofilm formation contributes to the severity of Staphylococcus aureus related infections. Biofilm 41 matrix is mainly composed by polysaccharide intercellular adhesion (PIA), proteins and extracellular 42 DNA (eDNA). By screening a mutant library of S. aureus, RpiRc was identified as a new regulator of 43 eDNA dependent biofilm formation. How RpiRc regulates biofilm and its role in S. aureus virulence 44 was studied in four different S. aureus strains. Deletion of RpiRc resulted in a pronounced decreased 45 eDNA dependent biofilm formation, but not PIA dependent biofilm formation. Decreased biofilm 46 formation was not related to increased autolysis, but was linked to extracellular compounds found in 47 the supernatant of mutant biofilms. Sln was identified as one of this compound. RpiRc deletion also 48 decreased...