RpiRc regulates RsbU to modulate eDNA-dependent biofilm formation and in vivo virulence of Staphylococcus aureus in a mouse model of catheter infection

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


Introduction
Biofilm is the most common mode of bacterial growth on medical devices and has also been reported RpiRc regulates RsbU to control eDNA amounts on mature biofilms 172 The regulation of eDNA amount in the biofilm of rpiRc mutant strains is not related to increased 173 induced autolysis ( Figure S4). Oxacillin induced autolysis also did not show differences for USA300 174 JE2 strain ( Figure S5), therefore suggesting another mechanism for eDNA regulation by RpiRc. SA113  (Table 1 and Tables S1 and S2), meaning that rpiRc may 182 regulate biofilm formation and eDNA through RsbU regulation. To confirm this hypothesis, rsbU was 183 deleted in UAMS-1 and SA564, wt and ΔrpiRc. Biofilm formation and eDNA amounts were measured 184 in these strains ( Figure 4). RsbU single deletion was responsible for moderate increased biofilm 185 formation in SA564 at 6h and 24h ( Figure 4B) as well as increased eDNA amounts at 6h and 24h for 186 SA564 ( Figure 4D) and 24h for UAMS-1 ( Figure 4C). rsbU deletion in rpiRc mutants resulted in a low 187 but significant biofilm formation increase at 6h for UAMS-1 ( Figure 4A) and at 24h for both strains 188 ( Figure 4A 198 RpiRc regulates the amount of eDNA on growing and mature biofilms but only affects mature biofilm   Figure S6). However no difference using 226 e-test could be observed with any of these antibiotics (Table S3).

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RpiRc regulates eDNA stability on growing and mature biofilms most probably by the regulation of an 228 extracellular nuclease. To test this hypothesis, both S. aureus extracellular nucleases nuc1 and nuc2 229 were deleted in SA564 wt and SA564 ΔrpiRc strains ( Figure S7 and  (Table 2). Some proteins identified did not match the selected 241 proteins weight analyzed (e.g around 35KDa). This is the case for SspB, Sbi, Atl and MecA. It is not 242 clear whether these proteins are more degraded or if they are over-produced (more peptides form 243 normal degradation found at lower weight). Three nucleases were also identified in the 244 supernatants, Nuc1, Sln and AdsA (Table 2). Nuc1, which was already tested by deletion and did not 245 reveal involved in rpiRc regulatory pathway, was over-produced in the wt supernatant, confirming 246 the deletion results ( Figure S7) and Nuc1 independent decreased eDNA amounts. Sln and AdsA are 247 over-produced in the mutant supernatant, suggesting one or both as potential targets for RpiRc 248 dependent eDNA regulation and biofilm formation. Sln had a weight of 33KDa while AdsA had a 249 weight of 83KDa (Table 2). For this reason, Sln was selected for further mutagenesis experiments.   Figure 10A). The amount of CFU recovered from the capsule showed more variation mostly for the 324 mutant strain with an inoculum at 1.10 4 ( Figure 10B). No differences could be observed statistically 325 significant between wt and mutant strain in the catheter and in the capsule. The amount of CFU/mL 326 recovered for mutant strains in the capsule between inoculum 1.10 4 and 2.10 5 is not statistically 327 different but present a trend for decreased CFU recovery with inoculum 2.10 5 . Further investigation 328 would be required to confirm any increased capsule colonization of USA300 JE2 ΔrpiRc strain with an 329 inoculum of 10 4 CFU. USA300 JE2 wt strain requires a minimum inoculum of 10 4 CFU to colonize host 330 tissues ( Figure 10C). The number of mice with this inoculum is low but the higher amount of mice 331 with an inoculum of 6.10 4 confirms the ability of USA300 wt to colonize tissues with a mean 1.10 3 332 CFU/mg. The mutant strain is also able to colonize host tissues but requires a higher inoculum. No 333 CFU counting is available with an inoculum of 10 3 . The mutant strain is not able to colonize host 334 tissues with an inoculum of 10 4 in contrary to wt strain ( Figure 10C). Mutant strain requires an 335 inoculum of 2.10 5 CFU to colonize the tissue (significant difference between 10 4 and 2.10 5 , p < 0.01). rather than eDNA production. G6P is also required for glycolysis as well as for Glc-N-6-P production.

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This molecule is then metabolized into UDP-Glc-NAc which is required for peptidoglycan production 355 but is also a precursor of the PIA (77
574    1220 Figure S10. Sequence of USA300 JE2 ΔrpiRc Nat7. Native rpiRc sequence with a RBS was cloned 1221 within the pRAB11 plasmid following method described in the M&M section of the manuscript.
1222 Table S1. Standard error of the mean for qRT-PCR 1223