Identification of Penicillin Binding Protein 4 (PBP4) as a critical factor for Staphylococcus aureus bone invasion during osteomyelitis in mice

Author summary Staphylococcus aureusis the most prevalent pathogen in osteomyelitis, and its infection of bone is difficult to cure.S.aureuscolonization of the osteocyte lacuno-canalicular network (OLCN) of cortical bone has been identified as a novel pathogenetic mechanism in chronic osteomyelitis. To elucidate factors involved in OLCN invasion, we conducted anin vitrogenetic screen that identifiedpbp4as a critical gene forS.aureuscell deformation and propagation through nanopores and demonstrated that PBP4 is critical for OLCN colonization in murine osteomyelitis. Thus, PBP4 inhibitors may be novel drugs to treat osteomyelitis in combination with standard of care antibiotics. Staphylococcus aureusinfection of bone is challenging to treat because it colonizes the osteocyte lacuno-canalicular network (OLCN) of cortical bone. To elucidate factors involved in OLCN invasion and identify novel drug targets, we completed a hypothesis-driven screen of 24S.aureustransposon insertion mutant strains for their ability to propagate through 0.5 mu m-sized pores in theMicrofluidicSiliconMembraneCanalicularArrays (mu SiM-CA), developed to modelS.aureusinvasion of the OLCN. This screen identified the uncanonicalS.aureustranspeptidase, penicillin binding protein 4 (PBP4), as a necessary gene forS.aureusdeformation and propagation through nanopores.In vivostudies revealed that Delta pbp4 infected tibiae treated with vancomycin showed a significant 12-fold reduction in bacterial load compared to WT infected tibiae treated with vancomycin (p<0.05). Additionally, Delta pbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Delta pbp4S.aureusfailed to invade and colonize the OLCN despite high bacterial loads on the implant and in adjacent tissues. Together, these results demonstrate that PBP4 is required forS.aureuscolonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN.