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

Abstract

Staphylococcus aureus infection 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 24 S. aureus transposon insertion mutant strains for their ability to propagate through 0.5 μm-sized pores in the Microfluidic Silicon Membrane Canalicular Arrays (μSiM-CA), developed to model S. aureus invasion of the OLCN. This screen identified the uncanonical S. aureus transpeptidase, penicillin binding protein 4 (PBP4), as a necessary gene for S. aureus deformation and propagation through nanopores. In vivo studies revealed that Δpbp4 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, Δpbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Δpbp4 S. aureus failed 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 for S. aureus colonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN.

Distinct vasculotropic versus osteotropic features ofS. agalactiaeversusS. aureusimplant‐associated bone infection in mice

“…All animal studies were performed in accordance with protocols approved by the University Committee on Animal Resources at the University of Rochester Medical Center and in accordance with the Animal Welfare Act. Tibial pin infection was performed as described previously 35,36,38,39 . Briefly, 6‐week‐old, female Balb/C mice were purchased from Jackson Laboratories and housed five per cage within their experimental group in two‐way housing on a 12‐h light/dark cycle.…”

“…Following 1‐week acclimatization, mice were anesthetized before surgery with xylazine (12 mg/kg) and ketamine (130 mg/kg) administered intraperitoneally and given preoperative slow‐release buprenorphine (0.5–1.0 mg/kg) administered subcutaneously by URMC veterinary staff. Pins were cut at 4 mm in length from stainless‐steel wire with a cross‐section of 0.2 mm × 0.5 mm (MicroDyne Technologies) and bent into an l ‐shaped implant as previously described 39 . The stainless‐steel pins were first sterilized, then inoculated by being submerged within an overnight culture of either USA300 or COH1 for 20 min at room temperature and then left to air dry for 5 min, resulting in an inoculation of approximately 5.0 × 105 CFU/ml.…”

“…X‐ray imaging was performed at Day 0 and 14 post‐infection using a Faxitron (Hologic, Marlborough, MA). Radiographs were used to characterize septic implant loosening where the number of pins completely dislodged from the bone was quantified relative to the number of pins stably fixed in the bone within each experimental group, as previously described 39 . Following sacrifice on Day 14, the infected tibiae, trans‐tibial implants and soft tissue were harvested and processed for CFU quantification or microcomputed tomography (µCT) imaging, followed by histology and transmission electron microscopy (TEM).…”

“…CFU quantification was performed as previously described 39 . Infected tibiae, transtibial implants and adjacent soft tissue were harvested, placed in sterile phosphate‐buffered saline (PBS) and tissues were homogenized using an IKA T‐10 handheld homogenizer.…”

“…µCT imaging and analysis were performed as previously described 39 . Infected tibiae were imaged ex vivo by µCT in a VivaCT 40 (Scanco Medical) with a 10.5 µm isotropic voxel size, using an integration time of 300 ms, energy of 55 kV, and intensity of 145 µA ( n = 5).…”