Staphylococcus aureus invasion of the osteocyte lacuno-canalicular network (OLCN) is a novel mechanism of bacterial persistence and immune evasion in chronic osteomyelitis. Previous work highlighted S. aureus cell wall transpeptidase, penicillin binding protein 4 (PBP4), and surface adhesin, S. aureus surface protein C (SasC), as critical factors for bacterial deformation and propagation through nanopores in vitro, representative of the confined canaliculi in vivo. Given these findings, we hypothesized that cell wall synthesis machinery and surface adhesins enable durotaxis- and haptotaxis-guided invasion of the OLCN, respectively. Here, we investigated select S. aureus cell wall synthesis mutants (Δpbp3, Δatl, and ΔmreC) and surface adhesin mutants (ΔclfA and ΔsasC) for nanopore propagation in vitro and osteomyelitis pathogenesis in vivo. In vitro evaluation in the microfluidic silicon membrane-canalicular array (μSiM-CA) showed pbp3, atl, clfA, and sasC deletion reduced nanopore propagation. Using a murine model for implant-associated osteomyelitis, S. aureus cell wall synthesis proteins were found to be key modulators of S. aureus osteomyelitis pathogenesis, while surface adhesins had minimal effects. Specifically, deletion of pbp3 and atl decreased septic implant loosening and S. aureus abscess formation in the medullary cavity, while deletion of surface adhesins showed no significant differences. Further, peri-implant osteolysis, osteoclast activity, and receptor activator of nuclear factor kappa-B ligand (RANKL) production were decreased following pbp3 deletion. Most notably, transmission electron microscopy (TEM) imaging of infected bone showed that pbp3 was the only gene herein associated with decreased submicron invasion of canaliculi in vivo. Together, these results demonstrate that S. aureus cell wall synthesis enzymes are critical for OLCN invasion and osteomyelitis pathogenesis in vivo.

Arrayed imaging reflectometry (AIR) is an optical biosensor platform for simple, multiplex measurement of antigenspecific antibody responses in patient blood samples. Here, we report the development of StaphAIR, an 8-plex Staphylococcus aureus antigen array on the AIR platform for profiling antigenspecific anti-S. aureus humoral immune responses. Initial validation experiments with mouse and humanized monoclonal antibodies against the S. aureus autolysin glucosaminidase (Gmd) domain, and subsequent testing with dilution series of pooled positive human serum confirmed analytically robust behavior of the array, with all antigens displaying Langmuir-type dose−response curves. Testing a cohort of 82 patients with S. aureus musculoskeletal infections (MSKI) and 30 healthy individuals enabled discrimination of individual patient responses to different S. aureus antigens, with statistical significance between osteomyelitis patients and controls obtained overall for four individual antigens (IsdA, IsdB, Gmd, and SCIN). Multivariate analyses of the antibody titers obtained from StaphAIR revealed its utility as a potential diagnostic tool for detecting S. aureus MSKI (area under the receiver operating characteristic curve (AUC) > 0.85). We conclude that StaphAIR has utility as a high-throughput immunoassay for studying and diagnosing osteomyelitis in patients.