Elucidating the Mechanisms of S. aureus Motility in Bone and Developing Interventions

Staphylococcus aureus is involved in 80% of all musculoskeletal infections (MSKI) costing $17,000–$150,000
per patient. Approximately 50% of these infections are caused by methicillin-resistant S. aureus (MRSA)
acquired in both hospital and community. With >1.5 million total joint replacements (TJR) performed each
year, the most rigorous prophylaxis and aseptic surgical techniques cannot reduce osteomyelitis (OM) rates
below 0.5%–2%. Treating established MSKI remains extremely challenging, with current rates of recurrent or
persistent infection following revision surgery still as high as 33%. The persistence of S. aureus infection is
attributed to its arsenal of immune evasion and antimicrobial resistance mechanisms. Despite great efforts to
develop solutions, treatment paradigms have not improved the poor clinical outcomes for OM patients over the
last four decades. However, our CoRTOBI paradigm-shifting discovery of S. aureus colonization of the
osteocyte lacuno-canalicular network (OLCN) of live cortical bone during OM in mice and patients may explain
why previous approaches for treating recurring bone infections have failed, and provide a new therapeutic
strategy for eliminating chronic OM. It also begs important questions about the mechanisms that: 1) enable
spherical S. aureus to deform into submicron-rod shaped bacteria to invade the OLCN, and 2) render
susceptible S. aureus strains refractory to antibiotics after OLCN invasion. Over the past four years we
developed a novel bone infection-on-chip utilizing silicon nanomembrane with submicron (~500 nm) array of
pores to simulate OLCN orifices (µSiM-CA). By targeted deletion of candidate genes, we identified cell wall
transpeptidase proteins, penicillin binding protein 4 (Pbp4), as essential for S. aureus propagation through
submicron channels of the µSiM-CA chips in vitro and then demonstrated that they inhibit OLCN colonization in
vivo. Moreover, we developed and performed a high throughput screening campaign to identify PBP4 inhibitors
(iPBP4). In this renewal, we will first demonstrate the efficacy of PBP4 small molecule inhibitors (iPBP4) in
abrogating the OLCN invasion in mouse models of osteomyelitis. We will then identify targets for OM therapy
based on gene expression changes that affords S. aureus adaptive tolerance to antibiotics in a novel µSiM-
OLCN Chip platform. Finally, we will test the premise that OLCN colonization likely involves many additional
factors other than PBP4, and that other chemical classes of OLCN colonization inhibitors can be identified by
empirically defining the genetic determinants. These potential targets can then be used to identify
corresponding putative therapeutics in a single screening approach. At the completion of this renewal program,
CoRTOBI will have: 1) validated recently discovered iPBP4 candidates and potentially new PBP-independent
hits against OLCN colonization, 2) a molecular genetic understanding of S. aureus refractory response to
antibiotics following OLCN colonization, and 3) translational methods for iPBP4 impregnated 3D-printed
scaffolds in one-stage revision surgery for bone infections.

Grant Number: 5P50AR072000-09
NIH Institute/Center: NIH
Principal Investigator: Hani Awad