AUTHOR=Masters Elysia A. , Muthukrishnan Gowrishankar , Ho Lananh , Gill Ann Lindley , de Mesy Bentley Karen L. , Galloway Chad A. , McGrath James L. , Awad Hani A. , Gill Steven R. , Schwarz Edward M. 

TITLE=Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.723498

DOI=10.3389/fmicb.2021.723498

ISSN=1664-302X

ABSTRACT=<p><italic>Staphylococcus aureus</italic> invasion of the osteocyte lacuno-canalicular network (OLCN) is a novel mechanism of bacterial persistence and immune evasion in chronic osteomyelitis. Previous work highlighted <italic>S. aureus</italic> cell wall transpeptidase, penicillin binding protein 4 (PBP4), and surface adhesin, <italic>S. aureus</italic> surface protein C (SasC), as critical factors for bacterial deformation and propagation through nanopores <italic>in vitro</italic>, representative of the confined canaliculi <italic>in vivo</italic>. 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 <italic>S. aureus</italic> cell wall synthesis mutants (Δpbp3, Δatl, and ΔmreC) and surface adhesin mutants (ΔclfA and ΔsasC) for nanopore propagation <italic>in vitro</italic> and osteomyelitis pathogenesis <italic>in vivo</italic>. <italic>In vitro</italic> evaluation in the microfluidic silicon membrane-canalicular array (μSiM-CA) showed <italic>pbp3</italic>, <italic>atl</italic>, <italic>clfA</italic>, and <italic>sasC</italic> deletion reduced nanopore propagation. Using a murine model for implant-associated osteomyelitis, <italic>S. aureus</italic> cell wall synthesis proteins were found to be key modulators of <italic>S. aureus</italic> osteomyelitis pathogenesis, while surface adhesins had minimal effects. Specifically, deletion of <italic>pbp3</italic> and <italic>atl</italic> decreased septic implant loosening and <italic>S. aureus</italic> 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 <italic>pbp3</italic> deletion. Most notably, transmission electron microscopy (TEM) imaging of infected bone showed that <italic>pbp3</italic> was the only gene herein associated with decreased submicron invasion of canaliculi <italic>in vivo</italic>. Together, these results demonstrate that <italic>S. aureus</italic> cell wall synthesis enzymes are critical for OLCN invasion and osteomyelitis pathogenesis <italic>in vivo</italic>.</p>