Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis

Abstract

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.

Antibacterial, Antibiofilm, and Antiviral Farnesol-Containing Nanoparticles Prevent Staphylococcus aureus from Drug Resistance Development

“…The interaction of the FSL NPs with the Gram-positive bacterium and the changes in the cell morphology and structure of the cells were further assessed using SEM ( Figure 3 B). S. aureus appear as smooth spheres with size ranging from 500 nm to 1 µm in diameter [ 30 ]. The exposure of the bacteria to empty NPs did not alter the bacterial cell morphology and the cell wall structure remained intact, similar to the control (non-treated bacteria).…”

Role of Animal Models to Advance Research of Bacterial Osteomyelitis

“…This finding suggests that implant microarchitecture is incredibly important and may offer bacteria a submicron reservoir to evade clearance by the immune system. The work of Masters et al ( 66 ) expanded upon these findings by investigating the role of S. aureus cell wall synthesis machinery and surface adhesins in OLCN invasion. The authors established a model of bacterial osteomyelitis by placing stainless steel pins inoculated with various mutant strains of S. aureus into the medial tibia of mice.…”

Osteocytes Serve as a Reservoir for Intracellular Persisting Staphylococcus aureus Due to the Lack of Defense Mechanisms

“…Numerous antimicrobials cannot penetrate and reach adequate concentrations for bactericidal efficacy in bone compared to soft tissue (Thompson and Townsend, 2011;Tuchscherr et al, 2016;Alder et al, 2020). Furthermore, some bacteria internalize and survive within bone cells for a long period of time, which contributes to insufficient treatments (Tuchscherr et al, 2016;De Mesy Bentley et al, 2018;Yang et al, 2018;Muthukrishnan et al, 2019;Masters et al, 2021). Access to the intracellular environment protects this pathogen from the action of the host immune response and antimicrobials, promoting persistence (Tuchscherr et al, 2011;Horn et al, 2018).…”

“…Although different studies have described that S. aureus can survive for long periods within osteoblasts (Kalinka et al, 2014;Ji et al, 2020;Bongiorno et al, 2021;Gunn et al, 2021;Marro et al, 2021), the mechanisms that may explain the persistence within bone cells are still unknown. In addition to osteoblasts, several studies have revealed osteocytes as possible niche for bacteria within bone tissue (De Mesy Bentley et al, 2018;Yang et al, 2018;Krauss et al, 2019;Masters et al, 2021). Recently, the persistence of S. aureus in bone tissue was associated with its capacity to invade the osteocyte lacunocanalicular network (OLCN; Masters et al, 2021).…”

“…In addition to osteoblasts, several studies have revealed osteocytes as possible niche for bacteria within bone tissue (De Mesy Bentley et al, 2018;Yang et al, 2018;Krauss et al, 2019;Masters et al, 2021). Recently, the persistence of S. aureus in bone tissue was associated with its capacity to invade the osteocyte lacunocanalicular network (OLCN; Masters et al, 2021). However, the mechanisms that contribute to the development of S. aureus persistence and immune evasion in chronic osteomyelitis are still unknown.…”