Introduction
Staphylococcus aureus is one of the main adaptable human pathogens, able to infect every organ and damage tissues, causing severe infections and able to resist antibiotics, in particular beta-lactams and methicillin. It is one of the main pathogens responsible for recurrent osteomyelitis (OM), accounting for more than 50%, and prosthetic joint infections (PJIs) (Wu et al ., 2019; Tuchscherret al ., 2016). PJIs can have a dramatic impact on a patient’s quality of life, often requiring surgical intervention and prosthesis removal, as well as prolonged antibiotics treatment (Stefani et al. , 2012; Purrello et al ., 2016; Moore et al ., 2017). These infections are often due to Healthcare-Associated Methicillin-Resistant S. aureus (HA-MRSA) belonging to Clonal Complex 5 (Peng et al., 2019; Muñoz-Gallego et al ., 2017; Perez-Montarelo et al. , 2018), and associated with Staphylococcal Chromosomal Cassette (SCCmec ) I and III (Hussain et al , 2009). Among them, ST239-III is probably the oldest pandemic MRSA clone, first discovered 1970, isolated in most countries all over the word and the most diffused in Europe and Italy (Moneke et al., 2018; Campanile et al. , 2015; Szymanek-Majchrzak et al ., 2018). The ST228-SCCmec I is one of the most diffused clones in Italy, associated with nosocomial infection (Campanile et al., 2012; Bongiorno et al., 2018).
The assessment of the interaction between S. aureus isolates and osteoblasts during PJIs and OM foresees three crucial events: adhesion, invasion and post-invasion, during which S. aureus controls the expression of adhesion and virulence determinants, with its large armamentarium of regulatory genes.
Many studies have been carried out in this field. Different authors have shown the role of many regulators involved in the invasion and adaptation to host tissue. The role of sig B in persistence and stress response, together with its link to sar A and consequently to its action on the agr locus was studied in osteoblasts, in anin-vivo model, using two different strains of S. aureus , in particular, the authors used the wild type and the defective strain for agr , sig B and sarA (Tuchscherr et al ., 2015). In fact, the global regulatory system - agr locus - cell density-dependent controls virulence factor expression. Through its effector molecule RNAIII, the agr locus controls the post-transcriptional regulation of proteins involved in cell-surface interaction and virulence cytotoxic factors (Paiter et al ., 2014).
Two other genes belonging to the SARA protein family are involved in the regulation of virulence genes: sarS (SarH1), whose expression is repressed by sar A and agr , is a repressor of hlaand etb and is a positive regulator of spa , androt , the “repressor of toxin”, it is a repressor of enterotoxin B (seb ) and alpha-toxin (hla ), and it is repressed by theagr effector RNAIII and SarA (Jenul and Horswill, 2018).
In the process of adhesion, cell surface proteins (adhesins) of the Microbial Surface Component Recognizing Adhesive Matrix Molecules (MSCRAMM), play an important role in the pathogenesis of osteoarticular (OM and PJ) infections. Among these, fibronectin binding proteins A and B (FnbA/B), fibrinogen‐binding protein clumping factors A and B (ClfA/B), the bone sialoprotein binding protein (Bbp), the collagen-adhesin (CNA) (Perez-Montarelo et al., 2018; Otsukaet al. , 2006) and SdrE, a serine–aspartate (SD) protein that anchors the cell-wall interacting with complement Factor H and facilitates colonization through adherence to the cell surface or extracellular matrix (ECM) components (Herr and Thorman, 2017).
S. aureus can invade endothelial cells and osteoblasts using cell surface integrin α5β1, binding Fn on the surface of human cells. In particular, as already demonstrated, FnBPA and FnBPB are involved not only in adhesion but also in internalization (Shnji et a l., 2011). Pore-forming proteins, such as Panton-Valentine leukocidine (PVL) and α- and δ-hemolysin (Hla and Hld) together with phenol-soluble modulins (PSMs), were able to induce local complications such as bone deformation, systemic complications such as severe sepsis in rabbit osteomyelitis, or neutrophil and osteoblast cytotoxicity in an ex vivo model. The hla gene was frequently present in strains associated with osteoarticular bacteremia (Perez-Montarelo et al. , 2018). PSMs are small peptides with amphipathic properties that destabilize the lipid bilayer, this activity is related to receptor-independent cytotoxicity to osteoblasts and specialized cells such as neutrophils. PSMs are also implicated in biofilm formation, in bacterial interference and in cell-cycle disruptions (Davido et al. , 2016).
S. aureus, during the infection process, is able to use alternative carbon sources and, in particular, Glucose-6-phosphate (G6P); the uptake of this alternative carbon source is possible using the hexose phosphate antiporter UhpT (Yang et al. , 2016).
After having observed that the interaction, internalization and persistence during an osteo-blast ex-vivo infection is a strain-dependent process, we selected two strains belonging to different genetic backgrounds showing a preliminary different ability to internalize as a model of infection in order to study how they differentially adapt strategies to react to changing environmental conditions and how they adjust their virulence factor expression at the different times of infection inside the MG-63 cell line.