The role of the msaABCR operon in implant-associated chronic osteomyelitis in Staphylococcus aureus USA300 LAC.
ABSTRACT: BACKGROUND:The msaABCR operon regulates several staphylococcal phenotypes such as biofilm formation, capsule production, protease production, pigmentation, antibiotic resistance, and persister cells formation. The msaABCR operon is required for maintaining the cell wall integrity via affecting peptidoglycan cross-linking. The msaABCR operon also plays a role in oxidative stress defense mechanism, which is required to facilitate persistent and recurrent staphylococcal infections. Staphylococcus aureus is the most frequent cause of chronic implant-associated osteomyelitis (OM). The CA-MRSA USA300 strains are predominant in the United States and cause severe infections, including bone and joint infections. RESULTS:The USA300 LAC strain caused significant bone damage, as evidenced by the presence of severe bone necrosis with multiple foci of sequestra and large numbers of multinucleated osteoclasts. Intraosseous survival and biofilm formation on the K-wires by USA300 LAC strains was pronounced. However, the msaABCR deletion mutant was attenuated. We observed minimal bone necrosis, with no evidence of intramedullary abscess and/or fibrosis, along reduced intraosseous bacterial population and significantly less biofilm formation on the K-wires by the msaABCR mutant. microCT analysis of infected bone showed significant bone loss and damage in the USA300 LAC and complemented strain, whereas the msaABCR mutant's effect was reduced. In addition, we observed increased osteoblasts response and new bone formation around the K-wires in the bone infected by the msaABCR mutant. Whole-cell proteomics analysis of msaABCR mutant cells showed significant downregulation of proteins, cell adhesion factors, and virulence factors that interact with osteoblasts and are associated with chronic OM caused by S. aureus. CONCLUSION:This study showed that deletion of msaABCR operon in USA300 LAC strain lead to defective biofilm in K-wire implants, decreased intraosseous survival, and reduced cortical bone destruction. Thus, msaABCR plays a role in implant-associated chronic osteomyelitis by regulating extracellular proteases, cell adhesions factors and virulence factors. However additional studies are required to further define the contribution of msaABCR-regulated molecules in osteomyelitis pathogenesis.
Project description:The staphylococcal accessory regulator (sarA) plays an important role in Staphylococcus aureus infections, including osteomyelitis, and the msaABCR operon has been implicated as an important factor in modulating expression of sarA Thus, we investigated the contribution of msaABCR to sarA-associated phenotypes in the S. aureus clinical isolates LAC and UAMS-1. Mutation of msaABCR resulted in reduced production of SarA and a reduced capacity to form a biofilm in both strains. Biofilm formation was enhanced in a LAC msa mutant by restoring the production of SarA, but this was not true in a UAMS-1 msa mutant. Similarly, extracellular protease production was increased in a LAC msa mutant but not a UAMS-1 msa mutant. This difference was reflected in the accumulation and distribution of secreted virulence factors and in the impact of extracellular proteases on biofilm formation in a LAC msa mutant. Most importantly, it was reflected in the relative impact of mutating msa as assessed in a murine osteomyelitis model, which had a significant impact in LAC but not in UAMS-1. In contrast, mutation of sarA had a greater impact on all of these in vitro and in vivo phenotypes than mutation of msaABCR, and it did so in both LAC and UAMS-1. These results suggest that, at least in osteomyelitis, it would be therapeutically preferable to target sarA rather than msaABCR to achieve the desired clinical result, particularly in the context of divergent clinical isolates of S. aureus.
Project description:Persister cells comprise a phenotypic variant that shows extreme antibiotic tolerance resulting in treatment failures of bacterial infections. While this phenomenon has posed a great threat in public health, mechanisms underlying their formation in Staphylococcus aureus remain largely unknown. Increasing evidences of the presence of persister cells in recalcitrant infections underscores the great urgency to unravel the mechanism by which these cells develop. Previously, we characterized msaABCR operon that plays roles in regulation of virulence, biofilm development and antibiotic resistance. We also characterized the function of MsaB protein and showed that MsaB is a putative transcription factor that binds target DNA in response to nutrients availability.In this study, we compared the number of persister cell in wild type, msaABCR deletion mutant and the complemented strain in two backgrounds USA300 LAC and Mu50. Herein, we report that msaABCR deletion mutant forms significantly less number of persister cells relative to wild type after challenge with various antibiotics in planktonic and biofilm growth conditions. Complementation of the msaABCR operon restored wild type phenotype. Combined antibiotic therapy along with msaABCR deletion significantly improves the killing kinetics of stationary phase and biofilm S. aureus cells. Transcriptomics analysis showed that msaABCR regulates several metabolic genes, transcription factors, transporters and enzymes that may play role in persister cells formation, which we seek to define in the future.This study presented a new regulator, msaABCR operon, that is involved in the persister cells formation, which is a poorly understood in S. aureus. Indeed, we showed that msaABCR deletion significantly reduces the persister cells formation in all growth phases tested. Although, we have not yet defined the mechanism, we have shown that msaABCR regulates several metabolic, transporters, and extracellular proteases genes that have been previously linked with persister cells formation in other bacterial systems. Taken together, this study showed that inactivation of the msaABCR operon enhances the effectiveness of antibiotics for the treatment of S. aureus infections, especially in context of persister cells.
Project description:We used a murine model of acute, posttraumatic osteomyelitis to evaluate the virulence of two divergent Staphylococcus aureus clinical isolates (the USA300 strain LAC and the USA200 strain UAMS-1) and their isogenic sarA mutants. The results confirmed that both strains caused comparable degrees of osteolysis and reactive new bone formation in the acute phase of osteomyelitis. Conditioned medium (CM) from stationary-phase cultures of both strains was cytotoxic to cells of established cell lines (MC3TC-E1 and RAW 264.7 cells), primary murine calvarial osteoblasts, and bone marrow-derived osteoclasts. Both the cytotoxicity of CM and the reactive changes in bone were significantly reduced in the isogenic sarA mutants. These results confirm that sarA is required for the production and/or accumulation of extracellular virulence factors that limit osteoblast and osteoclast viability and that thereby promote bone destruction and reactive bone formation during the acute phase of S. aureus osteomyelitis. Proteomic analysis confirmed the reduced accumulation of multiple extracellular proteins in the LAC and UAMS-1 sarA mutants. Included among these were the alpha class of phenol-soluble modulins (PSMs), which were previously implicated as important determinants of osteoblast cytotoxicity and bone destruction and repair processes in osteomyelitis. Mutation of the corresponding operon reduced the cytotoxicity of CM from both UAMS-1 and LAC cultures for osteoblasts and osteoclasts. It also significantly reduced both reactive bone formation and cortical bone destruction by CM from LAC cultures. However, this was not true for CM from cultures of a UAMS-1 psm? mutant, thereby suggesting the involvement of additional virulence factors in such strains that remain to be identified.
Project description:Staphylococcus aureus is an important human pathogen that causes nosocomial and community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host's immune response and antimicrobial agents. Biofilm development is very complex and involves several regulators that ensure cell survival on surfaces within the extracellular polymeric matrix. Previously, we identified the msaABCR operon as an additional positive regulator of biofilm formation. In this study, we define the regulatory pathway by which msaABCR controls biofilm formation. We demonstrate that the msaABCR operon is a negative regulator of proteases. The control of protease production mediates the processing of the major autolysin, Atl, and thus regulates the rate of autolysis. In the absence of the msaABCR operon, Atl is processed by proteases at a high rate, leading to increased cell death and a defect in biofilm maturation. We conclude that the msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm.
Project description:INTRODUCTION:In bone and joint infections (BJIs), bacterial toxins are major virulence factors: Panton-Valentine leukocidin (PVL) expression leads to severe local damage, including bone distortion and abscesses, while ?-hemolysin (Hla) production is associated with severe sepsis-related mortality. Recently, other toxins, namely phenol-soluble modulins (PSMs) expressed by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA300 (LAC WT) were shown to have ex vivo intracellular cytotoxic activity after S. aureus invasion of osteoblasts, but their in vivo contribution in a relatively PVL-sensitive osteomyelitis model remains poorly elucidated. MATERIALS AND METHODS:We compared the outcomes of experimental rabbit osteomyelitises induced with pvl+hla+psms+ LAC WT and its isogenic ?psm derivatives (LAC ?psm? and LAC ?psm??hld) using an inoculum of 3 × 108 CFUs. Mortality, hematogenous spread (blood culture, spleen and kidney), lung and bone involvements were assessed in two groups (non-survivors of severe sepsis and survivors sacrificed on day (D) 14). RESULTS:Severe sepsis-related mortality tended to be lower for ?psm derivatives (Kaplan-Meier curves, P = .06). Non-survivors' bone LAC-?psm? (6.9 log10 CFUs/g of bone, P = .04) or -?psm??hld (6.86 log10 CFUs/g of bone, P = .014) densities were significantly higher than LAC WT (6.43 log10 CFUs/g of bone). Conversely, lung ?psm??hld CFUs were significantly lower than LAC WT (P = .04). LAC ?psm?, ?psm??hld and WT induced similar bone damage in D14 survivors, with comparable bacterial densities (respectively: 5.89, 5.91, and 6.15 log10 CFUs/g of bone). Meanwhile, pulmonary histological scores of inflammation were significantly higher for LAC ?psm?- and ?psm??hld-infected rabbits compared to LAC WT (P = .04 and .01, respectively) but with comparable lung bacterial densities. CONCLUSION:Our experimental results showed that deactivating PSM peptides significantly limited bacterial dissemination from bone during the early phase of infection, but did not affect local severity of USA300 rabbit osteomyelitis.
Project description:Biofilms complicate treatment of Staphylococcus aureus (SA) wound infections. Previously, we determined alpha-toxin (AT)-promoted SA biofilm formation on mucosal tissue. Therefore, we evaluated SA wound isolates for AT production and biofilm formation on epithelium and assessed the role of AT in biofilm formation. Thirty-eight wound isolates were molecularly typed by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (ST), and spa typing. We measured biofilm formation of these SA isolates in vitro and ex vivo and quantified ex vivo AT production. We also investigated the effect of an anti-AT monoclonal antibody (MEDI4893*) on ex vivo biofilm formation by methicillin-resistant SA (USA 300 LAC) and tested whether purified AT rescued the biofilm defect of hla mutant SA strains. The predominant PFGE/ST combinations were USA100/ST5 (50%) and USA300/ST8 (33%) for methicillin-resistant SA (MRSA, n = 18), and USA200/ST30 (20%) for methicillin-susceptible SA (MSSA, n = 20). Ex vivo AT production correlated significantly with ex vivo SA wound isolate biofilm formation. Anti-alpha-toxin monoclonal antibody (MEDI4893*) prevented ex vivo biofilm formation by MRSA USA300 strain LAC. Wild-type AT rescued the ex vivo biofilm defect of non-AT producing SA strains. These findings provide evidence that AT plays a role in SA biofilm formation on epithelial surfaces and suggest that neutralization of AT may be useful in preventing and treating SA infections.
Project description:The emergence of serious infections due to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has fueled interest in the contributions of specific staphylococcal virulence factors to clinical disease. To assess the contributions of agr-dependent factors to the fate of organisms in polymorphonuclear neutrophils (PMN), we examined the consequences for organism and host cells of feeding PMN with wild-type CA-MRSA (LAC) or CA-MRSA (LAC agr KO) at different multiplicities of infection (MOIs). Phagocytosed organisms rapidly increased the transcription of RNAIII in a time- and MOI-dependent fashion; extracellular USA300 (LAC) did not increase RNAIII expression despite having the capacity to respond to autoinducing peptide-enriched culture medium. HOCl-mediated damage and intracellular survival were the same in the wild-type and USA300 (LAC agr KO). PMN lysis by ingested USA300 (LAC) was time- and MOI-dependent and, at MOIs >1, required ?-hemolysin (hla) as USA300 (LAC agr KO) and USA300 (LAC hla KO) promoted PMN lysis only at high MOIs. Taken together, these data demonstrate activation of the agr operon in human PMN with the subsequent production of ?-hemolysin and PMN lysis. The extent to which these events in the phagosomes of human PMN contribute to the increased morbidity and mortality of infections with USA300 (LAC) merits further study.
Project description:The relative impact of 23 mutations on biofilm formation was evaluated in the USA300, methicillin-resistant strain LAC. Mutation of sarA, atl, codY, rsbU, and sigB limited biofilm formation in comparison to the parent strain, but the limitation imposed by mutation of sarA was greater than that imposed by mutation of any of these other genes. The reduced biofilm formation of all mutants other than the atl mutant was correlated with increased levels of extracellular proteases. Mutation of fur- and mgrA-enhanced biofilm formation but in LAC had no impact on protease activity, nuclease activity, or accumulation of the polysaccharide intercellular adhesin (PIA). The increased capacity of these mutants to form a biofilm was reversed by mutation of sarA, and this was correlated with increased protease production. Mutation of sarA, mgrA, and sigB had the same phenotypic effect in the methicillin-sensitive strain UAMS-1, but mutation of codY increased rather than decreased biofilm formation. As with the UAMS-1 mgrA mutant, this was correlated with increased production of PIA. Examination of four additional clinical isolates suggests that the differential impact of codY on biofilm formation may be a conserved characteristic of methicillin-resistant versus methicillin-sensitive strains.
Project description:Osteomyelitis is a common manifestation of invasive Staphylococcus aureus infection. Pathogen-induced bone destruction limits antimicrobial penetration to the infectious focus and compromises treatment of osteomyelitis. To investigate mechanisms of S. aureus-induced bone destruction, we developed a murine model of osteomyelitis. Microcomputed tomography of infected femurs revealed that S. aureus triggers profound alterations in bone turnover. The bacterial regulatory locus sae was found to be critical for osteomyelitis pathogenesis, as Sae-regulated factors promote pathologic bone remodeling and intraosseous bacterial survival. Exoproteome analyses revealed the Sae-regulated protease aureolysin as a major determinant of the S. aureus secretome and identified the phenol-soluble modulins as aureolysin-degraded, osteolytic peptides that trigger osteoblast cell death and bone destruction. These studies establish a murine model for pathogen-induced bone remodeling, define Sae as critical for osteomyelitis pathogenesis, and identify protease-dependent exoproteome remodeling as a major determinant of the staphylococcal virulence repertoire.
Project description:Bone infections are a significant public health burden associated with morbidity and mortality in patients. Microbial biofilm pathogens are the causative agents in chronic osteomyelitis. Research on the pathogenesis of osteomyelitis has focused on indirect bone destruction by host immune cells and cytokines secondary to microbial insult. Direct bone resorption by biofilm pathogens has not yet been seriously considered. In this study, common osteomyelitis pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans) were grown as biofilms in multiple in vitro and ex vivo experiments to analyze quantitative and qualitative aspects of bone destruction during infection. Pathogens were grown as single or mixed species biofilms on the following substrates: hydroxyapatite, rat jawbone, or polystyrene wells, and in various media. Biofilm growth was evaluated by scanning electron microscopy and pH levels were monitored over time. Histomorphologic and quantitative effects of biofilms on tested substrates were analyzed by microcomputed tomography and quantitative cultures. All tested biofilms demonstrated significant damage to bone. Scanning electron microscopy indicated that all strains formed mature biofilms within 7 days on all substrate surfaces regardless of media. Experimental conditions impacted pH levels, although this had no impact on biofilm growth or bone destruction. Presence of biofilm led to bone dissolution with a decrease of total volume by 20.17±2.93% upon microcomputed tomography analysis, which was statistically significant as compared to controls (p <0.05, ANOVA). Quantitative cultures indicated that media and substrate did not impact biofilm formation (Kruskall-Wallis test, post-hoc Dunne's test; p <0.05). Overall, these results indicate that biofilms associated with osteomyelitis have the ability to directly resorb bone. These findings should lead to a more complete understanding of the etiopathogenesis of osteomyelitis, where direct bone resorption by biofilm is considered in addition to the well-known osteoclastic and host cell destruction of bone.