MsaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus.
ABSTRACT: 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: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:Peptidoglycan (PG) and wall teichoic acid (WTA) are the major staphylococcal cell wall components, and WTA biosynthesis has recently been explored for drug development. Targocil is a novel agent that targets the TarG subunit of the WTA translocase (TarGH) that transports WTA across the membrane to the wall. Previously we showed that targocil treatment of a methicillin-susceptible Staphylococcus aureus strain led to a rapid shut down of cellular autolysis. Targocil II, which targets the TarH subunit of TarGH, also resulted in a drastic decrease in autolysis. Here, we address the mechanism of targocil-mediated decreased autolysis. The mechanism is WTA dependent since targocil treatment decreased autolysis in methicillin-resistant strains but not in a WTA-deficient mutant. Similar to cellular autolysis, autolysin-retaining crude cell walls isolated from targocil-treated cells had vastly decreased autolytic activity compared to those from untreated cells. Purified cell walls from control and targocil-treated cells, which lack autolytic activity, were similarly susceptible to lysozyme and lysostaphin and had similar O-acetyl contents, indicating that targocil treatment did not grossly alter PG structure and chemistry. Purified cell walls from targocil-treated cells were highly susceptible to autolysin extracts, supporting the notion that targocil treatment led to decreased autolysin in the crude cell walls. Quantitative real-time PCR analysis revealed that the decrease in autolysis in the targocil-exposed cells was not due to transcriptional repression of the autolysin genes atl, lytM, lytN, and sle1 Zymographic analysis of peptidoglycan hydrolase profiles showed a deficiency of cell surface autolysins in targocil-treated cells but higher activity in cell membrane fractions. Here, we propose that the untranslocated WTA molecules in the targocil-exposed cells sequester Atl at the membrane, resulting in significantly decreased autolysis.
Project description:Sodium houttuyfonate (SH), an addition compound of sodium bisulfite and houttuynin, showed in vitro antibacterial activity against 21 Staphylococcus aureus (S. aureus) strains grown in planktonic cultures. Microarray results showed decreased levels of autolysin atl, sle1, cidA and lytN transcripts in the SH-treated strain as compared to the control strain, consistent with the induction of the autolytic repressors lrgAB and sarA and with the downregulation of the positive regulators agrA and RNAIII. Triton X-100-induced autolysis was significantly decreased by SH in S. aureus ATCC 25923, and quantitative bacteriolytic assays and zymographic analysis demonstrated SH-mediated reduction of extracellular murein hydrolase activity in these cells. Anti-biofilm assay showed that SH is poorly active against S. aureus grown in biofilm cultures, whereas SH diminished the amounts of extracellular DNA (eDNA) of S. aureus in a dose-dependent manner, which suggested that SH may impede biofilm formation by reducing the expression of cidA to inhibit autolysis and eDNA release in the early phase. Some of the microarray results were confirmed by real-time RT-PCR.
Project description:<i>Staphylococcus aureus</i> is a major human pathogen that causes chronic, systemic infections, and the recalcitrance of these infections is mainly due to the presence of persister cells, which are a bacterial subpopulation that exhibits extreme, yet transient, antibiotic tolerance accompanied by a transient halt in growth. However, upon cessation of antibiotic treatment, a resumption in growth of persister cells causes recurrence of infections and treatment failure. Previously, we reported the involvement of <i>msaABCR</i> in several important staphylococcal phenotypes, including the formation of persister cells. Additionally, observations of the regulation of several metabolic genes by the <i>msaABCR</i> operon in transcriptomics and proteomics analyses have suggested its role in the metabolic activities of <i>S. aureus</i>. Given the importance of metabolism in persister formation as our starting point, in this study we demonstrated how the <i>msaABCR</i> operon regulates energy metabolism and subsequent antibiotic tolerance. We showed that deletion of the <i>msaABCR</i> operon results in increased tricarboxylic acid (TCA) cycle activity, accompanied by increased cellular ATP content and higher NADH content in <i>S. aureus</i> cells. We also showed that <i>msaABCR</i> (through MsaB) represses the <i>ccpE</i> and <i>ndh</i> <sub>2</sub> genes, thereby regulating TCA cycle activity and the generation of membrane potential, respectively. Together, the observations from this study led to the conclusion that <i>msaABCR</i> operon deletion induces a metabolically hyperactive state, leading to decreased persister formation in <i>S. aureus</i>.
Project description:Vancomycin-intermediate Staphylococcus aureus (VISA) strains present an increasingly difficult problem in terms of public health. However, the molecular mechanism for this resistance is not yet understood. In this study, we define the role of the msaABCR operon in vancomycin resistance in three clinical VISA strains, i.e., Mu50, HIP6297, and LIM2. Deletion of the msaABCR operon resulted in significant decreases in the vancomycin MIC (from 6.25 to 1.56 ?g/ml) and significant reductions of cell wall thickness in strains Mu50 and HIP6297. Growth of the mutants in medium containing vancomycin at concentrations greater than 2 ?g/ml resulted in decreases in the growth rate, compared with the wild-type strains. Mutation of the msaABCR operon also reduced the binding capacity for vancomycin. We conclude that the msaABCR operon contributes to resistance to vancomycin and cell wall synthesis in S. aureus.
Project description:Streptococcus suis serotype 2 (S. suis 2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated atl and encoding a major autolysin of S. suis 2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular mass of 113 kDa and has a conserved N-acetylmuramoyl-l-alanine amidase domain. Recombinant Atl was expressed in Escherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the atl mutant was reduced ?30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the atl mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801.
Project description:Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.
Project description:Community-acquired, methicillin-resistant Staphylococcus aureus strains often cause localized infections in immunocompromised hosts, but some strains show enhanced virulence leading to severe infections even among healthy individuals with no predisposing risk factors. The genetic basis for this enhanced virulence has yet to be determined. S. aureus possesses a wide variety of virulence factors, the expression of which is carefully coordinated by a variety of regulators. Several virulence regulators have been well characterized, but others have yet to be thoroughly investigated. Previously, we identified the msa gene as a regulator of several virulence genes, biofilm development, and antibiotic resistance. We also found evidence of the involvement of upstream genes in msa function.To investigate the mechanism of regulation of the msa gene (renamed msaC), we examined the upstream genes whose expression was affected by its deletion. We showed that msaC is part of a newly defined four-gene operon (msaABCR), in which msaC is a non-protein-coding RNA that is essential for the function of the operon. Furthermore, we found that an antisense RNA (msaR) is complementary to the 5' end of the msaB gene and is expressed in a growth phase-dependent manner suggesting that it is involved in regulation of the operon.These findings allow us to define a new operon that regulates fundamental phenotypes in S. aureus such as biofilm development and virulence. Characterization of the msaABCR operon will allow us to investigate the mechanism of function of this operon and the role of the individual genes in regulation and interaction with its targets. This study identifies a new element in the complex regulatory circuits in S. aureus, and our findings may be therapeutically relevant.