Comparative evaluation of small molecules reported to be inhibitors of Staphylococcus aureus biofilm formation.

IMPORTANCE: Because biofilm formation is such a problematic feature of Staphylococcus aureus infections, much effort has been put into identifying biofilm inhibitors. However, the results observed with these compounds are often reported in isolation, and the methods used to assess biofilm formation vary between labs, making it impossible to assess relative efficacy and prioritize among these putative inhibitors for further study. The studies we report address this issue by directly comparing putative biofilm inhibitors using a consistent in vitro assay. This assay was previously shown to maximize biofilm formation, and the results observed with this assay have been proven to be relevant in vivo. Of the 19 compounds compared using this method, many had no impact on biofilm formation under these conditions. Indeed, only one proved effective at limiting biofilm formation without also inhibiting growth.

Delineating the Role of the msaABCR Operon in Staphylococcal Overflow Metabolism.

Staphylococcus aureus is an important human pathogen that can infect almost every organ system, resulting in a high incidence of morbidity and mortality. The msaABCR operon is an important regulator of several staphylococcal phenotypes, including biofilm development, cell wall crosslinking, antibiotic resistance, oxidative stress, and acute and chronic implant-associated osteomyelitis. Our previous study showed that, by modulating murein hydrolase activity, the msaABCR operon negatively regulates the proteases that govern cell death. Here, we report further elucidation of the mechanism of cell death, which is regulated by the msaABCR operon at the molecular level in the USA300 LAC strain. We showed that deletion of msaABCR enhances weak-acid-dependent cell death, because, in the biofilm microenvironment, this mutant strain consumes glucose and produces acetate and acetoin at higher rates than wild-type USA300 LAC strain. We proposed the increased intracellular acidification leads to increased cell death. MsaB, a dual-function transcription factor and RNA chaperone, is a negative regulator of the cidR regulon, which has been shown to play an important role in overflow metabolism and programmed cell death during biofilm development in S. aureus. We found that MsaB binds directly to the cidR promoter, which represses expression of the cidR regulon and prevents transcription of the cidABC and alsSD operons. In addition, we observed that pyruvate induced expression of the msaABCR operon (MsaB). The results reported here have enabled us to decipher the role of the msaABCR operon in staphylococcal metabolic adaption during biofilm development.

The msaABCR Operon Regulates Persister Formation by Modulating Energy Metabolism in Staphylococcus aureus.

Staphylococcus aureus 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 msaABCR in several important staphylococcal phenotypes, including the formation of persister cells. Additionally, observations of the regulation of several metabolic genes by the msaABCR operon in transcriptomics and proteomics analyses have suggested its role in the metabolic activities of S. aureus. Given the importance of metabolism in persister formation as our starting point, in this study we demonstrated how the msaABCR operon regulates energy metabolism and subsequent antibiotic tolerance. We showed that deletion of the msaABCR operon results in increased tricarboxylic acid (TCA) cycle activity, accompanied by increased cellular ATP content and higher NADH content in S. aureus cells. We also showed that msaABCR (through MsaB) represses the ccpE and ndh 2 genes, thereby regulating TCA cycle activity and the generation of membrane potential, respectively. Together, the observations from this study led to the conclusion that msaABCR operon deletion induces a metabolically hyperactive state, leading to decreased persister formation in S. aureus.

The role of the msaABCR operon in implant-associated chronic osteomyelitis in Staphylococcus aureus USA300 LAC.

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.

The Impacts of msaABCR on sarA-Associated Phenotypes Are Different in Divergent Clinical Isolates of Staphylococcus aureus.

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.

Analysis of the CodY RNome reveals RsaD as a stress-responsive riboregulator of overflow metabolism in Staphylococcus aureus.

In Staphylococcus aureus, the transcription factor CodY modulates the expression of hundreds of genes, including most virulence factors, in response to the availability of key nutrients like GTP and branched-chain amino acids. Despite numerous studies examining how CodY controls gene expression directly or indirectly, virtually nothing is known about the extent to which CodY exerts its effect through small regulatory RNAs (sRNAs). Herein, we report the first set of sRNAs under the control of CodY. We reveal that staphylococcal sRNA RsaD is overexpressed >20-fold in a CodY-deficient strain in three S. aureus clinical isolates and in S. epidermidis. We validated the CodY-dependent regulation of rsaD and demonstrated that CodY directly represses rsaD expression by binding the promoter. Using a combination of molecular techniques, we show that RsaD posttranscriptionally regulates alsS (acetolactate synthase) mRNA and enzyme levels. We further show that RsaD redirects carbon overflow metabolism, contributing to stationary phase cell death during exposure to weak acid stress. Taken together, our data delineate a role for CodY in controlling sRNA expression in a major human pathogen and indicate that RsaD may integrate nutrient depletion and other signals to mount a response to physiological stress experienced by S. aureus in diverse environments.

The msaABCR Operon Regulates the Response to Oxidative Stress in Staphylococcus aureus.

Staphylococcus aureus has evolved a complex regulatory network that controls a multitude of defense mechanisms against the deleterious effects of oxidative stress stimuli, subsequently leading to the pathogen's survival and persistence in the hosts. Previously, we characterized the msaABCR operon as a regulator of virulence, antibiotic resistance, and the formation of persister cells in S. aureus Deletion of the msaABCR operon resulted in the downregulation of several genes involved in resistance against oxidative stress. Notably, those included carotenoid biosynthetic genes and the ohr gene, which is involved in resistance against organic hydroperoxides. These findings led us to hypothesize that the msaABCR operon is involved in resisting oxidative stress generated in the presence of both H2O2 and organic hydroperoxides. Here, we report that a protein product of the msaABCR operon (MsaB) transcriptionally regulates the expression of the crtOPQMN operon and the ohr gene to resist in vitro oxidative stresses. In addition to its direct regulation of the crtOPQMN operon and ohr gene, we also show that MsaB is the transcriptional repressor of sarZ (repressor of ohr). Taken together, these results suggest that the msaABCR operon regulates an oxidative stress defense mechanism, which is required to facilitate persistent and recurrent staphylococcal infections. Moving forward, we plan to investigate the role of msaABCR in the persistence of S. aureus under in vivo conditions.IMPORTANCE This study shows the involvement of the msaABCR operon in resisting oxidative stress by Staphylococcus aureus generated under in vitro and ex vivo conditions. We show that MsaB regulates the expression and production of a carotenoid pigment, staphyloxanthin, which is a potent antioxidant in S. aureus We also demonstrate that MsaB regulates the ohr gene, which is involved in defending against oxidative stress generated by organic hydroperoxides. This study highlights the importance of msaABCR in the survival of S. aureus in the presence of various environmental stimuli that mainly exert oxidative stress. The findings from this study indicate the possibility that msaABCR is involved in the persistence of staphylococcal infections and therefore could be a potential antimicrobial target to overcome recalcitrant staphylococcal infections.

Role of the msaABCR Operon in Cell Wall Biosynthesis, Autolysis, Integrity, and Antibiotic Resistance in Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen in both community and health care settings. One of the challenges with S. aureus as a pathogen is its acquisition of antibiotic resistance. Previously, we showed that deletion of the msaABCR operon reduces cell wall thickness, resulting in decreased resistance to vancomycin in vancomycin-intermediate S. aureus (VISA). In this study, we investigated the nature of the cell wall defect in the msaABCR operon mutant in the Mu50 (VISA) and USA300 LAC methicillin-resistant Staphylococcus aureus (MRSA) strains. Results showed that msaABCR mutant cells had decreased cross-linking in both strains. This defect is typically due to increased murein hydrolase activity and/or nonspecific processing of murein hydrolases mediated by increased protease activity in mutant cells. The defect was enhanced by a decrease in teichoic acid content in the msaABCR mutant. Therefore, we propose that deletion of the msaABCR operon results in decreased peptidoglycan cross-linking, leading to increased susceptibility toward cell wall-targeting antibiotics, such as ß-lactams and vancomycin. Moreover, we also observed significantly downregulated transcription of early cell wall-synthesizing genes, supporting the finding that msaABCR mutant cells have decreased peptidoglycan synthesis. More specifically, the msaABCR mutant in the USA300 LAC strain (MRSA) showed significantly reduced expression of the murA gene, whereas the msaABCR mutant in the Mu50 strain (VISA) showed significantly reduced expression of glmU, murA, and murD Thus, we conclude that the msaABCR operon controls the balance between cell wall synthesis and cell wall hydrolysis, which is required for maintaining a robust cell wall and acquiring resistance to cell wall-targeting antibiotics, such as vancomycin and the ß-lactams.

Creep, recovery, and stress relaxation behavior of nanostructured bioactive calcium phosphate glass-POSS/polymer composites for bone implants studied under simulated physiological conditions.

The creep and recovery and the stress relaxation behaviors of poly(butylene adipate-co-terephthalate) (PBAT) and polyhydroxyalkanoates (PHA) binary blends incorporating 30 wt % of a mixture of trisilanolisobutyl polyhedral oligomeric silsesquioxanes (POSS) and calcium phosphate glass (CaP-g) were investigated under simulated physiological and human body temperature conditions. The synergistic effect of PHA and CaP-g/POSS filler remarkably improved the creep behavior of the PBAT matrix and decreased its residual strain, consequently enhancing its elastic recovery. A considerable increase of the relaxation modulus of the hybrid materials was also observed upon incorporation of PHA and CaP-g/POSS. The relaxation modulus of the neat PBAT sample increased from ~60 MPa to ~1600 MPa after addition of 30 wt % CaP-g/POSS and 70 wt % PHA. However, after exposure of the composites to the simulated human body conditions for 14 days, a drop of dynamic mechanical properties of the studied material systems was observed along with formation of a desirable calcium phosphate phase on the material surface. The long-term (i.e., up to 7 × 105 s) viscoelastic behavior of the studied materials was successfully predicted using the time-temperature superposition principle and the obtained creep strain and the relaxation modulus master curves were satisfactorily fitted to the Findley power law equation and the generalized Maxwell model, respectively. This study demonstrates a facile method for tailoring CaP-g/POSS bioactive glasses composition for bone-like apatite formation on biopolymer surfaces. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2419-2432, 2019.

MsaB and CodY Interact To Regulate Staphylococcus aureus Capsule in a Nutrient-Dependent Manner.

Staphylococcus aureus has a complex regulatory network for controlling the production of capsule polysaccharide. In S. aureus, capsule production is controlled by several regulators in response to various environmental stimuli. Previously, we described MsaB as a new regulator that specifically binds to the cap promoter in a growth phase- or nutrient-dependent manner. In addition to MsaB, several other regulators have also been shown to bind the same region. In this study, we examined the interactions between MsaB and other nutrient-sensing regulators (CodY and CcpE) with respect to binding to the cap promoter in a nutrient-dependent manner. We observed that msaABCR and ccpE interact in a complex fashion to regulate capsule production. However, we confirmed that ccpE does not bind cap directly. We also defined the regulatory relationship between msaABCR and CodY. When nutrients (branched-chain amino acids) are abundant, CodY binds to the promoter region of the cap operon and represses its transcription. However, when nutrient concentrations decrease, MsaB, rather than CodY, binds to the cap promoter. Binding of MsaB to the cap promoter activates transcription of the cap operon. We hypothesize that this same mechanism may be used by S. aureus to regulate other virulence factors.IMPORTANCE Findings from this study define the mechanism of regulation of capsule production in Staphylococcus aureus Specifically, we show that two key regulators, MsaB and CodY, coordinate their functions to control the expression of capsule in response to nutrients. S. aureus fine-tunes the production of capsule by coordinating the activity of several regulators and by sensing nutrient levels. This study demonstrates the importance of incorporating multiple inputs prior to the expression of costly virulence factors, such as capsule.

msaABCR operon is involved in persister cell formation in Staphylococcus aureus.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Antimicrobial Peptide Mimicking Primary Amine and Guanidine Containing Methacrylamide Copolymers Prepared by Raft Polymerization.

Naturally occurring antimicrobial peptides (AMPs) display the ability to eliminate a wide variety of bacteria, without toxicity to the host eukaryotic cells. Synthetic polymers containing moieties mimicking lysine and arginine components found in AMPs have been reported to show effectiveness against specific bacteria, with the mechanism of activity purported to depend on the nature of the amino acid mimic. In an attempt to incorporate the antimicrobial activity of both amino acids into a single water-soluble copolymer, a series of copolymers containing lysine mimicking aminopropyl methacrylamide (APMA) and arginine mimicking guanadinopropyl methacrylamide (GPMA) were prepared via aqueous RAFT polymerization. Copolymers were prepared with varying ratios of the comonomers, with degree of polymerization of 35-40 and narrow molecular weight distribution to simulate naturally occurring AMPs. Antimicrobial activity was determined against Gram-negative and Gram-positive bacteria under conditions with varying salt concentration. Toxicity to mammalian cells was assessed by hemolysis of red blood cells and MTT assays of MCF-7 cells. Antimicrobial activity was observed for APMA homopolymer and copolymers with low concentrations of GPMA against all bacteria tested, with low toxicity toward mammalian cells.

msaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus.

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.

Identification and characterization of an operon, msaABCR, that controls virulence and biofilm development in Staphylococcus aureus.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Phage-bacterium war on polymeric surfaces: can surface-anchored bacteriophages eliminate microbial infections?

These studies illustrate synthetic paths to covalently attach T1 and Φ11 bacteriophages (phages) to inert polymeric surfaces while maintaining the bacteriophage's biological activities capable of killing deadly human pathogens. The first step involved the formation of acid (COOH) groups on polyethylene (PE) and polytetrafluoroethylene (PTFE) surfaces using microwave plasma reactions in the presence of maleic anhydride, followed by covalent attachment of T1 and Φ11 species via primary amine groups. The phages effectively retain their biological activity manifested by a rapid infection with their own DNA and effective destruction of Escherichia coli and Staphylococcus aureus human pathogens. These studies show that simultaneous covalent attachment of two biologically active phages effectively destroy both bacterial colonies and eliminate biofilm formation, thus offering an opportunity for an effective combat against multibacterial colonies as well as surface detections of other pathogens.