The accessory gene regulator (agr) and the staphylococcal accessory regulator (sar) are central regulatory elements that control the production of Staphylococcus aureus virulence factors. To date, the functions of these loci have been defined almost exclusively using RN6390, which is representative of the laboratory strain 8325-4. However, RN6390 was recently shown to have a mutation in rsbU that results in a phenotype resembling that of a sigB mutant (I. Kullik et al., J. Bacteriol. 180:4814-4820, 1998). For that reason, it remains unclear whether the regulatory events defined in RN6390 are representative of the events that take place in clinical isolates of S. aureus. To address this issue, we generated mutations in the sarA and agr loci of three laboratory strains (RN6390, Newman, and S6C) and four clinical isolates (UAMS-1, UAMS-601, DB, and SC-1). Mutation of sarA in the cna-positive strains UAMS-1 and UAMS-601 resulted in an increased capacity to bind collagen, while mutation of agr had little impact. Northern blot analysis confirmed that the increase in collagen binding was due to increased cna transcription. Without exception, mutation of sarA resulted in increased production of proteases and a decreased capacity to bind fibronectin. Mutation of agr had the opposite effect. Although mutation of sarA resulted in a slight reduction in fnbA transcription, changes in the ability to bind fibronectin appeared to be more directly correlated with changes in protease activity. Lipase production was reduced in both sarA and agr mutants. While mutation of sarA in RN6390 resulted in reduced hemolytic activity, it had the opposite effect in all other strains. There appeared to be reduced levels of the sarC transcript in RN6390, but there was no difference in the overall pattern of sar transcription or the production of SarA. Although mutation of sarA resulted in decreased RNAIII transcription, this effect was not evident under all growth conditions. Taken together, these results suggest that studies defining the regulatory roles of sarA and agr by using RN6390 are not always representative of the events that occur in clinical isolates of S. aureus.Staphylococcus aureus is an opportunistic pathogen capable of causing a wide variety of infections. Its pathogenic diversity is due to its ability to produce a diverse array of virulence factors. These factors fall into two groups based on whether they remain associated with the cell surface or are exported into the extracellular milieu. In vitro, these two groups are globally and inversely regulated, with the surface proteins being produced during the exponential growth phase and the exoproteins being produced as cultures enter postexponential growth (33). This is consistent with the fact that the production of S. aureus virulence factors is responsive to a quorum-sensing signal (27). This signal exerts its regulatory effects through a two-component signal transduction system encoded by the accessory gene regulator (agr). Induction of agr results in increased expression of...
New methacrylate monomers containing pendant quaternary ammonium moieties based on 1,4-diazabicyclo-[2.2.2]-octane (DABCO) were synthesized. The DABCO group contains either a butyl or a hexyl pendant group comprising the hydrophobic segment of the monomers and one tether group to the methacrylate moiety. The monomers were homopolymerized in water by using 2,2Ј-azobis(2-methylpropionamide) dihydrochloride (V-50) as an initiator. The monomers and polymers were characterized by elemental analysis, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), FTIR, and 13 C-NMR. The antimicrobial activities of the corresponding small molecules (bis-quaternary ammonium monocarboxylates) and polymers were investigated against Staphylococcus aureus and Escherichia coli. Although the small molecules did not show any antimicrobial activity, the polymers were moderately effective against both Gram-positive and Gramnegative bacteria. The minimum inhibitory concentration (MIC) values of the polymers with butyl and hexyl hydrocarbon chains against S. aureus and E. coli were found to be 250 and 62.5 g/mL, respectively. The minimum bactericidal concentration (MBC) value for the polymer with the butyl group was higher than 1 mg/mL, whereas the MBC value for the polymer with hexyl group was found to be 62.5 g/mL. Thus, an increase of the alkyl chain length from 4 to 6 significantly increased the antimicrobial activity of the polymer.
BackgroundCommunity-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.ResultsTo 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.ConclusionThese 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.
Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.
We have developed a bioluminescent whole-cell biosensor that can be incorporated into biofilm ecosystems. RM4440 is a Pseudomonas aeruginosa FRD1 derivative that carries a plasmid-basedrecA-luxCDABE fusion. We immobilized RM4440 in an alginate matrix to simulate a biofilm, and we studied its response to UV radiation damage. The biofilm showed a protective property by physical shielding against UV C, UV B, and UV A. Absorption of UV light by the alginate matrix translated into a higher survival rate than observed with planktonic cells at similar input fluences. UV A was shown to be effectively blocked by the biofilm matrix and to have no detectable effects on cells contained in the biofilm. However, in the presence of photosensitizers (i.e., psoralen), UV A was effective in inducing light production and cell death. RM4440 has proved to be a useful tool to study microbial communities in a noninvasive manner.
New water-soluble methacrylate polymers with pendant quaternary ammonium (QA) groups were synthesized and used as antibacterial materials. The polymers with pendant QA groups were obtained by the reaction of the alkyl halide groups of a previously synthesized functional methacrylate homopolymer with various tertiary alkyl amines containing 12-, 14-, or 16-carbon alkyl chains. The structures of the functional polymer and the polymers with QA groups were confirmed with Fourier transform infrared and 1 H and 13 C NMR. The degree of conversion of alkyl halides to QA sites in each polymer was determined by 1 H NMR to be over 90% in all cases. The number-average molecular weight and polydispersity of the functional polymer were determined by size exclusion chromatography to be 32,500 g/mol and 2.25, respectively. All polymers were thermally stable up to 180 8C according to thermogravimetric analysis. The antibacterial activities of the polymers with pendant QA groups against Staphylococcus aureus and Escherichia coli were determined with broth-dilution and spread-plate methods. All the polymers showed excellent antibacterial activities in the range of 32-256 lg/mL. The antibacterial activity against S. aureus increased with an increase in the alkyl chain length for the ammonium groups, whereas the antibacterial activity against E. coli decreased with increasing alkyl chain length.
We recently demonstrated that mutation of sarA in clinical isolates of Staphylococcus aureus results in a phenotype that is distinct by comparison to sarA mutants generated in the laboratory strain RN6390 (J. S. Blevins, K. E. Beenken, M. O. Elasri, B. K. Hurlburt, and M. S. Smeltzer, Infect. Immun. 70: [470][471][472][473][474][475][476][477][478][479][480] 2002). This raises the possibility that studies demonstrating that RN6390 sarA mutants are attenuated do not accurately reflect the role of sarA in the pathogenesis of staphylococcal disease. To test this hypothesis, we used a murine model of musculoskeletal infection to assess the virulence of sarA and agr mutants generated in a clinical isolate of S. aureus (UAMS-1). By using this model, we confirmed that mutation of sarA and/or agr results in a reduced capacity to cause both septic arthritis and osteomyelitis.The accessory gene regulator (agr) and the staphylococcal accessory regulator (sar) are the two best-characterized loci responsible for modulating the expression of Staphylococcus aureus virulence factors (27). Mutation of either locus has been shown to result in attenuation of S. aureus in several models of staphylococcal disease (1,5,9,11,19,25). In most cases, characterization of these loci and evaluation of their role in pathogenesis were done with laboratory strain RN6390 and mounting evidence suggests that the regulatory events defined by using strain RN6390 are not representative of the events observed in clinical isolates of S. aureus (3, 10). Specifically, while RN6390 sarA mutants exhibit reduced hemolytic activity, mutation of sarA in other strains results in elevated hemolytic activity. Although sarA mutants generated in one such strain (DB) had reduced virulence in animal models of staphylococcal septic arthritis and endocarditis (11,25), neither DB nor RN6390 encodes the collagen binding adhesin (cna) and therefore neither binds collagen (20). This is relevant because the ability to bind collagen has also been associated with virulence in septic arthritis, osteomyelitis, and endocarditis models (14,21,31), and we have demonstrated that mutation of sarA results in elevated transcription of cna and an enhanced capacity to bind collagen (3,4,20).Recent reports have also identified several genotypic and phenotypic markers that appear to be characteristic of the most prominent S. aureus clinical isolates (6, 30). Included among these are the presence of cna and the absence of one of the two genes (fnbB) that encode fibronectin binding adhesins. These isolates also have a phenotype defined by a high binding capacity for host proteins and a relatively low level of exoprotein expression. Importantly, none of these characteristics have been observed in RN6390 (3,20). In addition, RN6390 was recently shown to have a deletion in rsbU, which encodes a positive regulator of the stress response sigma factor SigB (16,18,23). This is relevant because mutation of sigB results in reduced sar transcription and a reduced capacity to produce SarA (2, 17), a...
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