Staphylococcal Accessory Gene Regulator Research Articles

Pathogenic role of the staphylococcal accessory gene regulator quorum sensing system in atopic dermatitis.

The skin is home to various bacteria, archaea, fungi, and viruses, collectively referred to as the skin microbiota. Patients with certain skin diseases reportedly have unique skin "dysbiosis," a condition involving imbalanced microbiota, suggesting that dysbiosis in the skin may be either causal or a consequence of specific skin diseases. Atopic dermatitis (AD) is the most common allergic skin disease that affects 15-20% of children and 2-10% of adults worldwide. Both intrinsic genetic factors, such as susceptibility to type 2 inflammation or skin barrier dysfunction, and extrinsic environmental factors, such as air pollen and skin microbiota, contribute to AD. Staphylococcus aureus, which does not often colonize the skin of healthy individuals, is commonly identified in the lesional skin of patients with AD and is correlated with the disease flare. However, the role of S. aureus in the pathogenesis of AD has not been elucidated. Here, we discuss the pathological behavior of S. aureus, focusing on accessory gene regulator (Agr) quorum sensing, which is a fundamental bacterial cell-to-cell interaction mechanism that affects the behavior of S. aureus and other members of the microbial community. Importantly, beyond bacteria-bacteria interactions, the Agr quorum sensing system also regulates various virulence factors, which induce type 2 and IL-17-dependent skin inflammation in the host. Furthermore, the colonization of Agr-positive S. aureus in early life accelerates the development of pediatric AD. Finally, we aim to highlight the current efforts to establish novel therapeutic methods to ameliorate or prevent AD through Agr-targeted intervention.

Subinhibitory Concentrations of Fusidic Acid May Reduce the Virulence of S. aureus by Down-Regulating sarA and saeRS to Reduce Biofilm Formation and α-Toxin Expression

Staphylococcus aureus is an important pathogen in hospital and community infections. Fusidic acid is particularly effective in treating skin and wound infections caused by staphylococci. The purpose of our study was to clarify the effect of fusidic acid on the biofilm formation and α-toxin expression of S. aureus at subinhibitory concentrations [1/64, 1/32, and 1/16 × minimum inhibitory concentration (MIC)]. A total of 504 genes greater than a twofold or less than twofold change in expression of S. aureus effected by subinhibitory concentrations of fusidic acid were found, including 232 up-regulated genes and 272 down-regulated genes, which were determined by transcriptome sequencing. Our results showed subinhibitory concentrations of fusidic acid significantly inhibited the expression of hla, spa, icaA, and cidA at the mRNA level in clinical S. aureus strains tested. And subinhibitory concentrations of fusidic acid can significantly reduce the hemolysis activity and α-toxin production of S. aureus. In addition, the subinhibitory concentrations of fusidic acid significantly inhibited biofilm formation, autolysis, cell aggregation, and polysaccharide intercellular adhesin (PIA) production of S. aureus. Moreover, fusidic acid effectively reduces the damage of mouse skin lesion area. Furthermore, fusidic acid reduced the expression of the two-component regulatory system saeRS and staphylococcal accessory gene regulator (sarA). In conclusion, our results suggested that the subinhibitory concentrations of fusidic acid may reduce the virulence of S. aureus by down-regulating sarA and saeRS to reduce biofilm formation and α-toxin expression, which will provide a theoretical basis for the clinical treatment of S. aureus infection. This is the first report that fusidic acid has an inhibitory effect on the virulence of S. aureus, and this broadens the clinical application of fusidic acid.

Structure-Activity Relationship Studies of Small Molecule Modulators of the Staphylococcal Accessory Gene Regulator.

The accessory gene regulator (agr) quorum-sensing system is arguably the most important regulator of Staphylococcus virulence. The agr-system serves a crucial role in pathogenesis by triggering substantive gene expression alterations to up-regulate the production of a wide variety of virulence determinants such as exoenzymes (proteases, lipases, nucleases) and downregulate the expression of surface binding proteins. Accordingly, the agr-system represents a compelling target for the development of antivirulence therapeutics as potential adjuncts, or alternatives, to conventional bactericidal and bacteriostatic antibiotics. Despite this potential, to date, no agr-system inhibitors have progressed to the clinic; however, several promising lead compounds have been identified through screens of synthetic and natural product libraries. On the basis of the molecular components within the agr-system, the current contingent of regulating compounds can be clustered into three broad groups, AgrA-P3 activation inhibitors, AgrB-AgrD processing inhibitors, and AgrC-AIP interaction inhibitors. This review aims to provide an overview of the development, structure-activity-relationships, and limitations of compounds within each of these groups in addition to the current opportunities for developing next-generation anologs.

Biofilm production, quorum sensing system and analysis of virulence factors of Staphylococcus epidermidis collected from sheep milk samples

Intramammary infections are a serious problem in dairy sheep farming, and Staphylococcus epidermidis is one of the main etiological agents of ovine mastitis. In this work, 199 S. epidermidis isolates collected in Sardinia (Italy) from sheep milk samples and used for the preparation of inactivated autogenous vaccines were characterized with phenotypic and genotypic methods. The potential for biofilm production was evaluated phenotypically by assessing colony characteristics on Congo Red Agar (CRA), and genotypically by PCR for i) the icaA/D genes coding the polysaccharide intercellular adhesin (PIA) and ii) for the bhp, aap and embp genes coding biofilm accumulation proteins. Isolates were also investigated by PCR for the presence of autolysins (atlE and aae genes), microbial surface components recognizing adhesive matrix molecules (MSCRAMMs, sdrG and sdrF genes), enterotoxins (sea, seb, sec, sed and see genes), and the toxic shock syndrome toxin (tsst genes). In addition, the staphylococcal accessory gene regulator (agr), the most important locus responsible for the regulation of virulence factors, was characterized for the first time. As a result, only two isolates showed biofilm production on CRA and harbored the icaA/D genes. Four isolates, phenotypically classified as non-biofilm producers, possessed the icaA/D genes (n = 3) or only the icaA gene (n = 1). All isolates harbored the atlE, aae, embp and sdrG genes, but none harbored the genes encoding enterotoxins and tsst. Most isolates belonged to the agr-3se group (56.3%), followed by agr-1se (41.2%) and agr-2se (2.5%). All six isolates that harbored ica genes belonged to agr-1se.In conclusion, this study provides a detailed characterization of the putative virulence genes present in S. epidermidis strains circulating in Sardinian sheep. Because of their role in ovine mastitis pathogenesis, these might represent valuable candidates for vaccine development.

Regulation of saeRS, agrA and sarA on sasX Expression in Staphylococcus aureus

Background: The spread of Staphylococcus aureus and the types of infection caused by S. aureus are closely related to the secretion of a variety of adhesion proteins, which could be controlled by a variety of regulatory systems. However, for the newly discovered adhesion protein SasX, the regulatory mechanism is not completely clear. Objectives: The current study aimed at investigating the regulation of Staphylococcal accessory gene regulator A (agrA), Staphylococcal accessory regulator A (sarA), and two-component signal transduction system (saeRS) on the adhesion protein SasX. Methods: In this research, a saeRS mutant strain, a sarA mutant strain, and a agrA mutant strain were constructed by allelic replacement. In this study mRNA and protein expression levels of sasX in wild-type HS770 and knockout strains were studied to investigate the effects of regulatory factor saeRS, agrA, and sarA on adhesion protein SasX. Results: In contrast with the wild strain HS770, the transcriptional expression of sasX was highest at on the sixth hour time point in HS770ΔagrA and at nine and twelve hours in HS770ΔsarA. However, the sasX transcription level in HS770ΔsaeRS mutant strains had little change at different time points. Western-blot results suggested that the sasX expression level of wild strains was the highest at 6 hours; HS770ΔsaeRS mutation strains had no expression peak at 6 hours. The expression level of HS770ΔagrA mutant strains decreased at 6 hours of expression, however, increased at 9 hours and 12 hours; the expression level of HS770ΔsarA mutation knockout increased at three, six, nine, and twelve hours. Conclusions: All the results showed that agrA and sarA have negative regulation on sasX, but saeRS may not regulate sasX.

RNAIII suppresses the expression of LtaS via acting as an antisense RNA in Staphylococcus aureus.

RNAIII is known as the key effector of staphylococcal accessory gene regulator (agr) quorum-sensing system, which plays a central role in the pathogenesis of Staphylococcus aureus. As a regulatory RNA, RNAIII regulates multiple targets, including exoproteins and cell-wall-associated proteins. Lipoteichoic acid synthase (LtaS) is involved in the synthesis of lipoteichoic acid (LTA) that is one of the major components of cell wall. The chemical compound targeting to LtaS decreases S. aureus growth via blocking LTA production. Until now, the regulatory mechanism of LtaS expression is still not clear. The level of ltaS mRNA in S. aureus is analyzed by semi-quantitative RT-PCR analysis and qRT-PCR. The protein level of LtaS is determined by Western blotting. The putative interaction sites between RNAIII and LtaS mRNA are predicted. And LtaS-5ºUTR-lacZ and LtaS-5ºUTR-mutant-lacZ reporter vectors are constructed according to the putative interaction sites. Our data show that the expression of ltaS is regulated by RNAIII in S. aureus. The level of LtaS is significantly higher in the RNAIII deficient strain compared to its parent strain. In the further investigation, 5ºUTR of ltaS was predicted to be the putative interaction site of RNAIII. The results of detection of β-galactosidase activities suggest that RNAIII can inhibit the expression level of LtaS through acting on the 5ºUTR region of LtaS mRNA. Our finding presents that LtaS is another target of RNAIII and RNAIII suppresses the expression of LtaS via acting as an antisense RNA in S. aureus.

Rot and Agr system modulate fibrinogen-binding ability mainly by regulating clfB expression in Staphylococcus aureus NCTC8325

Staphylococcus aureus is an important human pathogen that causes a variety of diseases, ranging from localized skin infections to life-threatening systemic infections. The success of S. aureus as a pathogen is partly due to its ability to adhere to a wide range of host tissues by binding to host extracellular matrix proteins such as fibrinogen, fibronectin, and collagen. Staphylococcus aureus expresses two proteins that can bind specifically to fibrinogen, clumping factors A and B (ClfA and ClfB). Repressor of toxins (Rot) is known to be a global regulator of virulence gene expression in S. aureus. The translation of Rot is regulated by the staphylococcal accessory gene regulator (Agr) quorum-sensing system. In this study, we demonstrated that Rot and the Agr system in S. aureus NCTC8325 can affect the bacterial binding ability to human fibrinogen (Fg) under different bacterial growth phases. Our real-time RT-PCR results indicated that both Rot and the Agr system have no significant effect on clfA expression. However, Rot is an activator of clfB, and Agr/RNAIII can regulate clfB expression via Rot. Gel shift data further suggested that Rot might regulate clfB expression by directly binding to the promoter region of clfB. Moreover, Rot and the Agr system exhibited consistent regulatory effects on clfB transcription and bacterial Fg-binding ability, suggesting that Rot and the Agr system might affect bacterial Fg-binding ability mainly through regulating clfB transcription.

SarA inactivation reduces vancomycin-intermediate and ciprofloxacin resistance expression by Staphylococcus aureus

It is known that multiple genome-wide transcriptional changes often accompany the development of antimicrobial resistance and occur in response to challenge with antimicrobial agents. We now show that inactivation of the staphylococcal accessory gene regulator sarA, which controls at least tens of genes in Staphylococcus aureus, leads to dramatic reductions in vancomycin and ciprofloxacin resistance in vancomycin-intermediate and ciprofloxacin-resistant strains of S. aureus. This is particularly evident when judged by antimicrobial-gradient plate analysis or population analysis profiles. Whilst the intact sarA cistron is required for full vancomycin resistance expression by vancomycin-intermediate S. aureus (VISA), sarA expression as determined by quantitative real-time polymerase chain reaction was found to be VISA strain-dependent. Reductions in vancomycin resistance expression levels following sarA inactivation do not necessarily include an alteration in autolysis. Expression of sarR, the negative regulator of sarA, was downregulated in two VISA mutants, and transcription of the alternative sigma factor sigB was downregulated in one VISA strain. This study contributes to a growing body of evidence demonstrating the importance of loci previously identified to control virulence in the regulation of clinically relevant antibiotic resistance mechanisms.

Virulence Gene Expression in Human Community‐AcquiredStaphylococcus aureusInfection

Isolates of methicillin-resistant Staphylococcus aureus (MRSA) were once linked uniformly with hospital-associated infections; however, community-acquired MRSA (CA-MRSA) now represents an emerging threat worldwide. To examine the association of differential virulence gene expression with outcomes of human infection, we measured transcript levels of target staphylococcal genes directly in clinical samples from children with active known or suspected CA-MRSA infections. Virulence genes encoding secreted toxins, including Panton-Valentine leukocidin, were highly expressed during superficial and invasive CA-MRSA infections. In contrast, increased expression of surface-associated protein A was linked only with invasive disease. Comparisons with laboratory-grown corresponding clinical isolates revealed that tissue-specific expression profiles reflect the activity of the staphylococcal accessory gene regulator during human infection. These results represent the first demonstration of staphylococcal gene expression and regulation directly in human tissue. Such analysis will help to unravel the complex interactions between CA-MRSA and its host environmental niches during disease development.

Regulation of Rot Expression in Staphylococcus aureus

Repressor of toxins (Rot) is known to be a global regulator of virulence gene expression in Staphylococcus aureus. The function of Rot, but not the transcription of rot, is regulated by the staphylococcal accessory gene regulator (Agr) quorum-sensing system. In addition, the alternative sigma factor (sigma(B)) has a repressive effect on rot expression during the postexponential phase of growth. The transcriptional profiles of Rot in sigma(B)-positive and sigma(B)-negative strains in the postexponential and stationary phases of growth were compared. An upregulation of rot expression was observed during the stationary phase of growth, and this upregulation occurred in a sigma(B)-dependent manner. The effects of other staphylococcal transcriptional factors were also investigated. Electrophoretic mobility shift assays revealed that proteins present in staphylococcal lysates retarded the mobility of the rot promoter fragment and that the effect was reduced, but not eliminated, with lysates from strains lacking a functional SarS protein. A modest upregulation of rot expression was also observed in sarS-negative strains. Affinity purification of proteins binding to the rot promoter fragment, followed by N-terminal protein sequencing, identified the SarA and SarR proteins. Primer extension analysis of the rot promoter revealed a number of discreet products. However, these RNA species were not associated with identifiable promoter activity and likely represented RNA breakdown products. Loss of Rot function during the postexponential phase of growth likely involves degradation of the rot mRNA but not the inhibition of rot transcription.

Molecular Mechanisms of agr Quorum Sensing in Virulent Staphylococci

How to become virulent. The staphylococcal accessory gene-regulator (agr) quorum-sensing system is responsible for integrating growth-dependent signals with a complex genetic network to coordinate expression of a large variety of virulence factors. Our current understanding of the molecular mechanisms of agr activation and inhibition are described, along with implications for the in vivo role of agr in pathogenesis.

Inhibition of expression of a staphylococcal superantigen-like protein by a soluble factor from Lactobacillus reuteri

Lactobacillus reuteri RC-14 has previously been shown to inhibit Staphylococcus aureus infection in a rat surgical-implant model. To investigate the basis for this, communication events between the two bacterial species were examined. L. reuteri RC-14 and Staph. aureus Newman were grown in a co-culture apparatus that physically separates the two species, while allowing the passage of soluble compounds. Using two-dimensional gel electrophoresis (2D-E), protein expression changes in Staph. aureus were analysed in response to co-culture with medium alone, L. reuteri RC-14, and a Lactobacillus strain that did not inhibit Staph. aureus infection in the rat model. It was observed that one protein in particular, identified as staphylococcal superantigen-like protein 11 (SSL11), showed a dramatic decrease in expression in response to growth with L. reuteri RC-14. Genetic reporters that placed both gfp and lux under the transcriptional control of the SSL11 promoter confirmed the 2D-E results. Interestingly, using similar reporter gene experiments, it was observed that the Staph. aureus P3 promoter from the staphylococcal accessory gene regulator (agr) locus also showed a decrease in expression in response to growth in the presence of L. reuteri RC-14. It was further demonstrated that L. reuteri RC-14 supernatant contained small unidentified molecules that were able to repress the SSL11 and P3 promoters, but the repression of SSL11 occurred independently of the agr system. These results suggest that L. reuteri RC-14 has the potential to alter the virulence of Staph. aureus via secretion of cell-cell signalling molecules.

Heterogeneity of methicillin-resistant Staphylococcus aureus strains at a German university hospital during a 1-year period

Heterogeneous methicillin-resistant Staphylococcus aureus (MRSA) strains, including community-acquired MRSA strains, have been observed in Central Europe. The purpose of this study was to characterize by molecular methods MRSA isolated during the period 2002-2003 at the Otto-von-Guericke University Hospital in Magdeburg, Germany, and at a nearby chronic care facility. Strains were analyzed for their resistance phenotype. Selected isolates were typed by multilocus sequence typing (MLST), by a multiplex polymerase chain reaction (PCR) for the staphylococcal cassette chromosome mec (SCCmec), by an allele-specific PCR for the staphylococcal accessory gene regulator (agr), and by PCR for the presence of toxin genes (sea-sej, tsst-1, hlgA, C, and B, lukE/D, and luk-pvl). Of the 2,731 S. aureus isolates studied, 199 (7.3%) were MRSA, with a prevalence of 21.6%, 19.6%, and 12% in the department of dermatology, the chronic care facility, and the intensive care units. Six different sequence types (ST247, ST228, ST22, ST22a, ST225, and ST45) were observed. Of these, ST22, ST22a, and ST45 dominated (>50%) in the department of dermatology and the chronic care facility. Strains with these sequence types were usually not resistant to gentamicin and were associated with agr group I, the SCCmec type IV element, and the presence of the sec and sed toxin genes. ST228 strains were found mainly in the intensive care units and had a broader resistance phenotype and were associated with agr group II and the SCCmec type I element. All luk-pvl-positive MRSA isolates (n=8) belonged to agr group I and were typed as ST22 or ST45 and contained the SCCmec type I (n=1), type III (n=1), or type IV (n=6) element. The main observations of this study are in concordance with previously reported findings showing dissemination of MRSA in Central Europe. Through the multitude of applied methods, the data from this study contribute to a more precise knowledge about the heterogeneity of MRSA in a clinical setting. Rapid dissemination of MRSA clones at a university hospital was demonstrated, indicating that dissemination may depend on the environmental conditions within the individual departments.

Identification of the Putative Staphylococcal AgrB Catalytic Residues Involving the Proteolytic Cleavage of AgrD to Generate Autoinducing Peptide

The P2 operon of the staphylococcal accessory gene regulator (agr) encodes four genes (agrA, -B, -C, and -D) whose products compose a quorum sensing system: AgrA and AgrC resemble a two-component signal transduction system of which AgrC is a sensor kinase and AgrA is a response regulator; AgrD, a polypeptide that is integrated into the cytoplasmic membrane via an amphipathic alpha-helical motif in its N-terminal region, is the propeptide for an autoinducing peptide that is the ligand for AgrC; and AgrB is a novel membrane protein that involves in the processing of AgrD propeptide and possibly the secretion of the mature autoinducing peptide. In this study, we demonstrated that AgrB had endopeptidase activity, and identified 2 amino acid residues in AgrB (cysteine 84 and histidine 77) that might form a putative cysteine endopeptidase catalytic center in the proteolytic cleavage of AgrD at its C-terminal processing site. Computer analysis revealed that the cysteine and histidine residues were conserved among the potential AgrB homologous proteins, suggesting that the Agr quorum sensing system homologues might also exist in other Gram-positive bacteria.

Staphylococcal accessory gene regulator (sar) as a signature gene to detect enterotoxigenic staphylococci.

To evaluate the use of a staphylococcal accessory gene regulator (sar) as a means of detecting enterotoxigenic staphylococci. SarA gene-specific primers were designed and applied in PCR, which resulted in the detection of 49 sar-positive isolates from a total of 67 natural food isolates of staphylococci. Colony hybridization using PCR-generated Digoxigenin (DIG)-labelled sarA probe tested in spiked samples of khoa (a traditional heat-concentrated milk product) comprising a mixed microflora ensured the specificity of the probe. Validation experiments with the commercial samples of khoa also demonstrated the specificity of the probe. PCR characterization for enterotoxins A-D revealed the presence of at least one of the toxin-encoding genes in all the sarA-positive isolates tested. The study indicated that sarA gene could be an ideal marker gene either in colony hybridization or in PCR, for an effective detection of potentially enterotoxigenic strains of staphylococci in a food system. As an alternative to targeting the individual toxin genes, a regulatory gene responsible for controlling the synthesis of various virulence factors may be a suitable target gene for screening potentially toxigenic staphylococci in food system using nucleic acid-based methods.

Role of the staphylococcal accessory gene regulator (sar) in septic arthritis.

Staphylococcus aureus arthritis is a highly erosive disease in which both host and bacterial factors are of importance for its induction and progression. At the transcriptional level, three known loci act in regulating production of exoproteins and expression of cell wall structures. The aim of our study was to assess the role of the sar locus as a virulence determinant in the pathogenesis of septic arthritis. A recently established murine model of hematogenously spread S. aureus arthritis was employed. S. aureus strains, isogenic for the sar locus, were inoculated intravenously into NMRI mice, and the clinical, bacteriological, serological, and histopathological progression of the disease was studied. Within 1 week after inoculation of bacteria, the frequency of arthritis was 79% in the group of mice inoculated with the sar+ strain, whereas the corresponding frequency in sar mutants was 21% (P < 0.01). Mice inoculated with the sar+ staphylococcal strain exhibited a more pronounced T- and B-lymphocyte activation than those inoculated with the sar mutant, evidenced by splenomegaly, polyclonal B-cell activation, and high serum levels of interleukin 6 and gamma interferon. Also, infection with sar+ staphylococci induced a pronounced weight loss. To assess the relationship between clinical signs and spread of bacteria, we analyzed the homing pattern and persistence of S. aureus in host tissues. Kidneys and joints from sar+-inoculated subjects displayed a higher degree of bacterial persistence than other organs. Our results suggest that molecules controlled by the sar locus are important virulence determinants in the induction and progression of septic arthritis.