Defective pgsA contributes to increased membrane fluidity and cell wall thickening in Staphylococcus aureus with high-level daptomycin resistance.

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. Transcriptomics studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels. IMPORTANCE: The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to ß-lactams or vancomycin. Since daptomycin's activity is based on interaction with the negatively charged membrane of S. aureus, routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant S. aureus, including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene, pgsA, on membrane fluidity and cell wall thickness in S. aureus with high-level daptomycin resistance.

Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids over biosynthesized endogenous branched-chain fatty acids.

It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media, and when growing in vivo in an infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain. We show that incorporation of C18:1Δ9 and its elongation product C20:1Δ9 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol (PG) and diglycosyldiacylglycerol (DGDG) lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin; however, this was not an obligatory requirement for cold adaptation. Enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms.

Defective pgsA contributes to increased membrane fluidity and cell wall thickening in S. aureus with high-level daptomycin resistance.

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin-resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin-resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. RT-qPCR studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels.

Regulation of the Sae Two-Component System by Branched-Chain Fatty Acids in Staphylococcus aureus.

Staphylococcus aureus is a ubiquitous Gram-positive bacterium and an opportunistic human pathogen. S. aureus pathogenesis relies on a complex network of regulatory factors that adjust gene expression. Two important factors in this network are CodY, a repressor protein responsive to nutrient availability, and the SaeRS two-component system (TCS), which responds to neutrophil-produced factors. Our previous work revealed that CodY regulates the secretion of many toxins indirectly via Sae through an unknown mechanism. We report that disruption of codY results in increased levels of phosphorylated SaeR (SaeR~P) and that codY mutant cell membranes contain a higher percentage of branched-chain fatty acids (BCFAs) than do wild-type membranes, prompting us to hypothesize that changes to membrane composition modulate the activity of the SaeS sensor kinase. Disrupting the lpdA gene encoding dihydrolipoyl dehydrogenase, which is critical for BCFA synthesis, significantly reduced the abundance of SaeR, phosphorylated SaeR, and BCFAs in the membrane, resulting in reduced toxin production and attenuated virulence. Lower SaeR levels could be explained in part by reduced stability. Sae activity in the lpdA mutant could be complemented genetically and chemically with exogenous short- or full-length BCFAs. Intriguingly, lack of lpdA also alters the activity of other TCSs, suggesting a specific BCFA requirement managing the basal activity of multiple TCSs. These results reveal a novel method of posttranscriptional virulence regulation via BCFA synthesis, potentially linking CodY activity to multiple virulence regulators in S. aureus. IMPORTANCE Two-component systems (TCSs) are an essential way that bacteria sense and respond to their environment. These systems are usually composed of a membrane-bound histidine kinase that phosphorylates a cytoplasmic response regulator. Because most of the histidine kinases are embedded in the membrane, lipids can allosterically regulate the activity of these sensors. In this study, we reveal that branched-chain fatty acids (BCFAs) are required for the activation of multiple TCSs in Staphylococcus aureus. Using both genetic and biochemical data, we show that the activity of the virulence activator SaeS and the phosphorylation of its response regulator SaeR are reduced in a branched-chain keto-acid dehydrogenase complex mutant and that defects in BCFA synthesis have far-reaching consequences for exotoxin secretion and virulence. Finally, we show that mutation of the global nutritional regulator CodY alters BCFA content in the membrane, revealing a potential mechanism of posttranscriptional regulation of the Sae system by CodY.

Expression of Staphylococcal Virulence Genes In Situ in Human Skin and Soft Tissue Infections.

BACKGROUND: Staphylococcus aureus, the most common pathogen in skin and soft tissue infections (SSTI), harbors many well-characterized virulence genes. However, the expression of many of them in SSTIs is unknown. In this study, S. aureus virulence genes expressed in SSTI were investigated. METHODS: Fifty-three subjects presenting to the outpatient's care and emergency departments with a purulent SSTI at two medical centers in Wisconsin, USA, were enrolled in the study. Total mRNA was extracted from the purulent or swab materials, made into cDNA and sequenced on MiSeq platform. The relative cDNA counts to gmk and identifications of the transcripts were carried out with respect to USA300 reference genome and using SAMTOOLS v.1.3 and BWA, respectively. RESULT: A significantly higher cDNA count was observed for many of the virulence and regulatory gene transcripts in the pus samples compared to the swab samples relative to the cDNA counts for gmk, a housekeeping gene. They were for lukS-PV (18.6 vs. 14.2), isaA (13.4 vs. 8.5), ssaA (4.8 vs. 3.1), hlgC (1.4 vs. 1.33), atl (17.7 vs. 8.33), clfA (3.9 vs. 0.83), eno (6.04 vs. 3.16), fnbA (5.93 vs. 0.33), saeS (6.3 vs. 1.33), saeR (5.4 vs. 3.33) and agrC (5.6 vs. 1.5). CONCLUSIONS: A relative increase in the transcripts of several toxins, adhesion and regulatory genes with respect to a gmk in purulent materials suggests their role in situ during SSTIs, perhaps in an orchestrated manner.

Exogenous Fatty Acids Remodel Staphylococcus aureus Lipid Composition through Fatty Acid Kinase.

Staphylococcus aureus can utilize exogenous fatty acids for phospholipid synthesis. The fatty acid kinase FakA is essential for this utilization by phosphorylating exogenous fatty acids for incorporation into lipids. How FakA impacts the lipid membrane composition is unknown. In this study, we used mass spectrometry to determine the membrane lipid composition and properties of S. aureus in the absence of fakA We found the fakA mutant to have increased abundance of lipids containing longer acyl chains. Since S. aureus does not synthesize unsaturated fatty acids, we utilized oleic acid (18:1) to track exogenous fatty acid incorporation into lipids. We observed a concentration-dependent incorporation of exogenous fatty acids into the membrane that required FakA. We also tested how FakA and exogenous fatty acids impact membrane-related physiology and identified changes in membrane potential, cellular respiration, and membrane fluidity. To mimic the host environment, we characterized the lipid composition of wild-type and fakA mutant bacteria grown in mouse skin homogenate. We show that wild-type S. aureus can incorporate exogenous unsaturated fatty acids from host tissue, highlighting the importance of FakA in the presence of host skin tissue. In conclusion, FakA is important for maintaining the composition and properties of the phospholipid membrane in the presence of exogenous fatty acids, impacting overall cell physiology.IMPORTANCE Environmental fatty acids can be harvested to supplement endogenous fatty acid synthesis to produce membranes and circumvent fatty acid biosynthesis inhibitors. However, how the inability to use these fatty acids impacts lipids is unclear. Our results reveal lipid composition changes in response to fatty acid addition and when S. aureus is unable to activate fatty acids through FakA. We identify concentration-dependent utilization of oleic acid that, when combined with previous work, provides evidence that fatty acids can serve as a signal to S. aureus Furthermore, using mouse skin homogenates as a surrogate for in vivo conditions, we showed that S. aureus can incorporate host fatty acids. This study highlights how exogenous fatty acids impact bacterial membrane composition and function.

Impact of Deficiencies in Branched-Chain Fatty Acids and Staphyloxanthin in Staphylococcus aureus.

Staphylococcus aureus is a well-known human pathogen with the ability to cause mild superficial skin infections to serious deep-tissue infections, such as osteomyelitis, pneumonia, and infective endocarditis. A key to S. aureus infections and its pathogenicity is its ability to survive in adverse environments, especially at lower temperatures, by regulation of its cell membrane. Branched-chain fatty acids (BCFAs) and staphyloxanthin have been shown to regulate membrane fluidity and staphylococcal virulence. This study was conducted with the hypothesis that the simultaneous lack of BCFAs and staphyloxanthin will have a far greater implication on environmental survival and virulence of S. aureus. Lack of a functional branched-chain α-keto acid dehydrogenase (BKD) enzyme because of a mutation in the lpdA gene led to a decrease in the production of BCFAs, membrane fluidity, slower growth, and poor in vivo survival of S. aureus. A mutation in the crtM gene eliminated the production of staphyloxanthin but it did not affect membrane BCFA levels, fluidity, growth, or in vivo survival. A crtM:lpdA double mutant showed much slower growth and attenuation compared to individual mutants. The results of this study suggest that simultaneous targeting of the BCFA and staphyloxanthin biosynthetic pathways can be a strategy to control S. aureus infections.

Role of SigB and Staphyloxanthin in Radiation Survival of Staphylococcus aureus.

Staphylococcus aureus is a potent human pathogen. The virulence of this bacterium depends on a multitude of factors that it produces. One such virulence factor is the golden pigment, staphyloxanthin, which has been shown to protect the bacterium from oxidative stress. Expression of the staphyloxanthin biosynthetic pathway is dependent on SigB, a global stress response regulator in S. aureus. This study investigated the role of staphyloxanthin and SigB in protection of S. aureus from radiation damage. Using stationary-phase bacterial cells, it was determined that the staphyloxanthin-deficient (crt mutant) strain was significantly sensitive to UV radiation (~ threefold), but not sensitive to X-radiation. However, a SigB-deficient S. aureus that also lacks staphyloxanthin, was significantly sensitive to both UV- and X-radiation. To confirm that protection from X-radiation was due to hydroxyl radicals, effect of 3 M glycerol, a known hydroxyl scavenger, was also investigated. Glycerol increased the survival of the S. aureus sigB mutant to the wild-type level suggesting that the X-radiation sensitivity of these mutants was due to deficiency in scavenging hydroxyl radicals. In summary, SigB is critical for protection of S. aureus cells from radiation damage.

The Role of Methionine Sulfoxide Reductases in Oxidative Stress Tolerance and Virulence of Staphylococcus aureus and Other Bacteria.

Methionine sulfoxide reductases (MSRA1 and MSRB) are proteins overproduced in Staphylococcus aureus during exposure with cell wall-active antibiotics. Later studies identified the presence of two additional MSRA proteins (MSRA2 and MSRA3) in S. aureus. These MSR proteins have been characterized in many other bacteria as well. This review provides the current knowledge about the conditions and regulatory network that mimic the expression of these MSR encoding genes and their role in defense from oxidative stress and virulence.

Roles of pyruvate dehydrogenase and branched-chain α-keto acid dehydrogenase in branched-chain membrane fatty acid levels and associated functions in Staphylococcus aureus.

PURPOSE: Membrane fluidity to a large extent is governed by the presence of branched-chain fatty acids (BCFAs). Branched-chain α-keto acid dehydrogenase (BKD) is the key enzyme in BCFA synthesis. A Staphylococcus aureus BKD-deficient strain still produced substantial levels of BCFAs. Pyruvate dehydrogenase (PDH) with structural similarity to BKD has been speculated to contribute to BCFAs in S. aureus. METHODOLOGY: This study was carried out using BKD-, PDH- and BKD : PDH-deficient derivatives of methicillin-resistant S. aureus strain JE2. Differences in growth kinetics were evaluated spectrophotometrically, membrane BCFAs using gas chromatography and membrane fluidity by fluorescence polarization. Carotenoid levels were estimated by measuring A465 of methanol extracts from 48 h cultures. MIC values were determined by broth microdilution.Results/Key findings. BCFAs made up 50 % of membrane fatty acids in wild-type but only 31 % in the BKD-deficient mutant. BCFA level was ~80 % in the PDH-deficient strain and 38 % in the BKD : PDH-deficient strain. BKD-deficient mutant showed decreased membrane fluidity, the PDH-deficient mutant showed increased membrane fluidity. The BKD- and PDH-deficient strains grew slower and the BKD : PDH-deficient strain grew slowest at 37 °C. However at 20 °C, the BKD- and BKD : PDH-deficient strains grew only a little followed by autolysis of these cells. The BKD-deficient strain produced higher levels of staphyloxanthin. The PDH-deficient and BKD : PDH-deficient strains produced very little staphyloxanthin. The BKD-deficient strain showed increased susceptibility to daptomycin. CONCLUSION: The BCFA composition of the cell membrane in S. aureus seems to significantly impact cell growth, membrane fluidity and resistance to daptomycin.

Phylogenetic distribution and expression of a penicillin-binding protein homologue, Ear and its significance in virulence of Staphylococcus aureus.

PURPOSE: Staphylococcus aureus is an opportunistic human pathogen that can cause serious infections in humans. A plethora of known and putative virulence factors are produced by staphylococci that collectively orchestrate pathogenesis. Ear protein (Escherichia coli ampicillin resistance) in S. aureus is an exoprotein in COL strain, predicted to be a superantigen, and speculated to play roles in antibiotic resistance and virulence. The goal of this study was to determine if expression of ear is modulated by single nucleotide polymorphisms in its promoter and coding sequences and whether this gene plays roles in antibiotic resistance and virulence. METHODOLOGY: Promoter, coding sequences and expression of the ear gene in clinical and carriage S. aureus strains with distinct genetic backgrounds were analysed. The JE2 strain and its isogenic ear mutant were used in a systemic infection mouse model to determine the competiveness of the ear mutant.Results/Key findings. The ear gene showed a variable expression, with USA300FPR3757 showing a high-level expression compared to many of the other strains tested including some showing negligible expression. Higher expression was associated with agr type 1 but not correlated with phylogenetic relatedness of the ear gene based upon single nucleotide polymorphisms in the promoter or coding regions suggesting a complex regulation. An isogenic JE2 (USA300 background) ear mutant showed no significant difference in its growth, antibiotic susceptibility or virulence in a mouse model. CONCLUSION: Our data suggests that despite being highly expressed in a USA300 genetic background, Ear is not a significant contributor to virulence in that strain.

Effect of clpP and clpC deletion on persister cell number in Staphylococcus aureus.

Staphylococcus aureus is responsible for a wide variety of infections that include superficial skin and soft tissue infections, septicaemia, central nervous system infections, endocarditis, osteomyelitis and pneumonia. Others have demonstrated the importance of toxin-antitoxin (TA) modules in the formation of persisters and the role of the Clp proteolytic system in the regulation of these TA modules. This study was conducted to determine the effect of clpP and clpC deletion on S. aureus persister cell numbers following antibiotic treatment. Deletion of clpP resulted in a significant decrease in persister cells following treatment with oxacillin and erythromycin but not with levofloxacin and daptomycin. Deletion of clpC resulted in a decrease in persister cells following treatment with oxacillin. These differences were dependent on the antibiotic class and the CFU ml-1 in which the cells were treated. Persister revival assays for all the bacterial strains in these studies demonstrated a significant delay in resumption of growth characteristic of persister cells, indicating that the surviving organisms in this study were not likely due to spontaneous antibiotic resistance. Based on our results, ClpP and possibly ClpC play a role in persister cell formation or maintenance, and this effect is dependent on antibiotic class and the CFU ml-1 or the growth phase of the cells.

Functional importance of αIle-346 and αIle-348 in the catalytic sites of Escherichia coli ATP synthase.

We studied the functional role of highly conserved VISIT-DG sequence residues αIle-346 and αIle-348 in the catalytic sites of Escherichia coli F1Fo ATP synthase. αIle-346 is in close proximity, 2.98 and 3.63 Å, to the two known phosphate binding residues αR376 and ßR182; αIle-348 is situated within 3.66 Å from ßR182. Single or double mutants of both αI346 and αI348 resulted in a variable loss of oxidative phosphorylation and ATPase activity. Azide, fluoroaluminate, and fluoroscandium caused insignificant to significant inhibition of mutants. Whereas the wild-type enzyme was completely inhibited by NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole), a variable extent of inhibition was observed for αI346 and αI348 mutants. MgPi protection against NBD-Cl induced inhibition of wild-type, αI346, and αI348 demonstrated that, although strongly conserved, αI346 and αI348 have no direct role in phosphate binding. Insertion of Arginine in the form of αI346R/ßR182A, αI346R/αR376A, or αI348R/ßR182A was able to compensate for the absence of known phosphate-binding Arginine residues ßR182 and αR376. Results also suggest that αIle-346 and αIle-348 seem to have functional importance in upholding the phosphate-binding subdomain and transition state stabilization in the catalytic sites of E. coli ATP synthase.

Multiplex real-time PCR detection and relative quantification of periodontal pathogens.

Periodontitis is a chronic inflammatory disease, which is strongly associated with certain pathogenic bacteria. The aim of this study was to develop a real-time multiplex polymerase chain reaction (PCR) assay to detect and quantify bacterial species associated with periodontitis. We targeted detection and relative quantification of the following five bacterial species relevant to periodontal diseases: Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. The conserved regions of the genome of these species were targeted with oligos and TaqMan probes in real-time PCR assays. The species-specific TaqMan oligos and TaqMan probes showed no cross-amplification, and there was no loss of amplification yield in multiplex real-time PCR assays. All five bacterial targets were amplified analogous to the template concentrations used in these assays. This multiplex real-time PCR strategy could potentially be used to detect the bacterial species in periodontal pockets of patients with periodontal diseases. This assay may also serve as a quick tool for profiling and quantifying bacteria relevant to periodontal diseases and likely be a valuable tool for clinical translational research.

Regulation of Expression of Oxacillin-Inducible Methionine Sulfoxide Reductases in Staphylococcus aureus.

Cell wall-active antibiotics cause induction of a locus that leads to elevated synthesis of two methionine sulfoxide reductases (MsrA1 and MsrB) in Staphylococcus aureus. To understand the regulation of this locus, reporter strains were constructed by integrating a DNA fragment consisting of the msrA1/msrB promoter in front of a promoterless lacZ gene in the chromosome of wild-type and MsrA1-, MsrB-, MsrA1/MsrB-, and SigB-deficient methicillin-sensitive S. aureus strain SH1000 and methicillin-resistant S. aureus strain COL. These reporter strains were cultured in TSB and the cellular levels of ß-galactosidase activity in these cultures were assayed during different growth phases. ß-galactosidase activity assays demonstrated that the lack of MsrA1, MsrB, and SigB upregulated the msrA1/msrB promoter in S. aureus strain SH1000. In S. aureus strain COL, the highest level of ß-galactosidase activity was observed under the conditions when both MsrA1 and MsrB proteins were absent. The data suggest that the msrA1/msrB locus, in part, is negatively regulated by MsrA1, MsrB, and SigB in S. aureus.

Significance of four methionine sulfoxide reductases in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen and emergence of antibiotic resistance in clinical staphylococcal isolates raises concerns about our ability to control these infections. Cell wall-active antibiotics cause elevated synthesis of methionine sulfoxide reductases (Msrs: MsrA1 and MsrB) in S. aureus. MsrA and MsrB enzymes reduce S-epimers and R-epimers of methionine sulfoxide, respectively, that are generated under oxidative stress. In the S. aureus chromosome, there are three msrA genes (msrA1, msrA2 and msrA3) and one msrB gene. To understand the precise physiological roles of Msr proteins in S. aureus, mutations in msrA1, msrA2 and msrA3 and msrB genes were created by site-directed mutagenesis. These mutants were combined to create a triple msrA (msrA1, msrA2 and msrA3) and a quadruple msrAB (msrA1, msrA2, msrA3, msrB) mutant. These mutants were used to determine the roles of Msr proteins in staphylococcal growth, antibiotic resistance, adherence to human lung epithelial cells, pigment production, and survival in mice relative to the wild-type strains. MsrA1-deficient strains were sensitive to oxidative stress conditions, less pigmented and less adherent to human lung epithelial cells, and showed reduced survival in mouse tissues. In contrast, MsrB-deficient strains were resistant to oxidants and were highly pigmented. Lack of MsrA2 and MsrA3 caused no apparent growth defect in S. aureus. In complementation experiments with the triple and quadruple mutants, it was MsrA1 and not MsrB that was determined to be critical for adherence and phagocytic resistance of S. aureus. Overall, the data suggests that MsrA1 may be an important virulence factor and MsrB probably plays a balancing act to counter the effect of MsrA1 in S. aureus.

Regulation of Staphylococcal Superantigen-Like Gene, ssl8, Expression in Staphylococcus aureus strain, RN6390.

Staphylococcal superantigen-like (SSL) proteins, which are encoded by a cluster of eleven ssl genes, contribute to the Staphylococcus aureus virulence. Recently we reported ssl8 expression profiles in seven clinically important strains-MW2, USA300FPR3757, MSSA476, Newman, RN6390, Mu50, and N315-and showed the differential expression of ssl8 in Newman, RN6390, and USA300FPR3757 strains, despite harboring identical allelic forms of ssl8, suggesting the roles for different regulatory elements for this gene in different S. aureus strains. In this communication, using RN6390, a common laboratory S. aureus strain and its isogenic knockout mutant strains of agr, sae, sarA, sigB, rot, and the agr-/sigB (-) double mutant, we showed that SarA and Rot are inducer and repressor, respectively, for ssl8 expression in RN6390. This is in contrast to the Newman strain, where ssl8 is positively regulated by Sae but negatively by Agr, indicating the variable expression of ssl8 in clinical strains is more likely due to strain-specific regulatory elements.

Changes in rat spinal cord gene expression after inflammatory hyperalgesia of the joint and manual therapy.

CONTEXT: Mobilization of a joint affects local tissue directly but may also have other effects that are mediated through the central nervous system. OBJECTIVE: To identify differential gene expression in the spinal cords of rats with or without inflammatory joint injury after manual therapy or no treatment. METHODS: Rats were randomly assigned to 1 of 4 treatment groups: no injury and no touch (NI/NT), injury and no touch (I/NT), no injury and manual therapy (NI/MT), and injury and manual therapy (I/MT). We induced acute inflammatory joint injury in the rats by injecting carrageenan into an ankle. Rats in the no-injury groups did not receive carrageenan injection. One day after injury, rats received manual therapy to the knee of the injured limb. Rats in the no-touch groups were anesthetized without receiving manual therapy. Spinal cords were harvested 30 minutes after therapy or no touch, and spinal cord gene expression was analyzed by microarray for 3 comparisons: NI/NT vs I/NT, I/MT vs I/NT, and NI/NT vs NI/MT. RESULTS: Three rats were assigned to each group. Of 38,875 expressed sequence tags, 755 were differentially expressed in the NI/NT vs I/NT comparison. For the other comparisons, no expressed sequence tags were differentially expressed. Cluster analysis revealed that the differentially expressed sequence tags were over-represented in several categories, including ion homeostasis (enrichment score, 2.29), transmembrane (enrichment score, 1.55), and disulfide bond (enrichment score, 2.04). CONCLUSIONS: An inflammatory injury to the ankle of rats caused differential expression of genes in the spinal cord. Consistent with other studies, genes involved in ion transport were among those affected. However, manual therapy to the knees of injured limbs or to rats without injury did not alter gene expression in the spinal cord. Thus, evidence for central nervous system mediation of manual therapy was not observed.

Lack of a functional methionine sulfoxide reductase (MsrB) increases oxacillin and H2O2 stress resistance and enhances pigmentation in Staphylococcus aureus.

Staphylococcus aureus produces 3 MsrA enzymes (MsrA1, MsrA2, and MsrA3) and 1 MsrB enzyme. The genes encoding MsrA1 and MsrB are the first and second genes of a 4-gene operon in S. aureus. In a previous study, MsrA1-deficient S. aureus cells showed increased sensitivity to oxidative stress conditions in spite of a higher production of MsrB. In this study, an msrB mutant of S. aureus was created by site-directed mutagenesis that left the first gene of this locus, msrA1, intact. Studies with this mutant suggest that a deletion of MsrB increases resistance of S. aureus to H2O2 and oxacillin and that the mutant cells produce a higher level of carotenoids relative to wild-type S. aureus cells.

High Level Expression and Purification of Atl, the Major Autolytic Protein of Staphylococcus aureus.

Staphylococcus aureus is a major human and animal pathogen. Autolysins regulate the growth, turnover, cell lysis, biofilm formation, and the pathogenicity of S. aureus. Atl is the major autolysin in S. aureus. The biochemical and structural studies of staphylococcal Atl have been limited due to difficulty in cloning, high level overexpression, and purification of this protein. This study describes successful cloning, high level over-expression, and purification of two forms of fully functional Atl proteins. These pure proteins can be used to study the functional and structural properties of this important protein.