Evaluating Child Passenger Restraint Use Education.

BACKGROUND: Despite recommendations and laws for child restraint use in motor vehicles, evidence of low restraint use remains, and there is a lack of evidence addressing the effectiveness of restraint use education. OBJECTIVE: This project aims to measure the impact of an education initiative on child passenger restraint use. METHODS: This pre- and postintervention study was conducted in six elementary schools in a Southwestern U.S. metropolitan area over 5 months from October 2022 to March 2023. Motor vehicle restraint use was collected from occupants arriving at elementary schools during the morning drop-off times. Participants were provided one-on-one education regarding child passenger safety guidelines and state laws. Comparison data were collected 1-3 weeks later at the same schools to evaluate the education provided. RESULTS: A total of 1,671 occupants in 612 vehicles were observed across six schools, with 343 adults and 553 children preintervention and 306 adults and 469 children postintervention. Overall restraint adherence in children improved postintervention from 42.3% to 56.1%, a 32.6% increase (p = < .001). In the primary age group of 4-8 years, restraint adherence improved postintervention from 34.8% to 54.2%, a 55.8% increase (p = <.001). CONCLUSIONS: The study results demonstrate that one-on-one education increases child passenger restraint use.

Alcohol Misuse Screening Performance Improvement Initiative.

BACKGROUND: A trauma registry review of our trauma center's alcohol misuse screening compliance noted inconsistent screening and data collection methods, putting our American College of Surgeons trauma center reverification at risk for a deficiency. OBJECTIVE: The objective of this study was to evaluate an alcohol misuse screening improvement initiative on screening compliance in trauma patients. METHODS: This before and after analysis of a quality improvement initiative to improve alcohol misuse screening was conducted from 2019 to 2021 at a Southwestern U.S. Level II trauma center on admitted trauma patients aged 13 years and older. The multicomponent initiative included a change in the screening instrument and timing of application, implementing electronic medical record documentation screens, and educating staff. The primary outcome measure was screening adherence. RESULTS: A total of n = 4,734 patients were included in the study period. Alcohol misuse screening improved from 2.9% to 87.4% and remained sustained for over a year after the initiative's implementation. CONCLUSION: The performance improvement initiative improved the alcohol misuse screening process, resulting in consistent screenings exceeding the American College of Surgeons standards of 80% for verified trauma centers.

Association between ventilator-associated events and implementation of acute respiratory distress syndrome (ARDS) ventilator weaning protocol.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe and life-threatening condition that can occur in critically ill patients. Mechanical ventilation is a commonly used intervention with ARDS patients, but weaning patients off the ventilator can be challenging. An ARDSnet-like ventilator weaning protocol was implemented with the goal of reducing triggers for ventilator-associated events (VAEs). METHODS: The implementation of the new protocol was used to complete a retrospective investigation of patient outcomes for 1,233 ventilator periods. Periods were included between April and December 2022 for any ventilated patient lasting at least 4 days. National Health Care Safety Network VAE criteria were used to surveille the patient data. Triggers were based on the positive end-expiratory pressure increases or fraction of inspired oxygen (FiO2) increases. The preset weaning criteria was a reduction by 2 cmH2O per 24 hours. RESULTS: Of the total 1,233 individual ventilator periods, VAE criteria were met in 10%. Of the total 126 periods with VAE, 39.2% met the criteria for appropriate protocol implementation. There was a statistically significant relationship between VAE identification and implementation of the protocol. CONCLUSIONS: The implementation of a protocol for ventilator weaning affects the outcome of developing a VAE. The findings emphasize the importance of implementing the ARDS weaning protocol as a template to reduce the triggers for VAEs and improve overall patient outcomes.

Signal Biosynthesis Inhibition with Ambuic Acid as a Strategy To Target Antibiotic-Resistant Infections.

There has been major interest by the scientific community in antivirulence approaches against bacterial infections. However, partly due to a lack of viable lead compounds, antivirulence therapeutics have yet to reach the clinic. Here we investigate the development of an antivirulence lead targeting quorum sensing signal biosynthesis, a process that is conserved in Gram-positive bacterial pathogens. Some preliminary studies suggest that the small molecule ambuic acid is a signal biosynthesis inhibitor. To confirm this, we constructed a methicillin-resistant Staphylococcus aureus (MRSA) strain that decouples autoinducing peptide (AIP) production from regulation and demonstrate that AIP production is inhibited in this mutant. Quantitative mass spectrometric measurements show that ambuic acid inhibits signal biosynthesis (50% inhibitory concentration [IC50] of 2.5 ± 0.1 µM) against a clinically relevant USA300 MRSA strain. Quantitative real-time PCR confirms that this compound selectively targets the quorum sensing regulon. We show that a 5-µg dose of ambuic acid reduces MRSA-induced abscess formation in a mouse model and verify its quorum sensing inhibitory activity in vivo Finally, we employed mass spectrometry to identify or confirm the structure of quorum sensing signaling peptides in three strains each of S. aureus and Staphylococcus epidermidis and single strains of Enterococcus faecalis, Listeria monocytogenes, Staphylococcus saprophyticus, and Staphylococcus lugdunensis By measuring AIP production by these strains, we show that ambuic acid possesses broad-spectrum efficacy against multiple Gram-positive bacterial pathogens but does not inhibit quorum sensing in some commensal bacteria. Collectively, these findings demonstrate the promise of ambuic acid as a lead for the development of antivirulence therapeutics.

Differential regulation of staphylococcal virulence by the sensor kinase SaeS in response to neutrophil-derived stimuli.

Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Staphylococcus aureus uses the S. aureus exoprotein expression (sae) TCS to sense host signals and activate transcription of virulence factors essential to pathogenesis. Despite its importance, the mechanism by which the histidine kinase SaeS recognizes specific host stimuli is unknown. After mutagenizing the predicted extracellular loop of SaeS, we discovered one methionine residue (M31) was essential for the ability of S. aureus to transcribe sae target genes, including hla, lukAB/lukGH, and hlgA. This single M31A mutation also significantly reduced cytotoxicity in human neutrophils to levels observed in cells following interaction with ΔsaeS. Another important discovery was that mutation of two aromatic anchor residues (W32A and F33A) disrupted the normal basal signaling of SaeS in the absence of inducing signals, yet both mutant kinases had appropriate activation of effector genes following exposure to neutrophils. Although the transcriptional profile of aromatic mutation W32A was consistent with that of WT in response to human α-defensin 1, mutant kinase F33A did not properly transcribe the γ-toxin genes in response to this stimulus. Taken together, our results provide molecular evidence for how SaeS recognizes host signals and triggers activation of select virulence factors to facilitate evasion of innate immunity. These findings have important implications for signal transduction in prokaryotes and eukaryotes due to conservation of aromatic anchor residues across both of these domains and the important role they play in sensor protein structure and function.

Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.

Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.

The Staphylococcus aureus ArlRS two-component system is a novel regulator of agglutination and pathogenesis.

Staphylococcus aureus is a prominent bacterial pathogen that is known to agglutinate in the presence of human plasma to form stable clumps. There is increasing evidence that agglutination aids S. aureus pathogenesis, but the mechanisms of this process remain to be fully elucidated. To better define this process, we developed both tube based and flow cytometry methods to monitor clumping in the presence of extracellular matrix proteins. We discovered that the ArlRS two-component system regulates the agglutination mechanism during exposure to human plasma or fibrinogen. Using divergent S. aureus strains, we demonstrated that arlRS mutants are unable to agglutinate, and this phenotype can be complemented. We found that the ebh gene, encoding the Giant Staphylococcal Surface Protein (GSSP), was up-regulated in an arlRS mutant. By introducing an ebh complete deletion into an arlRS mutant, agglutination was restored. To assess whether GSSP is the primary effector, a constitutive promoter was inserted upstream of the ebh gene on the chromosome in a wildtype strain, which prevented clump formation and demonstrated that GSSP has a negative impact on the agglutination mechanism. Due to the parallels of agglutination with infective endocarditis development, we assessed the phenotype of an arlRS mutant in a rabbit combined model of sepsis and endocarditis. In this model the arlRS mutant displayed a large defect in vegetation formation and pathogenesis, and this phenotype was partially restored by removing GSSP. Altogether, we have discovered that the ArlRS system controls a novel mechanism through which S. aureus regulates agglutination and pathogenesis.

Staphopains modulate Staphylococcus aureus biofilm integrity.

Staphylococcus aureus is a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. The S. aureus genome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (ΔsigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ΔsigB mutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that an sspB scpA double mutant restored ΔsigB biofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment of S. aureus biofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved across S. aureus strain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulating S. aureus biofilm architecture.

Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis.

The CXC chemokine receptor 2 (CXCR2) on neutrophils, which recognizes chemokines produced at the site of infection, plays an important role in antimicrobial host defenses such as neutrophil activation and chemotaxis. Staphylococcus aureus is a successful human pathogen secreting a number of proteolytic enzymes, but their influence on the host immune system is not well understood. Here, we identify the cysteine protease Staphopain A as a chemokine receptor blocker. Neutrophils treated with Staphopain A are unresponsive to activation by all unique CXCR2 chemokines due to cleavage of the N-terminal domain, which can be neutralized by specific protease inhibitors. Moreover, Staphopain A inhibits neutrophil migration towards CXCR2 chemokines. By comparing a methicillin-resistant S. aureus (MRSA) strain with an isogenic Staphopain A mutant, we demonstrate that Staphopain A is the only secreted protease with activity towards CXCR2. Although the inability to cleave murine CXCR2 limits in-vivo studies, our data indicate that Staphopain A is an important immunomodulatory protein that blocks neutrophil recruitment by specific cleavage of the N-terminal domain of human CXCR2.

Alpha-toxin induces programmed cell death of human T cells, B cells, and monocytes during USA300 infection.

This investigation examines the influence of alpha-toxin (Hla) during USA300 infection of human leukocytes. Survival of an USA300 isogenic deletion mutant of hla (USA300Δhla) in human blood was comparable to the parental wild-type strain and polymorphonuclear leukocyte (PMN) plasma membrane permeability caused by USA300 did not require Hla. Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) following infection by USA300, USA300Δhla, and USA300Δhla transformed with a plasmid over-expressing Hla (USA300Δhla Comp) demonstrated this toxin plays a significant role inducing plasma membrane permeability of CD14(+), CD3(+), and CD19(+) PBMCs. Rapid plasma membrane permeability independent of Hla was observed for PMNs, CD14(+) and CD19(+) PBMCs following intoxication with USA300 supernatant while the majority of CD3(+) PBMC plasma membrane permeability induced by USA300 required Hla. Addition of recombinant Hla to USA300Δhla supernatant rescued CD3(+) and CD19(+) PBMC plasma membrane permeability generated by USA300 supernatant. An observed delay in plasma membrane permeability caused by Hla in conjunction with Annexin V binding and ApoBrdU Tunel assays examining PBMCs intoxicated with recombinant Hla or infected with USA300, USA300Δhla, USA300Δhla Comp, and USA300ΔsaeR/S suggest Hla induces programmed cell death of monocytes, B cells, and T cells that results in plasma membrane permeability. Together these findings underscore the importance of Hla during S. aureus infection of human tissue and specifically demonstrate Hla activity during USA300 infection triggers programmed cell death of human monocytes, T cells and B cells that leads to plasma membrane permeability.

Novel phenol-soluble modulin derivatives in community-associated methicillin-resistant Staphylococcus aureus identified through imaging mass spectrometry.

Staphylococcus aureus causes a wide range of human disease ranging from localized skin and soft tissue infections to potentially lethal systemic infections. S. aureus has the biosynthetic ability to generate numerous virulence factors that assist in circumventing the innate immune system during disease pathogenesis. Recent studies have uncovered a set of extracellular peptides produced by community-associated methicillin-resistant S. aureus (CA-MRSA) with homology to the phenol-soluble modulins (PSMs) from Staphylococcus epidermidis. CA-MRSA PSMs contribute to skin infection and recruit and lyse neutrophils, and truncated versions of these peptides possess antimicrobial activity. In this study, novel CA-MRSA PSM derivatives were discovered by the use of microbial imaging mass spectrometry. The novel PSM derivatives are compared with their parent full-length peptides for changes in hemolytic, cytolytic, and neutrophil-stimulating activity. A potential contribution of the major S. aureus secreted protease aureolysin in processing PSMs is demonstrated. Finally, we show that PSM processing occurs in multiple CA-MRSA strains by structural confirmation of additional novel derivatives. This work demonstrates that IMS can serve as a useful tool to go beyond genome predictions and expand our understanding of the important family of small peptide virulence factors.

Nuclease modulates biofilm formation in community-associated methicillin-resistant Staphylococcus aureus.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging contributor to biofilm-related infections. We recently reported that strains lacking sigma factor B (sigB) in the USA300 lineage of CA-MRSA are unable to develop a biofilm. Interestingly, when spent media from a USA300 sigB mutant was incubated with other S. aureus strains, biofilm formation was inhibited. Following fractionation and mass spectrometry analysis, the major anti-biofilm factor identified in the spent media was secreted thermonuclease (Nuc). Considering reports that extracellular DNA (eDNA) is an important component of the biofilm matrix, we investigated the regulation and role of Nuc in USA300. The expression of the nuc gene was increased in a sigB mutant, repressed by glucose supplementation, and was unaffected by the agr quorum-sensing system. A FRET assay for Nuc activity was developed and confirmed the regulatory results. A USA300 nuc mutant was constructed and displayed an enhanced biofilm-forming capacity, and the nuc mutant also accumulated more high molecular weight eDNA than the WT and regulatory mutant strains. Inactivation of nuc in the USA300 sigB mutant background partially repaired the sigB biofilm-negative phenotype, suggesting that nuc expression contributes to the inability of the mutant to form biofilm. To test the generality of the nuc mutant biofilm phenotypes, the mutation was introduced into other S. aureus genetic backgrounds and similar increases in biofilm formation were observed. Finally, using multiple S. aureus strains and regulatory mutants, an inverse correlation between Nuc activity and biofilm formation was demonstrated. Altogether, our findings confirm the important role for eDNA in the S. aureus biofilm matrix and indicates Nuc is a regulator of biofilm formation.

Proteolytic cleavage inactivates the Staphylococcus aureus lipoteichoic acid synthase.

Lipoteichoic acid (LTA) is a crucial cell envelope component in Gram-positive bacteria. In Staphylococcus aureus, the polyglycerolphosphate LTA molecule is synthesized by LtaS, a membrane-embedded enzyme with five N-terminal transmembrane helices (5TM domain) that are connected via a linker region to the C-terminal extracellular enzymatic domain (eLtaS). The LtaS enzyme is processed during bacterial growth, and the eLtaS domain is released from the bacterial membrane. Here we provide experimental evidence that the proteolytic cleavage following residues 215Ala-Leu-Ala217 is performed by the essential S. aureus signal peptidase SpsB, as depletion of spsB results in reduced LtaS processing. In addition, the introduction of a proline residue at the +1 position with respect to the cleavage site, a substitution known to inhibit signal peptidase-dependent cleavage, abolished LtaS processing at this site. It was further shown that the 5TM domain is crucial for enzyme function. The observation that the construction of hybrid proteins between two functional LtaS-type enzymes resulted in the production of proteins unable to synthesize LTA suggests that specific interactions between the 5TM and eLtaS domains are required for function. No enzyme activity was detected upon expression of the 5TM and eLtaS domains as separate fragments, indicating that the two domains cannot assemble postsynthesis to form a functional enzyme. Taken together, our data suggest that only the full-length LtaS enzyme is active in the LTA synthesis pathway and that the proteolytic cleavage step is used as a mechanism to irreversibly inactivate the enzyme.

Staphylococcus aureus metalloprotease aureolysin cleaves complement C3 to mediate immune evasion.

Complement is one of the first host defense barriers against bacteria. Activated complement attracts neutrophils to the site of infection and opsonizes bacteria to facilitate phagocytosis. The human pathogen Staphylococcus aureus has successfully developed ways to evade the complement system, for example by secretion of specific complement inhibitors. However, the influence of S. aureus proteases on the host complement system is still poorly understood. In this study, we identify the metalloprotease aureolysin as a potent complement inhibitor. Aureolysin effectively inhibits phagocytosis and killing of bacteria by neutrophils. Furthermore, we show that aureolysin inhibits the deposition of C3b on bacterial surfaces and the release of the chemoattractant C5a. Cleavage analyses show that aureolysin cleaves the central complement protein C3. Strikingly, there was a clear difference between the cleavages of C3 in serum versus purified conditions. Aureolysin cleaves purified C3 specifically in the α-chain, close to the C3 convertase cleavage site, yielding active C3a and C3b. However, in serum we observe that the aureolysin-generated C3b is further degraded by host factors. We pinpointed these factors to be factor H and factor I. Using an aureolysin mutant in S. aureus USA300, we show that aureolysin is essential and sufficient for C3 cleavage by bacterial supernatant. In short, aureolysin acts in synergy with host regulators to inactivate C3 thereby effectively dampening the host immune response.

Staphylococcus aureus TargetArray: comprehensive differential essential gene expression as a mechanistic tool to profile antibacterials.

The widespread emergence of antibiotic-resistant bacteria and a lack of new pharmaceutical development have catalyzed a need for new and innovative approaches for antibiotic drug discovery. One bottleneck in antibiotic discovery is the lack of a rapid and comprehensive method to identify compound mode of action (MOA). Since a hallmark of antibiotic action is as an inhibitor of essential cellular targets and processes, we identify a set of 308 essential genes in the clinically important pathogen Staphylococcus aureus. A total of 446 strains differentially expressing these genes were constructed in a comprehensive platform of sensitized and resistant strains. A subset of strains allows either target underexpression or target overexpression by heterologous promoter replacements with a suite of tetracycline-regulatable promoters. A further subset of 236 antisense RNA-expressing clones allows knockdown expression of cognate targets. Knockdown expression confers selective antibiotic hypersensitivity, while target overexpression confers resistance. The antisense strains were configured into a TargetArray in which pools of sensitized strains were challenged in fitness tests. A rapid detection method measures strain responses toward antibiotics. The TargetArray antibiotic fitness test results show mechanistically informative biological fingerprints that allow MOA elucidation.

Biofilm dispersal of community-associated methicillin-resistant Staphylococcus aureus on orthopedic implant material.

Orthopedic implant-related bacterial infections are associated with high morbidity that may lead to limb amputation and exert significant financial burden on the healthcare system. Staphylococcus aureus is a dominant cause of these infections, and increased incidence of community-associated methicillin-resistant S. aureus (CA-MRSA) is being reported. The ability of S. aureus to attach to the foreign body surface and develop a biofilm is an important determinant of resistance to antibiotic prophylaxis. To gain insight on CA-MRSA biofilm properties, USA300 biofilm maturation and dispersal was examined, and these biofilms were found to exhibit pronounced, quorum-sensing mediated dispersal from a glass surface. For comparison of biofilm maturation on different surface chemistries, USA300 biofilm growth was examined on glass, polycarbonate, and titanium, and minimal differences were apparent in thickness, total biomass, and substratum coverage. Importantly, USA300 biofilms grown on titanium possessed a functional dispersal mechanism, and the dispersed cells regained susceptibility to rifampicin and levofloxacin treatment. The titanium biofilms were also sensitive to proteinase K and DNaseI, suggesting the matrix is composed of proteinaceous material and extracellular DNA. These studies provide new insights on the properties of CA-MRSA biofilms on implant materials, and indicate that quorum-sensing dispersion could be an effective therapeutic strategy.

A Staphylococcus aureus fitness test platform for mechanism-based profiling of antibacterial compounds.

The emergence of drug-resistant bacteria coupled with the limited discovery of novel chemical scaffolds and druggable targets inspires new approaches to antibiotic development. Here we describe a chemical genomics strategy based on 245 Staphylococcus aureus antisense RNA strains, each engineered for reduced expression of target genes essential for S. aureus growth. Attenuation of gene expression can sensitize cells to compounds that inhibit the activity of a gene product or associated process. Pools of strains grown competitively in the presence of bioactive compounds generate characteristic profiles of strain sensitivities reflecting compound mechanism of action. Here, we validate this approach with a structurally and mechanistically diverse set of reference antibiotics and, in the accompanying paper in this issue of Chemistry & Biology (Huber et al., 2009), demonstrate its use in the discovery of new cell wall inhibitors.

Fluorescent reporters for Staphylococcus aureus.

With the emergence of Staphylococcus aureus as a prominent pathogen in community and healthcare settings, there is a growing need for effective reporter tools to facilitate physiology and pathogenesis studies. Fluorescent proteins are ideal as reporters for their convenience in monitoring gene expression, performing host interaction studies, and monitoring biofilm growth. We have developed a suite of fluorescent reporter plasmids for labeling S. aureus cells. These plasmids encode either green fluorescent protein (GFP) or higher wavelength reporter variants for yellow (YFP) and red (mCherry) labeling. The reporters were placed under control of characterized promoters to enable constitutive or inducible expression. Additionally, plasmids were assembled with fluorescent reporters under control of the agr quorum-sensing and sigma factor B promoters, and the fluorescent response with wildtype and relevant mutant strains was characterized. Interestingly, reporter expression displayed a strong dependence on ribosome binding site (RBS) sequence, with the superoxide dismutase RBS displaying the strongest expression kinetics of the sequences examined. To test the robustness of the reporter plasmids, cell imaging was performed with fluorescence microscopy and cell populations were separated using florescence-activated cell sorting (FACS), demonstrating the possibilities of simultaneous monitoring of multiple S. aureus properties. Finally, a constitutive YFP reporter displayed stable, robust labeling of biofilm growth in a flow-cell apparatus. This toolbox of fluorescent reporter plasmids will facilitate cell labeling for a variety of different experimental applications.

Modulation of yeast Sln1 kinase activity by the CCW12 cell wall protein.

The yeast Sln1p sensor kinase is best known as an osmosensor involved in the regulation of the hyperosmolarity glycerol mitogen-activated protein kinase cascade. Down-regulation of Sln1 kinase activity occurs under hypertonic conditions and leads to phosphorylation of the Hog1p mitogen-activated protein kinase and increased osmotic stress-response gene expression. Conditions leading to kinase up-regulation include osmotic imbalance caused by glycerol retention in the glycerol channel mutant, fps1 (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367). The hypothesis that Sln1p kinase activity is responsive to turgor was first suggested by the increased Sln1p kinase activity in mutants lacking Fps1p in which glycerol accumulation leads to water uptake. Also consistent with the turgor hypothesis is the observation that reduced turgor caused by treatment of cells with nystatin, a drug that increases membrane permeability and causes cell shrinkage, reduced Sln1p kinase activity (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367; Reiser, V., Raitt, D. C., and Saito, H. (2003) J. Cell Biol. 161, 1035-1040). The turgor hypothesis is revisited here in the context of the identification and characterization of the cell wall gene, CCW12, as a determinant of Sln1p activity. Results of this analysis suggest that the activity of the plasma membrane localized Sln1p is affected by the presence or absence of specific outer cell wall proteins and that this effect is independent of turgor.

Biosynthesis of Staphylococcus aureus autoinducing peptides by using the synechocystis DnaB mini-intein.

The Agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). The peptides are seven to nine residues in length and have the C-terminal five residues constrained in a thiolactone ring. We have developed a new method to generate AIP structures using an engineered DnaB mini-intein from Synechocystis sp. strain PCC6803. In the method, an oligonucleotide encoding the AIP is ligated to the intein and the fusion protein is expressed and purified by affinity chromatography. To produce the correct AIP structure, intein splicing is interrupted, allowing the cysteine side chain to catalyze thiolactone ring formation and release AIP from the resin. The technique is simple and robust, and we have successfully produced the three main classes of AIPs using the intein system. The intein-generated AIPs possessed the correct thiolactone ring modification based on biochemical analysis, and, importantly, all the samples were bioactive against S. aureus. The AIP activity was confirmed through Agr interference and activation profiling with developed S. aureus reporter strains. The simplicity of the method, benefits of DNA encoding, and scalable nature enable the production of S. aureus AIPs for many biological applications.