Redundant and Distinct Roles of Secreted Protein Eap and Cell Wall-Anchored Protein SasG in Biofilm Formation and Pathogenicity of Staphylococcus aureus.

Chronic and fatal infections caused by Staphylococcus aureus are sometimes associated with biofilm formation. Secreted proteins and cell wall-anchored proteins (CWAPs) are important for the development of polysaccharide-independent biofilms, but functional relationships between these proteins are unclear. In the present study, we report the roles of the extracellular adherence protein Eap and the surface CWAP SasG in S. aureus MR23, a clinical methicillin-resistant isolate that forms a robust protein-dependent biofilm and accumulates a large amount of Eap in the extracellular matrix. Double deletion of eap and sasG, but not single eap or sasG deletion, reduced the biomass of the formed biofilm. Mutational analysis demonstrated that cell wall anchorage is essential for the role of SasG in biofilm formation. Confocal laser scanning microscopy revealed that MR23 formed a rugged and thick biofilm; deletion of both eap and sasG reduced biofilm ruggedness and thickness. Although sasG deletion did not affect either of these features, eap deletion reduced the ruggedness but not the thickness of the biofilm. This indicated that Eap contributes to the rough irregular surface structure of the MR23 biofilm and that both Eap and SasG play roles in biofilm thickness. The level of pathogenicity of the Δeap ΔsasG strain in a silkworm larval infection model was significantly lower (P < 0.05) than those of the wild type and single-deletion mutants. Collectively, these findings highlight the redundant and distinct roles of a secreted protein and a CWAP in biofilm formation and pathogenicity of S. aureus and may inform new strategies to control staphylococcal biofilm infections.

Roles of lytic transglycosylases in biofilm formation and ß-lactam resistance in methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is responsible for numerous community outbreaks and is one of the most frequent causes of nosocomial infections with significant morbidity and mortality. While the function of lytic transglycosylases (LTs) in relation to cell division, biofilm formation, and antibiotic resistance has been determined for several bacteria, their role in S. aureus remains largely unknown. The only known LTs in S. aureus are immunodominant staphylococcal antigen A (IsaA) and Staphylococcus epidermidis D protein (SceD). Our study demonstrates that, in a strain of methicillin-resistant S. aureus (MRSA), IsaA and SceD contribute differently to biofilm formation and ß-lactam resistance. Deletion of isaA, but not sceD, led to decreased biofilm formation. Additionally, in isaA-deleted strains, ß-lactam resistance was significantly decreased compared to that of wild-type strains. Plasmid-based expression of mecA, a major determinant of ß-lactam resistance in MRSA, in an isaA-deleted strain did not restore ß-lactam resistance, demonstrating that the ß-lactam susceptibility phenotype is exhibited by isaA mutant regardless of the production level of PBP2a. Overall, our results suggest that IsaA is a potential therapeutic target for MRSA infections.

Detection of bacterial DNA from central venous catheter removed from patients by next generation sequencing: a preliminary clinical study.

BACKGROUND: Catheter-related infection (CRI) is one of the serious challenges in clinical practice. This preliminary clinical study aimed to examine whether next-generation sequencing (NGS) targeting 16S rDNA, which was PCR-amplified directly from the tip of a central venous catheter (CVC), can be used to identify causative pathogens in CRI, compared to the culture method. METHODS: Hospitalized patients, from whom a CVC had just been removed, were prospectively enrolled and divided into the CRI-suspected and routine removal groups. DNA was extracted from the sonication fluid of CVC specimens derived from patients. For analysis of bacterial composition by NGS, the V3-V4 fragments of bacterial 16S rDNA were PCR-amplified, followed by index PCR and paired-end sequencing on an Illumina MiSeq device. Conventional culture methods were also performed in the CRI-suspected group. RESULTS: Of CVCs collected from the 156 enrolled patients (114 men; mean age 65.6 years), a total of 14 specimens [nine out of 31 patients suspected with CRI and five out of 125 patients without infection symptoms (routine removal group)] were PCR-positive. In five patients with definite CRI, Staphylococcus was the most frequently detected genus by NGS (4/5 specimens), although no pathogens were detected by NGS in the one remaining specimen. The genera identified by NGS were consistent with those from conventional culture tests. There was high agreement between NGS and the culture method in the CRI-suspected group, with sensitivity and specificity values of 80.0% and 76.9%, respectively; meanwhile, the false-positive rate of NGS was as low as 4.0% in the routine removal group. Moreover, several genera, besides the genus identified by culture test, were detected in each patient with definite CRI and surgical site infection (SSI). Additionally, in one patient with SSI, Enterococcaceae were detected not only by NGS but also by abdominal abscess drainage culture. CONCLUSIONS: NGS targeting 16S rDNA was able to analyze the bacterial composition of CVC specimens and detect causative pathogens in patients with CRI and was therefore suggested as a promising diagnostic tool for CRI.

Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels.

The intrinsically stochastic dynamics of mRNA metabolism have important consequences on gene regulation and non-genetic cell-to-cell variability; however, no generally applicable methods exist for studying such stochastic processes quantitatively. Here, we describe the use of the amyloid-binding probe Thioflavin T (ThT) for monitoring RNA metabolism in vitro and in vivo. ThT fluoresced strongly in complex with bacterial total RNA than with genomic DNA. ThT bound purine oligoribonucleotides preferentially over pyrimidine oligoribonucleotides and oligodeoxyribonucleotides. This property enabled quantitative real-time monitoring of poly(A) synthesis and phosphorolysis by polyribonucleotide phosphorylase in vitro. Cellular analyses, in combination with genetic approaches and the transcription-inhibitor rifampicin treatment, demonstrated that ThT mainly stained mRNA in actively dividing Escherichia coli cells. ThT also facilitated mRNA metabolism profiling at the single-cell level in diverse bacteria. Furthermore, ThT can also be used to visualise transitions between non-persister and persister cell states, a phenomenon of isogenic subpopulations of antibiotic-sensitive bacteria that acquire tolerance to multiple antibiotics due to stochastically induced dormant states. Collectively, these results suggest that probing mRNA dynamics with ThT is a broadly applicable approach ranging from the molecular level to the single-cell level.

Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization.

Commensal bacteria are known to inhibit pathogen colonization; however, complex host-microbe and microbe-microbe interactions have made it difficult to gain a detailed understanding of the mechanisms involved in the inhibition of colonization. Here we show that the serine protease Esp secreted by a subset of Staphylococcus epidermidis, a commensal bacterium, inhibits biofilm formation and nasal colonization by Staphylococcus aureus, a human pathogen. Epidemiological studies have demonstrated that the presence of Esp-secreting S. epidermidis in the nasal cavities of human volunteers correlates with the absence of S. aureus. Purified Esp inhibits biofilm formation and destroys pre-existing S. aureus biofilms. Furthermore, Esp enhances the susceptibility of S. aureus in biofilms to immune system components. In vivo studies have shown that Esp-secreting S. epidermidis eliminates S. aureus nasal colonization. These findings indicate that Esp hinders S. aureus colonization in vivo through a novel mechanism of bacterial interference, which could lead to the development of novel therapeutics to prevent S. aureus colonization and infection.

Novel strategy for biofilm inhibition by using small molecules targeting molecular chaperone DnaK.

Biofilms are complex communities of microorganisms that attach to surfaces and are embedded in a self-produced extracellular matrix. Since these cells acquire increased tolerance against antimicrobial agents and host immune systems, biofilm-associated infectious diseases tend to become chronic. We show here that the molecular chaperone DnaK is important for biofilm formation and that chemical inhibition of DnaK cellular functions is effective in preventing biofilm development. Genetic, microbial, and microscopic analyses revealed that deletion of the dnaK gene markedly reduced the production of the extracellular functional amyloid curli, which contributes to the robustness of Escherichia coli biofilms. We tested the ability of DnaK inhibitors myricetin (Myr), telmisartan, pancuronium bromide, and zafirlukast to prevent biofilm formation of E. coli. Only Myr, a flavonol widely distributed in plants, inhibited biofilm formation in a concentration-dependent manner (50% inhibitory concentration [IC50] = 46.2 µM); however, it did not affect growth. Transmission electron microscopy demonstrated that Myr inhibited the production of curli. Phenotypic analyses of thermosensitivity, cell division, intracellular level of RNA polymerase sigma factor RpoH, and vulnerability to vancomycin revealed that Myr altered the phenotype of E. coli wild-type cells to make them resemble those of the isogenic dnaK deletion mutant, indicating that Myr inhibits cellular functions of DnaK. These findings provide insights into the significance of DnaK in curli-dependent biofilm formation and indicate that DnaK is an ideal target for antibiofilm drugs.

Immuno-electron microscopy of primary cell cultures from genetically modified animals in liquid by atmospheric scanning electron microscopy.

High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell's plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.

Staphylococcus epidermidis Esp degrades specific proteins associated with Staphylococcus aureus biofilm formation and host-pathogen interaction.

Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronic infections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassembles preformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesive and cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins of Esp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein (Esp(S235A)) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicillin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly. Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently used for testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed that Esp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellular adherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition, Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are involved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation by degrading specific proteins that are crucial for biofilm construction and host-pathogen interaction.

Effects of bacteriocins on methicillin-resistant Staphylococcus aureus biofilm.

Control of biofilms formed by microbial pathogens is an important subject for medical researchers, since the development of biofilms on foreign-body surfaces often causes biofilm-associated infections in patients with indwelling medical devices. The present study examined the effects of different kinds of bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by certain bacteria, on biofilms formed by a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA). The activities and modes of action of three bacteriocins with different structures (nisin A, lacticin Q, and nukacin ISK-1) were evaluated. Vancomycin, a glycopeptide antibiotic used in the treatment of MRSA infections, showed bactericidal activity against planktonic cells but not against biofilm cells. Among the tested bacteriocins, nisin A showed the highest bactericidal activity against both planktonic cells and biofilm cells. Lacticin Q also showed bactericidal activity against both planktonic cells and biofilm cells, but its activity against biofilm cells was significantly lower than that of nisin A. Nukacin ISK-1 showed bacteriostatic activity against planktonic cells and did not show bactericidal activity against biofilm cells. Mode-of-action studies indicated that pore formation leading to ATP efflux is important for the bactericidal activity against biofilm cells. Our results suggest that bacteriocins that form stable pores on biofilm cells are highly potent for the treatment of MRSA biofilm infections.

Glucose triggers ATP secretion from bacteria in a growth-phase-dependent manner.

ATP modulates immune cell functions, and ATP derived from gut commensal bacteria promotes the differentiation of T helper 17 (Th17) cells in the intestinal lamina propria. We recently reported that Enterococcus gallinarum, isolated from mice and humans, secretes ATP. We have since found and characterized several ATP-secreting bacteria. Of the tested enterococci, Enterococcus mundtii secreted the greatest amount of ATP (>2 µM/10(8) cells) after overnight culture. Glucose, not amino acids and vitamins, was essential for ATP secretion from E. mundtii. Analyses of energy-deprived cells demonstrated that glycolysis is the most important pathway for bacterial ATP secretion. Furthermore, exponential-phase E. mundtii and Enterococcus faecalis cells secrete ATP more efficiently than stationary-phase cells. Other bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, also secrete ATP in exponential but not stationary phase. These results suggest that various gut bacteria, including commensals and pathogens, might secrete ATP at any growth phase and modulate immune cell function.

Purification, crystallization and preliminary X-ray diffraction analysis of the Staphylococcus epidermidis extracellular serine protease Esp.

Esp, an extracellular serine protease from Staphylococcus epidermidis, has been shown to inhibit S. aureus biofilm formation and nasal colonization. The full-length 27 kDa pro-Esp was purified and digested with thermolysin to obtain mature Esp. The mature Esp containing 216 residues crystallized in space group P2(1), with unit-cell parameters a = 39.5, b = 61.2, c = 42.5 Å, ß = 98.2° and one molecule in the asymmetric unit, with an estimated solvent content of 42%. A diffraction data set has been collected to 1.8 Šresolution on a rotating-anode home-source facility.

Staphylococcus aureus utilizes environmental RNA as a building material in specific polysaccharide-dependent biofilms.

Biofilms are surface-bound microbial communities that are typically embedded in a matrix of self-produced extracellular polymeric substances and can cause chronic infections. Extracellular DNA is known to play a crucial role in biofilm development in diverse bacteria; however, the existence and function of RNA are poorly understood. Here, we show that RNA contributes to the structural integrity of biofilms formed by the human pathogen Staphylococcus aureus. RNase A dispersed both fresh and mature biofilms, indicating the importance of RNA at various stages. RNA-sequencing analysis demonstrated that the primary source of RNA in the biofilm matrix was the Brain Heart Infusion medium (>99.32%). RNA purified from the medium promoted biofilm formation. Microscopic and molecular interaction analyses demonstrated that polysaccharides were critical for capturing and stabilizing external RNA in biofilms, which contributes to biofilm organization. These findings provide a basis for exploring the role of externally derived substances in bacterial biofilm organization.

Antimicrobial strategy for targeted elimination of different microbes, including bacterial, fungal and viral pathogens.

The continuous emergence of microbial pathogens for which there are no effective antimicrobials threatens global health, necessitating novel antimicrobial approaches. Here, we present a targeted antimicrobial strategy that can be applied to various microbial pathogens. A photoimmuno-conjugate composed of an antibody against the target pathogen and a photoplastic phthalocyanine-derivative probe that generates photo-induced mechanical stress was developed based on photoimmuno-technology. This strategy, named as photoimmuno-antimicrobial strategy (PIAS), eliminates targeted pathogens, regardless of the target species or drug-resistance status. Specifically, PIAS acts on a broad range of microbes, including the bacterial pathogen Staphylococcus aureus, fungal pathogen Candida albicans, including their drug-resistant strains, and viral pathogen SARS-CoV-2, the causative agent of COVID-19. Furthermore, PIAS protects mice from fatal infections without damaging the non-targeted host microbiota and tissues. This study may contribute to the development of next-generation anti-infective therapies.

Small-Molecule-Induced Activation of Cellular Respiration Inhibits Biofilm Formation and Triggers Metabolic Remodeling in Staphylococcus aureus.

Staphylococcus aureus, a major pathogen of community-acquired and nosocomial-associated infections, forms biofilms consisting of extracellular matrix-embedded cell aggregates. S. aureus biofilm formation on implanted medical devices can cause local and systemic infections due to the dispersion of cells from the biofilms. Usually, conventional antibiotic treatments are not effective against biofilm-related infections, and there is no effective treatment other than removing the contaminated devices. Therefore, the development of new therapeutic agents to combat biofilm-related infections is urgently needed. We conducted high-throughput screening of S. aureus biofilm inhibitors and obtained a small compound, JBD1. JBD1 strongly inhibits biofilm formation of S. aureus, including methicillin-resistant strains. In addition, JBD1 activated the respiratory activity of S. aureus cells and increased the sensitivity to aminoglycosides. Furthermore, it was shown that the metabolic profile of S. aureus was significantly altered in the presence of JBD1 and that metabolic remodeling was induced. Surprisingly, these JBD1-induced phenotypes were blocked by adding an excess amount of the electron carrier menaquinone to suppress respiratory activation. These results indicate that JBD1 induces biofilm inhibition and metabolic remodeling through respiratory activation. This study demonstrates that compounds that enhance the respiratory activity of S. aureus may be potential leads in the development of therapeutic agents for chronic S. aureus-biofilm-related infections. IMPORTANCE Chronic infections caused by Staphylococcus aureus are characterized by biofilm formation, suggesting that methods to control biofilm formation may be of therapeutic value. The small compound JBD1 showed biofilm inhibitory activity and increased sensitivity to aminoglycosides and respiratory activity of S. aureus. Additionally, transcriptomic and metabolomic analyses demonstrated that JBD1 induced metabolic remodeling. All JBD1-induced phenotypes were suppressed by the extracellular addition of an excess amount of menaquinone, indicating that JBD1-mediated respiratory stimulation inhibits biofilm formation and triggers metabolic remodeling in S. aureus. These findings suggest a strategy for developing new therapeutic agents for chronic S. aureus infections.

Role of fibronectin-binding proteins A and B in in vitro cellular infections and in vivo septic infections by Staphylococcus aureus.

Fibronectin-binding protein A (FnBPA) and FnBPB are important adhesins for Staphylococcus aureus infection. We constructed fnbA and/or fnbB mutant strains from S. aureus SH1000, which possesses intact rsbU, and studied the role of these adhesins in in vitro and in vivo infections. In intravenous infection, all fnb mutants caused a remarkable reduction in the colonization rate in kidneys and the mortality rate of mice. fnbB mutant caused a more severe decrease in body weight than that caused by fnbA mutant. Serum levels of interleukin-6 and nuclear factor κB (NF-κB) activation in spleen cells were remarkably reduced in fnbA or fnbA fnbB mutant infections; however, there was no significant reduction in fnbB mutant infections. In in vitro cellular infection, FnBPA was shown to be indispensable for adhesion to and internalization by nonprofessional phagocytic cells upon ingestion by inflammatory macrophages and NF-κB activation. However, both FnBPs were required for efficient cellular responses. The results showed that FnBPA is more important for in vitro and in vivo infections; however, cooperation between FnBPA and FnBPB is indispensable for the induction of severe infection resulting in septic death.

Tissue binding patterns and in vitro effects of Campylobacter jejuni DNA-binding protein from starved cells.

Campylobacter jejuni (C. jejuni) is frequently associated with axonal Guillain-Barré syndrome (GBS). We reported that C. jejuni DNA-binding protein from starved cells (C-Dps) binds to and damages myelinated nerves in vivo. We studied the binding patterns of C-Dps to nervous tissues and its in vitro effects on neural cells. Immunohistochemically, C-Dps labeled the nodes of Ranvier, the outermost parts of internodal myelin and the basement membrane in the peripheral nerves, and neurons and myelin in the central nervous tissues. Its binding was blocked by sulfatide. C-Dps bound to the cell surfaces of nerve growth factor (NGF)-treated PC12 cells leading to dose-dependent LDH release, which was inhibited by either heat-denaturation of C-Dps or coincubation with an anti-C-Dps mAb. However, its binding to the surfaces of cultured NSC34 cells, S16 cells, or dorsal root ganglion cells, did not induce cytotoxicity. These findings suggest a possible involvement of C-Dps in C. jejuni-related GBS.

Hierarchical Model for the Role of J-Domain Proteins in Distinct Cellular Functions.

In Escherichia coli, the major bacterial Hsp70 system consists of DnaK, three J-domain proteins (JDPs: DnaJ, CbpA, and DjlA), and nucleotide exchange factor GrpE. JDPs determine substrate specificity for the Hsp70 system; however, knowledge on their specific role in bacterial cellular functions is limited. In this study, we demonstrated the role of JDPs in bacterial survival during heat stress and the DnaK-regulated formation of curli-extracellular amyloid fibers involved in biofilm formation. Genetic analysis demonstrate that only DnaJ is essential for survival at high temperature. On the other hand, either DnaJ or CbpA, but not DjlA, is sufficient to activate DnaK in curli production. Additionally, several DnaK mutants with reduced activity are able to complement the loss of curli production in E. coli ΔdnaK, whereas they do not recover the growth defect of the mutant strain at high temperature. Biochemical analyses reveal that DnaJ and CbpA are involved in the expression of the master regulator CsgD through the solubilization of MlrA, a DNA-binding transcriptional activator for the csgD promoter. Furthermore, DnaJ and CbpA also keep CsgA in a translocation-competent state by preventing its aggregation in the cytoplasm. Our findings support a hierarchical model wherein the role of JDPs in the Hsp70 system differs according to individual cellular functions.

Biogenesis of Salmonella enterica serovar typhimurium membrane vesicles provoked by induction of PagC.

Gram-negative bacteria ubiquitously release membrane vesicles (MVs) into the extracellular milieu. Although MVs are the product of growing bacteria, not of cell lysis or death, the regulatory mechanisms underlying MV formation remained unknown. We have found that MV biogenesis is provoked by the induction of PagC, a Salmonella-specific protein whose expression is activated by conditions that mimic acidified macrophage phagosomes. PagC is a major constituent of Salmonella MVs, and increased expression accelerates vesiculation. Expression of PagC is regulated at the posttranscriptional and/or posttranslational level in a sigmaS (RpoS)-dependent manner. Serial quantitative analysis has demonstrated that MV formation can accelerate when the quantity of the MV constituents, OmpX and PagC, rises. Overproduction of PagC dramatically impacts the difference in the relative amount of vesiculation, but the corresponding overproduction of OmpX was less pronounced. Quantitative examination of the ratios of PagC and OmpX in the periplasm, outer membrane, and MVs demonstrates that PagC is preferentially enriched in MVs released from Salmonella cells. This suggests that specific protein sorting mechanisms operate when MVs are formed. The possible role(s) of PagC-MV in host cells is discussed.

Isolation and identification of ATP-secreting bacteria from mice and humans.

In a recent report, ATP, which was possibly secreted by some intestinal bacteria, was shown to cause colitis in mice via Th17 cell differentiation. However, the ATP-secreting bacteria have not been isolated and identified. In the present study, we report that Enterococcus gallinarum, which is a vancomycin-resistant Gram-positive coccus isolated from mice and humans, secretes ATP.

Inhibition of endothelial interleukin-8 production and neutrophil transmigration by Staphylococcus aureus beta-hemolysin.

Neutrophils play a crucial role in the host response to infection with Staphylococcus aureus, which is a major human pathogen capable of causing life-threatening disease. Interleukin-8 (IL-8) is a potent chemoattractant and activator of neutrophils. We previously reported that S. aureus secretes a factor that suppresses IL-8 production by human endothelial cells. Here we isolated an inhibitor of IL-8 production from the supernatant and identified it as staphylococcal beta-hemolysin. Beta-hemolysin reduced IL-8 production without cytotoxicity to endothelial cells. Pretreatment with beta-hemolysin decreased the expression of both IL-8 mRNA and protein induced by tumor necrosis factor alpha (TNF-alpha). Migration of neutrophils across TNF-alpha-activated endothelium was also inhibited by beta-hemolysin. In contrast, beta-hemolysin had no effect on intercellular adhesive molecule 1 expression in activated endothelial cells. These results showed that beta-hemolysin produced by S. aureus interferes with inflammatory signaling in endothelial cells and may help S. aureus evade the host immune response.