Fatal hemorrhage induced by subtilase cytotoxin from Shiga-toxigenic Escherichia coli.

Subtilase cytotoxin (SubAB) is an AB(5) type toxin produced by a subset of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone BiP. The B subunit binds to a receptor on the cell surface. Although SubAB is lethal for mice, the cause of death is not clear. In this study, we demonstrate in mice that SubAB induced small bowel hemorrhage and a coagulopathy characterized by thrombocytopenia, prolonged prothrombin time and activated partial thromboplastin time. SubAB also induced inflammatory changes in the small intestine as detected by ¹8F-fluoro-2-deoxy-d-glucose positron emission tomography imaging and histochemical analysis. Using RT-PCR and ELISA, SubAB was shown to increase interleukin-6 in a time-dependent manner. Thus, our results indicate that death in SubAB-treated mice may be associated with severe inflammatory response and hemorrhage of the small intestine, accompanied by coagulopathy and IL6 production.

Identification of subtilase cytotoxin (SubAB) receptors whose signaling, in association with SubAB-induced BiP cleavage, is responsible for apoptosis in HeLa cells.

Subtilase cytotoxin (SubAB), which is produced by certain strains of Shiga-toxigenic Escherichia coli (STEC), causes the 78-kDa glucose-regulated protein (GRP78/BiP) cleavage, followed by induction of endoplasmic reticulum (ER) stress, leading to caspase-dependent apoptosis via mitochondrial membrane damage by Bax/Bak activation. The purpose of the present study was to identify SubAB receptors responsible for HeLa cell death. Four proteins, NG2, α2ß1 integrin (ITG), L1 cell adhesion molecule (L1CAM), and hepatocyte growth factor receptor (Met), were identified to be SubAB-binding proteins by immunoprecipitation and purification, followed by liquid chromatography-tandem mass spectrometry analysis. SubAB-induced Bax conformational change, Bax/Bak complex formation, caspase activation, and cell death were decreased in ß1 ITG, NG2, and L1CAM small interfering RNA-transfected cells, but unexpectedly, BiP cleavage was still observed. Pretreatment of cells with a function-blocking ß1 ITG antibody (monoclonal antibody [MAb] P5D2) enhanced SubAB-induced caspase activation; MAb P5D2 alone had no effect on caspase activation. Furthermore, we found that SubAB induced focal adhesion kinase fragmentation, which was mediated by a proteasome-dependent pathway, and caspase activation was suppressed in the presence of proteasome inhibitor. Thus, ß1 ITG serves as a SubAB-binding protein and may interact with SubAB-signaling pathways, leading to cell death. Our results raise the possibility that although BiP cleavage is necessary for SubAB-induced apoptotic cell death, signaling pathways associated with functional SubAB receptors may be required for activation of SubAB-dependent apoptotic pathways.

Subtilase cytotoxin induces apoptosis in HeLa cells by mitochondrial permeabilization via activation of Bax/Bak, independent of C/EBF-homologue protein (CHOP), Ire1alpha or JNK signaling.

Subtilase cytotoxin (SubAB) is an AB(5) cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum (ER) chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G(1) phase, and inducing caspase-dependent apoptosis via mitochondrial membrane damage in Vero cells. Here we investigated the mechanism of mitochondrial permeabilization in HeLa cells. SubAB-induced cytochrome c release into cytosol did not depend on mitochondrial permeability transition pore (PTP), since cyclosporine A did not suppress cytochrome c release. SubAB did not change the expression of anti-apoptotic Bcl-2 or Bcl-XL and pro-apoptotic Bax or Bak, but triggered Bax and Bak conformational changes and association of Bax with Bak. Silencing using siRNA of both bax and bak genes, but not bax, bak, or bim alone, resulted in reduction of cytochrome c release, caspase-3 activation, DNA ladder formation and cytotoxicity, indicating that Bax and Bak were involved in apoptosis. SubAB activated ER transmembrane transducers, Ire1alpha, and cJun N-terminal kinase (JNK), and induced C/EBF-homologue protein (CHOP). To investigate whether these signals were involved in cytochrome c release by Bax activation, we silenced ire1alpha, jnk or chop; however, silencing did not decrease SubAB-induced cytochrome c release, suggesting that these signals were not necessary for SubAB-induced mitochondrial permeabilization by Bax activation.

Resveratrol, a natural polyphenolic compound, inhibits cholera toxin-induced cyclic AMP accumulation in Vero cells.

Resveratrol (3,4',5-trihydroxystilbene), a natural polyphenol found in red grapes, berries and peanuts, exhibits anti-inflammatory, cell-growth modulatory, and anticarcinogenic effects. In this report, we show that resveratrol inhibited cholera toxin (CT)-induced cyclic AMP accumulation in Vero cells. Resveratrol suppressed the CT activity by suppressing the internalization of CT and traffic to the Golgi apparatus without affecting CT binding to cells. Further, resveratrol partially precipitated CT from solution, and suppressed CT ADP-ribosyltransferase activity. These data suggest that resveratrol may inhibit CT-induced activity directly by associating with CT and suppressing its enzymatic activity, and indirectly by inhibiting CT endocytosis into cells.

Novel subtilase cytotoxin produced by Shiga-toxigenic Escherichia coli induces apoptosis in vero cells via mitochondrial membrane damage.

Subtilase cytotoxin (SubAB) is an AB(5) cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G(1) phase. Here we show that SubAB, but not the catalytically inactive mutant SubAB(S272A), induced apoptosis in Vero cells, as detected by DNA fragmentation and annexin V binding. SubAB induced activation of caspase-3, -7, and -8. Caspase-3 appeared earlier than caspase-8, and by use of specific caspase inhibitors, it was determined that caspase-3 may be upstream of caspase-8. A general caspase inhibitor blocked SubAB-induced apoptosis, detected by annexin V binding. SubAB also stimulated cytochrome c release from mitochondria, which was not suppressed by caspase inhibitors. In HeLa cells, Apaf-1 small interfering RNA inhibited caspase-3 activation, suggesting that cytochrome c might form an apoptosome, leading to activation of caspase-3. These data suggested that SubAB induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage.

Effect of Gb3 in lipid rafts in resistance to Shiga-like toxin of mutant Vero cells.

Shiga-like toxin 1 (Stx1), produced by enterohemorrhagic Escherichia coli, binds to its receptor, globotriaosylceramide (Gb3), on target cell membranes, as a prerequisite for inducing host cell intoxication. To examine further toxin-receptor interactions, we established an Stx1-resistant clone of Vero cells by chemical mutagenesis. The mutant cells, expressed Gb3, but did not bind Stx1. These mutant cells were larger and had more Gb3 per cell than wild-type cells. Gb3 from both wild-type and mutant Vero cells was recovered in lipid rafts, isolated from cell lysates as detergent resistant membranes (DRMs); the DRMs derived from mutant cells had a lower density of Gb3 than did those from wild-type cells. Stx1 did not bind to the DRMs of mutant cells, both by ELISA and surface plasmon resonance. However, Stx1 bound to Gb3 separated by thin-layer chromatograms from the DRMs of mutant cells. The results indicate that not only presence of Gb3 but also Gb3 density on lipid rafts were important for Stx binding.

Subtilase cytotoxin, produced by Shiga-toxigenic Escherichia coli, transiently inhibits protein synthesis of Vero cells via degradation of BiP and induces cell cycle arrest at G1 by downregulation of cyclin D1.

Subtilase cytotoxin (SubAB) is a AB(5) type toxin produced by Shiga-toxigenic Escherichia coli, which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a subtilase-like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha). The phosphorylation of PERK and eIF2alpha was maximal at 30-60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress-inducible C/EBP-homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB-treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB-induced translational inhibition and continuous prolonged proteasomal degradation.

Inhibitory effects of various plant polyphenols on the toxicity of Staphylococcal alpha-toxin.

Staphylococcal alpha-toxin, known for its wide spectrum of biological activities, is involved in the pathogenesis of Staphylococcal infectious diseases. In recent years, various phytochemicals have been found to have antimicrobiological, including antibacterial, antiviral and antifungal, and antitoxic activities. We investigated whether several plant polyphenols inhibit alpha-toxin activities in vitro and in vivo. We found hop bract tannin (HBT) and apple condensed tannin (ACT) to exert inhibitory effects on alpha-toxin cytotoxicity. HBT also reduced the murine skin inflammatory effect and the lethality of alpha-toxin. These polyphenols formed aggregates with alpha-toxin and thereby inhibited its activities. Inhibition of alpha-toxin by HBT and ACT was dose dependent, suggesting that these polyphenols may be a useful adjunct to current treatments for alpha-toxin catalyzed Staphylococcal infectious diseases.

Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli.

Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 microg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was approximately 1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.

Helicobacter pylori induces mono-(adenosine 5'-diphosphate)-ribosylation in human gastric adenocarcinoma.

Mono-(adenosine 5'-diphosphate) (ADP)-ribosylation, which transfers an ADP-ribose from nicotinamide adenine dinucleotide (NAD) to an acceptor protein, is an important post-translational modification of cellular proteins. Several bacterial toxins are known to possess the mono-ADP-ribosyltransferase activity to catalyze this reaction as a possible pathogenic factor. Therefore, the aim of this study was to examine whether H. pylori may also induce mono-ADP-ribosylation in a human gastric mucosal protein in association with gastric cancer development. Tumorous and adjacent non-tumorous mucosal tissue specimens were obtained from the surgically removed stomachs of 5 patients with gastric adenocarcinoma, and then were homogenized into cytosolic and membranous fractions. Each homogenate or an H. pylori extract was assayed for mono-ADP-ribosylation with [adenylate-(32)P]-NAD and 3-aminobenzamide, a potent inhibitor of poly-ADP-ribosylation. The radiolabeled proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by radio-image analysis. In the extracts from H. pylori, a strain-dependent, endogenous radiolabeling of 70-kDa protein was detected. An assay of the membranous fractions from 5 gastric adenocarcinomas with the extract of OMH4, a clinical H. pylori isolate, revealed notable radiolabelings of 55- and 45-kDa proteins, which were not found without the OMH4 extract. In contrast, the radiolabelings were minimal in the membranous fractions from respective non-tumorous mucosae, and they were not detected in any of the examined cytosolic fractions. All three radiolabelings of 70-, 55-, and 45-kDa proteins were dependent on NAD, but not on ADP-ribose. Snake venom phosphodiesterase digestion of the 3 radiolabeled proteins released only AMP. We thus found that H. pylori had an enzymatic mono-ADP-ribosyltransferase activity which enabled it to modify the 55- and 45-kDa membranous proteins of human gastric adenocarcinoma, as well as the 70-kDa protein of H. pylori itself. The possible roles underlying our observations on carcinogenesis or development of human gastric carcinoma are yet to be elucidated.

Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin.

Some shiga toxin-producing Escherichia coli secrete a novel AB5 cytotoxin, named subtilase cytotoxin (SubAB), which induces vacuole formation in addition to cytotoxicity in susceptible cells. By immunoprecipitation with SubAB from Vero cells, we discovered proteins of 100 kDa, 135 kDa and 155 kDa as potential candidates for its receptor. These proteins were N-glycosylated in their extracellular domains, a modification that was necessary for interaction with SubAB. Biotinylated receptors were partially purified by Datura stramonium agglutinin affinity chromatography and avidin-agarose and analysed by TOF mass spectroscopy. The peptide sequences of p135 were identical to beta1 integrin, and its identification was confirmed with anti-integrin beta1 antibody. The p155 protein was identified as alpha2 integrin using anti-integrin alpha2 antibody. In addition, treatment of Vero cells with beta1 integrin RNAi before exposure to SubAB prevented vacuolating activity. These results suggested that SubAB recognizes alpha2beta1 integrin as a functional receptor; this first interaction may be an important key step leading to the SubAB-induced morphological changes in Vero cells.

Inhibitory effects of polyphenols on gastric injury by Helicobacter pylori VacA toxin.

BACKGROUND: Helicobacter pylori induces gastric damage and may be involved in the pathogenesis of gastric cancer. H. pylori-vacuolating cytotoxin, VacA, is one of the important virulence factors, and is responsible for H. pylori-induced gastritis and ulceration. The aim of this study is to assess whether several naturally occurring polyphenols inhibit VacA activities in vitro and in vivo. MATERIALS AND METHODS: Effects of polyphenols on VacA were quantified by the inhibition of: 1, vacuolation; 2, VacA binding to AZ-521 or G401 cells or its receptors; 3, VacA internalization. Effects of hop bract extract (HBT) containing high molecular weight polymerized catechin on VacA in vivo were investigated by quantifying gastric damage after oral administration of toxins to mice. RESULTS: HBT had the strongest inhibitory activity among the polyphenols investigated. HBT inhibited, in a concentration-dependent manner: 1, VacA binding to its receptors, RPTP(alpha) and RPTP(beta); 2, VacA uptake; 3, VacA-induced vacuolation in susceptible cells. In addition, oral administration of HBT with VacA to mice reduced VacA-induced gastric damage at 48 hours. In vitro, VacA formed a complex with HBT. CONCLUSIONS: HBT may suppress the development of inflammation and ulceration caused by H. pylori VacA, suggesting that HBT may be useful as a new type of therapeutic agent for the prevention of gastric ulcer and inflammation caused by VacA.

Acidic conditions enhance bactericidal effects of sodium bisulfite on Helicobacter pylori.

BACKGROUND: The Brucella broth medium, which is often used for the cultivation of microaerobic bacteria including Helicobacter pylori. It contains sodium bisulfite to decrease oxygen content in the medium. The growth of H. pylori, however, is inhibited by sodium bisulfite. In this study, the effect of sodium bisulfite was compared with several antioxidants and quantified under acidic conditions, mimicking the gastric environment. METHODS: Growth of H. pylori in the presence of several antioxidants was evaluated at OD655 nm. Effect of sodium bisulfite on H. pylori under acidic conditions was evaluated by measuring colony forming units (cfu). RESULTS: Under neutral conditions, sodium bisulfite was a more potent suppressor of H. pylori. Resveratrol, a polyphenol found in wine, exhibited the most potent inhibitory activity. To quantify the effect of sodium bisulfite on H. pylori under acidic conditions, the bacteria were grown at 37 degrees C for 30 minutes in 0.15 mol/l HCl/KCl (pH 2.0) with or without urea and sodium bisulfite. Sodium bisulfite (0.5 mmol/l) did not affect the viability at neutral pH 7.0, however, it killed H. pylori under acidic conditions, even if urea, the key substance enabling H. pylori to survive under acidic conditions, was present. The bacteria, which had been incubated under acidic conditions in the presence of urea, could survive a subsequent 30 minute-incubation at pH 2.0 without urea. Presence of sodium bisulfite, however, in the subsequent 30 minute-incubation, killed the bacteria. CONCLUSIONS: The bactericidal effect of sodium bisulfite on H. pylori was greater under acidic conditions and independent of urease activity.

Differential activities of plant polyphenols on the binding and internalization of cholera toxin in vero cells.

Plant polyphenols, RG-tannin, and applephenon had been reported to inhibit cholera toxin (CT) ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. We report here the effect of those polyphenols on the binding and entry of CT into Vero cells. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon but not RG-tannin. These observations were confirmed by fluorescence microscopy using Cy3-labeled CT. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CTA, and CTB from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization.

BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity.

Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is associated mainly with the trans-Golgi network. In the present study, we have revealed that another population of BIG2 is associated with the recycling endosome and found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We also have shown that BIG2 has an exchange activity toward class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF exaggerates the BIG2(E738K)-induced tubulation of endosomal membranes. These observations together indicate that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs.

Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin.

Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused by VacA. To define the region of RPTPbeta involved in VacA binding, we made mutants of human cDNA RPTPbeta-B, a short receptor form of RPTPbeta. Immunoprecipitation experiments to assess VacA binding to RPTPbeta-B mutants indicated that five residues (QTTQP) at positions 747-751 of the extracellular domain of RPTPbeta-B (which is commonly retained in RPTPbeta-A, a long form of RPTPbeta) play a crucial role in its interaction with VacA, resulting in vacuolation as well as Git-1 phosphorylation. Transfected cells expressing deletion mutant Delta752, which lacks QTTQP, or the double point mutant Delta747 (T748A,T749A) had diminished vacuolation in response to VacA. Treatment of RPTPbeta-B and Delta747 (which have QTTQP at 747-751) with neuraminidase and O-glycosidase diminished their VacA binding, whereas chondroitinase ABC did not have an effect. No inhibitory effect of pleiotrophin, a natural RPTPbeta ligand, on VacA binding to RPTPbeta-B or Delta747 was observed, supporting the conclusion that the extracellular region of RPTPbeta-B responsible for VacA binding is different from that involved in binding pleiotrophin. These data define the region in the RPTPbeta extracellular domain critical for VacA binding, in particular the sequence QTTQP at positions 747-751 with crucial threonines at positions 748 and 749 and are consistent with a role for terminal sialic acids possibly because of threonine glycosylation.

Purification, cloning and characterization of variant LukE-LukD with strong leukocidal activity of staphylococcal bi-component leukotoxin family.

Staphylococcus aureus produces bi-component leukotoxins composed of non-associated soluble proteins, S and F. Neither S nor F component alone is cytotoxic, but components together are active. These include Panton-Valentine leukocidin (PVL), gamma-hemolysin, LukE-LukD and others. Purification of leukotoxin from Staphylococcus aureus V8 strain (ATCC 27733) which does not have PVL genes, identified an F component with 100% identical to that of PVL in the first twenty-five N-terminal amino acids. Molecular cloning of this toxin obtained 2,595 nucleotides sequences containing two novel open reading frames for S and F. Deduced amino acid sequences of the S and F were respectively 91 and 94% identical to those of LukE and LukD. These were named variant of LukE-LukD (LukEv-LukDv). The activity of the recombinant LukEv-LukDv to rabbit leukocytes was similar to that of recombinant PVL. LukEv-LukDv was hemolytic to rabbit red blood cells although the activity was only 8% of gamma-hemolysin, but PVL was not. These activities were quite different from the LukE-LukD which was reported no hemolytic and poorly cytotoxic to leukocytes compared to PVL. The lukEv-IukDv was found in 87% of clinical isolates of Staphylococcus aureus but lukE-lukD was not detected. These data demonstrate the existence of variant LukE-LukD in V8 strain (ATCC 27733).

Calcium-regulated exocytosis of dense-core vesicles requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide binding site opener at the plasma membrane.

The ADP ribosylation factor (ARF) GTP binding proteins are believed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. Here we show that ARF6 is specifically associated with dense-core secretory granules in neuroendocrine PC12 cells. Stimulation with a secretagogue triggers the recruitment of secretory granules to the cell periphery and the concomitant activation of ARF6 by the plasma membrane-associated guanine nucleotide exchange factor, ARF nucleotide binding site opener (ARNO). Expression of the constitutively inactive ARF6(T27N) mutant inhibits secretagogue-dependent exocytosis from PC12 cells. Using a mutant of ARF6 specifically impaired for PLD1 stimulation, we find that ARF6 is functionally linked to phospholipase D (PLD)1 in the exocytotic machinery. Finally, we show that ARNO, ARF6, and PLD1 colocalize at sites of exocytosis, and we demonstrate direct interaction between ARF6 and PLD1 in stimulated cells. Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore.

Destructive pulmonary embolism in a patient with community-acquired staphylococcal bacteremia.

We report a 17-year-old man with destructive pulmonary embolism caused by Staphylococcus aureus bacteremia. The patient was not immunocompromised and had neither underlying diseases nor risk factors, such as concomitant influenza viral infection, which exacerbate staphylococcal infections. The rapid and extensive progression of pulmonary involvement in all lung fields make this a rare case; there have been few reports in the literature describing a similar radiographic appearance in patients with community-acquired staphylococcal bacteremia. In-vitro studies did not demonstrate the production of enterotoxins or toxic shock syndrome toxin 1 (TSST-1) by the isolated strain, but genetic analysis detected Panton-Valentine leukocidine gene from the strain. Subsequent empyema with bilateral pneumothorax was prolonged because of superinfection with both methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Optional surgical treatments, including thoracostomy and thoracopneumoplasty, finally improved his condition.