Staphylococcal γ-hemolysins induce IL-4 production in murine basophils.

Basophils are known to produce a large amount of IL-4 in response to stimuli and play a role in the initiation and propagation of type 2 inflammations. S. aureus secretes a series of pore-forming toxins: α-hemolysin, γ-hemolysins, and leukocidins. In this study, we examined the effects of α-hemolysin, γ-hemolysins (HlgAB and HlgCB), and leukocidins (LukAB, LukED, and Panton-Valentine leukocidin) on the function of basophils. All pore-forming toxins except for Panton-Valentine leukocidin bound to murine bone marrow-derived basophils (BMBs). HlgAB and LukED but not other toxins evoked the leakage of lactate dehydrogenase from BMBs at the concentration of 30 µg/ml γ-hemolysins, HlgAB and HlgCB, induced the secretion of IL-4 in BMBs at concentrations above 3.3 µg/ml. LukAB did not induce, and Hla and LukED induced only a small amount of IL-4. HlgBΔstem, the 5 amino acids deletion mutant of HlgB in the stem region, diminished IL-4 secretion by HlgAB and HlgCB in BMBs. These results suggest that the cell damage and the induction of IL-4 in basophils by HlgAB require pore formation. The induction of IL-4 by γ-hemolysins was also observed in fleshly isolated murine basophils. These results demonstrate a novel function of γ-hemolysins, the induction of IL-4 in basophils, in an IgE-independent manner.

LukS-PV inhibits hepatocellular carcinoma cells migration by downregulating HDAC6 expression.

BACKGROUND: Hepatocellular carcinoma (HCC) is a clinically common malignant tumor worldwide. LukS-PV is the S component of Panton-Valentine leukocidin secreted by Staphylococcus aureus, which has shown anti-cancer activity. Based on previous findings, this study investigated the effects of LukS-PV on HCC migration and the potential molecular mechanisms involving acetylation pathways. METHODS: After treating HCC cells with different concentrations of LukS-PV, we used scratch assays to determine the mobility of the cancer cells. Western blots were used to determine the expression levels of migration-related proteins. Quantitative proteomic sequencing was used to evaluate proteomic changes in target proteins. Immunoprecipitation and liquid chromatography coupled with tandem mass spectrometry analyses were used to validate the binding of related target proteins. RESULTS: LukS-PV inhibited HCC cell migration in a concentration-dependent manner. In addition, LukS-PV attenuated the expression of histone deacetylase (HDAC)6, which is highly expressed in HCC cells. Further studies showed that LukS-PV increased the acetylation level of α-tubulin by down-regulating HDAC6, which resulted in the inhibition of HCC cell migration. CONCLUSION: Taken together, our data revealed a vital role of LukS-PV in suppressing HCC cell migration by down-regulating HDAC6 and increasing the acetylation level of α-tubulin.

Prevalence of Panton Valentine Leucocidin gene containing Staphylococcus aureus in pus samples from Paediatric patients.

Staphylococcus (Staph) aureus containing Panton Valentine Leucocidin (PVL) gene are spreading in the whole world. This gene encodes PVL toxin that has lytic effect on WBCs contributing to the low immunity of the body. It also causes pus formation in various places of the body. This study was conducted to understand the effect of PVL positive Staph aureus in causing purulent infections in children between the age of one day to 15 years. Pus samples from various sites of the body from children between the age of one day to 15 years were taken. The number of pus samples containing Staph aureus was 45. These were collected over a period of one year, from October 2, 2017 to September 30, 2018, at the Shaikh Zayed Hospital, Lahore. A total of 27 (60%) PVL samples were positive Staph aureus. Prevalence of PVL gene was noted to be high in MSSA 9(64%), wound swabs 18(75%), in isolates from orthopaedic department 6(75%), indoor 21(63%), and in males 18(66%). Our study showed that most of the Staph aureus samples that were obtained from pus samples from children had PVL gene in their genome. This percentage is very high. To control its spread, we need to treat not only the patients but also their close contacts. The main objective to conduct this study was to assess the prevalence of PVL positive Staph aureus strain in our local setup. Paediatric age group was selected because it is the most vulnerable group and pus samples were chosen because this strain causes recurrent purulent infections.

LukS-PV induces cell cycle arrest and apoptosis through p38/ERK MAPK signaling pathway in NSCLC cells.

Non-small-cell lung cancer (NSCLC) accounts for nearly 85% of lung cancer cases. LukS-PV, one of the two components of Panton-Valentine leucocidin (PVL), is produced by Staphylococcus aureus. The present study showed that LukS-PV can induce apoptosis in human acute myeloid leukemia (AML) lines (THP-1 and HL-60). However, the role of LukS-PV in NSCLC is unclear. In this study, we treated NSCLC cell lines A549 and H460 and a normal lung cell line, 16HBE, with LukS-PV and investigated the biological roles of LukS-PV in NSCLC. Cells were treated with varying concentrations of LukS-PV and cell viability was evaluated by CCK8 and EdU assay. Flow cytometry was used to detect cell apoptosis and analyze the cell cycle, and the expression of apoptosis and cell cycle-associated proteins and genes were identified by western blotting analysis and qRT-polymerase chain reaction, respectively. We found that LukS-PV inhibited the proliferation of NSCLC cells but had little cytotoxicity in normal lung cells. LukS-PV induced NSCLC cell apoptosis and increased the BAX/BCL-2 ratio, triggering S-phase arrest in A549 and H460 cells while increasing P21 expression and decreasing CDK2, cyclin D1, and cyclin A2 expression. We also observed increased P-p38 and P-ERK in NSCLC cells treated with LukS-PV. Treatment of NSCLC with LukS-PV combined with p38 and ERK inhibitors reversed the pro-apoptotic and pro-cell cycle arrest effects of LukS-PV. Overall, these findings indicate that LukS-PV has anti-tumor effects in NSCLC and may contribute to the development of anti-cancer agents.

Quantitative proteomic analysis reveals that Luks-PV exerts antitumor activity by regulating the key proteins and metabolic pathways in HepG2 cells.

Hepatocellular carcinoma (HCC) is a complicated and poor prognosis cancer, necessitating the development of a potential treatment strategy. In this study, we initially revealed that LukS-PV belonged to leukocidin family performs an anti-HCC action. Then, we used liquid chromatography-mass spectrometry (LC/MS) to compare protein expression profiles of the LukS-PV-treated human HCC cell lines HepG2 and the control cells. GO annotations and Kyoto Encyclopedia of Genes and Genomes pathway analysis were carried out of differential expression followed by protein-protein interactome, to explore the underlying cancer suppressor mechanisms of LukS-PV for human HCC. A total of 88 upregulated proteins and 46 downregulated proteins were identified. The top 10 proteins identified by the MCC method are FN1, APP, TIMP1, nucleobindin-1, GOLM1, APLP2, CYR61, CD63, ENG, and CD9. Our observation on protein expression indicated that LukS-PV produces a signature affecting central carbon metabolism in cancer, galactose metabolism, and fructose and mannose metabolism pathways. The results give a functional effects and molecular mechanism insight, following LukS-PV treatment.

LukS-PV-Regulated MicroRNA-125a-3p Promotes THP-1 Macrophages Differentiation and Apoptosis by Down-Regulating NF1 and Bcl-2.

BACKGROUND/AIMS: LukS-PV is a component of Panton-Valentine leukocidin (PVL). We have previously demonstrated that LukS-PV potently promoted differentiation and induced apoptosis in THP-1 cells. However, the precise mechanisms of these actions remain unknown. MicroRNAs (miRs) play important roles in cellular differentiation and apoptosis. This study aimed to investigate the role of miR-125a-3p in LukS-PV-regulated differentiation and apoptosis and its underlying mechanism in THP-1 cells. METHODS: MicroRNA profiling analyses were conducted to determine differential miRNA expression levels in THP-1 cells treated with LukS-PV. Cell differentiation and apoptosis were measured in THP-1 cells by gain-of-function and loss-of-function experiments. Bioinformatics analysis and luciferase reporter assays were used to confirm the targets of miR-125a-3p. The effects of the miR-125a-3p targets on cellular differentiation were determined by knocking them down. RESULTS: MiR-125a-3p was up-regulated after treating the human monocytic leukaemia cell line THP-1 with LukS-PV. In addition, miR-125a-3p positively regulated apoptosis and differentiation in THP-1 cells treated with LukS-PV. Concordantly, luciferase reporter assays confirmed that neurofibromatosis type 1 (NF1) and B-cell lymphoma 2 (Bcl-2) were direct target genes of miR-125a-3p. Moreover, NF1 knockdown in THP-1 cells significantly promoted differentiation in vitro. Finally, the extracellular signal-regulated kinase (ERK) pathway, a downstream target of NF1, was activated after NF1 knockdown. CONCLUSIONS: These findings confirm that miR-125a-3p is involved in LukS-PV-mediated cell differentiation and apoptosis in THP-1 cells.

Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection.

Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγnull (NSG) mice engrafted with human CD34+ umbilical cord blood cells. These "humanized" NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL+ versus isogenic PVL- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL+ S. aureus but not PVL- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.

New Strategies Targeting Virulence Factors of Staphylococcus aureus and Pseudomonas aeruginosa.

Morbidity, mortality, and economic burden of nosocomial pneumonia caused by Staphylococcus aureus and Pseudomonas aeruginosa remain high in mechanically ventilated and hospitalized patients despite the use of empirical antibiotic therapy or antibiotics against specific classes of pathogens and procedures to reduce nosocomial infections in hospital settings. Newer agents that neutralize or inhibit specific S. aureus or P. aeruginosa virulence factors may eliminate or reduce the risk for developing pneumonia before or during mechanical ventilation and may improve patient outcomes through mechanisms that differ from those of antibiotics. In this article, we review the types, mechanisms of action, potential advantages, and stage of development of antivirulence agents (AVAs) that hold promise as alternative preventive or interventional therapies against S. aureus­ and P. aeruginosa­associated nosocomial pneumonias. We also present and discuss challenges to the effective utilization of AVAs separately from or in addition to antibiotics and the design of clinical trials and meaningful study end points.

Improved Protection in a Rabbit Model of Community-Associated Methicillin-Resistant Staphylococcus aureus Necrotizing Pneumonia upon Neutralization of Leukocidins in Addition to Alpha-Hemolysin.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), especially the USA300 pulsotype, is a frequent cause of skin and soft tissue infections and severe pneumonia. Despite appropriate antibiotic treatment, complications are common and pneumonia is associated with high mortality. S. aureus strains express multiple cytotoxins, including alpha-hemolysin (Hla) and up to five bicomponent leukocidins that specifically target phagocytic cells for lysis. CA-MRSA USA300 strains carry the genes for all six cytotoxins. Species specificity of the leukocidins greatly contributes to the ambiguity regarding their role in S. aureus pathogenesis. We performed a comparative analysis of the leukocidin susceptibility of human, rabbit, and mouse polymorphonuclear leukocytes (PMNs) to assess the translational value of mouse and rabbit S. aureus models. We found that mouse PMNs were largely resistant to LukSF-PV, HlgAB, and HlgCB and susceptible only to LukED, whereas rabbit and human PMNs were highly sensitive to all these cytotoxins. In the rabbit pneumonia model with a USA300 CA-MRSA strain, passive immunization with a previously identified human monoclonal antibody (MAb), Hla-F#5, which cross-neutralizes Hla, LukSF-PV, HlgAB, HlgCB, and LukED, provided full protection, whereas an Hla-specific MAb was only partially protective. In the mouse USA300 CA-MRSA pneumonia model, both types of antibodies demonstrated full protection, suggesting that Hla, but not leukocidin(s), is the principal virulence determinant in mice. As the rabbit recapitulates the high susceptibility to leukocidins characteristic of humans, this species represents a valuable model for assessing novel, cytotoxin-targeting anti-S. aureus therapeutic approaches.

Staphylococcus aureus leukotoxin GH promotes formation of neutrophil extracellular traps.

Staphylococcus aureus secretes numerous virulence factors that facilitate evasion of the host immune system. Among these molecules are pore-forming cytolytic toxins, including Panton-Valentine leukocidin (PVL), leukotoxin GH (LukGH; also known as LukAB), leukotoxin DE, and γ-hemolysin. PVL and LukGH have potent cytolytic activity in vitro, and both toxins are proinflammatory in vivo. Although progress has been made toward elucidating the role of these toxins in S. aureus virulence, our understanding of the mechanisms that underlie the proinflammatory capacity of these toxins, as well as the associated host response toward them, is incomplete. To address this deficiency in knowledge, we assessed the ability of LukGH to prime human PMNs for enhanced bactericidal activity and further investigated the impact of the toxin on neutrophil function. We found that, unlike PVL, LukGH did not prime human neutrophils for increased production of reactive oxygen species nor did it enhance binding and/or uptake of S. aureus. Unexpectedly, LukGH promoted the release of neutrophil extracellular traps (NETs), which, in turn, ensnared but did not kill S. aureus. Furthermore, we found that electropermeabilization of human neutrophils, used as a separate means to create pores in the neutrophil plasma membrane, similarly induced formation of NETs, a finding consistent with the notion that NETs can form during nonspecific cytolysis. We propose that the ability of LukGH to promote formation of NETs contributes to the inflammatory response and host defense against S. aureus infection.

Combined action of influenza virus and Staphylococcus aureus panton-valentine leukocidin provokes severe lung epithelium damage.

Staphylococcus aureus necrotizing pneumonia is a life-threatening disease that is frequently preceded by influenza infection. The S. aureus toxin Panton-Valentine leukocidin (PVL) is most likely causative for necrotizing diseases, but the precise pathogenic mechanisms of PVL and a possible contribution of influenza virus remain to be elucidated. In this study, we present a model that explains how influenza virus and PVL act together to cause necrotizing pneumonia: an influenza infection activates the lung epithelium to produce chemoattractants for neutrophils. Upon superinfection with PVL-expressing S. aureus, the recruited neutrophils are rapidly killed by PVL, resulting in uncontrolled release of neutrophil proteases that damage the airway epithelium. The host counteracts this pathogen strategy by generating PVL-neutralizing antibodies and by neutralizing the released proteases via protease inhibitors present in the serum. These findings explain why necrotizing infections mainly develop in serum-free spaces (eg, pulmonary alveoli) and open options for new therapeutic approaches.

Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence.

Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in hlgCB expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on hlgC promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of hlgCB mRNA strongly impaired hlgC translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. IMPORTANCE S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in pvl-negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of hlgCB mRNA induce the differential expression of hlgCB, drastically impacting hlgC mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.

FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins.

Staphylococcus aureus causes severe infections such as pneumonia and sepsis depending on the pore-forming toxin Panton-Valentine leukocidin (PVL). PVL kills and induces inflammation in macrophages and other myeloid cells by interacting with the human cell surface receptor, complement 5a receptor 1 (C5aR1). C5aR1 expression is tighly regulated and may thus modulate PVL activity, although the mechanisms involved remain incompletely understood. Here, we used a genome-wide CRISPR/Cas9 screen and identified F-box protein 11 (FBXO11), an E3 ubiquitin ligase complex member, to promote PVL toxicity. Genetic deletion of FBXO11 reduced the expression of C5aR1 at the mRNA level, whereas ectopic expression of C5aR1 in FBXO11-/- macrophages, or priming with LPS, restored C5aR1 expression and thereby PVL toxicity. In addition to promoting PVL-mediated killing, FBXO11 dampens secretion of IL-1ß after NLRP3 activation in response to bacterial toxins by reducing mRNA levels in a BCL-6-dependent and BCL-6-independent manner. Overall, these findings highlight that FBXO11 regulates C5aR1 and IL-1ß expression and controls macrophage cell death and inflammation following PVL exposure.

LukS-PV inhibits the proliferation of hepatocellular carcinoma cells by maintaining FOXO3 stability via the PI3K/AKT signaling pathway.

Hepatocellular carcinoma(HCC) is one of the common malignant tumors. LukS-PV is the S component of Panton-Valetine leukocidin(PVL) secreted by Staphylococcus aureus. Forkhead box O3 (FOXO3) is a member of the FOXO subfamily of transcription factors that acts as a tumor suppressor. In this study, we investigated the role of LukS-PV on the proliferation of HCC and explored possible mechanisms. We treated HCC cells with various concentrations of LukS-PV and evaluated the effect of LukS-PV on cell viability using the cell counting kit-8 and colony formation assays. Real-time PCR and western blot assays were used to analyze mRNA and protein expression levels, respectively. Immunofluorescence staining was performed to examine the intracellular localization of FOXO3. The expression of FOXO3 and its downstream target genes were analyzed by immunohistochemical staining. The protein synthesis inhibitor cycloheximide and the proteosome inhibitor MG132 were used to explore the potential mechanisms by which LukS-PV regulated FOXO3. We demonstrated that LukS-PV inhibited the proliferation of HCC cells in a concentration dependent manner. LukS-PV upregulated FOXO3 expression both in vitro and in vivo. Moreover, LukS-PV facilitated the entry of FOXO3 into the nucleus and, subsequently, regulated the transcription of downstream target genes. In addition, we discovered that LukS-PV decreased the expression of phosphorylated FOXO3 through the PI3K/AKT signaling pathway and maintained FOXO3 protein stability via the ubiquitin-proteasome pathway. Taken together, our data indicated that LukS-PV exert anticancer activities through FOXO3. LukS-PV may be a promising candidate for HCC treatment.

Panton-Valentine Leucocidin of Staphylococcus aureus Induces Oxidative Stress and Neurotransmitter Imbalance in a Retinal Explant Model.

Purpose: Endophthalmitis models have reported the virulent role of Panton-Valentine leucocidin (PVL) secreted by Staphylococcus aureus on the retina. PVL targets retinal ganglion cells (RGCs), expressing PVL membrane receptor C5aR. Interactions between PVL and retinal cells lead to glial activation, retinal inflammation, and apoptosis. In this study, we explored oxidative stress and retinal neurotransmitters in a rabbit retinal explant model incubated with PVL. Methods: Reactive oxygen species (ROS) production in RGCs has been assessed with fluorescent probes and immunohistochemistry. Nuclear magnetic resonance (NMR) spectroscopy quantified retinal concentrations of antioxidant molecules and neurotransmitters, and concentrations of neurotransmitters released in the culture medium. Quantifying the expression of some pro-inflammatory and anti-inflammatory factors was performed using RT-qPCR. Results: PVL induced a mitochondrial ROS production in RGCs after four hours' incubation with the toxin. Enzymatic sources of ROS, involving nicotinamide adenine dinucleotide phosphate-oxidase and xanthine oxidase, were also activated after four hours in PVL-treated retinal explants. Retinal antioxidants defenses, that is, glutathione, ascorbate and taurine, decreased after two hours' incubation with PVL. Glutamate retinal concentrations and glutamate release in the culture medium remained unaltered in PVL-treated retinas. GABA, glycine, and acetylcholine (Ach) retinal concentrations decreased after PVL treatment. Glycine release in the culture medium decreased, whereas Ach release increased after PVL treatment. Expression of proinflammatory and anti-inflammatory cytokines remained unchanged in PVL-treated explants. Conclusions: PVL activates oxidative pathways and alters neurotransmitter retinal concentrations and release, supporting the hypothesis that PVL could induce a neurogenic inflammation in the retina.

Vibrio pore-forming leukocidin activates pyroptotic cell death via the NLRP3 inflammasome.

Cell death mechanisms are central to combat infections and to drive inflammation. The inflammasome controls infection through activation of caspase-1 leading to either IL-1ß dependent inflammation, or pyroptotic cell death in infected cells. Hemolysins, which are pore-forming toxins (PFTs), alter the permeability of the host target membrane, often leading to cell death. We previously discovered a leukocidin domain-containing PFT produced by the Gram-negative bacterium Vibrio proteolyticus, named VPRH. VPRH constitutes a distinct, understudied class within the leukocidin superfamily, which is distributed among several photogenic Vibrios. Since PFTs of other pathogens were shown to activate the inflammasome pathway, we hypothesized that VPRH-induced cell death is mediated by direct activation of the inflammasome in mammalian immune host cells. Indeed, we found that VPRH induced a two-step cell death in macrophages. The first, a rapid step, was mediated by activating the NLRP3 inflammasome, leading to caspase-1 activation that resulted in IL-1ß secretion and pyroptosis. The second step was independent of the inflammasome; however, its mechanism remains unknown. This study sets the foundation for better understanding the immunological consequences of inflammasome activation by a new leukocidin class of toxins, which may be shared between marine bacteria and give rise to new pathogenic isolates.

LukS-PV induces apoptosis in acute myeloid leukemia cells mediated by C5a receptor.

LukS-PV is one of the two components of Panton-Valentine leucocidin (PVL). Our previous study showed that LukS-PV can induce apoptosis in human acute myeloid leukemia (AML) THP-1 and HL-60 cells. C5aR (C5a receptor) is the receptor for PVL, but whether C5aR plays a key role in LukS-PV induced apoptosis is unclear. The aim of this study was to establish whether C5aR plays a physiological role in apoptosis of leukemia cells induced by LukS-PV. We investigated the role of C5aR in leukemia cell apoptosis induced by LukS-PV by pretreatment of THP-1 and HL-60 cells with C5aR antagonist and transfection to knockdown C5aR in THP-1 cells or overexpress C5aR in Jurkat cells before treatment with LukS-PV. Cell apoptosis was analyzed by staining with Annexin V/propidium iodide or Annexin V-PE/7-AAD. Mitochondrial membrane potential (MMP) was determined using JC-1 dye. The expression of apoptosis-associated genes and proteins was identified by qRT-polymerase chain reaction and Western blotting analysis, respectively. As the C5aR antagonist concentration increased, the rate of apoptosis induced by LukS-PV decreased, the MMP increased, and expression of pro-apoptotic Bax and Bak genes and proteins was downregulated while that of anti-apoptotic Bcl-2 and Bcl-x genes and proteins was upregulated. Knockdown of C5aR also decreased LukS-PV-induced THP-1 cell apoptosis. LukS-PV did not induce apoptosis of Jurkat cells, which have no endogenous C5aR expression; however, LukS-PV did induce apoptosis in Jurkat cells after overexpression of C5aR. Correspondingly, the MMP decreased and Bax and Bak were upregulated while Bcl-2 and Bcl-x were downregulated. LukS-PV can induce apoptosis in AML cells by targeting C5aR. C5aR may be a potential therapeutic target for AML and LukS-PV is a candidate targeted drug for the treatment of AML.

Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils.

Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus that has recently been associated with necrotizing pneumonia. In the present study, we report that in vitro, PVL induces polymorphonuclear cell death by necrosis or by apoptosis, depending on the PVL concentration. PVL-induced apoptosis was associated with a rapid disruption of mitochondrial homeostasis and activation of caspase-9 and caspase-3, suggesting that PVL-induced apoptosis is preferentially mediated by the mitochondrial pathway. Polymorphonuclear cell exposure to PVL leads to mitochondrial localization of the toxin, whereas Bax, 1 of the 2 essential proapoptotic members of the Bcl-2 family, was still localized in the cytosol. Addition of PVL to isolated mitochondria induced the release of the apoptogenic proteins cytochrome c and Smac/DIABLO. Therefore, we suggest that PVL, which belongs to the pore-forming toxin family, could act at the mitochondrion level by creating pores in the mitochondrial outer membrane. Furthermore, LukS-PV, 1 of the 2 components of PVL, was detected in lung sections of patients with necrotizing pneumonia together with DNA fragmentation, suggesting that PVL induces apoptosis in vivo and thereby is directly involved in the pathophysiology of necrotizing pneumonia.

Panton-Valentine Leukocidin associated with S. aureus osteomyelitis activates platelets via neutrophil secretion products.

Globalization and migration promote the spread of Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus strains. The toxin PVL is linked to the development of thrombosis in association with osteomyelitis. The mechanisms by which PVL drives thrombosis development are however still unknown. We demonstrate that PVL-damaged neutrophils activate platelets via neutrophil secretion products, such as α-defensins and the myeloperoxidase product HOCl, as well as the formation of HOCl-modified proteins. Neutrophil damage by PVL is blocked by anti-PVL-antibodies, explaining why especially young osteomyelitis patients with a low antibody titre against PVL suffer from thrombotic complications. Platelet activation in the presence of PVL-damaged neutrophils is prevented by α-defensin inhibitors and by glutathione and resveratrol, which are both inhibitors of HOCl-modified protein-induced platelet activation. Remarkably, intravenously infused glutathione also prevents activation of human platelets in an ex vivo assay. We here describe a new mechanism of PVL-neutrophil-platelet interactions, which might be extrapolated to other toxins that act on neutrophils. Our observations may make us think about new approaches to treat and/or prevent thrombotic complications in the course of infections with PVL-producing S. aureus strains.

Infective pyomyositis and myositis in children in the era of community-acquired, methicillin-resistant Staphylococcus aureus infection.

BACKGROUND: Cases of pyomyositis and myositis have been increasing in frequency at Texas Children's Hospital (Houston) since 2000. The increase appears to correlate with the emergence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA). METHODS: The medical records of patients with pyomyositis and myositis hospitalized at Texas Children's Hospital during the period from January 2000 through December 2005 were reviewed. Available S. aureus isolates were obtained for susceptibility testing, to determine the presence of pvl (lukS-PV and lukF-PV), and for pulsed-field gel electrophoresis analysis. RESULTS: Forty-five previously healthy children with bacterial pyomyositis or myositis were analyzed. The causes were S. aureus (in 57.8% of children) and Streptococcus pyogenes (in 2.2%); 40.0% of children had negative culture results. The number of cases increased between 2000 and 2005, primarily as a result of an increase in the prevalence of community-acquired MRSA. The mean patient age was 5.5 years (range, 0.06-15 years). The thigh (40.0% of children) and pelvis (28.9%) were the most commonly affected sites. The mean abscess diameter was 3.5 cm. Eighteen children required at least 1 muscle drainage procedure. Of the 24 available S. aureus isolates (15 community-acquired MRSA isolates and 9 community-acquired, methicillin-susceptible S. aureus [MSSA] isolates), 16 were found to be USA300 by pulsed-field gel electrophoresis, and 17 carried pvl. Patients with community-acquired MRSA, USA300, and/or pvl-positive strains required more drainage procedures than did those with community-acquired MSSA, non-USA300, and/or pvl-negative strains (81% vs. 40% [P=.05], 82% vs. 29% [P=.02], and 81% vs. 38% [P=.07], respectively). CONCLUSIONS: Community-acquired MRSA is an increasing cause of pyomyositis and myositis in children. Community-acquired MRSA, USA300, pvl-positive S. aureus isolates caused more severe disease than did community-acquired MSSA, non-USA300, and pvl-negative isolates, respectively.