Staphylococcus aureus α-Toxin Induces Acid Sphingomyelinase Release From a Human Endothelial Cell Line.
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ABSTRACT:
SUBMITTER: Krones D
PROVIDER: S-EPMC8358437 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:During sepsis, small blood vessels can become occluded by large platelet aggregates of poorly understood etiology. During Staphylococcal aureus infection, sepsis severity is linked to the bacterial α-toxin (α-hemolysin, AT) through unclear mechanisms. In this study, we visualized intravascular events in the microcirculation and found that intravenous AT injection induces rapid platelet aggregation, forming dynamic micro-thrombi in the microcirculation. These aggregates are retained in the liver sinusoids and kidney glomeruli, causing multi-organ dysfunction. Acute staphylococcal infection results in sequestration of most bacteria by liver macrophages. Platelets are initially recruited to these macrophages and help eradicate S. aureus. However, at later time points, AT causes aberrant and damaging thrombosis throughout the liver. Treatment with an AT neutralizing antibody (MEDI4893∗) prevents platelet aggregation and subsequent liver damage, without affecting the initial and beneficial platelet recruitment. Thus, AT neutralization may represent a promising approach to combat staphylococcal-induced intravascular coagulation and organ dysfunction.
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Project description:Resistance and tolerance are two defense strategies employed by the host against microbial threats. Autophagy-mediated degradation of bacteria has been extensively described as a major resistance mechanism. Here we find that the dominant function of autophagy proteins during infections with the epidemic community-associated methicillin-resistant Staphylococcus aureus USA300 is to mediate tolerance rather than resistance. Atg16L1 hypomorphic mice (Atg16L1(HM)), which have reduced autophagy, were highly susceptible to lethality in both sepsis and pneumonia models of USA300 infection. Autophagy confers protection by limiting the damage caused by α-toxin, particularly to endothelial cells. Remarkably, Atg16L1(HM) mice display enhanced survival rather than susceptibility upon infection with α-toxin-deficient S. aureus. These results identify an essential role for autophagy in tolerance to Staphylococcal disease and highlight how a single virulence factor encoded by a pathogen can determine whether a given host factor promotes tolerance or resistance.
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Project description:Staphylococcus aureus infection is one of the leading causes of morbidity in hospitalized patients in the United States, an effect compounded by increasing antibiotic resistance. The secreted agent hemolysin alpha toxin (Hla) requires the receptor A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10) to mediate its toxic effects. We hypothesized that these effects are in part regulated by Notch signaling, for which ADAM10 activation is essential. Notch proteins function in developmental and pathological angiogenesis via the modulation of key pathways in endothelial and perivascular cells. Thus, we hypothesized that Hla would activate Notch in vascular cells. Human umbilical vein endothelial cells were treated with recombinant Hla (rHla), Hla-H35L (genetically inactivated Hla), or Hank's solution (HBSS), and probed by different methods. Luciferase assays showed that Hla (0.01 µg/mL) increased Notch activation by 1.75?±?0.5-fold as compared to HBSS controls (p?<?0.05), whereas Hla-H35L had no effect. Immunocytochemistry and Western blotting confirmed these findings and revealed that ADAM10 and ?-secretase are required for Notch activation after inhibitor and siRNA assays. Retinal EC in mice engineered to express yellow fluorescent protein (YFP) upon Notch activation demonstrated significantly greater YFP intensity after Hla injection than controls. Aortic rings from Notch reporter mice embedded in matrix and incubated with rHla or Hla-H35L demonstrate increased Notch activation occurs at tip cells during sprouting. These mice also had higher skin YFP intensity and area of expression after subcutaneous inoculation of S. aureus expressing Hla than a strain lacking Hla in both EC and pericytes assessed by microscopy. Human liver displayed strikingly higher Notch expression in EC and pericytes during S. aureus infection by immunohistochemistry than tissues from uninfected patients. In sum, our results demonstrate that the S. aureus toxin Hla can potently activate Notch in vascular cells, an effect which may contribute to the pathobiology of infection with this microorganism.
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Project description:UnlabelledPulmonary edema associated with increased vascular permeability is a severe complication of Staphylococcus aureus-induced sepsis and an important cause of human pathology and death. We investigated the role of the mammalian acid sphingomyelinase (Asm)/ceramide system in the development of lung edema caused by S. aureus. Our findings demonstrate that genetic deficiency or pharmacologic inhibition of Asm reduced lung edema in mice infected with S. aureus. The Asm/ceramide system triggered the formation of superoxide, resulting in degradation of tight junction proteins followed by lung edema. Treatment of infected mice with amitriptyline, a potent inhibitor of Asm, protected mice from lung edema caused by S. aureus, but did not reduce systemic bacterial numbers. In turn, treatment with antibiotics reduced bacterial numbers but did not protect mice from lung edema. In contrast, only the combination of antibiotics and amitriptyline inhibited both pulmonary edema and bacteremia protecting mice from lethal sepsis and lung dysfunction suggesting the combination of both drugs as novel treatment option for sepsis.Key messagesAntibiotics are often insufficient to cure S. aureus-induced sepsis. S. aureus induces lung edema via the Asm/ceramide system. Genetic deficiency of Asm inhibits lung dysfunction upon infection with S. aureus. Pharmacologic inhibition of Asm reduces lung edema induced by S. aureus. Antibiotics plus amitriptyline protect mice from lung edema and lethal S. aureus sepsis.
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Project description:Biofilms complicate treatment of Staphylococcus aureus (SA) wound infections. Previously, we determined alpha-toxin (AT)-promoted SA biofilm formation on mucosal tissue. Therefore, we evaluated SA wound isolates for AT production and biofilm formation on epithelium and assessed the role of AT in biofilm formation. Thirty-eight wound isolates were molecularly typed by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (ST), and spa typing. We measured biofilm formation of these SA isolates in vitro and ex vivo and quantified ex vivo AT production. We also investigated the effect of an anti-AT monoclonal antibody (MEDI4893*) on ex vivo biofilm formation by methicillin-resistant SA (USA 300 LAC) and tested whether purified AT rescued the biofilm defect of hla mutant SA strains. The predominant PFGE/ST combinations were USA100/ST5 (50%) and USA300/ST8 (33%) for methicillin-resistant SA (MRSA, n = 18), and USA200/ST30 (20%) for methicillin-susceptible SA (MSSA, n = 20). Ex vivo AT production correlated significantly with ex vivo SA wound isolate biofilm formation. Anti-alpha-toxin monoclonal antibody (MEDI4893*) prevented ex vivo biofilm formation by MRSA USA300 strain LAC. Wild-type AT rescued the ex vivo biofilm defect of non-AT producing SA strains. These findings provide evidence that AT plays a role in SA biofilm formation on epithelial surfaces and suggest that neutralization of AT may be useful in preventing and treating SA infections.
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Project description:Staphylococcus aureus is a leading cause of human bacterial infection, causing a wide spectrum of disease ranging from skin and soft tissue infections to life-threatening pneumonia and sepsis. S. aureus toxins play an essential role in disease pathogenesis, contributing to both immunomodulation and host tissue injury. Prominent among these toxins are the membrane-active pore-forming cytolysin alpha-toxin (Hla) and the amphipathic ?-helical phenol-soluble modulin (PSM) peptides. As deletion of either the hla or psm locus leads to a phenotypically similar virulence defect in skin and soft tissue infection, we sought to determine the relative contribution of each locus to disease pathogenesis. Here we show that production of Hla can be modulated by PSM expression. An S. aureus mutant lacking PSM expression exhibits a transcriptional delay in hla mRNA production and therefore fails to secrete normal levels of Hla at early phases of growth. This leads to attenuation of virulence in vitro and in murine skin and lung models of infection, correlating with reduced recovery of Hla from host tissues. Production of Hla and restoration of staphylococcal virulence can be achieved in the psm mutant by plasmid-driven overexpression of hla. Our study suggests the coordinated action of Hla and PSMs in host tissue during early pathogenesis, confirming a major role for Hla in epithelial injury during S. aureus infection. These findings highlight the possibility that therapeutics targeting PSM production may simultaneously prevent Hla-mediated tissue injury.
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Project description:The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo. Staphylococcus aureus has a propensity to form multicellular communities known as biofilms. While growing in a biofilm, S. aureus displays increased tolerance to nutrient deprivation, antibiotic insult, and even host immune challenge. Previous studies have shown that S. aureus biofilms thwart host immunity in part by preventing macrophage phagocytosis. It remained unclear whether this was influenced solely by the considerable size of biofilms or whether molecules were also actively secreted to circumvent macrophage-mediated phagocytosis. This is the first report to demonstrate that S. aureus biofilms inhibit macrophage phagocytosis and induce macrophage death through the combined action of leukocidin AB and alpha-toxin. Loss of leukocidin AB and alpha-toxin expression resulted in enhanced S. aureus biofilm clearance in a mouse model of orthopedic implant infection, suggesting that these toxins could be targeted therapeutically to facilitate biofilm clearance in humans.
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Project description:Sex bias in innate defense against Staphylococcus aureus skin and soft tissue infection (SSTI) is dependent on both estrogen production by the host and S. aureus secretion of the virulence factor, α-hemolysin (Hla). The impact of estrogen signaling on the immune system is most often studied in terms of the nuclear estrogen receptors ERα and ERβ. However, the potential contribution of the G protein-coupled estrogen receptor (GPER) to innate defense against infectious disease, particularly with respect to skin infection, has not been addressed. Using a murine model of SSTI, we found that GPER activation with the highly selective agonist G-1 limits S. aureus SSTI and Hla-mediated pathogenesis, effects that were absent in GPER knockout mice. Specifically, G-1 reduced Hla-mediated skin lesion formation and pro-inflammatory cytokine production, while increasing bacterial clearance. In vitro, G-1 reduced surface expression of the Hla receptor, ADAM10, in a human keratinocyte cell line and increased resistance to Hla-mediated permeability barrier disruption. This novel role for GPER activation in skin innate defense against infectious disease suggests that G-1 may have clinical utility in patients with epithelial permeability barrier dysfunction or who are otherwise at increased risk of S. aureus infection, including those with atopic dermatitis or cancer.
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Project description:Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.
| S-EPMC4895005 | biostudies-literature
Project description:Immunomodulatory cytotoxins are prominent virulence factors produced by Staphylococcus aureus, a leading cause of bacterial sepsis, skin infection, and pneumonia. S. aureus α-toxin is a pore-forming toxin that utilizes a widely expressed receptor, ADAM10, to injure the host epithelium, endothelium, and immune cells. As each host tissue is characterized by a unique composition of resident cells and recruited immune cells, the outcome of α-toxin-mediated injury may depend on the infected tissue environment. Utilizing myeloid lineage-specific Adam10 knockout mice, we show that α-toxin exerts tissue-specific effects on innate immunity to staphylococcal infection. Loss of ADAM10 expression exacerbates skin infection, yet affords protection against lethal pneumonia. These diverse outcomes are not related to altered immune cell recruitment, but rather correlate with a defect in toxin-induced IL-1β production. Extension of these studies through analysis of ADAM10 double-knockout mice affecting both the myeloid lineage and either the skin or lung epithelium highlight the prominence of toxin-induced injury to the epithelium in governing the outcome of infection. Together, these studies provide evidence of tissue specificity of pore-forming cytotoxin action in the modulation of host immunity, and illustrate that the outcome of infection is a collective manifestation of all effects of the toxin within the tissue microenvironment.
| S-EPMC4464663 | biostudies-literature