Search

OBJECTIVES: We aimed at determining whether specific S. aureus strains cause infective endocarditis (IE) in the course of Staphylococcus aureus bacteraemia (SAB). METHODS: A genome-wide association study (GWAS) including 924 S. aureus genomes from IE (274) and non-IE (650) SAB patients from international cohorts was conducted, and a subset of strains was tested with two experimental animal models of IE, one investigating the early step of bacterial adhesion to inflamed mice valves, the second evaluating the local and systemic developmental process of IE on mechanically-damaged rabbit valves. RESULTS: The genetic profile of S. aureus IE and non-IE SAB strains did not differ when considering single nucleotide polymorphisms, coding sequences, and k-mers analysed in GWAS. In the murine inflammation-induced IE model, no difference was observed between IE and non-IE SAB strains both in terms of adhesion to the cardiac valves and in the propensity to cause IE; in the mechanical IE-induced rabbit model, there was no difference between IE and non-IE SAB strains regarding the vegetation size and CFU. CONCLUSION: All strains of S. aureus isolated from SAB patients must be considered as capable of causing this common and lethal infection once they have accessed the bloodstream. ispartof: JOURNAL OF INFECTION vol:86 issue:2 pages:123-133 ispartof: location:England status: published

11 pag, 6 figs. Coordinates for the model of SrrB DHp-CA region solved by X-ray crystallography have been deposited in the Protein Data Bank, https://www.rcsb.org/ (ID code 6PAJ). This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921307117/-/DCSupplemental., Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis., This work was supported by funding from the National Institutes of Health (NIH) and National Institute of Allergy and Infectious Diseases (NIAID) to E.J.F. and P.M.S. (NIAID Grant AI135305). J.M.B. was funded by the NIH (NIAID Grant AI139100-01) and US Department of Agriculture Multistate Reseach Fund (Project NE−1028). W.S.-P. was supported by NIH (NIAID Grant AI134692-03). The J.K.M. lab was supported by the Canadian Institutes of Health Research (Grant PJT-166050). A.M. was supported by grant BIO2016-78571-P from the Ministerio de Economia y Competitividad (Spain).

and KeywordsAimsInfective endocarditis (IE) complicates 10-20% of Staphylococcus aureus bacteraemia (SAB). We aimed to determine whether IE strains of S. aureus are genotypically different or behave differently in experimental endocarditis models as compared to non-IE SAB strains.Methods and ResultsWe conducted a genome wide association study (GWAS) of 924 S. aureus genomes from IE (274) and non-IE (650) SAB patients, and tested a subset of strains in two experimental animal models of IE, one studying the early step of bacterial adhesion to inflamed mice valves, the second evaluating the local and systemic developmental process of IE on mechanically damaged rabbit valves. The genetic profile of S. aureus IE and non-IE SAB strains did not differ when considering single nucleotide polymorphisms, coding sequences and k-mers analyses in GWASs. In the inflammation-induced IE model in mice no difference was observed between IE and non-IE SAB strains both in adhesion to the cardiac valves and in the propensity to cause IE; in the mechanical IE-induced rabbit model, there was no difference between IE and non-IE SAB strains regarding vegetation size and CFU.ConclusionS. aureus isolates from SAB patients with and without IE were indistinguishable, by GWAS and by two in vivo models of IE. Thus, S. aureus strain variation is not the primary determinant of IE. Pending the possible identification of host factors predisposing to IE, all strains of S. aureus must be considered in patients as capable of causing this common, lethal infection once they have accessed the bloodstream.Translational PerspectiveStaphylococcus aureus endocarditis (IE) is a deadly complication of S. aureus bacteraemia (SAB). Beyond well-identified host related IE risk factors, whether bacterial features may influence the occurrence of IE in the course of bacteraemia remain elusive. We analysed the genomes of 924 S. aureus strains from IE and non-IE SAB and compared some in two in vivo IE models. We demonstrated that the propensity of S. aureus to cause IE in the course of bacteraemia does not depend on the intrinsic genetic or virulence factors of S. aureus. These findings are of importance for the management of S. aureus bacteraemia.

The superantigen staphylococcal enterotoxin C (SEC) is critical for Staphylococcus aureus infective endocarditis (SAIE) in rabbits. Superantigenicity, its hallmark function, was proposed to be a major underlying mechanism driving SAIE but was not directly tested. With the use of S. aureus MW2 expressing SEC toxoids, we show that superantigenicity does not sufficiently account for vegetation growth, myocardial inflammation, and acute kidney injury in the rabbit model of native valve SAIE. These results highlight the critical contribution of an alternative function of superantigens to SAIE. In support of this, we provide evidence that SEC exerts antiangiogenic effects by inhibiting branching microvessel formation in an ex vivo rabbit aortic ring model and by inhibiting endothelial cell expression of one of the most potent mediators of angiogenesis, VEGF-A. SEC’s ability to interfere with tissue revascularization and remodeling after injury serves as a mechanism to promote SAIE and its life-threatening systemic pathologies.

Infective endocarditis (IE) is one of the most feared and lethal diseases caused by

Infective endocarditis (IE) is one of the most feared and lethal diseases caused by Staphylococcus aureus. Once established, the infection is fast-progressing and tissue destructive. S. aureus of the clonal complex 5 (CC5) commonly cause IE yet are severely understudied. IE results from bacterial colonization and formation of tissue biofilms (known as vegetations) on injured or inflamed cardiac endothelium. S. aureus IE is promoted by adhesins, coagulases, and superantigens, with the exotoxins and exoenzymes likely contributing to tissue destruction and dissemination. Expression of the large repertoire of virulence factors required for IE and sequelae is controlled by complex regulatory networks. We investigated the temporal expression of the global regulators agr (RNAIII), rot, sarS, sarA, sigB, and mgrA in 8 invasive CC5 isolates and established intrinsic expression patterns associated with IE outcomes. We show that vegetation formation, as tested in the rabbit model of IE, inversely correlates with RNAIII and sarA expression during growth in Todd-Hewitt broth (TH). Large vegetations with severe sequelae arise from strains with high-level expression of colonization factors but slower transition towards expression of the exotoxins. Overall, strains proficient in vegetation formation, a hallmark of IE, exhibit lower expression of RNAIII and sarA. Simultaneous high expression of RNAIII, sarA, sigB, and mgrA is the one phenotype assessed in this study that fails to promote IE. Thus, RNAIII and sarA expression that provides for rheostat control of colonization and virulence genes, rather than an on and off switch, promote both vegetation formation and lethal sepsis.

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.

The superantigen staphylococcal enterotoxin C (SEC) is critical for Staphylococcus aureus infective endocarditis (SAIE) in rabbits. Superantigenicity, its hallmark function, was proposed to be a major underlying mechanism driving SAIE but was not directly tested. With the use of S. aureus MW2 expressing SEC toxoids, we show that superantigenicity does not sufficiently account for vegetation growth, myocardial inflammation, and acute kidney injury in the rabbit model of native valve SAIE. These results highlight the critical contribution of an alternative function of superantigens to SAIE. In support of this, we provide evidence that SEC exerts anti-angiogenic effects by inhibiting branching microvessel formation in an ex vivo rabbit aortic ring model and by inhibiting endothelial cell expression of one of the most potent mediators of angiogenesis, VEGF-A. SEC’s ability to interfere with tissue re-vascularization and remodeling after injury serves as a mechanism to promote SAIE and its life-threatening systemic pathologies.

β-Toxin is a sphingomyelinase hemolysin that significantly contributes to Staphylococcus aureus pathogenesis. In most S. aureus isolates the prophage ϕSa3int inserts into the β-toxin gene hlb, inactivating it, but human and experimental infections give rise to β-toxin-producing variants. However, it remained to be established whether ϕSa3mw excises in response to specific environmental cues, restoring the β-toxin gene sequence. This is not only of fundamental interest but also critical when designing intervention strategies and therapeutics. We provide evidence that ϕSa3mw actively excises, allowing the conditional expression of β-toxin. ϕSa3int prophages may play a novel and largely uncharacterized role in S. aureus pathogenesis as molecular regulatory switches that promote bacterial fitness and adaptation to the challenges presented by the mammalian host., Phage regulatory switches (phage-RSs) are a newly described form of active lysogeny where prophages function as regulatory mechanisms for expression of chromosomal bacterial genes. In Staphylococcus aureus, ϕSa3int is a widely distributed family of prophages that integrate into the β-toxin structural gene hlb, effectively inactivating it. However, β-toxin-producing strains often arise during infections and are more virulent in experimental infective endocarditis and pneumonia infections. We present evidence that in S. aureus MW2, ϕSa3mw excision is temporally and differentially responsive to growth conditions relevant to S. aureus pathogenesis. PCR analyses of ϕSa3mw (integrated and excised) and of intact hlb showed that ϕSa3mw preferentially excises in response to hydrogen peroxide-induced oxidative stress and during biofilm growth. ϕSa3mw remains as a prophage when in contact with human aortic endothelial cells in culture. A criterion for a prophage to be considered a phage-RS is the inability to lyse host cells. MW2 grown under phage-inducing conditions did not release infectious phage particles by plaque assay or transmission electron microscopy, indicating that ϕSa3mw does not carry out a productive lytic cycle. These studies highlight a dynamic, and perhaps more sophisticated, S. aureus-prophage interaction where ϕSa3int prophages provide a novel regulatory mechanism for the conditional expression of virulence factors. IMPORTANCE β-Toxin is a sphingomyelinase hemolysin that significantly contributes to Staphylococcus aureus pathogenesis. In most S. aureus isolates the prophage ϕSa3int inserts into the β-toxin gene hlb, inactivating it, but human and experimental infections give rise to β-toxin-producing variants. However, it remained to be established whether ϕSa3mw excises in response to specific environmental cues, restoring the β-toxin gene sequence. This is not only of fundamental interest but also critical when designing intervention strategies and therapeutics. We provide evidence that ϕSa3mw actively excises, allowing the conditional expression of β-toxin. ϕSa3int prophages may play a novel and largely uncharacterized role in S. aureus pathogenesis as molecular regulatory switches that promote bacterial fitness and adaptation to the challenges presented by the mammalian host.

Staphylococcus aureus infective endocarditis (IE) is a fast-progressing and tissue-destructive infection of the cardiac endothelium. The superantigens (SAgs) toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin C (SEC), and the toxins encoded by the enterotoxin gene cluster ( egc ) play a novel and essential role in the etiology of S. aureus IE. Recent studies indicate that SAgs act at the infection site to cause tissue pathology and promote vegetation growth. The underlying mechanism of SAg involvement has not been clearly defined. In SAg-mediated responses, immune cell priming is considered a primary triggering event leading to endothelial cell activation and altered function. Utilizing immortalized human aortic endothelial cells (iHAECs), we demonstrated that TSST-1 directly activates iHAECs, as documented by upregulation of vascular and intercellular adhesion molecules (VCAM-1 and ICAM-1). TSST-1-mediated activation results in increased monolayer permeability and defects in vascular reendothelialization. Yet stimulation of iHAECs with TSST-1 fails to induce interleukin-8 (IL-8) and IL-6 production. Furthermore, simultaneous stimulation of iHAECs with TSST-1 and lipopolysaccharide (LPS) inhibits LPS-mediated IL-8 and IL-6 secretion, even after pretreatment with either of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β. IL-8 suppression is not mediated by TSST-1 binding to its canonical receptor major histocompatibility complex class II (MHC-II), supporting current evidence for a nonhematopoietic interacting site on SAgs. Together, the data suggest that TSST-1 differentially regulates cell-bound and secreted markers of endothelial cell activation that may result in dysregulated innate immune responses during S. aureus IE. Endothelial changes resulting from the action of SAgs can therefore directly contribute to the aggressive nature of S. aureus IE and development of life-threatening complications.