Alice G. Cheng

Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues

Staphylococcus aureus infections are associated with abscess formation and bacterial persistence; however, the genes that enable this lifestyle are not known. We show here that following intravenous infection of mice, S. aureus disseminates rapidly into organ tissues and elicits abscess lesions that develop over weeks but cannot be cleared by the host. Staphylococci grow as communities at the center of abscess lesions and are enclosed by pseudocapsules, separating the pathogen from immune cells. By testing insertional variants in genes for cell wall‐anchored surface proteins, we are able to infer the stage at which these molecules function. Fibrinogen‐binding proteins ClfA and ClfB are required during the early phase of staphylococcal dissemination. The heme scavenging factors IsdA and IsdB, as well as SdrD and protein A, are necessary for abscess formation. Envelope‐associated proteins, Emp and Eap, are either required for abscess formation or contribute to persistence. Fluorescence microscopy revealed Eap deposition within the pseudocapsule, whereas Emp was localized within staphylococcal abscess communities. Antibodies directed against envelope‐associated proteins generated vaccine protection against staphylococcal abscess formation. Thus, staphylococci employ envelope proteins at discrete stages of a developmental program that enables abscess formation and bacterial persistence in host tissues.—Cheng, A. G., Kim, H. K., Burts, M. L., Krausz, T., Schneewind, O., Missiakas, D. M. Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues. FASEB J. 23, 3393–3404 (2009). www.fasebj.org

Nontoxigenic protein A vaccine for methicillin-resistant Staphylococcus aureus infections in mice

The current epidemic of hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections has caused significant human morbidity, but a protective vaccine is not yet available. Prior infection with S. aureus is not associated with protective immunity. This phenomenon involves staphylococcal protein A (SpA), an S. aureus surface molecule that binds to Fcγ of immunoglobulin (Ig) and to the Fab portion of VH3-type B cell receptors, thereby interfering with opsonophagocytic clearance of the pathogen and ablating adaptive immune responses. We show that mutation of each of the five Ig-binding domains of SpA with amino acid substitutions abolished the ability of the resulting variant SpAKKAA to bind Fcγ or Fab VH3 and promote B cell apoptosis. Immunization of mice with SpAKKAA raised antibodies that blocked the virulence of staphylococci, promoted opsonophagocytic clearance, and protected mice against challenge with highly virulent MRSA strains. Furthermore, SpAKKAA immunization enabled MRSA-challenged mice to mount antibody responses to many different staphylococcal antigens.

Contribution of Coagulases towards Staphylococcus aureus Disease and Protective Immunity

The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule. Using histochemical staining, we identified prothrombin and fibrin within abscesses and pseudocapsules. S. aureus secretes two clotting factors, coagulase (Coa) and von Willebrand factor binding protein (vWbp). We report here that Coa and vWbp together are required for the formation of abscesses. Coa and vWbp promote the non-proteolytic activation of prothrombin and cleavage of fibrinogen, reactions that are inhibited with specific antibody against each of these molecules. Coa and vWbp specific antibodies confer protection against abscess formation and S. aureus lethal bacteremia, suggesting that coagulases function as protective antigens for a staphylococcal vaccine.

A play in four acts: Staphylococcus aureus abscess formation

Staphylococcus aureus is an important human pathogen that causes skin and soft tissue abscesses. Abscess formation is not unique to staphylococcal infection and purulent discharge has been widely considered a physiological feature of healing and tissue repair. Here we present a different view, whereby S. aureus deploys specific virulence factors to promote abscess lesions that are distinctive for this pathogen. In support of this model, only live S. aureus is able to form abscesses, requiring genes that act at one or more of four discrete stages during the development of these infectious lesions. Protein A and coagulases are distinctive virulence attributes for S. aureus, and humoral immune responses specific for these polypeptides provide protection against abscess formation in animal models of staphylococcal disease.

IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge

Staphylococcus aureus is the most frequent cause of bacteremia and hospital-acquired infection, however a vaccine that prevents staphylococcal disease is currently not available. Two sortase-anchored surface proteins, IsdA and IsdB, have been identified as subunit vaccines that, following active immunization, protect experimental animals against intravenous challenge with staphylococci. Here we investigate the molecular basis of this immunity and report that, when passively transferred to naïve mice, purified antibodies directed against IsdA or IsdB protected against staphylococcal abscess formation and lethal intravenous challenge. When added to mouse blood, IsdA- or IsdB-specific antibodies did not promote rapid opsonophagocytic killing of wild-type staphylococci. Antibodies directed against IsdA interfered with heme-binding and IsdB antibodies perturbed the ability of this surface protein to bind hemoglobin. As the structural genes for isdA and isdB are required for heme-iron scavenging during the pathogenesis of infection, we hypothesize that IsdA and IsdB antibodies may at least in part provide protection against staphylococci by interfering with the pathogens heme-iron scavenging mechanisms.

A New Oxidative Sensing and Regulation Pathway Mediated by the MgrA Homologue SarZ in Staphylococcus aureus

Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses from the human pathogen Staphylococcus aureus. Among these, a thiol‐based oxidation sensing pathway through a global regulator MgrA controls the virulence and antibiotic resistance of the bacterium. Herein, we report a new thiol‐based oxidation sensing and regulation system that is mediated through a parallel global regulator SarZ. SarZ is a functional homologue of MgrA and is shown to affect the expression of ∼87 genes in S. aureus. It uses a key Cys residue, Cys‐13, to sense oxidative stress and to co‐ordinate the expression of genes involved in metabolic switching, antibiotic resistance, peroxide stress defence, virulence, and cell wall properties. The discovery of this SarZ‐mediated regulation, mostly independent from the MgrA‐based regulation, fills a missing gap of oxidation sensing and response in S. aureus.

Golden Pigment Production and Virulence Gene Expression Are Affected by Metabolisms in Staphylococcus aureus

The pathogenesis of staphylococcal infections is multifactorial. Golden pigment is an eponymous feature of the human pathogen Staphylococcus aureus that shields the microbe from oxidation-based clearance, an innate host immune response to infection. Here, we screened a collection of S. aureus transposon mutants for pigment production variants. A total of 15 previously unidentified genes were discovered. Notably, disrupting metabolic pathways such as the tricarboxylic acid cycle, purine biosynthesis, and oxidative phosphorylation yields mutants with enhanced pigmentation. The dramatic effect on pigment production seems to correlate with altered expression of virulence determinants. Microarray analysis further indicates that purine biosynthesis impacts the expression of approximately 400 genes involved in a broad spectrum of functions including virulence. The purine biosynthesis mutant and oxidative phosphorylation mutant strains exhibit significantly attenuated virulence in a murine abscess model of infection. Inhibition of purine biosynthesis with a known small-molecule inhibitor results in altered virulence gene expression and virulence attenuation during infection. Taken together, these results suggest an intimate link between metabolic processes and virulence gene expression in S. aureus. This study also establishes the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival.

Coagulases as Determinants of Protective Immune Responses against Staphylococcus aureus

ABSTRACT During infection, Staphylococcus aureus secretes two coagulases (Coa and von Willebrand factor binding protein [vWbp]), which, following an association with host prothrombin and fibrinogen, form fibrin clots and enable the establishment of staphylococcal disease. Within the genomes of different S. aureus isolates, coagulase gene sequences are variable, and this has been exploited for a classification of types. We show here that antibodies directed against the variable prothrombin binding portion of coagulases confer type-specific immunity through the neutralization of S. aureus clotting activity and protection from staphylococcal disease in mice. By combining variable portions of coagulases from North American isolates into hybrid Coa and vWbp proteins, a subunit vaccine that provided protection against challenge with different coagulase-type S. aureus strains in mice was derived.

Inhibition of staphylothrombin by dabigatran reduces Staphylococcus aureus virulence

Summary.  Background: Staphylocoagulase and von Willebrand binding protein (VWbp) bind to prothrombin to form the staphylothrombin complex that converts fibrinogen into fibrin. Objectives: To study the role of staphylothrombin and its inhibition by dabigatran on Staphylococcus aureus virulence. Methods: We studied the effect of staphylothrombin inhibition on bacterial attachment to polystyrene surfaces, leukocyte activation and bactericidal activity for S. aureus ATCC 25923, S. aureus Newman, and staphylocoagulase‐ and VWbp‐negative S. aureus Newman mutants in the presence or absence of prothrombin and fibrinogen. We measured the abscess size after subcutaneous (s.c.) injection of S. aureus ATCC 25923 and S. aureus Newman, as well as an S. aureus Newman mutant strain lacking staphylocoagulase and VWbp, in mice treated with either dabigatran or placebo. Results: Staphylothrombin‐mediated fibrin increased the association of S. aureus to polystyrene surfaces and reduced the bactericidal activity of leukocytes. The absence or inhibition of staphylothrombin decreased the bacterial association, enhanced leukocyte activation and reduced bacterial survival in vitro. Abscess size was smaller in mice treated with dabigatran or infected with a coagulase‐negative mutant. Conclusion: Inhibition or the absence of staphylothrombin reduced S. aureus virulence in in vitro and in vivo models.

The Giant Protein Ebh Is a Determinant of Staphylococcus aureus Cell Size and Complement Resistance

Staphylococcus aureus USA300, the clonal type associated with epidemic community-acquired methicillin-resistant S. aureus (MRSA) infections, displays the giant protein Ebh on its surface. Mutations that disrupt the ebh reading frame increase the volume of staphylococcal cells and alter the cross wall, a membrane-enclosed peptidoglycan synthesis and assembly compartment. S. aureus ebh variants display increased sensitivity to oxacillin (methicillin) as well as susceptibility to complement-mediated killing. Mutations in ebh are associated with reduced survival of mutant staphylococci in blood and diminished virulence in mice. We propose that Ebh, following its secretion into the cross wall, contributes to the characteristic cell growth and envelope assembly pathways of S. aureus, thereby enabling complement resistance and the pathogenesis of staphylococcal infections.