The role of two-component regulatory systems of Streptococcus pyogenes in virulence

The ability of Streptococcus pyogenes to adapt to different host environments represents a key feature during the infection process of this important human pathogen. Two-component regulatory systems (TCS) act as sensors and communicators of environmental changes and their contribution to streptococcal pathogenesis was investigated in the scope of this dissertation. Therefore, five streptococcal mutants with deletions in two-component systems were constructed and investigated for different virulence behaviour in comparison to the parental strain. The mutants deficient in the TCS FasCA, TCS09, TCS07, or Irr/Ihk were less resistant to killing by human phagocytic cells. In a mouse model of skin infection, the TCS07 and Irr/Ihk deletion strains showed reduced virulent behaviour, expressed by survival of infected animals and a rapid clearing of the mutants from systemic organs and bloodstream in comparison to the wildtype strain. Furthermore, using a mouse model of intraperitoneal infection, enhanced killing of the Irr/Ihk mutant within murine PMNs could be demonstrated. On the molecular level, the TCS07 mutant lacked transcription of two major antiphagocytic virulence factors, the M-protein and SIC. In the Irr/Ihk mutant, transcription of these factors was not affected, however a cleavage of the M-proteins N-terminus from the surface of the mutant was observed, resulting in release of a 28-kDa fragment. Absence of full-length M protein in both mutants was accompanied by a loss of fibrinogen binding capability. Furthermore, both failed to induce encapsulation with hyaluronic acid under in vivo conditions as it was observed for the wildtype strain.Die Fähigkeit der Adaptation an verschiedene Wirtsnischen verleiht Streptococcus pyogenes einen wesentlichen Vorteil während des Infektionsprozesses. In dieser Dissertation wurde die Rolle von regulatorischen Zwei-Komponenten Systemen (TCS) als Sensoren und Regulatoren der adaptiven Genexpression von S. pyogenes untersucht und ihre Bedeutung für die Virulenz dieses pathogenen Bakteriums bestimmt. Fünf Streptokokken Mutanten mit Deletionen in verschiedenen TCS wurden konstruiert und ihr Virulenzverhalten im Vergleich zum parentalen Stamm untersucht. In einem Bakterizidversuch führten dabei Deletionen in den TCS FasCA, TCS07, TCS09 und Irr/Ihk führten zu einer verminderten Fähigkeit der Streptokokken, Abtötung durch humane phagozytische Zellen zu entkommen. Weitere Untersuchungen der TCS07 und der Irr/Ihk Mutanten in einem murinem Hautinfektionsmodell zeigten eine verminderte Virulenz beider Mutanten, die sich durch Überleben Mutanten-infizierten Mäuse und einer schnellen Eliminierung der Bakterien aus systemischen Organen und Blut ausdrückte. In einem intraperitonealem Mausinfektionsmodell konnte zusätzlich die erhöhte Abtötung der Irr/Ihk Mutante durch murine PMNs gezeigt werden. Das Fehlen zweier antiphagozytischer Virulenzfaktoren in der TCS07 Mutante konnten durch Transkriptionsanalyse der TCS07 Mutante festgestellt werden. In der Irr/Ihk Mutante fand auf posttranslationaler Ebene eine Abspaltung des Streptokokken-M-Proteins und Verlust der Fibrinogen-Bindungsfähigkeit statt. Expressionsdifferenzen der antiphagozytisch wirkenden Hyaluronsäurekapsel konnte ex vivo zwischen Wildtyp und Mutanten beobachtet werden, wobei lediglich der Wildtyp eine starke Bakapselung aufwies

Anthrax Lethal Factor Cleavage of Nlrp1 Is Required for Activation of the Inflammasome

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1β and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT

The crystal sructure of Bacillus cereus HblL1

The Hbl toxin is a three-component haemolytic complex produced by Bacillus cereus sensu lato strains and implicated as a cause of diarrhoea in B. cereus food poisoning. While the structure of the HblB component of this toxin is known, the structures of the other components are unresolved. Here, we describe the expression of the recombinant HblL1 component and the elucidation of its structure to 1.36 Å. Like HblB, it is a member of the alpha-helical pore-forming toxin family. In comparison to other members of this group, it has an extended hydrophobic beta tongue region that may be involved in pore formation. Molecular docking was used to predict possible interactions between HblL1 and HblB, and suggests a head to tail dimer might form, burying the HblL1 beta tongue region

Inflammasome Sensor Nlrp1b-Dependent Resistance to Anthrax Is Mediated by Caspase-1, IL-1 Signaling and Neutrophil Recruitment

Bacillus anthracis infects hosts as a spore, germinates, and disseminates in its vegetative form. Production of anthrax lethal and edema toxins following bacterial outgrowth results in host death. Macrophages of inbred mouse strains are either sensitive or resistant to lethal toxin depending on whether they express the lethal toxin responsive or non-responsive alleles of the inflammasome sensor Nlrp1b (Nlrp1bS/S or Nlrp1bR/R, respectively). In this study, Nlrp1b was shown to affect mouse susceptibility to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1bS/S alleles (which allow activation of caspase-1 and IL-1β release in response to anthrax lethal toxin challenge) effectively controlled bacterial growth and dissemination when compared to mice having Nlrp1bR/R alleles (which cannot activate caspase-1 in response to toxin). Nlrp1bS-mediated resistance to infection was not dependent on the route of infection and was observed when bacteria were introduced by either subcutaneous or intravenous routes. Resistance did not occur through alterations in spore germination, as vegetative bacteria were also killed in Nlrp1bS/S mice. Resistance to infection required the actions of both caspase-1 and IL-1β as Nlrp1bS/S mice deleted of caspase-1 or the IL-1 receptor, or treated with the Il-1 receptor antagonist anakinra, were sensitized to infection. Comparison of circulating neutrophil levels and IL-1β responses in Nlrp1bS/S,Nlrp1bR/R and IL-1 receptor knockout mice implicated Nlrp1b and IL-1 signaling in control of neutrophil responses to anthrax infection. Neutrophil depletion experiments verified the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection, and establish roles for Nlrp1bS, caspase-1, and IL-1β in countering anthrax infection

Molecular Typing of Staphylococcus aureus Isolated from Patients with Autosomal Dominant Hyper IgE Syndrome

Autosomal dominant hyper IgE syndrome (AD-HIES) is a primary immunodeficiency caused by a loss-of-function mutation in the Signal Transducer and Activator of Transcription 3 (STAT3). This immune disorder is clinically characterized by increased susceptibility to cutaneous and sinopulmonary infections, in particular with Candida and Staphylococcus aureus. It has recently been recognized that the skin microbiome of patients with AD-HIES is altered with an overrepresentation of certain Gram-negative bacteria and Gram-positive staphylococci. However, these alterations have not been characterized at the species- and strain-level. Since S. aureus infections are influenced by strain-specific expression of virulence factors, information on colonizing strain characteristics may provide insights into host-pathogen interactions and help guide management strategies for treatment and prophylaxis. The aim of this study was to determine whether the immunodeficiency of AD-HIES selects for unique strains of colonizing S. aureus. Using multi-locus sequence typing (MLST), protein A (spa) typing, and PCR-based detection of toxin genes, we performed a detailed analysis of the S. aureus isolates (n = 13) found on the skin of twenty-one patients with AD-HIES. We found a low diversity of sequence types, and an abundance of strains that expressed methicillin resistance, Panton-Valentine leukocidin (PVL), and staphylococcal enterotoxins K and Q (SEK, SEQ). Our results indicate that patients with AD-HIES may often carry antibiotic-resistant strains that harbor key virulence factors

Bacterial Exotoxins: How bacteria fight the immune system

The goal of this research topic was to gather current knowledge on the interaction of bacterial exotoxins and effector proteins with the host immune system. The following 16 research and review articles in this special issue describe mechanisms of immune modification and evasion and provide an overview over the complexity of bacterial toxin interaction with different cells of the immune system

Vaccination equally enables both genetically susceptible and resistant mice to control infection with group A streptococci.

There is substantial evidence that host genetic factors are important in determining susceptibility to infection with group A streptococci (GAS). Several studies have revealed that, similarly to humans, a genetic component may be important in determining susceptibility to GAS infection in mice. Thus, C3H/HeN mice are much more susceptible to streptococcal infection than BALB/c mice. We have determined here whether vaccination makes genetically susceptible mice as capable as genetically resistant mice to control GAS infection. Resistant BALB/c and susceptible C3H/HeN mice were immunized either systemically with heat-killed GAS or through the mucosal route with an M protein-based subunit vaccine, and challenged with live bacteria. Vaccination elicited in both mouse strains similar levels of bactericidal anti-GAS IgG antibodies and also antigen-specific mucosal IgA. Vaccination provided mice of both strains with an increased and equal capacity to express immunity against GAS as indicated by the reduced level of bacteria in the organs and the ability of vaccinated mice to survive infection. Protection in vaccinated mice was dependent on the presence of T cell-dependent bactericidal antibodies as shown by the ability of serum elicited in immunocompetent mice but not of serum elicited in T cell-deficient nu/nu mice to passively transfer anti-GAS immunity. In conclusion, the results presented here demonstrated that the presence of anti-GAS specific, T cell-dependent bactericidal antibodies elicited after vaccination overcomes the innate genetic susceptibility of C3H/HeN mice and makes both resistant and susceptible mice equally capable of controlling GAS infection

Protein FOG is a moderate inducer of MIG/CXCL9, and group G streptococci are more tolerant than group A streptococci to this chemokine's antibacterial effect

Streptococcus dysgalactiae subsp. equisimilis (group G streptococci; GGS) cause disease in humans but are often regarded as commensals in comparison with Streptococcus pyogenes (group A streptococci; GAS). The current study investigated the degree and kinetics of the innate immune response elicited by the two species. This was assessed as expression of the chemokine MIG/CXCL9 and bacterial susceptibility to its bactericidal effect. No significant difference in MIG/CXCL9 expression from THP-1 or Detroit 562 cells was observed when comparing whole GGS or GAS as stimuli. The study demonstrates that protein FOG was released from the bacterial surface directly and by neutrophil elastase. Expression of MIG/CXCL9 following stimulation with soluble M proteins of the two species (the recently described protein FOG of GGS and protein M1 of GAS) was reduced for protein FOG in both the monocytic and the epithelial cell line. When the antibacterial effects of MIG/CXCL9 were examined in conditions of increased ionic strength, MIG/CXCL9 killed GAS more efficiently than GGS. Also in the absence of MIG/CXCL9, GGS were more tolerant to increased salt concentrations than GAS. In summary, both GGS and GAS evoke MIG/CXCL9 expression but they differ in susceptibility to its antibacterial effects. This may in part explain the success of GGS as a commensal and its potential as a pathogen