The genus Kingella comprises four common species: the commensals K. oralis and K. denitrificans, the emerging paediatric pathogen K. kingae and the novel species K. negevensis. Improved diagnostic methods have led to the recognition of K. kingae as a major cause of septic arthritis in young children. The key virulence factor responsible for the pathogenesis of K. kingae is its cytotoxin RtxA, which is thought to facilitate host invasion. After binding to target cells, RtxA inserts into the host cell membrane and forms ion-conducting membrane pores that disrupt normal cell physiology and ultimately lead to cell death. In this study, we analysed the pro-inflammatory response of oral epithelial cells to a clinical isolate of K. kingae, its isogenic RtxA-deficient mutant and the commensals K. oralis and K. denitrificans, which do not produce RtxA. The results show that infection of cells with K. kingae, but not with the RtxA-deficient mutant and the commensal species, leads to increased expression and secretion of certain pro-inflammatory cytokines and chemokines. Furthermore, the RtxA-producing K. kingae, but not the RtxA-deficient mutant, upregulated the expression of DEFB4A and SLPI genes encoding antimicrobial peptides. These findings demonstrate that the RtxA toxin induces an innate immune response in oral epithelial cells.

• Keywords
• Innate immunity, Kingella kingae, Oral epithelial cells, Repeats-in-toxin, RtxA,
• Publication type
• Journal Article MeSH

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind β2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for β2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the β2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in Tm), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the Tm closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by β2 integrins.

• Keywords
• RTX toxin, acylated segment, adenylate cyclase toxin, cytotoxicity, hydrogen/deuterium exchange, thermal stability, tryptophan residue, α-hemolysin, β(2) integrins,
• MeSH
• Adenylate Cyclase Toxin * chemistry   genetics   metabolism   MeSH
• CD18 Antigens * genetics   metabolism   MeSH
• Bordetella pertussis MeSH
• Cell Membrane metabolism   MeSH
• Erythrocytes metabolism   MeSH
• Conserved Sequence MeSH
• Tryptophan * chemistry   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylate Cyclase Toxin * MeSH
• CD18 Antigens * MeSH
• Tryptophan * MeSH

The pertussis agent Bordetella pertussis produces a number of virulence factors, of which the filamentous hemagglutinin (FhaB) plays a role in B. pertussis adhesion to epithelial and phagocytic cells. Moreover, FhaB was recently found to play a crucial role in nasal cavity infection and B. pertussis transmission to new hosts. The 367 kDa FhaB protein translocates through an FhaC pore to the outer bacterial surface and is eventually processed to a ~220 kDa N-terminal FHA fragment by the SphB1 protease. A fraction of the mature FHA then remains associated with bacterial cell surface, while most of FHA is shed into the bacterial environment. Previously reported indirect evidence suggested that FHA, or its precursor FhaB, may bind the β2 integrin CD11b/CD18 of human macrophages. Therefore, we assessed FHA binding to various cells producing or lacking the integrin and show that purified mature FHA does not bind CD11b/CD18. Further results then revealed that the adhesion of B. pertussis to cells does not involve an interaction between the bacterial surface-associated FhaB and/or mature FHA and the β2 integrin CD11b/CD18. In contrast, FHA binding was strongly inhibited at micromolar concentrations of heparin, corroborating that the cell binding of FHA is ruled by the interaction of its heparin-binding domain with sulfated glycosaminoglycans on the cell surface.

• Keywords
• Bordetella pertussis, CD11b/CD18, adenylate cyclase toxin, filamentous hemagglutinin, heparin, integrin,
• MeSH
• CD18 Antigens MeSH
• Bacterial Adhesion MeSH
• Adhesins, Bacterial metabolism   MeSH
• Bordetella pertussis * metabolism   MeSH
• Virulence Factors, Bordetella MeSH
• Glycosaminoglycans MeSH
• Hemagglutinins metabolism   MeSH
• Heparin MeSH
• Integrins MeSH
• Humans MeSH
• Macrophage-1 Antigen MeSH
• Whooping Cough * MeSH
• Peptide Hydrolases MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• CD18 Antigens MeSH
• Adhesins, Bacterial MeSH
• Virulence Factors, Bordetella MeSH
• Glycosaminoglycans MeSH
• Hemagglutinins MeSH
• Heparin MeSH
• Integrins MeSH
• Macrophage-1 Antigen MeSH
• Peptide Hydrolases MeSH

The Gram-negative bacterium Kingella kingae is part of the commensal oropharyngeal flora of young children. As detection methods have improved, K. kingae has been increasingly recognized as an emerging invasive pathogen that frequently causes skeletal system infections, bacteremia, and severe forms of infective endocarditis. K. kingae secretes an RtxA cytotoxin, which is involved in the development of clinical infection and belongs to an ever-growing family of cytolytic RTX (Repeats in ToXin) toxins secreted by Gram-negative pathogens. All RTX cytolysins share several characteristic structural features: (i) a hydrophobic pore-forming domain in the N-terminal part of the molecule; (ii) an acylated segment where the activation of the inactive protoxin to the toxin occurs by a co-expressed toxin-activating acyltransferase; (iii) a typical calcium-binding RTX domain in the C-terminal portion of the molecule with the characteristic glycine- and aspartate-rich nonapeptide repeats; and (iv) a C-proximal secretion signal recognized by the type I secretion system. RTX toxins, including RtxA from K. kingae, have been shown to act as highly efficient 'contact weapons' that penetrate and permeabilize host cell membranes and thus contribute to the pathogenesis of bacterial infections. RtxA was discovered relatively recently and the knowledge of its biological role remains limited. This review describes the structure and function of RtxA in the context of the most studied RTX toxins, the knowledge of which may contribute to a better understanding of the action of RtxA in the pathogenesis of K. kingae infections.

• Keywords
• Kingella kingae, RTX toxin, RtxA, membrane, pore-forming, β2 integrins,
• Publication type
• Journal Article MeSH
• Review MeSH