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.

• Klíčová slova
• RTX toxin, acylated segment, adenylate cyclase toxin, cytotoxicity, hydrogen/deuterium exchange, thermal stability, tryptophan residue, α-hemolysin, β(2) integrins,
• MeSH
• adenylátcyklasový toxin * chemie   genetika   metabolismus   MeSH
• antigeny CD18 * genetika   metabolismus   MeSH
• Bordetella pertussis MeSH
• buněčná membrána metabolismus   MeSH
• erytrocyty metabolismus   MeSH
• konzervovaná sekvence MeSH
• tryptofan * chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• antigeny CD18 * MeSH
• tryptofan * 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.

• Klíčová slova
• Kingella kingae, RTX toxin, RtxA, membrane, pore-forming, β2 integrins,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) that catalyzes the conversion of intracellular ATP to cAMP and through its signaling annihilates the bactericidal activities of host sentinel phagocytes. In parallel, CyaA permeabilizes host cells by the formation of cation-selective membrane pores that account for the hemolytic activity of CyaA. The pore-forming activity contributes to the overall cytotoxic effect of CyaA in vitro, and it has previously been proposed to synergize with the cAMP-elevating activity in conferring full virulence on B. pertussis in the mouse model of pneumonic infection. CyaA primarily targets myeloid phagocytes through binding of their complement receptor 3 (CR3, integrin αMβ2, or CD11b/CD18). However, with a reduced efficacy, the toxin can promiscuously penetrate and permeabilize the cell membrane of a variety of non-myeloid cells that lack CR3 on the cell surface, including airway epithelial cells or erythrocytes, and detectably intoxicates them by cAMP. Here, we used CyaA variants with strongly and selectively enhanced or reduced pore-forming activity that, at the same time, exhibited a full capacity to elevate cAMP concentrations in both CR3-expressing and CR3-non-expressing target cells. Using B. pertussis mutants secreting such CyaA variants, we show that a selective enhancement of the cell-permeabilizing activity of CyaA does not increase the overall virulence and lethality of pneumonic B. pertussis infection of mice any further. In turn, a reduction of the cell-permeabilizing activity of CyaA did not reduce B. pertussis virulence any importantly. These results suggest that the phagocyte-paralyzing cAMP-elevating capacity of CyaA prevails over the cell-permeabilizing activity of CyaA that appears to play an auxiliary role in the biological activity of the CyaA toxin in the course of B. pertussis infections in vivo.

• Klíčová slova
• Bordetella pertussis, RTX toxin, adenylate cyclase toxin, cAMP intoxication, lung colonization, lung inflammation, pore-forming activity, virulence,
• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• AMP cyklický metabolismus   MeSH
• Bordetella pertussis patogenita   fyziologie   MeSH
• fagocyty metabolismus   mikrobiologie   MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• ovce MeSH
• permeabilita buněčné membrány MeSH
• pertuse metabolismus   mikrobiologie   patologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• AMP cyklický MeSH

Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.

• MeSH
• adenylátcyklasový toxin genetika   MeSH
• Bordetella bronchiseptica * genetika   metabolismus   MeSH
• Bordetella pertussis * genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• hemolýza MeSH
• infekce bakteriemi rodu Bordetella mikrobiologie   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• THP-1 buňky MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH

The whooping cough agent Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) that through its large carboxy-proximal Repeat-in-ToXin (RTX) domain binds the complement receptor 3 (CR3). The RTX domain consists of five blocks (I-V) of characteristic glycine and aspartate-rich nonapeptides that fold into five Ca2+-loaded parallel β-rolls. Previous work indicated that the CR3-binding structure comprises the interface of β-rolls II and III. To test if further portions of the RTX domain contribute to CR3 binding, we generated a construct with the RTX block II/III interface (CyaA residues 1132-1294) linked directly to the C-terminal block V fragment bearing the folding scaffold (CyaA residues 1562-1681). Despite deletion of 267 internal residues of the RTX domain, the Ca2+-driven folding of the hybrid block III/V β-roll still supported formation of the CR3-binding structure at the interface of β-rolls II and III. Moreover, upon stabilization by N- and C-terminal flanking segments, the block III/V hybrid-comprising constructs competed with CyaA for CR3 binding and induced formation of CyaA toxin-neutralizing antibodies in mice. Finally, a truncated CyaAΔ1295-1561 toxin bound and penetrated erythrocytes and CR3-expressing cells, showing that the deleted portions of RTX blocks III, IV, and V (residues 1295-1561) were dispensable for CR3 binding and for toxin translocation across the target cell membrane. This suggests that almost a half of the RTX domain of CyaA is not involved in target cell interaction and rather serves the purpose of toxin secretion.

• Klíčová slova
• Bordetella pertussis, CD11b/CD18 integrin receptor, RTX toxin, adenylate cyclase toxin,
• MeSH
• acylace MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• Bordetella pertussis patogenita   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• epitopy metabolismus   MeSH
• lidé MeSH
• makrofágový antigen 1 chemie   metabolismus   MeSH
• neutralizující protilátky metabolismus   MeSH
• proteinové domény MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• THP-1 buňky MeSH
• vápník metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• epitopy MeSH
• makrofágový antigen 1 MeSH
• neutralizující protilátky MeSH
• vápník MeSH

The Gram-negative coccobacillus Kingella kingae is increasingly recognized as an important invasive pediatric pathogen that causes mostly bacteremia and skeletal system infections. K. kingae secretes an RtxA toxin that belongs to a broad family of the RTX (Repeats in ToXin) cytotoxins produced by bacterial pathogens. Recently, we demonstrated that membrane cholesterol facilitates interaction of RtxA with target cells, but other cell surface structures potentially involved in toxin binding to cells remain unknown. We show that deglycosylation of cell surface structures by glycosidase treatment, or inhibition of protein N- and O-glycosylation by chemical inhibitors substantially reduces RtxA binding to target cells. Consequently, the deglycosylated cells were more resistant to cytotoxic activity of RtxA. Moreover, experiments on cells expressing or lacking cell surface integrins of the β2 family revealed that, unlike some other cytotoxins of the RTX family, K. kingae RtxA does not bind target cells via the β2 integrins. Our results, hence, show that RtxA binds cell surface oligosaccharides present on all mammalian cells but not the leukocyte-restricted β2 integrins. This explains the previously observed interaction of the toxin with a broad range of cell types of various mammalian species and reveals that RtxA belongs to the group of broadly cytolytic RTX hemolysins.

• Klíčová slova
• Kingella kingae, RTX toxin, RtxA, oligosaccharides, β2 integrins,
• MeSH
• antigeny CD18 metabolismus   MeSH
• bakteriální toxiny metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• buněčná smrt MeSH
• buněčné linie MeSH
• glykosidhydrolasy metabolismus   MeSH
• glykosylace MeSH
• Kingella kingae metabolismus   MeSH
• lidé MeSH
• makrofágy metabolismus   MeSH
• myši MeSH
• oligosacharidy chemie   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD18 MeSH
• bakteriální toxiny MeSH
• glykosidhydrolasy MeSH
• oligosacharidy MeSH