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.

• 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

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

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.

• 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

Bordetella pertussis whole-cell vaccines (wP) caused a spectacular drop of global pertussis incidence, but since the replacement of wP with acellular pertussis vaccines (aP), pertussis has resurged in developed countries within 7 to 12 years of the change from wP to aP. In the mouse infection model, we examined whether addition of further protective antigens into the aP vaccine, such as type 2 and type 3 fimbriae (FIM2/3) with outer membrane lipooligosaccharide (LOS) and/or of the adenylate cyclase toxoid (dACT), which elicits antibodies neutralizing the CyaA toxin, could enhance the capacity of the aP vaccine to prevent colonization of the nasal mucosa by B. pertussis. The addition of the toxoid and of the opsonizing antibody-inducing agglutinogens modestly enhanced the already high capacity of intraperitoneally-administered aP vaccine to elicit sterilizing immunity, protecting mouse lungs from B. pertussis infection. At the same time, irrespective of FIM2/3 with LOS and dACT addition, the aP vaccination ablated the natural capacity of BALB/c mice to clear B. pertussis infection from the nasal cavity. While wP or sham-vaccinated animals cleared the nasal infection with similar kinetics within 7 weeks, administration of the aP vaccine promoted persistent colonization of mouse nasal mucosa by B. pertussis.

• Publikační typ
• časopisecké články MeSH

In a wide range of organisms, from bacteria to humans, numerous proteins have to be posttranslationally acylated to become biologically active. Bacterial repeats in toxin (RTX) cytolysins form a prominent group of proteins that are synthesized as inactive protoxins and undergo posttranslational acylation on ε-amino groups of two internal conserved lysine residues by co-expressed toxin-activating acyltransferases. Here, we investigated how the chemical nature, position, and number of bound acyl chains govern the activities of Bordetella pertussis adenylate cyclase toxin (CyaA), Escherichia coli α-hemolysin (HlyA), and Kingella kingae cytotoxin (RtxA). We found that the three protoxins are acylated in the same E. coli cell background by each of the CyaC, HlyC, and RtxC acyltransferases. We also noted that the acyltransferase selects from the bacterial pool of acyl-acyl carrier proteins (ACPs) an acyl chain of a specific length for covalent linkage to the protoxin. The acyltransferase also selects whether both or only one of two conserved lysine residues of the protoxin will be posttranslationally acylated. Functional assays revealed that RtxA has to be modified by 14-carbon fatty acyl chains to be biologically active, that HlyA remains active also when modified by 16-carbon acyl chains, and that CyaA is activated exclusively by 16-carbon acyl chains. These results suggest that the RTX toxin molecules are structurally adapted to the length of the acyl chains used for modification of their acylated lysine residue in the second, more conserved acylation site.

• MeSH
• acyltransferasy metabolismus   MeSH
• Bacteria metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• buněčné linie MeSH
• hemolyziny metabolismus   MeSH
• mastné kyseliny metabolismus   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) and the α-hemolysin (HlyA) of Escherichia coli belong to the family of cytolytic pore-forming Repeats in ToXin (RTX) cytotoxins. HlyA preferentially binds the αLβ2 integrin LFA-1 (CD11a/CD18) of leukocytes and can promiscuously bind and also permeabilize many other cells. CyaA bears an N-terminal adenylyl cyclase (AC) domain linked to a pore-forming RTX cytolysin (Hly) moiety, binds the complement receptor 3 (CR3, αMβ2, CD11b/CD18, or Mac-1) of myeloid phagocytes, penetrates their plasma membrane, and delivers the AC enzyme into the cytosol. We constructed a set of CyaA/HlyA chimeras and show that the CyaC-acylated segment and the CR3-binding RTX domain of CyaA can be functionally replaced by the HlyC-acylated segment and the much shorter RTX domain of HlyA. Instead of binding CR3, a CyaA1-710/HlyA411-1024 chimera bound the LFA-1 receptor and effectively delivered AC into Jurkat T cells. At high chimera concentrations (25 nm), the interaction with LFA-1 was not required for CyaA1-710/HlyA411-1024 binding to CHO cells. However, interaction with the LFA-1 receptor strongly enhanced the specific capacity of the bound CyaA1-710/HlyA411-1024 chimera to penetrate cells and deliver the AC enzyme into their cytosol. Hence, interaction of the acylated segment and/or the RTX domain of HlyA with LFA-1 promoted a productive membrane interaction of the chimera. These results help delimit residues 400-710 of CyaA as an "AC translocon" sufficient for translocation of the AC polypeptide across the plasma membrane of target cells.

• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• antigen-1 spojený s lymfocytární funkcí metabolismus   MeSH
• Bordetella * MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• cytosol metabolismus   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• makrofágový antigen 1 metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• THP-1 buňky MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Two distinct conformers of the adenylate cyclase toxin (CyaA) appear to accomplish its two parallel activities within target cell membrane. The translocating conformer would deliver the N-terminal adenylyl cyclase (AC) enzyme domain across plasma membrane into cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities then involve a membrane-interacting 'AC-to-Hly-linking segment' (residues 400 to 500). Here, we report the NMR structure of the corresponding CyaA411-490 polypeptide in dodecylphosphocholine micelles and show that it consists of two α-helices linked by an unrestrained loop. The N-terminal α-helix (Gly418 to His439) remained solvent accessible, while the C-terminal α-helix (His457 to Phe485) was fully enclosed within detergent micelles. CyaA411-490 weakly bound Ca2+ ions (apparent KD 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 μM) the CyaA411-490 polypeptide formed stable conductance units in artificial lipid bilayers with applied voltage, suggesting its possible transmembrane orientation in the membrane-inserted toxin. Mutagenesis revealed that two clusters of negatively charged residues within the 'AC-to-Hly-linking segment' (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA in function of calcium concentration.

• MeSH
• adenylátcyklasový toxin chemie   metabolismus   MeSH
• AMP cyklický metabolismus   MeSH
• biologický transport genetika   MeSH
• Bordetella pertussis chemie   metabolismus   MeSH
• buněčná membrána chemie   metabolismus   MeSH
• hemolýza genetika   MeSH
• konformace proteinů, alfa-helix genetika   MeSH
• lidé MeSH
• lipidové dvojvrstvy chemie   metabolismus   MeSH
• permeabilita buněčné membrány genetika   MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA).

• MeSH
• bakteriální proteiny izolace a purifikace   toxicita   MeSH
• cytotoxiny izolace a purifikace   toxicita   MeSH
• detergenty chemie   MeSH
• erytrocyty účinky léků   MeSH
• Escherichia coli metabolismus   MeSH
• hemolýza MeSH
• hemolyziny izolace a purifikace   toxicita   MeSH
• lidé MeSH
• lipopolysacharidy analýza   MeSH
• močovina chemie   MeSH
• nádorové buněčné linie MeSH
• oktoxynol chemie   MeSH
• ovce MeSH
• THP-1 buňky MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of pathogenic Bordetellae delivers its adenylyl cyclase (AC) enzyme domain into the cytosol of host cells and catalyzes uncontrolled conversion of cellular ATP to cAMP. In parallel, the toxin forms small cation-selective pores that permeabilize target cell membrane and account for the hemolytic activity of CyaA on erythrocytes. The pore-forming domain of CyaA is predicted to consist of five transmembrane α-helices, of which the helices I, III, IV and V have previously been characterized. We examined here the α-helix II that is predicted to form between residues 529 to 549. Substitution of the glycine 531 residue by a proline selectively reduced the hemolytic capacity but did not affect the AC translocating activity of the CyaA-G531P toxin. In contrast, CyaA toxins with alanine 538 or 546 replaced by diverse residues were selectively impaired in the capacity to translocate the AC domain across cell membrane but remained fully hemolytic. Such toxins, however, formed pores in planar asolectin bilayer membranes with a very low frequency and with at least two different conducting states. The helix-breaking substitution of alanine 538 by a proline residue abolished the voltage-activated increase of membrane activity of CyaA in asolectin bilayers. These results reveal that the predicted α-helix comprising the residues 529 to 549 plays a key role in CyaA penetration into the target plasma membrane and pore-forming activity of the toxin.

• MeSH
• adenylátcyklasový toxin chemie   genetika   toxicita   MeSH
• Bordetella enzymologie   MeSH
• buněčná membrána účinky léků   MeSH
• erytrocyty účinky léků   MeSH
• hemolýza MeSH
• konformace proteinů, alfa-helix MeSH
• kultivované buňky MeSH
• myši MeSH
• ovce MeSH
• substituce aminokyselin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Kingella kingae is a member of the commensal oropharyngeal flora of young children. Improvements in detection methods have led to the recognition of K. kingae as an emerging pathogen that frequently causes osteoarticular infections in children and a severe form of infective endocarditis in children and adults. Kingella kingae secretes a membrane-damaging RTX (Repeat in ToXin) toxin, RtxA, which is implicated in the development of clinical infections. However, the mechanism by which RtxA recognizes and kills host cells is largely unexplored. To facilitate structure-function studies of RtxA, we have developed a procedure for the overproduction and purification of milligram amounts of biologically active recombinant RtxA. Mass spectrometry analysis revealed the activation of RtxA by post-translational fatty acyl modification on the lysine residues 558 and/or 689 by the fatty-acyltransferase RtxC. Acylated RtxA was toxic to various human cells in a calcium-dependent manner and possessed pore-forming activity in planar lipid bilayers. Using various biochemical and biophysical approaches, we demonstrated that cholesterol facilitates the interaction of RtxA with artificial and cell membranes. The results of analyses using RtxA mutant variants suggested that the interaction between the toxin and cholesterol occurs via two cholesterol recognition/interaction amino acid consensus motifs located in the C-terminal portion of the pore-forming domain of the toxin. Based on our observations, we conclude that the cytotoxic activity of RtxA depends on post-translational acylation of the K558 and/or K689 residues and on the toxin binding to cholesterol in the membrane.

• MeSH
• acylace MeSH
• bakteriální toxiny genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• buněčné linie MeSH
• cholesterol metabolismus   MeSH
• Kingella kingae enzymologie   genetika   MeSH
• lidé MeSH
• lysin chemie   MeSH
• posttranslační úpravy proteinů * MeSH
• rekombinantní proteiny metabolismus   MeSH
• transaminasy genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH