Modification of the RTX domain cap by acyl chains of adapted length rules the formation of functional hemolysin pores.

The acylated pore-forming Repeats in ToXin (RTX) cytolysins α-hemolysin (HlyA) and adenylate cyclase toxin (CyaA) preferentially bind to β integrins of myeloid leukocytes but can also promiscuously bind and permeabilize cells lacking the β integrins. We constructed a HlyA/CyaA chimera that was acylated either by the toxin-activating acyltransferase CyaC, using sixteen carbon-long (C16) acyls, or by the HlyC acyltransferase using fourteen carbon-long (C14) acyls. Cytolysin assays with the C16- or C14-acylated HlyA/CyaA chimeric toxin revealed that the RTX domain of CyaA can functionally replace the RTX domain of HlyA only if it is modified by C16-acyls on the Lys983 residue of CyaA.

The adenylate cyclase toxin RTX domain follows a series templated folding mechanism with implications for toxin activity.

Folding of the Repeats-in-toxin (RTX) domain of the bacterial adenylate cyclase toxin-hemolysin (CyaA) is critical to its toxin activities and the virulence of the whooping cough agent Bordetella pertussis. The RTX domain (RD) contains five RTX blocks (RTX-i to RTX-v) and their folding is driven by the binding of calcium. However, the detailed molecular mechanism via which the folding signal transmits within the five RTX blocks remains unknown.

A conserved tryptophan in the acylated segment of RTX toxins controls their β integrin-independent cell penetration.

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind β 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 β 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 β integrin CR3.

Selective Enhancement of the Cell-Permeabilizing Activity of Adenylate Cyclase Toxin Does Not Increase Virulence of .

The whooping cough agent, , 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 in the mouse model of pneumonic infection.

Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins.

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.

Almost half of the RTX domain is dispensable for complement receptor 3 binding and cell-invasive activity of the Bordetella adenylate cyclase toxin.

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 Ca-loaded parallel β-rolls. Previous work indicated that the CR3-binding structure comprises the interface of β-rolls II and III.

Acellular Pertussis Vaccine Inhibits Clearance from the Nasal Mucosa of Mice.

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 . 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 infection.

Acyltransferase-mediated selection of the length of the fatty acyl chain and of the acylation site governs activation of bacterial RTX toxins.

In a wide range of organisms, from bacteria to humans, numerous proteins have to be posttranslationally acylated to become biologically active. Bacterial epeats in oin (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 adenylate cyclase toxin (CyaA), α-hemolysin (HlyA), and cytotoxin (RtxA).

Retargeting from the CR3 to the LFA-1 receptor uncovers the adenylyl cyclase enzyme-translocating segment of adenylate cyclase toxin.

The adenylate cyclase toxin-hemolysin (CyaA) and the α-hemolysin (HlyA) of belong to the family of cytolytic pore-forming Repeats in ToXin (RTX) cytotoxins. HlyA preferentially binds the αβ 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, αβ, CD11b/CD18, or Mac-1) of myeloid phagocytes, penetrates their plasma membrane, and delivers the AC enzyme into the cytosol.

Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin.

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).

Rapid Purification of Endotoxin-Free RTX Toxins.

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 . Such preparations are, however, typically contaminated by high amounts of lipopolysaccharide (LPS or endotoxin).

Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella adenylate cyclase toxin.

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.

Cytotoxic activity of Kingella kingae RtxA toxin depends on post-translational acylation of lysine residues and cholesterol binding.

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.

Adenylate Cyclase Toxin Does Not Possess a Phospholipase A Activity; Serine 606 and Aspartate 1079 Residues Are Not Involved in Target Cell Delivery of the Adenylyl Cyclase Enzyme Domain.

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) plays a crucial role in virulence and airway colonization capacity of the whooping cough agent . The toxin penetrates target cell membranes and exhibits three distinct biological activities. A population of CyaA conformers forms small cation-selective pores that permeabilize the cell membrane for potassium efflux, which can provoke colloid-osmotic (oncotic) cell lysis.

Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes.

, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αβ₂) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by .

The conserved tyrosine residue 940 plays a key structural role in membrane interaction of Bordetella adenylate cyclase toxin.

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) translocates its adenylate cyclase (AC) enzyme domain into target cells in a step that depends on membrane cholesterol content. We thus examined what role in toxin activities is played by the five putative cholesterol recognition amino acid consensus (CRAC) motifs predicted in CyaA hemolysin moiety. CRAC-disrupting phenylalanine substitutions had no impact on toxin activities and these were not inhibited by free cholesterol, showing that the putative CRAC motifs are not involved in cholesterol binding.

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis.

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αβ integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes.

The extent of the temperature-induced membrane remodeling in two closely related species reflects their adaptation to diverse environmental niches.

Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens and Although represents a pathogen strictly adapted to the human body temperature, causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement.

Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin.

The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane.

Calcium-Driven Folding of RTX Domain β-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts.

Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls.

Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella adenylate cyclase toxin.

Adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, α(M)β(2) or Mac-1). CyaA translocates its adenylate cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis.

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions.

Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme.

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA targets myeloid phagocytes expressing the complement receptor 3 (CR3, known as αMβ2 integrin CD11b/CD18 or Mac-1) and translocates by a poorly understood mechanism directly across the cytoplasmic membrane into cell cytosol of phagocytes an adenylyl cyclase(AC) enzyme. This binds intracellular calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP.