PURPOSE OF REVIEW: RTX toxin action often defines the outcome of bacterial infections. Here, we discuss the progress in understanding the impacts of RTX toxin activities on host immunity. RECENT FINDINGS: Bordetella pertussis CyaA activity paralyzes sentinel phagocytic cells by elevating cellular cAMP levels and blocks differentiation of infiltrating monocytes into bactericidal macrophages, promoting also de-differentiation of resident alveolar macrophages into monocyte-like cells. Vibrio cholerae multifunctional autoprocessing repeats-in-toxins (MARTX), through Rho inactivating and α/β-hydrolase (ABH) domain action blocks mitogen-activated protein kinase signaling in epithelial cells and dampens the inflammatory responses of intestinal epithelia by blocking immune cell recruitment. The action of actin crosslinking effector domain and Ras/Rap1-specific endopeptidase (RRSP) domains of MARTX compromises the phagocytic ability of macrophages. Aggregatibacter actinomycetemcomitans LtxA action triggers neutrophil elastase release into periodontal tissue, compromising the epithelial barrier and promoting bacterial spreads into deeper tissue. SUMMARY: Action of RTX toxins enables bacterial pathogens to cope with the fierce host immune defenses. RTX toxins often block phagocytosis and bactericidal reactive oxygen species and NO production. Some RTX toxins can reprogram the macrophages to less bactericidal cell types. Autophagy is hijacked for example by the activity of the V. cholerae ABH effector domain of the MARTX protein. Subversion of immune functions by RTX toxins thus promotes bacterial survival and proliferation in the host.

• MeSH
• adaptivní imunita MeSH
• Bacteria metabolismus   patogenita   MeSH
• bakteriální toxiny toxicita   MeSH
• buňky NK účinky léků   MeSH
• dendritické buňky účinky léků   MeSH
• epitelové buňky účinky léků   MeSH
• lidé MeSH
• makrofágy účinky léků   MeSH
• monocyty účinky léků   MeSH
• virulence 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

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

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: alphahemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size - 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities alpha for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV(1000)) were found to be more suitable for distinguishing between high alpha values whereas smaller ones (LUV(100)) were more appropriate for discriminating an all-or-none leakage (alpha=0) from the graded leakage with low values of alpha. While disrupting LUV(1000), CyaA caused a highly cation-selective leakage (alpha~15) whereas its mutated form with decreased channel K(+)/Cl(-) selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K+E516K) exhibited much lower selectivity (alpha approximately 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.

• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• Bordetella pertussis fyziologie   MeSH
• buněčná membrána fyziologie   MeSH
• elektroforéza v agarovém gelu MeSH
• Escherichia coli fyziologie   MeSH
• fluorescenční spektrometrie MeSH
• hemolyziny metabolismus   MeSH
• kinetika MeSH
• liposomy metabolismus   MeSH
• permeabilita buněčné membrány fyziologie   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• Publikační typ
• práce podpořená grantem MeSH

Repeats-in-Toxin (RTX) proteins of Gram-negative bacteria are excreted through the type I secretion system (T1SS) that recognizes non-cleavable C-terminal secretion signals. These are preceded by arrays of glycine and aspartate-rich nonapeptide repeats grouped by four to eight β strands into blocks that fold into calcium-binding parallel β-roll structures. The β-rolls are interspersed by linkers of variable length and sequence and the organization of multiple RTX repeat blocks within large RTX domains remains unknown. Here we examined the structure and function of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA) that is composed of five β-roll RTX blocks. We show that the non-folded RTX repeats maintain the stability of the CyaA polypeptide in the Ca2+-depleted bacterial cytosol and thereby enable its efficient translocation through the T1SS apparatus. The efficacy of secretion of truncated CyaA constructs was dictated by the number of retained RTX repeat blocks and depended on the presence of extracellular Ca2+ ions. We further describe the crystal structure of the RTX blocks IV-V of CyaA (CyaA1372-1681) that consists of a contiguous assembly of two β-rolls that differs substantially from the arrangement of the RTX blocks observed in RTX lipases or other RTX proteins. These results provide a novel structural insight into the architecture of the RTX domains of large RTX proteins and support the "push-ratchet" mechanism of the T1SS-mediated secretion of very large RTX proteins.

• MeSH
• adenylátcyklasový toxin chemie   genetika   metabolismus   MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• bakteriální toxiny chemie   genetika   metabolismus   MeSH
• Bordetella pertussis metabolismus   MeSH
• cytosol metabolismus   MeSH
• gramnegativní bakterie metabolismus   MeSH
• konformace proteinů MeSH
• sbalování proteinů MeSH
• sekreční systém typu I MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens.

• MeSH
• adenylátcyklasový toxin chemie   metabolismus   MeSH
• bakteriální toxiny chemie   metabolismus   MeSH
• Bordetella pertussis chemie   enzymologie   MeSH
• buněčné linie MeSH
• gramnegativní bakterie chemie   metabolismus   MeSH
• molekulární modely MeSH
• myši MeSH
• sbalování proteinů MeSH
• sekreční systém typu I metabolismus   MeSH
• sekundární struktura proteinů MeSH
• transport proteinů MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.

• MeSH
• Actinobacillus pleuropneumoniae metabolismus   MeSH
• adenylátcyklasový toxin antagonisté a inhibitory   chemie   metabolismus   MeSH
• bakteriální proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• Bordetella pertussis metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• erytrocyty metabolismus   MeSH
• hemolýza * MeSH
• hemolyziny antagonisté a inhibitory   chemie   metabolismus   MeSH
• hexokinasa MeSH
• kultivované buňky MeSH
• lipidové dvojvrstvy metabolismus   MeSH
• makrofágy MeSH
• myši MeSH
• osmotický tlak MeSH
• permeabilita buněčné membrány MeSH
• pyridoxalfosfát analogy a deriváty   MeSH
• rosanilinová barviva MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (Hly) (CyaA, ACT, or AC-Hly) is a cytotoxin of the RTX (repeat in toxin) family. It delivers into target cells an AC domain that catalyzes uncontrolled conversion of ATP to cAMP, a key signaling molecule subverting phagocyte functions. CyaA utilizes a heavily N-glycosylated beta(2) integrin receptor CD11b/CD18 (alpha(M)beta(2), Mac-1, or CR3). We show that deglycosylation of cell surface proteins by glycosidase treatment, or inhibition of protein N-glycosylation by tunicamycin, ablates CyaA binding and penetration of CD11b-expressing cells. Furthermore, binding of CyaA to cells was strongly inhibited in the presence of free saccharides occurring as building units of integrin oligosaccharide complex, whereas saccharides absent from integrin oligosaccharide chains failed to inhibit CyaA binding to CD11b/CD18-expressing cells. CyaA, hence, selectively recognized sugar residues of N-linked oligosaccharides of integrins. Moreover, glycosylation of CD11a/CD18, another receptor of the beta(2) integrin family, was also essential for cytotoxic action of other RTX cytotoxins, the leukotoxin of Aggregatibacter actinomycetemcomitans (LtxA) and the Escherichia coli alpha-Hly (HlyA). These results show that binding and killing of target cells by CyaA, LtxA, and HlyA depends on recognition of N-linked oligosaccharide chains of beta(2) integrin receptors. This sets a new paradigm for action of RTX cytotoxins.

• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• antigeny CD11b metabolismus   MeSH
• antigeny CD18 metabolismus   MeSH
• bakteriální proteiny MeSH
• bakteriální toxiny metabolismus   MeSH
• Bordetella enzymologie   chemie   patogenita   MeSH
• financování organizované MeSH
• glykosylace MeSH
• hemolyziny MeSH
• lidé MeSH
• oligosacharidy metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé 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 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