A conserved tryptophan in the acylated segment of RTX toxins controls their ß2 integrin-independent cell penetration.

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind ß2 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 ß2 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 ß2 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 ß2 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 ß2 integrins.

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 Ca2+-loaded parallel ß-rolls. Previous work indicated that the CR3-binding structure comprises the interface of ß-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 ß-roll still supported formation of the CR3-binding structure at the interface of ß-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.

Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue.

Bordetella bronchiseptica and Bordetella pertussis are closely related respiratory pathogens that evolved from a common bacterial ancestor. While B. bronchiseptica has an environmental reservoir and mostly establishes chronic infections in a broad range of mammals, B. pertussis is a human-specific pathogen causing acute pulmonary pertussis in infants and whooping cough illness in older humans. Both species employ a type III secretion system (T3SS) to inject a cytotoxic BteA effector protein into host cells. However, compared to the high BteA-mediated cytotoxicity of B. bronchiseptica, the cytotoxicity induced by B. pertussis BteA (Bp BteA) appears to be quite low and this has been attributed to the reduced T3SS gene expression in B. pertussis. We show that the presence of an alanine residue inserted at position 503 (A503) of Bp BteA accounts for its strongly attenuated cytotoxic potency. The deletion of A503 from Bp BteA greatly enhanced the cytotoxic activity of B. pertussis B1917 on mammalian HeLa cells and expression of Bp BteAΔA503 was highly toxic to Saccharomyces cerevisiae cells. Vice versa, insertion of A503 into B. bronchiseptica BteA (Bb BteA) strongly decreased its cytotoxicity to yeast and HeLa cells. Moreover, the production of Bp BteAΔA503 increased virulence of B. pertussis B1917 in the mouse model of intranasal infection (reduced LD50) but yielded less inflammatory pathology in infected mouse lungs at sublethal infectious doses. This suggests that A503 insertion in the T3SS effector Bp BteA may represent an evolutionary adaptation that fine-tunes B. pertussis virulence and host immune response.

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

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 αLß2 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, αMß2, 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.

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 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.

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

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 αMß2, 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.

Bordetellapertussis Acetylome is Shaped by Lysine Deacetylase Bkd1.

Post-translational modifications of proteins enable swift physiological adaptation of cells to altered growth conditions and stress. Aside from protein phosphorylation, acetylation on ε-amino groups of lysine residues (N-ε-lysine acetylation) represents another important post-translational modification of proteins. For many bacterial pathogens, including the whooping cough agent Bordetella pertussis, the role and extent of protein acetylation remain to be defined. We expressed in Escherichia coli the BP0960 and BP3063 genes encoding two putative deacetylases of B. pertussis and show that BP0960 encodes a lysine deacetylase enzyme, named Bkd1, that regulates acetylation of a range of B. pertussis proteins. Comparison of the proteome and acetylome of a Δbkd1 mutant with the proteome and acetylome of wild-type B. pertussis (PRIDE ID. PXD016384) revealed that acetylation on lysine residues may modulate activities or stabilities of proteins involved in bacterial metabolism and histone-like proteins. However, increased acetylation of the BvgA response regulator protein of the B. pertussis master virulence-regulating BvgAS two-component system affected neither the total levels of produced BvgA nor its phosphorylation status. Indeed, the Δbkd1 mutant was not impaired in the production of key virulence factors and its survival within human macrophages in vitro was not affected. The Δbkd1 mutant exhibited an increased growth rate under carbon source-limiting conditions and its virulence in the in vivo mouse lung infection model was somewhat affected. These results indicate that the lysine deacetylase Bkd1 and N-ε-lysine acetylation primarily modulate the general metabolism rather than the virulence of B. pertussis.

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 ß2 integrins of myeloid leukocytes but can also promiscuously bind and permeabilize cells lacking the ß2 integrins. We constructed a HlyA1-563/CyaA860-1706 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 C16-monoacylated HlyA/CyaA chimera was as pore-forming and cytolytic as native HlyA, whereas the C14-acylated chimera exhibited very low pore-forming activity. Hence, the capacity of the RTX domain of CyaA to support the insertion of the N-terminal pore-forming domain into the target cell membrane, and promote formation of toxin pores, strictly depends on the modification of the Lys983 residue by an acyl chain of adapted length.

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. 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.

A Mutation Upstream of the rplN-rpsD Ribosomal Operon Downregulates Bordetella pertussis Virulence Factor Production without Compromising Bacterial Survival within Human Macrophages.

The BvgS/BvgA two-component system controls expression of ∼550 genes of Bordetella pertussis, of which, ∼245 virulence-related genes are positively regulated by the BvgS-phosphorylated transcriptional regulator protein BvgA (BvgA∼P). We found that a single G-to-T nucleotide transversion in the 5'-untranslated region (5'-UTR) of the rplN gene enhanced transcription of the ribosomal protein operon and of the rpoA gene and provoked global dysregulation of B. pertussis genome expression. This comprised overproduction of the alpha subunit (RpoA) of the DNA-dependent RNA polymerase, downregulated BvgA and BvgS protein production, and impaired production and secretion of virulence factors by the mutant. Nonetheless, the mutant survived like the parental bacteria for >2 weeks inside infected primary human macrophages and persisted within infected mouse lungs for a longer period than wild-type B. pertussis These observations suggest that downregulation of virulence factor production by bacteria internalized into host cells may enable persistence of the whooping cough agent in the airways.IMPORTANCE We show that a spontaneous mutation that upregulates transcription of an operon encoding ribosomal proteins and causes overproduction of the downstream-encoded α subunit (RpoA) of RNA polymerase causes global effects on gene expression levels and proteome composition of Bordetella pertussis Nevertheless, the resulting important downregulation of the BvgAS-controlled expression of virulence factors of the whooping cough agent did not compromise its capacity to persist for prolonged periods inside primary human macrophage cells, and it even enhanced its capacity to persist in infected mouse lungs. These observations suggest that the modulation of BvgAS-controlled expression of virulence factors may occur also during natural infections of human airways by Bordetella pertussis and may possibly account for long-term persistence of the pathogen within infected cells of the airways.

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 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).

Bordetella pertussis and Bordetella bronchiseptica filamentous hemagglutinins are processed at different sites.

Filamentous hemagglutinin (FHA) mediates adherence and plays an important role in lower respiratory tract infections by pathogenic Bordetellae. The mature FHA proteins of B. pertussis (Bp-FHA) and the B. bronchiseptica (Bb-FHA) are generated by processing of the respective FhaB precursors by the autotransporter subtilisin-type protease SphB1. We have used bottom-up proteomics with differential 16O/18O labeling and show that despite high-sequence conservation of the corresponding FhaB segments, the mature Bp-FHA (~ 230 kDa) and Bb-FHA (~ 243 kDa) proteins are processed at different sites of FhaB, after the Ala-2348 and Lys-2479 residues, respectively. Moreover, protease surface accessibility probing by on-column (on-line) digestion of the Bp-FHA and Bb-FHA proteins yielded different peptide patterns, revealing structural differences in the N-terminal and C-terminal domains of the Bp-FHA and Bb-FHA proteins. These data indicate specific structural variations between the highly homologous FHA proteins.