Characterization of Post-Translational Modifications and Cytotoxic Properties of the Adenylate-Cyclase Hemolysin Produced by Various Bordetella pertussis and Bordetella parapertussis Isolates

Abstract

Bordetella pertussis and Bordetella parapertussis are the causal agents of whooping cough in humans. They produce diverse virulence factors, including adenylate cyclase-hemolysin (AC-Hly), a secreted toxin of the repeat in toxins (RTX) family with cyclase, pore-forming, and hemolytic activities. Post-translational modifications (PTMs) are essential for the biological activities of the toxin produced by B. pertussis. In this study, we compared AC-Hly toxins from various clinical isolates of B. pertussis and B. parapertussis, focusing on (i) the genomic sequences of cyaA genes, (ii) the PTMs of partially purified AC-Hly, and (iii) the cytotoxic activity of the various AC-Hly toxins. The genes encoding the AC-Hly toxins of B. pertussis and B. parapertussis displayed very limited polymorphism in each species. Most of the sequence differences between the two species were found in the C-terminal part of the protein. Both toxins harbored PTMs, mostly corresponding to palmitoylations of the lysine 860 residue and palmoylations and myristoylations of lysine 983 for B. pertussis and AC-Hly and palmitoylations of lysine 894 and myristoylations of lysine 1017 for B. parapertussis AC-Hly. Purified AC-Hly from B. pertussis was cytotoxic to macrophages, whereas that from B. parapertussis was not.

Keywords: Bordetella parapertussis; Bordetella pertussis; PTMs; adenylate cyclase hemolysin.

Figure 1

(A) Protein migration pattern, (B) Western blot with a polyclonal anti-Bp-AC-Hly antibody, (C) Western blot with a polyclonal anti-Bp-FHA antibody (lane 1: Tohama, lane 2: FR4624, lane 3: FR5133, lane 4: FR5187, lane 5: CIP1672, lane 6: FR5388, lane 7: FR5392, lane 8: Bpp12822, lane 9: BPP1, lane 10: FR3728, lane 11: FR5840) Full length AC-Hly is surrounded. Bp = B. pertussis; Bpp = B. parapertussis; AC-Hly = adenylate cyclase hemolysin; and FHA = filamentous hemagglutinin.

Figure 2

Post-translational modifications (PTMs) observed for Bp-Ac-Hly ((A) PTMs on K-860; (B) PTMs on K-983). From the modifications Specific Peptides output table of the MaxQuant results, intensities of the unmodified and modified peptides of interest (peptide forms) were extracted. For each lysine residue (K-860 and K-983), intensities were summed by peptide form and normalized against the sum of all extracted intensities per involved lysine. These calculated percentages were plotted on the bar chart representation: Myristoylation (light grey), Palmitoyalation (intermediate grey), no PTMs (dark). The intensities of peptide ions used in the bar charts (Figure 2 and Figure 3) do not necessarily directly correlate with the actual amount of each corresponding peptide since ionization efficiencies can vary with the presence of post-translational modifications. However, myristoylation and palmitoylation are comparable modifications that occur on the same amino acid (Lysine), and, thus, we can postulate that the ionization efficiencies of the corresponding peptides should not be very different.

Figure 3

PTMs observed for Bpp-Ac-Hly ((A) PTMs on K-894; (B) PTMs on K-1017). From the modifications Specific Peptides output table of the MaxQuant results, intensities of the unmodified and modified peptides of interest (peptide forms) were extracted. For each lysine residue (K-894 and K-1017), intensities were summed by peptide form and normalized against the sum of all extracted intensities per involved lysine. These calculated percentages were plotted on the bar chart representation: Myristoylation (light grey), Palmitoyalation (intermediate grey), Palmitoleylation (hatched), no PTMs (dark). The intensities of peptide ions used in the bar charts (Figure 2 and Figure 3) do not necessarily directly correlate with the actual amount of each corresponding peptide since ionization efficiencies can vary with the presence of post-translational modifications. However, myristoylation, palmitoylation, and palmitoleylation are comparable modifications that occur on the same amino acid (Lysine), and, thus, we can postulate that the ionization efficiencies of the corresponding peptides should not be very different.

Figure 3

PTMs observed for Bpp-Ac-Hly ((A) PTMs on K-894; (B) PTMs on K-1017). From the modifications Specific Peptides output table of the MaxQuant results, intensities of the unmodified and modified peptides of interest (peptide forms) were extracted. For each lysine residue (K-894 and K-1017), intensities were summed by peptide form and normalized against the sum of all extracted intensities per involved lysine. These calculated percentages were plotted on the bar chart representation: Myristoylation (light grey), Palmitoyalation (intermediate grey), Palmitoleylation (hatched), no PTMs (dark). The intensities of peptide ions used in the bar charts (Figure 2 and Figure 3) do not necessarily directly correlate with the actual amount of each corresponding peptide since ionization efficiencies can vary with the presence of post-translational modifications. However, myristoylation, palmitoylation, and palmitoleylation are comparable modifications that occur on the same amino acid (Lysine), and, thus, we can postulate that the ionization efficiencies of the corresponding peptides should not be very different.

Figure 4

Percentage of J774.A1 cells lysed by Bp-AC-Hly. The results presented are the mean ± standard deviation of three different experiments.