Identification of the minimum region of Bordetella pertussis Vag8 required for interaction with C1 inhibitor

Abstract

An autotransporter of Bordetella pertussis , virulence‐associated gene 8 (Vag8), binds and inactivates the complement regulator, C1 inhibitor (C1‐Inh), and plays a role in evasion of the complement system. However, the molecular interaction between Vag8 and C1‐Inh remains unclear. Here, we localized the minimum region of Vag8 required for interaction with C1‐Inh by examining the differently truncated Vag8 derivatives for the ability to bind and inactivate C1‐Inh. The truncated Vag8 containing amino‐acid residues 102–548, but not 102–479 and 202–648, showed the full activity of intact Vag8, suggesting that the separate 102–202 and 548–648 amino‐acid regions of Vag8 mediate the interaction with C1‐Inh.

The Mechanism of Pertussis Cough Revealed by the Mouse-Coughing Model

“…5D ). In addition, the truncated fragment of Vag8 ranging from amino acid positions 102 to 479 (ΔC479), which is unable to bind and inactivate C1-Inh, did not increase the number of coughs in mice inoculated with LOS and PTx; in contrast, other types of truncated Vag8, which retain the ability to inhibit C1-Inh increased the number of coughs, similar to Vag8 ( 49 ) ( Fig. 5E to G ).…”

“…The expression vectors for histone acetyltransferase (HAT)-tagged recombinant Vag8 proteins, pCold II-HAT- vag8 Tohama and pCold II-HAT- vag8 18323 , and pCold II-HAT- vag8 102–596, 102–548, and 102–479 , which were constructed as previously described ( 49 ), were introduced into E. coli BL21(DE3). The Vag8 proteins were expressed by incubation at 15°C for 24 h in the presence of 1 mM isopropyl-β- d -thiogalactopyranoside (IPTG).…”

Immunization with autotransporter Vag8 prevents coughing induced by Bordetella pertussis infection in mice

“…The passenger domain of Vag8 (Vag8 p ) is cleaved and liberated into the extracellular milieu 8,9 . The liberated Vag8 inactivates C1 inhibitor, which negatively regulates bradykinin generation in the kallikrein–kinin system, to upregulate bradykinin levels, resulting in exacerbation of the cough response 6,10 . In the present study, we investigated the protective effects of immunization with the Vag8 p against B. pertussis –induced coughing in mice.…”

“…The recombinant proteins were expressed in Escherichia coli DH5α or BL21 (DE3) and purified by nickel affinity chromatography as described previously. 10 We intraperitoneally injected mice with recombinant proteins (0.15, 1.5, or 15 µg/mouse) in combination with 1/25th the human dose of DPT-IPV or alum (2 mg) and assessed the cough production and the bacterial colonization of the tracheas and lungs after the intranasal inoculation of B. pertussis 18323 on the same schedule as described earlier. We confirmed the production of anti-Vag8, anti-Phg, and anti-SphB2 IgG antibodies in Vag8 p -, Phg-, and SphB2-immunized mice, respectively, by ELISA 3 (Figure 2a) and observed that immunization with Vag8 p did not affect the levels of anti-PTx IgG antibody in DPT-IPV-immunized mice (Figure 2b).…”

“…PTx enhances the bradykinin-induced sensitization of TRPV1, which evokes the cough reflex, while Vag8 upregulates bradykinin levels. 6,10,15 Therefore, it is conceivable that immunization with PTx and Vag8, which respectively target distinct steps in the cough reflex pathway, synergizes protection against cough Whether Vag8 immunization contributes to protection against B. pertussis infection is still controversial. Several groups have proposed Vag8 as a possible candidate for protective antigens against B. pertussis infection based on the observation that immunization with Vag8 p significantly reduces the bacterial colonization in respiratory organs of BALB/c mice, [3][4][5] an observation inconsistent with our current results in C57BL/6 mice.…”