Abstract
Bordetella pertussis employs numerous strategies to evade the immune system, including the ability to resist killing via complement. Previously we have shown that B. pertussis binds a complement regulatory protein, C1 esterase inhibitor (C1inh) to its surface in a Bvg-regulated manner (i.e. during its virulence phase), but the B. pertussis factor was not identified. Here we set out to identify the B. pertussis C1inh-binding factor. Using a serum overlay assay, we found that this factor migrates at approximately 100 kDa on an SDS-PAGE gel. To identify this factor, we isolated proteins of approximately 100 kDa from wild type strain BP338 and from BP347, an isogenic Bvg mutant that does not bind C1inh. Using mass spectrometry and bioinformatics, we identified the autotransporter protein Vag8 as the putative C1inh binding protein. To prove that Vag8 binds C1inh, vag8 was disrupted in two different B. pertussis strains, namely BP338 and 18–323, and the mutants were tested for their ability to bind C1inh in a surface-binding assay. Neither mutant strain was capable of binding C1inh, whereas a complemented strain successfully bound C1inh. In addition, the passenger domain of Vag8 was expressed and purified as a histidine-tagged fusion protein and tested for C1inh-binding in an ELISA assay. Whereas the purified Vag8 passenger bound C1inh, the passenger domain of BrkA (a related autotransporter protein) failed to do so. Finally, serum assays were conducted to compare wild type and vag8 mutants. We determined that vag8 mutants from both strains were more susceptible to killing compared to their isogenic wild type counterparts. In conclusion, we have discovered a novel role for the previously uncharacterized protein Vag8 in the immune evasion of B. pertussis. Vag8 binds C1inh to the surface of the bacterium and confers serum resistance.
Highlights
Complement is a critical and multifaceted host defense system comprised of a series of proteins—many of which are zymogens— found largely in plasma [1] and on respiratory mucosal surfaces [2,3]
We have previously shown that B. pertussis is capable of binding human C1 esterase inhibitor (C1inh) to its surface and that this is dependent on signal transduction by the BvgAS two-component system, the master virulence regulatory system of Bordetella species
In the present study we showed that disruption of vag8 abolishes binding of C1inh and that complementing a virulence-activated gene 8 (Vag8) mutant with a plasmid encoding a full-length vag8 open reading frame (ORF) under control of the non-native promoter Pcpn10 partially restores the C1inh-binding phenotype
Summary
Complement is a critical and multifaceted host defense system comprised of a series of proteins—many of which are zymogens— found largely in plasma [1] and on respiratory mucosal surfaces [2,3]. To protect the host from the effector functions of complement and prevent rapid consumption of complement components in response to trivial stimuli, all three activation pathways of complement are tightly regulated at different stages by a number of complement regulatory proteins [4]. Bordetella pertussis, the causative agent of whooping cough, has been shown to express a variety of virulence-associated factors that in concert enable the bacteria to colonize the mucosa of the upper respiratory tract in humans. Expression of most virulence-associated factors in B. pertussis is positively regulated by the BvgAS two component system [5,6]. Apart from adherence of the bacteria to the ciliated respiratory epithelium, many of these factors help the bacteria to evade or modulate host immune defenses [7,8].
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