agglutinates guinea pig erythrocytes, and loss of this property via mutation results in attenuation (2
). However, the mechanism by which hemagglutination is carried out remains ill defined. Here we show that the characteristics of hemagglutination, explanted tracheal ring binding, and turkey poult colonization are all linked in their requirement for the products of the hagA
genes. Further, we present evidence for the direct involvement of HagB in erythrocyte agglutination and tracheal ring binding.
We discovered the hagA
genes in the course of characterizing 20 hemagglutination-negative miniTn5
insertion mutants and report here the locations of 10 insertions that were demonstrably unique. All isolated mutants had lesions in either hagA
. The hagA
genes were transcriptionally divergent and had no orthologs in any other published Bordetella
To better characterize the HagA and HagB products, we constructed in-frame, unmarked deletions in hagA
. As expected, the deletion mutants were hemagglutination negative. Additionally, complementation experiments revealed that each gene produced a unique trans
-acting product required for hemagglutination, explanted tracheal ring binding, and turkey poult tracheal colonization. Part of the success of the complementations, especially those performed in vivo
(in the absence of antibiotic-contributed selective pressure for plasmid maintenance), was likely due to the stability of the pLAFR5 vector, a property noted previously (32
In order to obtain evidence for the direct involvement of one or both of the HagA and HagB products in attachment, we generated polyclonal HagA and HagB antisera. The antisera raised were each demonstrably specific for proteins of the predicted size of HagA or HagB present in our outer envelope-enriched fraction in Western blots. However, antiserum raised to HagA did not block hemagglutination or impede explanted tracheal ring attachment by strain 197N any differently than preimmune serum, even though we could additionally confirm (using immunoblots) the reactivity of the HagA antiserum with the isolated immunizing protein (our unpublished observations). In contrast to the HagA antiserum, the HagB antiserum inhibited both hemagglutination and tracheal ring binding.
We inferred from the foregoing that HagB directly binds a component or components common to the surface of guinea pig erythrocytes and explanted turkey tracheal cells. We found no support for a direct role for HagA in receptor binding. However, our results do not rule out the possibility that HagA is also directly involved, because the ability of each protein to elicit antibodies that specifically block attachment could vary significantly. This could be due to differences in sensitivity to their isolation procedures or other factors unique to each protein.
Bioinformatic analysis places HagA and HagB in a family of two-component secretory pairs (12
). With regard to HagA, recent analysis of B. pertussis
FhaC (a protein required for the export of filamentous hemagglutinin, FhaB), has reinforced predicted structural similarities between HagA and FhaC (4
). Also, portions of HagA show characteristics of proteins that facilitate exposition of hemagglutinins and hemolysins from other microorganisms (4
). With regard to HagB, bioinformatic data indicate that strong similarities exist between portions of FhaB and other hemagglutinins from bacteria outside the bordetellae (4
Our complementation experiments told us that HagB was synthesized in the hagA mutant and vice versa. However, we did not detect HagB in our hagA mutant outer envelope-enriched fraction. One way to explain this observation is that HagA was necessary for the retention or stabilization of the HagB product. If that is the case, there was no reciprocity in this relationship because HagB was not required for the stability or retention of HagA. Consistent with the bioinformatic inferences, HagA may act to facilitate the proper localization and presentation of HagB via direct interactions characteristic of two-component regulators. However, additional means will need to be employed to unequivocally place HagA and HagB in the same or different membranes and subsequently address the possibility that HagA directly interacts with HagB (e.g., coimmunoprecipitation). Also, tracheal and erythrocyte competitive binding studies with purified HagA and HagB could help reinforce the present conclusions regarding the different roles of the proteins determined here with antisera.
We note that the results presented here (and additional unpublished observations) did not lend support to the earlier findings of Moore and Jackwood (22
), who proposed that hemagglutination in B. avium
is associated with a 41-kDa protein, as well as a periodate-sensitive component. However, our results do not rule out the possibility that there are additional factors required for hemagglutination that were not detected by the mutagenic methods we employed to discover HagA and HagB.
In B. avium
, hemagglutination is essential for turkey poult colonization (33
). In no other member of the bordetellae is the loss of hemagglutinating ability so profoundly attenuating. The HagB hemagglutinin may prove to be a beneficial adjunct in vaccine formulations aimed at preventing tracheal colonization. Targeting this molecule could reduce the level of subclinical carriage and reduce disease incidence in commercial turkey operations. In addition, the surface location of HagB may make this product a good candidate to expose foreign antigens as gene fusions. This measure could facilitate the use of B. avium
as a live oral poultry vaccine platform to protect poultry against other infectious agents.