Serendipitous Discovery of Immunoglobulin-Binding Autotransporter in Bordetella

[Sikotic™]

Serendipitous Discovery of an Immunoglobulin-Binding Autotransporter in Bordetella Species


The introduction to the paper gives a description of the three strains of Bordetella that are being tested: Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. They are thought to be organisms of the same species because they cause mammalian respiratory ailments and have gene similarities; however, they differ dramatically in their effects to host organisms. B. pertussis and B. parapertussis differ from B. bronchiseptica because they cause whooping cough and infect humans while B. bronchiseptica is found in all types of mammalian species without causing disease for a longer period of time. Autotranporter genes are believed to be encoded in all three strains of Bordetella. One of the genes looked at is associated with the initiation of filamentous hemagglutinin, which is important for cell attachment. By making antibodies against filamentous hemagglutinin, an unknown autotransporter protein was accidentally discovered that is regulated positively by the Bvg system in B. bronchiseptica.

After creating antibodies against filamentous hemagglutinin for the Bordetella strains, a Western blot was performed and an unknown Bvg- positive protein associated with B. bronchiseptica was located in the results. The unknown protein was believed to be located in the outer membrane which led the conductors of the experiment to believe it was an autotransporter. After performing various experiments including the creation of suicide plasmids and in-frame deletions, batB was the gene responsible for making the 180kDa protein. Though almost identical, there are slight differences in the transcription start sites between the three stains of Bordetella. Characterizations of the protein in question were discovered through a database of other protein sequences. Discoveries included that it shared sequences similar to other autotransporters and Immunoglobulin proteases. Anti-590 and anti-802 antibodies were made and tested against B. bronchiseptica genes, which was suspected of having BatB, and RBAT, a strain without the batB gene. Only B. bronchiseptica was detected by the antibodies to confirm that BatB is the protein in question. Results for B. pertussis and B. parapertussis were not as unanimous.

To determine batB’s transcription regulation in association with the Bvg system, PCR was performed on strains with Bvg-positive, Bvg-positive phase locked, a combination of Bvg-positive and negative and Bvg-negative strands. Results showed transcription in the Bvg-positive and Bvg-positive locked strains, a small amount of transcription in the combination of Bvg- positive and negative strain and no transcription in the Bvg-negative strain. This evidence suggests that batB is positively-regulated in the BvgAS phosphorelay.

The extent of batB transcription was tested amongst all three strains next through PCR. Results determined that both B. bronchiseptica and B. parapertussis had active transcription in Bvg-positive, but not Bvg- negative scenarios. On the other hand, B. pertussis did not have activity under both types of Bvg conditions. To test if there were any differences in the genes of B. pertussis and the other two Bordetella strains, a plasmid with an altered lacZ promoter region was added to all three strains. Results showed that both B. bronchiseptica and B. parapertussis both showed β-galactosidase activity and B. pertussis did not, indicating that B pertussis has differences within its promoter region.

To determine whether the BatB autotransporter is critical in the accumulation of colonies in the respiratory tract of mammals, rats were treated with both B. bronchiseptica and the RBAT strain without the batb gene. Both types were able to colonize the respiratory tracts after a period of two to three weeks indicating that BatB is not important in respiratory tract colonization.

To test if BatB protein is effective in the resistance of removal by inflammation, mice were treated with B. bronchiseptica and the batB- functionless RBAT. Both strains were counted for their colony forming units periodically. Results showed distinctly lower numbers of colonies in the lungs and trachea for the RBAT compared to B. bronchiseptica. To see if the RBAT acted differently due to the inactivity of batB, a plasmid containing batB was added to the RBAT strain. Result show that the altered RBAT and B. bronchiseptica had similar results which gives evidence that BatB is important in reducing inflammatory response. Another experiment was done to see if missing peptides in B. parapertussis had anything to do with the operation of BatB. The batB gene in B. bronchiseptica was removed and the number colonies were accounted for periodically. The results show that the modified B. bronchiseptica had lower amounts of colonies in the lungs and trachea than the wild-type B. bronchiseptica, but slightly higher than RBAT. This leads us to believe that B. parapertussis has slight BatB function.

In order to see if BatB has some sort of similarities to immunoglobulin proteases, a Western blot was done with B. bronchiseptica and RBAT with immunoglobulins from various mammals. Each and every mammal immunoglobulin tested showed BatB presence on the Western blot for B. bronchiseptica. To see whether BatB bound to immunoglobulins or if antigen- specific immunoglobulins bound to BatB, a Western blot was done after B. bronchiseptica and RBAT were treated with Fc and Fab fragments from mouse immunoglobulin. B. bronchiseptica showed up signifying that BatB binds to immunoglobulins.

Tests were also performed by injecting young mice without immunoglobulin-producing capability with B. bronchiseptica, RBAT and B. bronchiseptica minus the batB nucleotides in equal amounts. The colony forming units were recorded periodically. Although B. bronchiseptica numbers were slightly higher to start off with, towards the end of the experiment the numbers between all three strands were very similar. This evidence suggests that BatB’s effectiveness is due in part to immunoglobulin, B cells and T cells normally present within older mice.

In order to see once and for all if there are differences between the types of BatB formed in all three Bordetella strands, all three strands were treated with anti-BatB antibody. Regular-sized BatB was present in B. bronchiseptica. Smaller BatB was present in B. parapertussis. No BatB was found in B. pertussis. Evidence shows that only the full-sized BatB are found in B. bronchiseptica.

Through the battery of tests and experiments demonstrated, it can be seen that BatB is an important indicator in the resilience and host specificity within a mammal by Bordetella. This explains why B. bronchiseptica infection is long-term compared to B. pertussis and B. parapertussis. This accidental discovery sheds light on this autotransporter’s ability to bind to immunoglobulins and prevent inflammatory responses. It has also been discovered that B. bronchiseptica’s colonization of the lower respiratory tract is dependent on the presence of immunoglobulins and other factors in a mammal’s immune system.

[Al Bundy]

english?

[Sikotic™]

That was my brief summary of the topic lol