Principal Investigator: Maor Bar Peled
Grant Title: Boosting immune response against Bordetella bronchiseptica EPS facilitatesclearance.
Abstract
Many pathogenic bacteria produce exopolysaccharide (EPS) as a shield to avoid hostrecognitions. Early work have shown that Bordetella bronchiseptica (Bb) EPS is important for
efficient transmission between hosts. Here we show that two unique activated sugars, UDP-Nacetyl-glucosaminouronate (UDP-GlcNAcA) and UDP-N-acetyl-galactosaminouronate (UDPGalNAcA), are involved in EPS biosynthesis. When mice are inoculated with Bb wild type they
produced high level of IgM against EPS while Bb EPS mutants lack anti-EPS IgM, suggesting afaster clearance of Bb wild type than Bb EPS mutant. TLR4 mutant mice cleared both Bb wild type
and Bb EPS mutant similarly, suggesting that TLR4 is involved in EPS-targeting clearance.
Objectives:
1- To determine function of selective genes involved in EPS biosynthesis;
2- To initiate and characterize EPS chemical structure; and3- Use mice model to determine the biological role the EPS.
Methods:- Gene encoding specific EPS enzyme were cloned and expressed in E.coli. Recombinant
enzymes were purified and used for enzyme assay. Enzyme’s products analyses wereperformed by mass spectrometry and by proton NMR.
- EPS was isolated from bacteria culture grown under different conditions and defined media.
The EPS was isolated by chemical fractionation followed by ion exchange. The EPS was wasproven hard to hydrolyzed and therefore subjected to various hydrolysis condition andderivatization prior being detected by GC-MS and by HPLC-PAD detection.
- Mice were infected with known amounts of bacteria (wild type and mutant) and samples wereanalyzed t different day post infection.
Results:Two Bordetella genes bb24 and bb25 were shown to encode enzymes that sequentially convert
UDP-N-acetyl-glucosamine (UDP-GlcNAc) to UDP-GlcNAcA and then 4-epimerization to UDPGalNacA. The products of both enzymes confirmed by LC-MS, GC-MS and by NMR. These rarehighly charged amino-uronic acids suggest an EPS straucture that will resist chemical hydrolysis.
Different hydrolyses condition were tested. To further evaluate EPS role, Bordetella mutats
lacking the entire EPS operon or lacking gene expressing a protein predicted to be involved in EPS
transloaction across membranes were generated. These bacterial wild type and mutants wereused to intranasally inoculate mice and mice were subsequently exaimined.Polysaccharides areusually not very immunogenic. In order to trigger anti-EPS immune response, mice were
inoculated with large amounts of Bb wild type or ΔtEPS mutant (negative control). Periodically
post inoculation, bacteria colonization level was determined and mice serum were collected totest for antibody response. Compared to ΔtEPS, RB50 was found to trigger higher total IgM levelat day 7 and day 14 post inoculation. Serum from RB50 infected mice were found to be crossreactive to Salmonella typhi Vi antigen, which is also composed of GalNAcA, while serum fromΔtEPS infected mice were negative to Vi, proposing that anti-EPS IgM is present in serum of RB50infected mice. Further studies will revealed the fine chemical structure of Bb EPS and evaluatedthe contribution of anti-EPS immune response during clearance of Bb from respiratory tract.