Acinetobacter baumannii is an opportunistic pathogen and can cause severe disease in immune-suppressed and/or injured patients. It is an extreme-drug resistant bacterium with the ability to form biofilms thereby significantly increasing resistance to treatment. Because of the extreme drug resistance and relatively unknown immunological profile of A. baumannii new treatment options are needed. A. baumannii has been reported to express a Biofilm Associated Protein (BAP); a high molecular weight protein composed of multiple repeat modules and thought to be surface exposed on planktonic bacterium and upregulated in biofilm. While it is unknown if BAP has any role in in vivo infection of humans, the repeats of BAP proteins are thought to function in intercellular adhesion to support the mature biofilm and thus represent potential targets for immunotherapeutic intervention. Herein my thesis is aimed at trying to verify that BAP is surface exposed, upregulated in biofilm and to prove a role for BAP in pathogenesis, as well as investigating A. baumannii interactions with components of the innate immune system in vitro. Consensus synthetic peptides corresponding to the major internal repeats of BAP were designed and conjugated to carrier proteins and recombinant proteins were manufactured to correspond to the non-repetitive N and C terminals of the protein for murine immunization and assay development. Serum from immunized mice was collected and analyzed in ELISA and western immunoblot to determine reactivity with planktonic and biofilm whole organism. Anti-serum to whole bacteria was also tested in opsonisation assays to determine direct killing ability of serum on bacteria in vitro. Anti-serum to whole bacteria showed direct killing of the organism in vitro when in high concentrations (diluted 1/10), relative to pre-immune serum, but was less effective in lower concentrations (diluted 1/50). Despite generating antibody reagents to multiple domains and epitopes spanning the published BAP sequence, we were unable to confirm that BAP is expressed by A. baumannii as reported by others. However, if BAP is indeed expressed in A. baumannii our DNA and immunochemical data collectively suggest that BAP is potentially mosaic in this pathogen.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:MWU.1993/4998 |
| Date | 15 December 2011 |
| Creators | Murray, Brenda-Lee L. |
| Contributors | Berry, Jody (Medical Microbiology), Alfa, Michelle (Medical Microbiology) de Kievit, Teresa (Microbiology) Zhanel, George (Medical Microbiology) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Detected Language | English |
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