Graduation Year

2005

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology

Major Professor

My Lien Dao.

Co-Major Professor

Co-adviser:

Keywords

Mutans streptococcus, Prime-boost, Saliva, Secretory iga, Wapa

Abstract

Streptococcus mutans is the main causative agent of dental caries, which is a widespread infectious disease. A number of surface molecules are involved in the pathogenicity of this organism, including adherence and aggregation factors. The wall-associated protein A (WapA) of Streptococcus mutans GS-5 was previously demonstrated to be a sucrose-dependent adherence and aggregation factor, and is a larger precursor to extracellular antigen A (AgA), a candidate antigen for a dental caries vaccine.The full-length wapA gene and a C-terminal truncated version agA encoding the AgA were cloned into the mammalian expression vector pcDNA 3.1/V5/His-TOPO. The above constructs were mixed with a cationic lipid and used to transfect Chinese hamster ovary (CHO) cells. Transient expression of the wapA and agA genes was observed at 24 h post-transfection, as shown by Western immunoblot analysis.

In CHO, cells WapA containing the membrane and wall-spanning region was found in apoptotic bodies, whereas the soluble AgA, which lacked the hydrophobic region, was found in extracellular medium. A higher salivary IgA level was observed in mice immunized with the pcDNA-wapA vaccine as compared to those immunized with the pcDNA-agA vaccine. Furthermore, the anti-WapA antibody inhibited S. mutans sucrose-dependent adherence, suggesting potential protection of the tooth against S. mutans colonization, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs that are absent from AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not.

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