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The study of cell surface components on Porphyromonas gingivalis

University of British Columbia

Porphyromonas gingivalis plays a major role in mixed bacterial infections of the periodontium. The objective of the study was to clone genes encoding, and characterize cell surface components of P. gingivalis as potential virulence factors of this organism. AP. gingivalis ATCC 33277 genomic library was generated. Clones were screened by biological assays for haemagglutination and proteolytic activity, and by a colony immunoassay using antiserum raised to P. gingivalis whole cells. The recombinant clones Escherichia coli BA1 and E. coli BA3 were identified and chosen for further study. E. coli BA1 exhibited enhanced haemagglutinating activity compared to an E. coliJM83/pUC18 control strain. The increase in haemagglutinating activity was believed to be due to the presence of a recombinant P. gingivalis haemagglutinin molecule. Recombinant protein was not detected in E. coli BA1 samples, however transfer of the cloned DNA fragment into a high-level expression system resulted in the identification of two recombinant proteins of 24 and33 k Da. Immunological studies failed to show evidence of a relationship between the recombinant proteins and P. gingivalis. The cloned proteins did not exhibit haemagglutinating activity. The recombinant proteins and antibodies raised to the recombinant proteins did not inhibit P. gingivalis haemagglutinating activity. The NH2-terminal amino acid sequences for the24 and 33 k Da cloned proteins were determined. A protein database search did not reveal any marked similarity between the recombinant proteins and other listed protein sequences. As attempts to characterize the cloned proteins did not indicate a clear association with P. gingivalis, clone E. coli BA1 was not studied further. The basis of the enhanced haemagglutinating activity shown by BA1 remains undetermined. Clone E. coli BA3 highly expressed a 48 kDa cloned protein (designated rPgAg1) which was strongly reactive with antiserum raised to P. gingivalis whole cells. The cell surface location of the native PgAg1 molecule was demonstrated by immunogold labelling of intact P. gingivalis cells using antibodies raised to the recombinant protein (arPgAg1). The recombinant protein was purified to homogeneity, and the native PgAg1 protein partially purified from P. gingivalis. The NH2-terminal amino acid sequence was determined for the recombinant and native proteins. This was identical to the NH2-terminal amino acid sequence reported for a 47 kDa immunodominant antigen of P. gingivalis. Immunological studies using periodontal case-control sera indicated that the 47 kDa molecule was a surface-associated antigen which invokes a strong immunological response in the host. The nucleotide sequence of the pgagl gene was determined. A database search using the deduced amino acid sequence revealed the PgAg1 protein to be highly homologous to glutamate dehydrogenases isolated from various sources. Analysis of purified recombinant protein and partially purified native protein demonstrated that PgAg1 did function as a NAD-dependent glutamate dehydrogenase enzyme. A PgAg1-deficient isogenic mutant of P. gingivalis was generated by homologousre combination between a suicide vector carrying a deletion-disrupted allele of pgagl andP. gingivalis chromosomal DNA. The mutant strain (P. gingivalis E51) lacked NAD-dependent glutamate dehydrogenase activity, did not surface-label in an immunogold labelling assay using arPgAg 1 antibodies, and exhibited a longer generation time than the wild-type strain. This study has identified and characterized a cell surface component (designated PgAg1) of P. gingivalis which has the capacity to function as an NAD-dependent glutamated ehydrogenase enzyme. A mutant P. gingivalis strain deficient in PgAg1 grew poorly in comparison to the wild-type strain. This indicates that PgAg1 does play a significant role in the growth of this organism.

Thesis/Dissertation

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2008-09-17

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http://hdl.handle.net/2429/2189

Microbiology and Immunology

University of British Columbia

UBCV

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