SUMMARYTreponema denticola, a periodontal pathogen, binds the complement regulatory protein, Factor H (FH). FhbB (FH binding protein B) is the sole FH binding protein produced by T.denticola. The interaction of FhbB with FH is unique in that FH is bound to the cell and then cleaved by the T.denticola protease, dentilisin. A ~50kDa product generated by dentilisin cleavage is retained at the cell surface. Until this study, a direct role for the FhbB-FH interaction in complement evasion and serum sensitivity has not been demonstrated. Here we assess the serum resistance of T.denticola strain 35405 (Td35405wt) and isogeneic mutants deficient in dentilisin (Td35405-CCE) and FhbB production (Td35405)fhbB), respectively. Both dentilisin and FhbB have been postulated to be key virulence factors that mediate complement evasion. Consistent with conditions in the sub-gingival crevice, an environment with a significant concentration of complement, Td35405wt was resistant to serum concentrations as high as 25%. Deletion of fhbB (Td35405)fhbB), which resulted in the complete loss of FH binding ability, but not inactivation of dentilisin activity (Td35405-CCE), rendered T.denticola highly sensitive to 25% human serum with 80% of the cells being disrupted after 4 hours of incubation. Heat treatment of the serum to inactivate complement confirmed that killing was mediated by complement. These results indicate that the FH-FhbB interaction is required for serum resistance while dentilisin is not. This report provides new insight into the novel complement evasion mechanisms of T. denticola.

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The nucleotide sequence of a 2777 bp DNA segment containing the Bacteroides fragilis glnA gene was determined. The B. fragilis glnA open reading frame of 2187 bp encoded a glutamine synthetase (GS) subunit of 729 amino acid residues with a calculated Mr of 82,827. The apparent Mr of the GS subunit determined by SDS-PAGE was approximately 75,000. A single mRNA transcription start point was identified upstream of the B. fragilis glnA open reading frame. The B. fragilis GS subunit is approximately 270 and 400 amino acids longer than the GSI and GSII subunits, respectively, of other prokaryotes and eukaryotes. The GSI and GSII holoenzymes are dodecamers and octamers respectively, whereas the GS of B. fragilis is a hexamer. Although GSI and GSII subunits show amino acid similarity in five conserved regions, this similarity is not strongly conserved in the B. fragilis GS. The GS of B. fragilis is not regulated by adenylylation and lacks the adenylylation site. It also lacks the Trp residue associated with the active site in GSI and GSII enzymes from other prokaryotes and eukaryotes.

A Butyrivibrio fibrisolvens glnA gene encoding glutamine synthetase (GS) was cloned on a recombinant plasmid pGS4 which enabled Escherichia coligfnA deletion mutants to utilize (NH4),S04 as a sole source of nitrogen. The nucleotide sequence of a 2423 bp DNA segment containing the GS-coding region of B.fibrisolvens was determined and the complete amino acid sequence (701 residues) was deduced. Comparisons of the derived B.fibrisolvens GS protein sequence with the amino acid sequences of GS from other bacteria indicate that it is the second reported example of a type III GS, originally identified in the obligate anaerobe Bacteroides frugilis. The presence of GS in B. jibrisolvens cells and the regulation of the cloned GS in E. coli cells was demonstrated by Western blot analysis.

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