Actinobacillus actinomycetemcomitans apaH is implicated in invasion of epithelial cells. MARIA SAARELA, JOAN LIPPMANN, DIANE HUTCHINS MEYER and PAULA M. FIVES-TAYLOR.

Actinobacillus actinomycetemcomitans(Aa) is associated as a causative agent of periodontitis. The virulence of Aa may be due, in part, to its ability to adhere to and invade epithelial cells. A plasmid library containing Aa Y4 genomic DNA was screened with Y4 polyclonal antisera to identify genes which may be associated with cell surface components. Plasmid DNA from the positive clones was used to transform non-invasive E.coli. A strain containing a 2.1 kb Aa DNA fragment was identified as conferring on E.coli the ability to invade KB oral epithelial cells (VT1102) using gentamicin protection assays and immunofluorescence microscopy. In this study, the 2.1 kb fragment was sequenced and analyzed revealing two open reading frames. These ORF's were 70% homologous to the ksgA and apaH genes within a complex operon in E.coli. apaH encodes the diadenosine 5',5'''-P1, P4-tetraphosphate-hydrolase which cleaves the 'alarmone', diadenosine tetraphosphate (Ap4A). Ap4A also controls the timing of cell division in E.coli where low levels of Ap4A, caused by an increase in ApaH, results in delayed cell division (Nishimura, 1997). Microscopic analysis of VT1102 revealed a filamentous phenotype suggesting delayed cell division. The Aa apaH gene encoded a putative protein containing an RGD sequence, while E.coli apaH encodes an RAD sequence. RGD sequences have been implicated in invasion of other species. In our assay, RGD peptide inhibited invasion of E.coli VT1102 by 80%. These data demonstrate that the 2.1 kb Aa DNA fragment contains the apaH gene and suggest this construct may be implicated in invasion and delayed cell division of VT1102. A mutation of this gene in Aa would confirm this work. This research was supported by NIH-NADR grant RO1DE09760.