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. 2012 Feb;18(1):128-34.
doi: 10.1016/j.anaerobe.2011.10.005. Epub 2011 Nov 11.

Both the unique and repeat regions of the Porphyromonas gingivalis hemagglutin A are involved in adhesion and invasion of host cells

Myriam Bélanger  1 , Emil Kozarov, Hong Song, Joan Whitlock, Ann Progulske-Fox
Affiliations

Both the unique and repeat regions of the Porphyromonas gingivalis hemagglutin A are involved in adhesion and invasion of host cells

Myriam Bélanger et al. Anaerobe. 2012 Feb.

Abstract

Porphyromonas gingivalis is one of the major etiologic agents of adult periodontitis and has been associated with cardiovascular diseases. It expresses multiple hemagglutinins that are significant virulence factors and play an important role in bacterial attachment and invasion of host cells. The objective of this study was to determine the impact of P. gingivalis hemagglutinin A (HagA) on the attachment to and invasion of human coronary artery endothelial cells (HCAEC) and gingival epithelial cells (GEC). Bacterial strains expressing the HagA protein (or subunits), including Escherichia coli carrying plasmid pEKS5, E. coli carrying plasmid ST2, and Salmonella enterica serovar Typhimurium with plasmid pNM1.1 were used in this study. The strains were tested for their ability to attach to and invade HCAEC and GEC using antibiotic protection assays. In addition, the unique 5' N-terminal non-repeated segment of HagA was purified in recombinant form and a monoclonal antibody was created against the polypeptide. The monoclonal antibody against the unique portion of HagA was tested for inhibitory activity in these assays. The attachment of both E. coli strains expressing HagA fragment to host cells was significantly increased compared to their respective controls. However, they did not invade GEC or HCAEC. Interestingly, HagA expression in the Salmonella strain increased both adherence to and invasion of HCAEC, which may be due to the presence of the entire hagA ORF. A monoclonal antibody against the unique 5' N-terminal portion of HagA reduced invasion. Further experiments are needed to determine the role of the unique and the repeat segments of P. gingivalis HagA.

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Figures

Fig. 1
Fig. 1
Diagram depicting the various portions of the hagA gene that have been used in this study. In P. gingivalis 381, the 7,887 bp hagA gene include 4 direct repeats (HArep) represented as shaded areas. The whole hagA gene has been cloned into S. enterica pNM1.1 whereas only portions of the hagA gene, as illustrated above, have been cloned into strains E. coli-ST2 (nt 705-3872; see [37]), E. coli-pEKS5 (nt 705-3872), and E. coli HagAU (nt 1-1233; see [37]). The nt numbers are provided for each clone in relationship to the HagA molecule depicted at the top of the Figure.
Fig. 2
Fig. 2
Attachment of different E. coli strains to (a) GEC and (b) HCAEC. E. coli-pUC8 and E.coli-ST2 (Group 1) and E. coli-pUC19b and E. coli-pEKS5 (Group 2) were compared. Results are reported as the average (± standard deviation) of the attachment to host cells. Using GEC or HCAEC, a statistically significant difference (P<0.05) between the E. coli control and E. coli expressing HagA strains from each group was observed.
Fig. 3
Fig. 3
Salmonella enterica strains attachment (a) and invasion (b) to HCAEC. S. enterica control (solid bar) and pNM1.1 were compared. Results are reported as the average (± standard deviation) of the attachment or invasion to host cells. A statistically significant difference (P<0.05) between the both strains was observed during both the attachment and invasion steps.
Fig. 4
Fig. 4
Transmission electron micrographs of wild-type (a) and HagA mutant (b) of P. gingivalis after a 1 h incubation with mAb HL1899 and followed by labeling with gold particles conjugated to a mouse IgG antibody.
Fig. 5
Fig. 5
Inhibition of the (a) attachment or (b) invasion of P. gingivalis 381 to HCAEC by pre-incubating cells with various concentrations of BSA (solid bars) or mAb HL1899 (dotted bars). Results are reported as the average (± standard deviation) of the attachment or invasion to host cells. The adhesion and invasion values for each P. gingivalis group without inhibitors were individually normalized to 100% and used to as control to derive the other adhesion and invasion values. The asterisk indicates a statistically significant difference (P<0.01) between that group and the group without inhibitors.
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