Fig. 1.

HusA is important for utilisation of haem/porphyrin and intracellular survival of P. gingivalis. a Representative planktonic growth curves of P. gingivalis wild-type W83 strain (blue circles), husA deleted mutant ΔhusA (burgundy squares), and husA complemented mutant ΔhusA⁺ (orange triangles) under iron-limited conditions (100 µM iron chelator dipyridyl) supplemented with different haem/porphyrin sources, including haemoglobin (at 0.5 µM), haemin (at 2 µM), protoporphyrin-IX (PPIX; at 2 µM) and coproporphyrin III dihydrochloride (at 2 µM). Schematics for the different porphyrins used are shown with the following notations: Fe for iron, M for methyl group, V for vinyl group, P for propionate group and A for acetate group. b HusA protein expression and sub-cellular distribution were evaluated by immunoblotting using polyclonal anti-HusA antibodies. The bacterial biomass was adjusted to OD₆₀₀ of 0.5. c Representative immunofluorescence images of P. gingivalis invading H413 gingival epithelial cells at 24 h post infection. Confocal laser scanning microscopic (CLSM) images represent epithelial cells infected by W83 or ΔhusA mutant, respectively. Higher magnifications of DIC images represent the differential interference contrast images of field of interest. P. gingivalis appeared as green and the nuclei of epithelial cells appeared as red. Scale bar, 10 µm. d Comparison of intracellular viability of P. gingivalis wild type and mutants quantified by colony forming units (CFU ×10⁵) per flask of epithelial cells. Three independent experiments with triplicate assays were conducted. Box plot shows the median value, 25th and 75th percentiles (box limits), maximum and minimum values (whiskers) and outliers (dots; **p < 0.001). e Comparison of husA gene expression of P. gingivalis in medium and within epithelial cells. Relative transcription of the husA gene was normalised against the housekeeping gene 16S rRNA. Results represent mean value ± standard deviation of three independent experiments