Fig. 5
A prototype DPIX-conjugated antibiotic binds HusA and inhibits P. gingivalis. a The chemical structure of the deuteroporphyrin-lysine-metronidazole adduct, P8. b NMR signal intensity changes (black) and 1HN CSPs (red/green) for HusA upon addition of P8 (measured from 15N-HSQC spectra shown in Supplementary Figure 21). c In silico docking of P8 to the energy minimized structure of HusA (conformer set 1), with residues experiencing NMR signal loss (orange) or CSPs (red/green) mapped to the structure (colouring as described in Fig. 2d). d Scaled and corrected fluorescence at 335 nm during titration of 1 μM of HusA with increasing concentrations of P8, haemin or DPIX. e Comparison of P8 inhibitory effect towards P. gingivalis wild-type W83 and husA mutants using checkerboard analysis. Serial dilutions of antibiotics (horizontal axis) were applied to a 96-well plate with different P. gingivalis strains (vertical axis). Serial dilutions of DPIX were also supplemented into wild-type W83 group to evaluate the competitive effect against P8. Absorption at OD600 of each well was recorded after 24 h of incubation. The OD600 of each strain grown in the absence of antibiotics was defined as 100% growth, which was applied to normalise the reading collected from other wells. The normalised percentage of each well was plotted in the checkerboard. Metronidazole alone and in combination with PPIX or DPIX was applied as control. f Intracellular killing assay of P8 antibiotics. Gingival H413 epithelial cells were infected by P. gingivalis W83 and then treated with P8 at various concentrations. Recovered bacteria were determined by CFU count. Metronidazole and DMSO were used as control. 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). g Iron restriction induced HusA expression and enhanced P8 growth inhibition of P. gingivalis in the presence of 100-µM dipyridyl. Wild-type W83 was susceptible to P8 at 1 µM, a concentration below the MIC; error bars, standard deviations
