Fluorescence- and 2D gel-based bismuth-proteome of Porphyromonas gingivalis

Abstract

OBJECTIVES: Our recent work shows that the growth of the ‘keystone’ periodontal pathogen Porphyromonas gingivalis could be inhibited by bismuth-based anti-Helicobacter pylori drugs. This study aimed to identify bismuth-associating proteins within P. gingivalis using an intracellular fluorescent probe (Bi3+-TRACER), and explore the underlying molecular mechanisms of bismuth-based inhibition of the bacterium. METHODS: Bi3+-TRACER was prepared by mixing bismuth citrate with TRACER at 1:1 molar ratio. P. gingivalis (ATCC 33277) was cultured in liquid medium at 37°C anaerobically for three days. Bacteria were washed three times and concentrated by PBS, and then added 50 μM of final concentration of Bi3+-TRACER with 30-min incubation. The bacterial suspension was diluted for confocal fluorescence imaging. Bacteria were further irradiated by UV light at 365 nm for 10 min before lysis by sonication, and the labeled bacterial lysates were then subjected to 2D-PAGE gel separation of fluorescence-labeling proteins. The fluorescent spots were imaged, and the gels were further analyzed by silver staining. The corresponding lit-up spots were excised and subjected to peptide mass fingerprinting for protein identification. RESULTS: The Bi3+-TRACER could light up P. gingivalis cells in vivo emitting blue fluorescence with little cytotoxicity. On 2D-PAGE gels, proteins in P. gingivalis cellular lysates were separated based on pI and molecular weights. Multiple protein spots in silver-stained gels were noticed. By comparison, lit-up spots were excised and identified. The identified potential bismuth-associating proteins included Arg- and Lys-gingipains, hemagglutinin, NAD-specific glutamate dehydrongease, Mn/Fe-SOD, thioredoxin and electron transfer flavoprotein subunit α with high fidelity. CONCLUSIONS: The present study suggests that the multiple bismuth-associating proteins identified may be involved in the underlying mechanisms of bismuth-based inhibition of P. gingivalis. Further study is warranted to solidify the details of bismuth action on P. gingivalis for potential clinical applications.