Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

Abstract

Despite a growing interest in using probiotic microorganisms to prevent disease, the mechanisms by which probiotics exert their action require further investigation. Porphyromonas gingivalis is an important pathogen implicated in the development of periodontitis. We isolated several strains of Lactobacillus delbrueckii from dairy products and examined their ability to inhibit P. gingivalis growth in vitro. We observed strain-specific inhibition of P. gingivalis growth in vitro. Whole-genome sequencing of inhibitory and noninhibitory strains of L. delbrueckii revealed significant genetic differences supporting the strain specificity of the interaction. Extracts of the L. delbrueckii STYM1 inhibitory strain contain inhibitory activity that is abolished by treatment with heat, proteinase K, catalase, and sodium sulfite. We purified the inhibitory protein(s) from L. delbrueckii STYM1 extracts using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration chromatography. Pyruvate oxidase was highly enriched in the purified samples. Lastly, we showed that purified, catalytically active, recombinant pyruvate oxidase is sufficient to inhibit P. gingivalis growth in vitro without the addition of cofactors. Further, using a saturated transposon library, we isolated transposon mutants of P. gingivalis in the feoB2 (PG_1294) gene that are resistant to killing by inhibitory L. delbrueckii, consistent with a mechanism of hydrogen peroxide production by pyruvate oxidase. Our results support the current understanding of the importance of strain selection, not simply species selection, in microbial interactions. Specific L. delbrueckii strains or their products may be effective in the treatment and prevention of P. gingivalis-associated periodontal disease. IMPORTANCE P. gingivalis is implicated in the onset and progression of periodontal disease and associated with some systemic diseases. Probiotic bacteria represent an attractive preventative therapy for periodontal disease. However, the efficacy of probiotic bacteria can be variable between studies. Our data support the known importance of selecting particular strains of bacteria for probiotic use, not simply a single species. Specifically, in the context of probiotic intervention of periodontitis, our data suggest that high-level expression of pyruvate oxidase with hydrogen peroxide production in L. delbrueckii could be an important characteristic for the design of a probiotic supplement or a microbial therapeutic.

Hydrogen peroxide-producing pyruvate oxidase from Lactobacillus delbrueckii is catalytically activated by phosphotidylethanolamine

“…Enhanced hydrogen peroxide production by L. delbrueckii could be beneficial in a complex microbial community. In fact, hydrogen peroxide production by Pox has been shown to be important in the inhibition of dental pathogens like P. gingivalis [16]. The pentameric structure of L. delbrueckii Pox is different from what has been observed with E. coli Pox and L. plantarum Pox [1,4].…”

“…First, we wanted to determine the biochemical properties of the L. delbrueckii Pox enzyme. We previously purified L. delbrueckii Pox and used it for subsequent testing [16]. We determined the k m values for pyruvate and phosphate and the k cat value.…”

“…Streptococcus gordonii and Streptococcus sanguinis are known to produce enough hydrogen peroxide primarily through Pox to inhibit the oral pathogen Streptococcus mutans [15]. Recently, Lactobacillus delbrueckii strains harboring intact pox genes were shown to produce enough hydrogen peroxide to inhibit the growth of the oral pathogen Porphyromonas gingivalis [16]. The characterization of hydrogen peroxide-producing strains and their respective encoded hydrogen peroxide-producing enzymes could be beneficial in the development of novel treatments for diseases like periodontitis.…”

Antibacterial activity of plant species used for oral health against Porphyromonas gingivalis

“…Freeze dried P. gingivalis strain ATCC1 33277 was rehydrated in Brain Heart Infusion (BHI) media supplemented with 5 μg/mL hemin (Alfa Caesar1, Heysham, UK) and 1 μg/mL menadione (Alfa Caesar1, Heysham, UK). Bacteria were grown on supplemented BHI (sBHI) agar plates (5 μg/mL hemin, 1 μg/mL menadione) and supplemented blood agar plates (5% defibrinated sheep's blood (Hemostat Laboratories, Dixon, CA), 5 μg/mL hemin, 1 μg/mL menadione, 2 g/L yeast extract) [30][31][32]. Hemin stock was prepared by dissolving 250 mg hemin in 5 mL of 1M NaOH and 495 mL deionized distilled water (ddH 2 O) for a final concentration of 0.5 mg/mL.…”

“…The minimum inhibitory concentration (MIC) was defined as the lowest concentration at which 90% of growth was inhibited (corresponding to the lowest concentration of no visible growth in the well) compared with vehicle control, as previously reported [33,34], and the IC 50 was defined as the lowest concentration at which 50% of growth was inhibited. As there is no standard method described in the CLSI (Clinical and Laboratory Standards Institute) guidelines for growing and evaluating the MIC of P. gingivalis, we followed previously described methods with some modifications [30][31][32]. Briefly, the bacterial culture previously incubated for 48 hours was standardized to a final concentration of 10 6 CFU/mL in sBHI using a Cytation 3 multimode plate reader (Biotek1, Winooski, VT) by change in optical density (OD 600 nm), and confirmed by colony plate counts.…”