Microbial pathogenesis · 2023
Fumarate and nitrite reduction by Prevotella nigrescens and Prevotella buccae isolated from Chronic Periodontitis patients.
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Sharma Geetika, Garg Nancy, Hasan Shamimul, Saffarini Daad, Shirodkar Sheetal
Аннотация
OBJECTIVE: This study is an investigation of anaerobic nitrite and fumarate reduction/respiration abilities of two characterised Prevotella species namely Prevotella nigrescens (SS6B) and Prevotella buccae (GS6B) isolated from the periodontal pockets of chronic periodontitis (ChP) patients.
METHODS: Isolation and identification of the periodontal bacteria from 20 patients showing clinical symptoms of ChP. Characterisation of anaerobic nitrite and fumarate reduction was done in P. nigrescens (SS6B) and P. buccae (GS6B) using reduction assays, inhibition assays with use of specific inhibitors, growth assays and enzyme activity assays. Degenerate PCR was used to detect and amplify nitrite reductase (nrfA) and fumarate reductase (frdA) gene sequences in these Prevotella isolates. In addition, molecular and in silico analysis of the amplified anaerobic reductase gene sequences was performed using NCBI conserved domain analysis, Interpro database and MegaX.
RESULTS: We provided experimental evidence for presence of active nitrite and fumarate reductase activities through enzyme activity, reduction, inhibitor and growth assays. Moreover, we were able to detect presence of 505 bps nrfA gene fragment and 400 bps frdA gene fragment in these Prevotella spp. These fragments show similarity to multiheme ammonia forming cytochrome c nitrite reductases and fumarate reductases flavoprotein subunit, respectively.
CONCLUSION: Anaerobic nitrite and fumarate respiration abilities in P. nigrescens and P. buccae isolates appear to be important for detoxification process and growth, respectively.
METHODS: Isolation and identification of the periodontal bacteria from 20 patients showing clinical symptoms of ChP. Characterisation of anaerobic nitrite and fumarate reduction was done in P. nigrescens (SS6B) and P. buccae (GS6B) using reduction assays, inhibition assays with use of specific inhibitors, growth assays and enzyme activity assays. Degenerate PCR was used to detect and amplify nitrite reductase (nrfA) and fumarate reductase (frdA) gene sequences in these Prevotella isolates. In addition, molecular and in silico analysis of the amplified anaerobic reductase gene sequences was performed using NCBI conserved domain analysis, Interpro database and MegaX.
RESULTS: We provided experimental evidence for presence of active nitrite and fumarate reductase activities through enzyme activity, reduction, inhibitor and growth assays. Moreover, we were able to detect presence of 505 bps nrfA gene fragment and 400 bps frdA gene fragment in these Prevotella spp. These fragments show similarity to multiheme ammonia forming cytochrome c nitrite reductases and fumarate reductases flavoprotein subunit, respectively.
CONCLUSION: Anaerobic nitrite and fumarate respiration abilities in P. nigrescens and P. buccae isolates appear to be important for detoxification process and growth, respectively.