Oral Communication presented at the ";Forum des Jeunes Chercheurs";, Brest (France) 2011

Bonnaure-Mallet, M

Guyodo, H

Le Pottier, L

Leclerc, J

Martin, B

Meuric, V

Tamanai-Shacoori, Z

English

Leclerc, Julia

Martin, Bénédicte

Tamanai-Shacoori, Zohreh

Le Pottier, Laetitia

Guyodo, Hélène

Meuric, Vincent

Bonnaure-Mallet, Martine

HAL-Rennes 1

Involvement of respiratory chain in biofilm formation inporphyromonas gingivalis.Julia Leclerc, Be´ne´dicte Martin, Zohreh Tamanai-Shacoori, Laetitia LePottier, He´le`ne Guyodo, Vincent Meuric, Martine Bonnaure-MalletTo cite this version:Julia Leclerc, Be´ne´dicte Martin, Zohreh Tamanai-Shacoori, Laetitia Le Pottier, He´le`ne Guy-odo, et al.. Involvement of respiratory chain in biofilm formation in porphyromonas gingivalis..Bulletin- Groupement International pour la Recherche Scientifique en Stomatologie et Odon-tologie, Groupement International pour la Recherche Stomatologie et Odontologie, 2011, 50(2), pp.5-6. <inserm-00826678>HAL Id: inserm-00826678http://www.hal.inserm.fr/inserm-00826678Submitted on 3 Jun 2013HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.L’archive ouverte pluridisciplinaire HAL, estdestine´e au de´poˆt et a` la diffusion de documentsscientifiques de niveau recherche, publie´s ou non,e´manant des e´tablissements d’enseignement et derecherche franc¸ais ou e´trangers, des laboratoirespublics ou prive´s.5Bull Group Int Rech Sci Stomatol Odontol. 50(2): 5-6 (2011)SHORT COMMUNICATIONEA1254 “Microbiologie – Risques infectieux”, Université Rennes 1, 2 avenue Prof. L. Bernard, Ren-nes, France. julia.leclerc@etu.univ-rouen.fr J. Leclerc, B. Martin, Z. Tamanai-Shacoori, L. Le Pottier, H. Guyodo, V. Meuric and M. Bonnaure-Mallet.O-2. INVOLVEMENT OF RESPIRATORY CHAIN IN BIOFILM FORMA-TION IN PORPHYROMONAS GINGIVALISKey wordsPorphyromonas gingivalis - periodontal disea-VH¨F\G$%¨IQUELRÀOPIRUPDWLRQIntroductionPorphyromonas gingivalis (P.gingivalis) is a gram-negative strict-anaerobe bacterium and constitutes one of the main pathogen respon-sible for periodontal diseases (2). Periodontal GLVHDVHVDUHFKURQLF LQÁDPPDWRU\ LQIHFWLRQVLQGXFHGE\EDFWHULDRUJDQLVHGLQELRÀOP0RUHthan 500 bacteria species take part in this bio-ÀOPLQFOXGLQJ3JLQJLYDOLVAlthough strict-anaerobe, P. gingivalis is able to endure the presence of low oxygen concen-tration. Previous in silico studies comparing on P. gingivalis and Escherichia coli meta-bolisms showed that some genes like cydAB (encoding for cytochrome bd) or fnr (fumarate nitrate reduction regulator) were involved in aerobic respiratory and aero-anaerobic switch respectively (1).The present work is based on the creation of P. gingivalis mutants for the operon cydAB and the gene fnr and their characterization using various approaches: gene expression, JURZWKVWXGLHVDQGELRÀOPIRUPDWLRQMaterials and MethodsStrains and culture conditions:3JLQJLYDOLV$7&&DQGLWVLVRJHQLFPX-tants were grown in anaerobic chamber (10 % CO2, 10 % H2 and 80 % N2). P. gingivalis and Streptococcus gordonii DL1 pCM18 stra-LQ *)3PXWZHUHJURZQ LQHQULFKHGEUDLQheart infusion (BHI) medium and on Columbia DJDUZLWKKRUVHEORRGHQULFKHGE\\HDVWH[-WUDFWKHPLQDQGPHQDGLRQHDW&Mutants construction: The central part of the cydAB operon or the fnr gene was deleted and replaced by inser-tion of the Erythromycin cassette via double homologous recombination of the suicide vec-tor pSK-erm.Gene expression and polar effect:Reverse transcription-PCR was used to am-plify the different mutated genes and their ÁDQNLQJJHQHVRQWRWDO51$H[WUDFWHGIURP3gingivalis wild-type and mutants strains.Growth:The growth of each P. gingivalis strain was VWXGLHG LQ DQDHURELF FKDPEHU DW & E\measure of Optic Density at 600 nm over time.)RUPDWLRQRIVWDWLFELRÀOPFormation of mono-bacterial (P. gingivalis) and bi-bacterial (P. gingivalis and S. gordonii) ELRÀOPVZDVVWXGLHGXVLQJ%LRÀOP5LQJ7HVWPHWKRG%LRÀOP&RQWURO)UDQFH)RUPDWLRQRIG\QDPLFELRÀOP)RUPDWLRQ RI ELEDFWHULDO ELRÀOP IRUPDWLRQwith S. gordonii-GFP and different strains of P. gingivalis was analyzed under continuous ÁRZ RQ JODVV FRYHUVOLSV /XGLQ &KDPEHULife Imaging Services). To identify P. gingivalis DVSHFLÀFLPPXQRVWDLQLQJZDVXVHG%LRÀOPVwere visualised by confocal microscopy (Lei-ca DMI 6000 CS, Sp5) and images analysed DQG TXDQWLÀHG ZLWK ,PDJH - DQG &RPVWDW associate with Matlab softwares.ResultsGene expression studies showed that when fnr gene and cydAB operon were deleted, ÁDQNLQJJHQHVERUGHULQJZHUHVWLOOH[SUHVVHG6Bull Group Int Rech Sci Stomatol Odontol. 50(2): 5-6 (2011)in the same way in the mutants as in the wild-type strains. Growth of mutant strains was not PRGLÀHGE\WKHPXWDWLRQVDVFRPSDUHGWRWKHwild-type strain. Analysis of static bi-bacterial ELRÀOP ZLWK 3 JLQJLYDOLV DQG 6 JRUGRQLL GLGnot show any difference between mutant stra-LQV DQG ZLOGW\SH VWUDLQ ELRÀOPV ZHUH FRP-SOHWHO\IRUPHGDIWHUKRXUV$QDO\VLVRIVWDWLFPRQREDFWHULDO ELRÀOP VKRZHG WKDW PXWDQWVWUDLQV LQGXFHGWKH IRUPDWLRQRIDELRÀOPDI-ter 5 hours whereas wild-type strain needed KRXUV+RZHYHUWKHELRÀOPIRUPHGE\¨IQUstrain seemed to start losing its structure after KRXUV4XDQWLÀFDWLRQRI3JLQJLYDOLVLQG\QD-PLFELRÀOP7DEOHVKRZHGWKDWWKH¨IQUVWUDLQIRUPHGD WKLFNHUELRÀOPZLWKKLJKHUELRPDVVWKDW ZLOG W\SH VWUDLQ ¨F\G$% PXWDQW VWUDLQseemed to have a similar behaviour to wild-type strain.Discussion%RWKPXWDQWVZHUHFRQVWUXFWHGDQGFRQÀUPHGby RT-PCR and there was no polar effect on JHQHV ÁDQNLQJ WKH PXWDWLRQ 7KH PXWDWLRQVhad no effect on bacterial growth in anaerobic condition as expected. However, these genes may be involved in bacterial growth under va-riable nutrients or in the presence of few quan-tity of oxygen. Thus future experiment will be performed in different conditions like oxygen SUHVHQFHDQGLQPRGLÀHGFXOWXUHPHGLD,Q VWDWLF ELRÀOP VWXGLHV WKH DFFHOHUDWLRQ RIELRÀOPSURGXFWLRQIRUPXWDQWVWUDLQVFRXOGEHH[SODLQHGE\DQ LQÁXHQFHRIR[LGRUHGXFWLRQstatus of the bacterium that could modify gene expression implicated in virulence such as adhesion capacities (a mechanism already VKRZQIRUÀPEULDHIRUH[DPSOH7REHFOR-ser to in vivo conditions we used the dynamic ELRÀOPPRGHOVWXGLHVDQGVKRZHGWKDWLQWKHpresence of S. gordonii (a primary coloniser LQ SHULRGRQWDO ELRÀOPV ¨IQU VWUDLQ PDGH DWKLFNHUELRÀOP7KHSXWDWLYH LQÁXHQFHRI WKLVtranscriptional regulator Fnr on gene involved in P. gingivalis respiration and growth could H[SODLQWKHKLJKHUYROXPHIRXQGLQ¨IQUPXOWLEDFWHULDELRÀOPV)RUWKH¨F\G$%VWUDLQWKHUHis no observable difference on dynamic bio-ÀOPIRUPDWLRQZLWKZLOGW\SHVWUDLQConclusionAs Fnr is a transcriptional factor, the effect of its mutation seems to be more pleiotropic compared to the mutation of cydAB, which is only involved in oxygen metabolism. These preliminary results give us a better unders-WDQGLQJRIIDFWRUVLQYROYHGLQELRÀOPIRUPDWLRQduring periodontal diseases depending on P. gingivalis. These preliminary results should be repeated and require further studies in or-der to understand the precise role of genes in P. gingivalis metabolism.Acknowledgements / fundingsThis work was supported by Conseil Régio-nal de Bretagne, Rennes Métropole, Europe-Feder and Fondation des Gueules Cassées.References1. Meuric V et al. (2010) Future Microbiol, 5  3HULDVDP\6HWDO-%DFWHULRO(22), 6804-6811. 5RVDQ %HW DO  0LFUREHV ,QIHFW  :X-HWDO )(060LFURELRO/HWW2008 May, 282 (2), 188-95WT-1 WT-2 :7 Cyd-1 Cyd-2 &\G Fnr-1 Fnr-2 )QUTotal biomass (µm/µm2) 6,5 2,9  5,6 5,8  4,2 12,2 14,1Maximum thickness (µm)     29,2    7DEOH4XDQWLÀFDWLRQRI3JLQJLYDOLVG\QDPLFELRÀOPVWXGLHGXQGHUÁRZFRQGLWLRQV%LRPDVVHVDWWULEXWHGWR3JLQJLYDOLVDQGPD[LPDOELRÀOPWKLFNQHVVHVZHUHVKRZQIRUZLOGW\SHDQGERWKPXWDQWVVWUDLQV¨F\G$%DQG¨IQULQWKUHHGLIIHUHQWFRQIRFDOLPDJHDFTXLVLWLRQVDIWHUKRXUVRIELRÀOPIRUPDWLRQ%LRÀOPVZHUHÀ[HGE\IRUPDOGH-hyde before observation.

Involvement of respiratory chain in biofilm formation in porphyromonas gingivalis.

Revistes CientÃ­fiques de la Universitat de Barcelona

INVOLVEMENT OF RESPIRATORY CHAIN IN BIOFILM FORMATION IN PORPHYROMONAS GINGIVALIS

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INVOLVEMENT OF RESPIRATORY CHAIN IN BIOFILM FORMATION IN PORPHYROMONAS GINGIVALIS

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HAL-Rennes 1

Revistes CientÃfiques de la Universitat de Barcelona

HAL-Inserm

INVOLVEMENT OF RESPIRATORY CHAIN IN BIOFILM FORMATION IN PORPHYROMONAS GINGIVALIS

Abstract

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HAL-Rennes 1

Revistes CientÃ­fiques de la Universitat de Barcelona

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Revistes CientÃfiques de la Universitat de Barcelona