Real-time acquisition of drug resistance by DNA transfer in live cells
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1 eal-time acquisition of drug resistance by DNA transfer in live cells Christian LESELIN (INSEM-CNS) Cell-to-cell DNA transfer team (AIP-Avenir FINOVI) MMSB - Molecular Microbiology and Structural Biochemistry unit, Lyon JOUNEE INFECIONS NOSOCIMIALES - FINOVI et LYONBIOPOLE - 14 novembre 2017
2 BACEIAL CONJUGAION DNA conjugation apid evolution of bacterial genomes Donor ecipient DNA conjugation Phenotypic conversion Dissemination Donor ecipient ransconjugant Acquisition of new metabolic properties (Drug resistance, Pathogenicity, Environmental adaptations, Symbiotic life-style)
3 Importance de la conjugaison bactérienne IMPOANCE OF GENE ANSFE IN DUG ESISANCE DISSEMINAION Spread of Drug resistance a global priority Conjugative plasmids carry resistance to all classes of antibiotics: Penicillins, Sulfonamids, glycopeptide, polymyxin, MD plasmids (Infection and carriage) Cellular organisation, regulation and chronology of conjugational DNA transfer? Establishment of newly acquired properties (drug resistance)?
4 Cell-to-cell DNA ransfer reporter system pars F ANSCONJUGAN [c; LAC+; ParB-mCh focus] [cs; LAC+; diffuse ParB-mCh] DONO [c; LAC-] pars/parb-gfp ransfer X F ParB-mCherry ParB-mCherry c cs c Live cells microscopy: Snapshots and Microfluidics Louis Pasteur Études sur la Bière (1876) 140 years + funding
![Cell-to-cell DNA ransfer reporter system pars F ANSCONJUGAN [c; LAC+; ParB-mCh focus] [cs; LAC+; diffuse ParB-mCh] DONO [c; LAC-] pars/parb-gfp ransfer X F](/docs-images/92/109230666/images/5-10.jpg "ParB-mCherry ParB-mCherry c cs c Live cells microscopy: Snapshots and Microfluidics DNA Dissemination Proteins dynamic DONO ANSCONJ.")
5 Cell-to-cell DNA ransfer reporter system pars F ANSCONJUGAN [c; LAC+; ParB-mCh focus] [cs; LAC+; diffuse ParB-mCh] DONO [c; LAC-] pars/parb-gfp ransfer X F ParB-mCherry ParB-mCherry c cs c Live cells microscopy: Snapshots and Microfluidics DNA Dissemination Proteins dynamic DONO ANSCONJ. Mobility Localisation Quantification
![focus] pars pars/parb-gfp ransfer X ANSCONJUGAN [c; LAC+; ParB-mCh focus;](/docs-images/92/109230666/images/6-7.jpg "sfgfp] ParB-mCherry P laciq1-sfgfp ParB-mCherry Expression profiles of")
6 Cell-to-cell DNA ransfer + gene expression reporter systems [cs; LAC+; diffuse ParB-mCh] DONO [c; LAC-, sfgfp] ANSCONJUGAN [c; LAC+; ParB-mCh focus] pars pars/parb-gfp ransfer X ANSCONJUGAN [c; LAC+; ParB-mCh focus; sfgfp] ParB-mCherry P laciq1-sfgfp ParB-mCherry Expression profiles of newly acquired genes? Production of sfgfp after plasmid transfer 14 cellules Intensité de fluorescence sfgfp (UA) Fluorescence des cellules donneuses 25 cellules 800 ransfert du plasmide emps (en min) P laciq1-sfgfp pars/parb-gfp Gene expression P laciq1-sfgfp ParB-mCherry
7 evealing the intracellular dynamic of conjugation machineries or resistance factors eta-mch ranslating our molecular knowledge to the cellular context Interplay between cell-to-cell interactions and host-range Understanding the conversion of a commensal into a drug resistant bacterium
8 Acquisition of tetracycline resistance by conjugation Møller et al., BMC 2006 DONO [c ; eta-mcherry] pars tet teta-mcherry mcherry Møller et al., BMC 2006
9 eta-mcherry localisation in absence of tetracycline (3D-SIM) MG1655 F-n10 teta-mcherry M9 Glucose 0.2%, 30ºC (3D-SIM)
10 Simultaneous visualisation of eta and etracycline in live cells: Efflux Pump / Substrate DONO [c ; eta-mcherry] pars tet teta-mcherry mcherry
11 Visualisation of eta production and etracycline efflux in real-time F teta-mcherry c 10 µg/ml M9 Glucose 0.2%, 30ºC; 30min/frame
12 Visualisation of eta production and etracycline efflux in real-time F teta-mcherry c 10 µg/ml M9 Glucose 0.2%, 30ºC; 30min/frame
13 eta initial level determines the level of efflux/resistance 2,800 n= etamcherry intracellular signal (a.u.) 2,600 2,400 2,200 2,000 1,800 1,600 1,400 1,200 1, F teta-mcherry c 100 µg/ml M9 Glucose 0.2%, 30ºC 3 min/frame 0 No induction 2H 4H 8H 16H 2H 4H 8H 16H Ac 0.2 µg/ml c 10 µg/ml 1.00E+01 Viability loss on etracycline plates 3.75E E+00 More eta means 1.88E+00 more resistance 1.88E E+00 0 => because of immediate drug efflux 2.50E E E E E E-03 Axis itle 1.00E-03 c0 c400 c E E E E E E E E E E E-07 No induction 2H 1.25E-07 4H 8H 16H 2H 4H 8H 16H Ac 0.2 µg/ml c 10 µg/ml
14 Conjugation rescues wild type cells (cs) even in presence of tetracycline Pre incubation with etracycline tet teta Conjugation in presence of tetracycline D tet teta X Increasing doses of c in µg/ml 100% 1 Preincubation with c10µg/ml Conjugation efficiency 80% 60% 40% 20% 0% c 0 c 0.5 c 1 c 10 c 25 c 100 tet teta Conjugation efficiency No c 0 min 10 min 30 min 60 min
15 ransfer of tetracycline resistance: eta production in transconjugant cells DONO [c ; eta-mcherry] [diffuse ParB-GFP] ANSCONJUGAN [ParB-mGFP focus] ANSCONJUGAN [ParB-mGFP focus eta-mcherry] pars tet teta-mcherry X DNA ransfer pars/parb-gfp tet teta-mcherry Gene expression pars/parb-gfp tet et teta-mcherry ParB-GFP ParB-GFP eta D eta-mch intracellular fluorescence (a.u.) 0,01 eta quantification in transconjugant cells Zygotic induction Wollman and Jacob, 1954 Donor: wild-type ecipient: wild-type Donor: Δtet ecipient: wild-type Donor: Δtet ecipient: tet +++ n= D 0 1H 2H 3H 4H eta quantification in transconjugants after 4 hours
16 ransfer of tetracycline resistance: eta production in transconjugant cells DONO [c ; eta-mcherry] [diffuse ParB-GFP] ANSCONJUGAN [ParB-mGFP focus] ANSCONJUGAN [ParB-mGFP focus eta-mcherry] pars tet teta-mcherry X DNA ransfer pars/parb-gfp tet teta-mcherry Gene expression pars/parb-gfp tet et teta-mcherry ParB-GFP ParB-GFP eta D eta-mch intracellular fluorescence (a.u.) 0,01 eta quantification in transconjugant cells Zygotic induction Wollman and Jacob, 1954 eta-mch intracellular fluorescence (a.u.) 0,01 eta quantification in transconjugant cells in presence of c 10 n= D 0 1H 2H 3H 4H n= D 0 1H 2H 3H 4H
17 escue of etracycline sensitive cells by conjugation c - c sensitive cells - Bacteriostatic effect of etracycline through inhibition of translation
18 escue of etracycline sensitive cells by conjugation pars et* tet teta eta c D - c sensitive cells - Bacteriostatic effect of etracycline through inhibition of translation - Cells in which translation is inhibited can still receive the plasmid and produce eta
19 escue of etracycline sensitive cells by conjugation D pars et* tet teta eta c - c resistant cells - Bacteriostatic effect of etracycline through inhibition of translation - Cells in which translation is inhibited can still receive the plasmid and produce eta and contribute to the exponential spread of the conjugative plasmid
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