JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Biologia

Brug af enkeltormdata til at kvantificere heterogenitet i Caenorhabditis elegans-bakterielle interaktioner

Published: July 22, 2022
doi:
10.3791/64027
, ,
1Department of Biology,Emory University, 2Department of Physics,Emory University

Summary

Denne protokol beskriver en 96-brønds forstyrrelse af individuelle bakterielt koloniserede Caenorhabditis elegans efter kold lammelse og overfladeblegning for at fjerne eksterne bakterier. Den resulterende suspension er belagt på agarplader for at muliggøre nøjagtig kvantificering af bakteriebelastningen i et stort antal individuelle orme.

Abstract

Nematoden Caenorhabditis elegans er et modelsystem for værtsmikrobe- og værtsmikrobiominteraktioner. Mange undersøgelser til dato bruger batchfordøjelser snarere end individuelle ormprøver til at kvantificere bakteriebelastning i denne organisme. Her hævdes det, at den store interindividuelle variabilitet, der ses i bakteriel kolonisering af C. elegans tarm, er informativ, og at batchfordøjelsesmetoder kasserer information, der er vigtig for nøjagtig sammenligning på tværs af betingelser. Da beskrivelsen af variationen i disse prøver kræver et stort antal individer, etableres en bekvem 96-brønds pladeprotokol til forstyrrelse og kolonibelægning af individuelle orme.

Introduction

Heterogenitet i værtsmikrobeforeninger observeres allestedsnærværende, og variation mellem individer anerkendes i stigende grad som en medvirkende faktor i processer på befolkningsniveau fra konkurrence og sameksistens1 til sygdomsoverførsel 2,3,4. I C. elegans er “skjult heterogenitet” inden for isogene populationer blevet observeret gentagne gange, hvor underpopulationer af individer viser forskellige fænotyper i varmechokrespons 5,6, aldring og levetid 7,8,9,10,11 og mange andre aspekter af fysiologi og udvikling12 . De fleste analyser, der forsøger at identificere subpopulationsstruktur, giver bevis for to underpopulationer i eksperimentelle populationer af isogene, synkroniserede orme 5,7,8, selvom andre data antyder muligheden for fordeling af træk inden for befolkningen snarere end forskellige grupper 7,12,13 . Af relevans her observeres betydelig heterogenitet i tarmpopulationer selv inden for isogene populationer af orme koloniseret fra en fælles kilde til mikrober 13,14,15,16, og denne heterogenitet kan skjules af batchfordøjelsesmålingerne, der er meget udbredt 17,18,19,20 til bakteriel kvantificering i ormen.

Dette arbejde præsenterer data, der tyder på et behov for større afhængighed af enkeltormmålinger i værtsmikrobeforening samt protokoller for at øge nøjagtigheden og gennemstrømningen i enkeltormforstyrrelse. Disse protokoller er designet til at lette mekanisk forstyrrelse af et stort antal individuelle C. elegans til kvantificering af levedygtig bakteriebelastning, samtidig med at de giver bedre repeterbarhed og lavere indsats pr. Prøve end pestlebaseret forstyrrelse af individuelle orme. Et anbefalet tarmrensningstrin, hvor orme får lov til at fodre med varmedræbt E. coli inden forberedelsen til afbrydelse, er inkluderet for at minimere bidrag fra nyligt indtagne og andre forbigående (ikke-klæbende) bakterier. Disse protokoller inkluderer en koldlammelsesmetode til rengøring af neglebåndet med en lavkoncentrationsoverfladeblegebehandling; overfladeblegning kan bruges som et forberedende trin i enkeltormforstyrrelse eller som en metode til fremstilling af levende, eksternt kimfrie orme. Denne overfladeblegningsmetode er tilstrækkelig til at fjerne en bred vifte af eksterne mikrober, og koldbehandling giver et alternativ til konventionel levamisolbaseret lammelse; mens levamisol vil blive foretrukket til koldfølsomme eksperimenter, minimerer koldlammelse bidrag til farlige affaldsstrømme og muliggør hurtig genoptagelse af normal aktivitet. Mens den fulde protokol beskriver et laboratorieforsøg, hvor orme koloniseres med kendte bakterier, kan procedurerne for rengøring af orme og enkeltormforstyrrelse let anvendes på orme isoleret fra vilde prøver eller koloniseret i mikrokosmoseksperimenter. De protokoller, der er beskrevet her, producerer levende bakterier ekstraheret fra ormtarmen, der er egnede til plettering og kvantificering af kolonidannende enheder (CFU’er) i individuelle orme; til sekventeringsbaseret analyse af tarmsamfundet bør efterfølgende cellelysis- og nukleinsyreekstraktionstrin tilføjes til disse protokoller.

Protocol

Orme, der blev brugt i disse eksperimenter, blev opnået fra Caenorhabditis Genetic Center, som finansieres af NIH Office of Research Infrastructure Programs (P40 OD010440). Bristol N2 er vildtypen. DAF-2/IGF-mutanter daf-16(mu86) I (CGC CF1038) og daf-2(e1370) III (CGC CB1370) bruges til at illustrere forskelle i tarmbakteriebelastning. HT115(DE3) E. coli , der bærer pos-1 RNAi-vektoren , er fra Ahringer-biblioteket21. MYb-…

Representative Results

Blegestilisering af levende ormeOverfladeblegede orme er effektivt fri for eksterne bakterier, indtil bevægeligheden vender tilbage, og udskillelsen genoptages. Under de betingelser, der anvendes her, observeres hurtig udryddelse af bakterier i buffer (figur 1A-C, supplerende figur 2, video 1) uden at forstyrre de tarmassocierede bakterier i koldlammede orme (figur 1D-F, video 2</st…

Discussion

Her præsenteres data om fordelene ved enkeltormkvantificering af bakteriebelastning i C. elegans sammen med en 96-brønds afbrydelsesprotokol for at muliggøre hurtig og konsekvent erhvervelse af store datasæt af denne type. Sammenlignet med eksisterende metoder33 tillader disse protokoller måling af tarmmikrobielle samfund med højere kapacitet i ormen.

Denne tilgang har plettering som et hastighedsbegrænsende trin og er ikke virkelig “high-throughput”. Cy…

Declarações

The authors have nothing to disclose.

Acknowledgements

Forfatterne vil gerne anerkende H. Schulenberg og C. LaRock for deres generøse deling af bakteriestammer, der anvendes i disse eksperimenter. Dette arbejde blev støttet af finansiering fra Emory University og NSF (PHY2014173).

Materials

96-well flat-bottom polypropylene plates, 300 uL Evergreen Labware 290-8350-03F
96-well plate sealing mat, silicon, square wells (AxyMat) Axygen AM-2ML-SQ
96-well plates, 2 mL, square wells Axygen P-2ML-SQ-C-S
96-well polypropylene plate lids Evergreen Labware 290-8020-03L
Agar Fisher Scientific 443570050
Bead mill adapter set for 96-well plates QIAGEN 119900 Adapter plates for use with two 96-well plates on the TissueLyser II
Bead mill tissue homogenizer (TissueLyser II) QIAGEN 85300 Mechanical homogenizer for medium to high-throughput sample disruption
BioSorter Union Biometrica By quotation Large object sorter equipped with a 250 micron focus for C. elegans
Bleach, commercial, 8.25% sodium hypochlorite Clorox
Breathe-Easy 96-well gas permeable sealing membrane Diversified Biotech BEM-1 Multiwell plate gas permeable polyurethane membranes. Thin sealing film is permeable to O2, CO2, and water vapors and is UV transparent down to 300 nm. Sterile, 100/box.
Calcium chloride dihydrate Fisher Scientific AC423525000
Cholesterol VWR AAA11470-30
Citric acid monohydrate Fisher Scientific AC124910010
Copper (II) sulfate pentahydrate Fisher Scientific AC197722500
Corning 6765 LSE Mini Microcentrifuge Corning  COR-6765
Disodium EDTA Fisher Scientific 409971000
DL 1,4 Dithiothreitol, 99+%, for mol biology, DNAse, RNAse and Protease free, ACROS Organics Fisher Scientific 327190010
Eppendorf 1.5 mL microcentrifuge tubes, natural Eppendorf
Eppendorf 5424R microcentrifuge Eppendorf 5406000640 24-place refrigerated benchtop microcentrifuge
Eppendorf 5810R centrifuge with rotor S-4-104 Eppendorf 22627040 3L benchtop centrifuge with adaptors for 15-50 mL tubes and plates
Eppendorf plate bucket (x2), for Rotor S-4-104 Eppendorf 22638930
Ethanol 100% Fisher Scientific BP2818500
Glass beads, 2.7 mm Life Science Products LS-79127
Glass beads, acid-washed, 425-600 µm Sigma G877-500G
Glass plating beads VWR 76005-124
Hydrochloric acid VWR BDH7204-1
Iron (II) sulfate heptahydrate Fisher Scientific 423731000
Kimble Kontes pellet pestle motor DWK Life Sciences 749540-0000
Kimble Kontes polypropylene pellet pestles and microtubes, 0.5 mL DWK Life Sciences 749520-0590
Leica DMi8 motorized inverted microscope with motorized stage Leica 11889113
Leica LAS X Premium software Leica 11640687
Magnesium sulfate heptahydrate Fisher Scientific AC124900010
Manganese(II) chloride tetrahydrate VWR 470301-706
PARAFILM M flexible laboratory sealing film Amcor PM996
Peptone Fisher Scientific BP1420-500
Petri dishes, round, 10 cm VWR 25384-094
Petri dishes, round, 6 cm VWR 25384-092
Petri dishes, square, 10 x 10 cm VWR 10799-140
Phospho-buffered saline (1X PBS) Gold Bio P-271-200
Polypropylene autoclave tray, shallow Fisher Scientific 13-361-10
Potassium hydroxide Fisher Scientific AC134062500
Potassium phosphate dibasic Fisher Scientific BP363-1
Potassium phosphate monobasic Fisher Scientific BP362-1
R 4.1.3/RStudio 2022.02.0 build 443 R Foundation n/a
Scoop-type laboratory spatula, metal VWR 470149-438
Silicon carbide 36 grit MJR Tumblers n/a Black extra coarse silicon carbide grit. Available in 0.5-5 lb sizes from this vendor.
Sodium dodecyl sulfate Fisher Scientific BP166-100
Sodium hydroxide VWR BDH7247-1
Sodium phosphate dibasic anhydrous Fisher Scientific BP332-500
Sodum chloride Fisher Scientific BP358-1
Sucrose Fisher Scientific AC419760010
Tri-potassium citrate monohydrate Fisher Scientific AC611755000
Triton X-100 Fisher Scientific BP151-100
Zinc sulfate heptahydrate Fisher Scientific AC205982500
DOWNLOAD MATERIALS LIST

Referências

  1. Armitage, D. W., Jones, S. E. How sample heterogeneity can obscure the signal of microbial interactions. The ISME Journal. 13 (11), 2639-2646 (2019).
  2. Stephenson, J., et al. Host heterogeneity affects both parasite transmission to and fitness on subsequent hosts. Philosophical Transactions of the Royal Society B: Biological Sciences. 372 (1719), 20160093 (2017).
  3. VanderWaal, K. L., Ezenwa, V. O. Heterogeneity in pathogen transmission: mechanisms and methodology. Functional Ecology. 30 (10), 1606-1622 (2016).
  4. Dwyer, G., Elkinton, J. S., Buonaccorsi, J. P. Host heterogeneity in susceptibility and disease dynamics: tests of a mathematical model. The American Naturalist. 150 (6), 685-707 (1997).
  5. Wu, D., Rea, S. L., Yashin, A. I., Johnson, T. E. Visualizing hidden heterogeneity in isogenic populations of C. elegans. Experimental Gerontology. 41 (3), 261-270 (2006).
  6. Yashin, A. I., et al. Heat shock changes the heterogeneity distribution in populations of Caenorhabditis elegans does it tell us anything about the biological mechanism of stress response. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences. 57 (3), 83-92 (2002).
  7. Zhao, Y., et al. Two forms of death in ageing Caenorhabditis elegans. Nature Communications. 8 (1), 1-8 (2017).
  8. Eckley, D. M., et al. Molecular characterization of the transition to mid-life in Caenorhabditis elegans. AGE. 35 (3), 689-703 (2012).
  9. Rea, S. L., Wu, D., Cypser, J. R., Vaupel, J. W., Johnson, T. E. A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans. Nature Genetics. 37 (8), 894-898 (2005).
  10. Kinser, H. E., Mosley, M. C., Plutzer, I. B., Pincus, Z. Global, cell non-autonomous gene regulation drives individual lifespan among isogenic C. elegans. eLife. , (2021).
  11. Churgin, M. A., et al. Longitudinal imaging of Caenorhabditis elegans in a microfabricated device reveals variation in behavioral decline during aging. eLife. 6, 26652 (2017).
  12. Perez, M. F., Francesconi, M., Hidalgo-Carcedo, C., Lehner, B. Maternal age generates phenotypic variation in Caenorhabditis elegans. Nature. 552 (7683), 106-109 (2017).
  13. Baeriswyl, S., et al. Modulation of aging profiles in isogenic populations of Caenorhabditis elegans by bacteria causing different extrinsic mortality rates. Biogerontology. 11 (1), 53 (2009).
  14. Taylor, M., Vega, N. M. Host immunity alters community ecology and stability of the microbiome in a Caenorhabditis elegans model. mSystems. 6 (2), 00608-00620 (2021).
  15. Diaz, S. A., Restif, O. Spread and transmission of bacterial pathogens in experimental populations of the nematode Caenorhabditis elegans. Applied and Environmental Microbiology. 80 (17), 5411-5418 (2014).
  16. Twumasi-Boateng, K., Berg, M., Shapira, M. Automated separation of C. elegans variably colonized by a bacterial pathogen. Journal of Visualized Experiments: JoVE. (85), e51090 (2014).
  17. Ortiz, A., Vega, N. M., Ratzke, C., Gore, J. Interspecies bacterial competition regulates community assembly in the C. elegans intestine. The ISME Journal. 15 (7), 2131-2145 (2021).
  18. Berg, M., et al. TGFβ/BMP immune signaling affects abundance and function of C. elegans gut commensals. Nature Communications. 10 (1), 604 (2019).
  19. Portal-Celhay, C., Blaser, M. J. Competition and resilience between founder and introduced bacteria in the Caenorhabditis elegans gut. Infection and Immunity. 80 (3), 1288-1299 (2012).
  20. Scott, E., Holden-Dye, L., O’Connor, V., Wand, M. E. Intra strain variation of the effects of gram-negative ESKAPE pathogens on intestinal colonization, host viability, and host response in the model organism Caenorhabditis elegans. Frontiers in Microbiology. 10, 3113 (2020).
  21. Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., Ahringer, J. Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biology. 2 (1), (2001).
  22. Dirksen, P., et al. The native microbiome of the nematode Caenorhabditis elegans: gateway to a new host-microbiome model. BMC Biology. 14, 38 (2016).
  23. Vega, N. M., Allison, K. R., Samuels, A. N., Klempner, M. S., Collins, J. J. Salmonella typhimurium intercepts Escherichia coli signaling to enhance antibiotic tolerance. Proceedings of the National Academy of Sciences. 110 (35), 14420-14425 (2013).
  24. Stiernagle, T. Maintenance of C. elegans. WormBook. , (2006).
  25. Tabara, H., et al. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell. 99 (2), 123-132 (1999).
  26. Ahringer, J. Reverse genetics. WormBook. , (2006).
  27. Rual, J. -. F., et al. Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Research. 14 (10), 2162-2168 (2004).
  28. Revtovich, A. V., et al. Development and characterization of high-throughput Caenorhabditis elegans – Enterococcus faecium infection model. Frontiers in Cellular and Infection Microbiology. 11, 667327 (2021).
  29. Anderson, Q. L., Revtovich, A. V., Kirienko, N. V. A high-throughput, high-content, liquid-based C. elegans pathosystem. JoVE (Journal of Visualized Experiments. (137), e58068 (2018).
  30. Scholz, M., Dinner, A. R., Levine, E., Biron, D. Stochastic feeding dynamics arise from the need for information and energy. Proceedings of the National Academy of Sciences. 114 (35), 9261-9266 (2017).
  31. Wu, T., et al. Pheromones modulate learning by regulating the balanced signals of two insulin-like peptides. Neuron. 104 (6), 1095-1109 (2019).
  32. Ching, T. -. T., Hsu, A. -. L. Solid plate-based dietary restriction in Caenorhabditis elegans. Journal of Visualized Experiments: JoVE. (51), e2701 (2011).
  33. Walker, A. C., Bhargava, R., Vaziriyan-Sani, A. S., Brust, A. S., Czyz, D. M. Quantification of bacterial loads in Caenorhabditis elegans. Bio-protocol. 12 (2), 4291-4291 (2022).
  34. Manjarrez, J. R., Mailler, R. Stress and timing associated with Caenorhabditis elegans immobilization methods. Heliyon. 6 (7), 04263 (2020).
  35. Zhang, S., Banerjee, D., Kuhn, J. R. Isolation and culture of larval cells from C. elegans. PLoS ONE. 6 (4), 0019505 (2011).
  36. Garsin, D. A., et al. Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens. Science. 300 (5627), 1921 (2003).
  37. Thutupalli, S., et al. Farming and public goods production in Caenorhabditis elegans populations. Proceedings of the National Academy of Sciences. 114 (9), 2289-2294 (2017).
  38. Ly, K., Reid, S. J., Snell, R. G. Rapid RNA analysis of individual Caenorhabditis elegans. MethodsX. 2, 59-63 (2015).
  39. Johnke, J., Dirksen, P., Schulenburg, H. Community assembly of the native C. elegans microbiome is influenced by time, substrate, and individual bacterial taxa. Environmental Microbiology. 22 (4), 1265-1279 (2020).
  40. Vega, N. M., Gore, J. Stochastic assembly produces heterogeneous communities in the Caenorhabditis elegans intestine. PLOS Biology. 15 (3), 2000633 (2017).
  41. Gulyas, L., Powell, J. R. Cold shock induces a terminal investment reproductive response in C. elegans. Scientific Reports. 12 (1), 1338 (2022).
  42. Jiang, W., et al. A genetic program mediates cold-warming response and promotes stress-induced phenoptosis in C. elegans. eLife. 7, 35037 (2018).
  43. Robinson, J. D., Powell, J. R. Long-term recovery from acute cold shock in Caenorhabditis elegans. BMC Cell Biology. 17 (1), 2 (2016).

Tags

Keywords: Caenorhabditis ElegansBacterial InteractionsSingle-worm DataMechanical Disruption96-well FormatMedium ThroughputQuantificationWorm MeasurementsM9TX01OP50SDS/DTT SolutionChemical PermeabilizationWorm Cuticle
check_url/pt/64027?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Taylor, M. N., Spandana Boddu, S., Vega, N. M. Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions. J. Vis. Exp. (185), e64027, doi:10.3791/64027 (2022).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image