Den nematodeprøveudtagning Caenorhabditis Elegans - en alsidig In Vivo Model til at studere Host-mikrobe interaktioner
Summary
Vi præsenterer her, nematode Caenorhabditis elegans som en alsidig host model til at studere mikrobielle interaktion.
Abstract
Vi demonstrere en metode ved hjælp af Caenorhabditis elegans som model vært for at studere mikrobielle interaktion. Mikrober er indført via kosten gør tarmen den primære placering for sygdom. Ødelægge tarmen strukturelt og funktionelt efterligner pattedyr tarmene og er gennemsigtige, hvilket gør den modtagelig til mikroskopisk undersøgelse af kolonisering. Her viser vi, at patogener kan forårsage sygdom og død. Vi er i stand til at identificere mikrobielle mutanter, der viser ændrede virulens. Dens bevarede medfødte svar til biotiske understreger gør C. elegans et fremragende system at sonde facetter host medfødte immun interaktioner. Vi viser, at værter med mutationer i gen, dobbelt oxidase ikke kan producere reaktive ilt arter og er i stand til at modstå mikrobielle fornærmelse. Vi demonstrere yderligere alsidighed af præsenteres overlevelse analyse ved at vise, at det kan bruges til at studere virkningerne af hæmmere af mikrobiel vækst. Denne analyse kan også bruges til at opdage svampe virulens faktorer som mål for udviklingen af roman svampedræbende stoffer, samt give mulighed for at afdække yderligere host-mikrobe interaktioner. Udformningen af dette assay egner sig godt til høj overførselshastighed hele-genom skærme, mens evne til cryo-Bevar orme til fremtidig brug gør det en omkostningseffektiv og attraktive hele dyr model til at studere.
Introduction
C. elegans har været brugt som en kraftfuld model organisme i mere end 50 år. I 1960 ‘ erne banebrydende sydafrikanske biolog Sydney Brenner brugen af C. elegans at studere neuronal udvikling, baner vejen for en lang slægt af forskere til at studere forskellige aspekter af celle og dyr biologi i nematoder. Denne slægt omfatter nobelpristagere Craig Mello og Andrew Fire for deres RNAi arbejde1, Robert Horvitz og John Sulston for deres arbejde på orgel udvikling og apoptose2,3,4, og Martin Chalfie for hans arbejde på grøn fluorescerende proteiner5. Selvom denne model organisme har traditionelt været brugt til at studere molekylære og udviklingsmæssige biologi, over de sidste 15 år, er forskere begyndt at bruge C. elegans at undersøge forskellige menneskelige patogener herunder Pseudomonas biologi aeruginosa, Staphylococcus aureusog Salmonella enterica Serratia marcescens6,7,8,9,10. Disse undersøgelser afslørede, at mange af de mekanismer, der er involveret i den menneskelige-patogen interaktion er bevaret i nematoder, men også at der er nogle immunitet mekanismer, der er unikke for denne model organisme11,12. I naturen, C. elegans møder en række trusler fra indtagne patogener i jorden og det har givet en stærk selektivt pres til at udvikle og vedligeholde en sofistikeret medfødte immunsystem i sin intestinal lumen. Mange af de gener og mekanismer involveret i beskyttelse af intestinal lumen er orkestreret af yderst bevares elementer, som også findes i højere pattedyr11,13. C. elegans repræsenterer derfor et stor model til at studere gastrointestinale patogener som Salmonella enterica14, Shigella boydii15eller Vibrio kolera16.
Her fremhæve vi det bemærkelsesværdige alsidighed af C. elegans som model vært at studere smitstoffer som C. albicans. C. elegans som model vært giver mulighed for high throughput screening for virulens, der er mindre dyre og tidskrævende end en musemodel, som er almindeligt brugt til at studere candidiasis42.
I denne undersøgelse, vi viser, at denne model og assosiated overlevelse assay kan være pålideligt anvendes til at studere vært medfødte immun effektorer vigtigt at modvirke infektioner, patogen determinanter, der drive virulens, og farmakologiske forbindelser der kan gribe ind i patogenesen. Ulig tidligere beskrevet assays, denne metode giver et middel til at studere eksponering en patogen over levetiden af dyr fra larve scenen til voksenalderen, snarere end kun voksenalderen død43,44. I Resumé, vores C. elegans – er C. albicans model et alsidigt og kraftfuldt værktøj, der kan bruges ikke kun til at studere det genetiske grundlag, at drive infektion og immunitet, men også at identificere nye stoffer til terapeutisk intervention.
Protocol
Representative Results
Discussion
Metoder til paavisning C. elegans infektion og overlevelse over livslange eksponering for C. albicans , vi har beskrevet kan ændres for at teste en anden patogen. Flydende kulturer af en anden bakterier eller svamp kan lavet og fodres til C. elegans på en lignende måde. Derudover seriel infektioner kan analyseres ved først at udsætte larve til et patogen, som beskrevet, og derefter overføre dyr ind på en ny plade der indeholder en separat patogen efter at have nået voksenalderen.
<p…Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev udført på og støttet af Worcester Polytechnic Institute.
Materials
| Agar (granulated, bacterilogical grade) | Apex BioResearch Products | 20-248 | |
| Aluminum Wire (95% Pt, 32 Gauge) | Genesee Scientific | 59-1M32P | |
| Axiovision Zeiss Inverted Microscope | Axiovision Zeiss | ||
| Bacto-Peptone | Fisher BioReagants | BP1420-500 | |
| C. elegans strain Bli-3 | Caenorhabditis Genetics Center | Bli-3(e767) CB767 | |
| Calcium Chloride | Fisher Scientific | BP51-250 | |
| Cholesterol, Sigma Grade, minimum 99% | Sigma | C8667-25G | |
| Disposable Culture Tubes (20 x 150 mm) | FIsherBrand | 14-961-33 | |
| Dissection Microscope (NI-150 High Intensity Illuminator) | Nikon Instrument Inc. | ||
| E. coli | Caenorhabditis Genetics Center | OP50 | |
| GraphPad Prism (Survival Curve Analysis Software) | GraphPad Software | ||
| LB Broth (Miller's) | Apex BioResearch Products | 11-120 | |
| Magnesium Sulfate | Fisher Scientific | 10034-99-8 | |
| Medium Petri Dishes (35 X 10 mm) | Falcon | 353001 | |
| Potassium Phosphate monobasic | Sigma | P0662-500G | |
| Sodium Chloride | Fisher Scientific | BP358-1 | |
| Sodium Phosphate | Fisher Scientific | BP332-500 | |
| Wildtype C. albicans SC5314 | ATCC | SC5314 | |
| Wildtype C. elegans | Caenorhabditis Genetics Center | N2 |
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