Автоматизированная Разделение<em> С. Элеганс</em> Переменно колонизирована бактериальным патогеном

Summary

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage: sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

Abstract

Wormsorter является инструментом аналогично машине FACS, который используется в исследованиях Caenorhabditis Элеганс, как правило, для сортировки червей на основе экспрессии флуоресцентного репортера. Здесь мы выделяем альтернативный использование этого инструмента, для сортировки червей в соответствии с их степенью колонизации в GFP-экспрессирующих патогена. Эта новая использование позволило нам рассмотреть вопрос о взаимосвязи между колонизации червячного кишечника и индукции иммунного ответа. В то время как С. Элеганс иммунные ответы на различных патогенов были зарегистрированы, это еще неизвестно, что инициирует их. Два основных возможностей (которые не являются взаимоисключающими) являются признание патоген-ассоциированных молекулярных моделей, и обнаружение повреждений, вызванных инфекцией. Чтобы различать две возможности, воздействие патогена должны быть отделены от ущерба, который он вызывает. Wormsorter включен разделение червей, которые были широко-колонизирована Грама-нegative возбудитель синегнойная палочка, с ущербом, вероятно, вызванной патогеном нагрузки, от червей, которые были так же, подвергающихся, но не так, или незначительно, колонизировали. Эти различные группы населения были использованы для оценки взаимосвязи между возбудителя нагрузки и индукции транскрипции иммунных реакций. Полученные результаты свидетельствуют о том, что два разобщены, подтверждающие возможность признания патогена.

Introduction

Automatic worm sorting is much like FACS, operating by measuring a fluorescent signal in a worm (typically provided by transgenic expression of reporter proteins) as it passes straightened in a tube, allowing redirection either to a collection tube/well or to a waste container according to gating parameters set by the researcher¹. The wormsorter can facilitate research in many ways; an example of employing it as an analytical tool is a study that followed spatiotemporal patterns of promoter activity for almost 1,000 genes².
However, the major use of the wormsorter is in genetic screens, following target gene expression levels or localization of fluorescent protein along the axis of the worm^3-5.

Here, we describe a new application for the wormsorter, in following colonization of the worm by a fluorescently tagged pathogen. With this as a tool we focused on the relationship between pathogen colonization/load and the immune response, to gain new insights into the mechanisms responsible for initiation of immune responses in the worm.

In virtually all organisms studied to date, initiation of innate immune responses to microbial pathogens depends on recognition of pathogen-associated molecular patterns (PAMPs), and/or danger/damage-associated molecular patterns (DAMPs)^6,7. The first are conserved microbial structures that include components of the microbial cell wall, its flagellum, or its lipid bilayer⁶; the second, include both released molecules (e.g. ATP⁸), altered proteins or other markers of altered cellular processes^9,10. Both types of signals are recognized by proteins designated as pattern recognition receptors (PRRs), which upon specific binding of a pattern molecule activate a chain of events leading to a protective response. C. elegans has been extremely useful as a tractable model to dissect various aspects of host-pathogen interactions, but one thing that is not well understood is how immune responses are initiated in the worm. None of the putative receptors that are orthologous to pattern recognition receptors (PRRs) in other organisms have been shown to bind PAMPs, and many of the orthologs of PRRs that are pivotal for immune responses in other organisms show a surprisingly limited contribution to worm pathogen responses and resistance. For example, the Drosophila Toll receptor, which is essential for resisting Gram positive pathogens, is represented in C. elegans by a sole homolog, tol-1, which contributes to protection from the Gram negative pathogen Salmonella Typhimurium¹¹, but not from other tested Gram-negative, or -positive pathogens^11,12 . These observations, combined with data indicating that immune responses could be induced by disrupting cellular protein translation has led some to suggest that C. elegans primarily detects DAMPs^9,13,14. Nevertheless, reports describing ability of dead pathogens to induce immune responses suggest that PAMP binding may be have an important role in pathogen recognition in C. elegans^15,16. Previous work focusing on immune responses in age-synchronized genetically identical C. elegans populations, demonstrated large individual variability in intestinal colonization by the bacterial Gram-negative pathogen Pseudomonas aeruginosa.

However, transcriptional profiling studies treated these variably-colonized populations as one entity^17,18. Taking advantage of this variability, we developed a protocol focusing an automated wormsorter to separate differentially colonized populations of Caenorhabditis elegans exposed to GFP-expressing P. aeruginosa. Examining gene expression in differently-colonized populations facilitated assessment of the relationship between pathogen load (and the associated damage) and immune responses and provided new insights about pathogen recognition in C. elegans¹⁹. Below we describe the protocol, which could be applied to sort worms infected with any fluorescently labeled pathogen.

To potential users it should be noted that the number of worms required to be sorted out depends on the nature of the subsequent analyses and protocols in use. For example, in the case of microarray gene expression analysis, >1,000 worms will be required to obtain enough RNA, if standard protocols are used, but ~100 worms would suffice if amplification is employed, allowing fast collection of material and thus minimizing stress to the worms.

Protocol

1. Obtaining a Synchronized Culture of Young Adult Animals Grow worms on several NGM plates seeded with OP50-1 E. coli bacteria (10x concentrated from a saturated culture) until many worms have reached the gravid stage. Treat gravid animals with egg-prep solution to obtain a synchronized culture (eggs). Plate eggs on several 60-mm NGM plates seeded with 10x concentrated OP50-1 at a density of roughly 150-200 eggs/plate. Incubate plates at 25 °C f…

Representative Results

When age-matched, genetically identical C. elegans are exposed to P. aeruginosa, a wide distribution is observed in levels of colonization (Figure 1A). With the help of the protocol described here efficient separation of noncolonized from colonized worms can be achieved (Figure 1B). Unlike noncolonized worms, colonized worms show signs of damage, such as sluggishness and reduced defecation19. The latter may be a reason why worms colonized by P. aerug…

Discussion

The method we describe takes advantage of fluorescent labeling of entities outside of the worm, to follow interactions between the worm and its environment. In the case we present, separation was based on labeling of a pathogen and was employed to separate worms with heavy pathogen load from those with no (or light) load. Subsequent gene expression analysis found no difference in immune responses between the two groups suggesting that they were independent of pathogen load. The signal that initiates the response was show…

Materials

M9 Buffer	Prepared in house	Recipe at wormbook.org
Rifampicin	Sigma	R3501
Egg prep solution	Prepared in house	50ml water ; 40ml bleach ; 10ml of 10N Sodium Hydroxide
NGM plates	Prepared in house	Recipe at wormbook.org
SKP plates	Prepared in house	Recipe same as NGM only 0.35% peptone instead of 0.25%
Control test particles	Union Biometrica	310-5071-001