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Nov 2019

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Bacterial Lawn Avoidance and Bacterial Two Choice Preference Assays in Caenorhabditis elegans
秀丽隐杆线虫的细菌草坪趋避和细菌双选择参照实验   

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Abstract

Physical avoidance of pathogens is a crucial defense strategy used by the host to reduce pathogen infection. Hosts display the use of multiple strategies to sense and avoid pathogens, ranging from olfaction to sensing of damage caused by pathogen infection. Understanding various mechanisms of pathogen avoidance has the potential to uncover conserved host defense responses that are important against pathogen infections. Here, we describe protocols for studying pathogen lawn avoidance behavior as well as a change of bacterial preferences in the model nematode Caenorhabditis elegans. Besides, we describe the protocol for measuring preferences for pathogenic and nonpathogenic bacteria after training of the animals on pathogenic bacteria. These assays can be implemented in discovering various mechanisms of host learning that result in the avoidance of pathogens.

Keywords: Pathogen avoidance (病菌趋避), Learning (学习), Bacterial infection (细菌感染), Avoidance behavior (趋避行为), Defense response (防御反应)

Background

A host uses multiple strategies to defend itself against pathogen infections (Medzhitov et al., 2012). Physical avoidance of pathogens is one of the various defense strategies used by the host (Medzhitov et al., 2012; Kavaliers et al., 2019; Singh and Aballay, 2020). Different sensory mechanisms, including chemosensation and elicitation of pain by nociceptor neurons upon detection of bacterial toxins, lead to avoidance behaviors. A deeper understanding of the mechanisms leading to pathogen avoidance holds the potential to uncover conserved host defense responses that are important against pathogen infections.

The nematode C. elegans has been widely used to understand pathogen avoidance behavior and associative learning. C. elegans appears to use multiple mechanisms to learn about pathogens resulting in elicitation of avoidance behaviors (Singh and Aballay, 2020). C. elegans can sense bacterial metabolites (Tran et al., 2017) as well as perturbations in core cellular activities (Melo and Ruvkun, 2012). Moreover, infection of the intestine can modulate neuroendocrine signaling to elicit avoidance behaviors (Singh and Aballay, 2019a and 2019b). Therefore, studying pathogen avoidance behavior and associative learning in C. elegans can help in deciphering various strategies used by a host in sensing pathogens.

Materials and Reagents

  1. 35 mm Petri dishes (Tritech Research, catalog number: T3500 )
  2. 60 mm Petri dishes (Tritech Research, catalog number: T3315 )
  3. 100 mm Petri dishes (Tritech Research, catalog number: T3361 )
  4. 14 ml polypropylene round-bottom tube (VWR, catalog number: 60819-761 )
  5. 15 ml centrifuge tube (VWR, catalog number: 21008-216 )
  6. 10 ml syringe (BD, BactoTM, catalog number: 309604 )
  7. 0.2 μm sterile syringe filter (VWR, catalog number: 28145-501 )
  8. Escherichia coli strain OP50 (Caenorhabditis Genetics Center (CGC))
  9. E. coli strain HT115 containing control RNAi plasmid (L4440) (Source BioScience, Ahringer C. elegans RNAi Collection)
  10. E. coli strain HT115 expressing dsRNA for any gene of interest (Source BioScience, Ahringer C. elegans RNAi Collection)
  11. Pseudomonas aeruginosa strain PA14 (Liberati et al., 2006)
  12. C. elegans wild type Bristol N2 strain and/or any mutants of interest (CGC)
  13. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Anatrace Product, catalog number: I1003 100 GM )
  14. Sodium chloride (NaCl) (Fisher Bioreagents, catalog number: BP358-1 )
  15. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 746495-500G )
  16. Magnesium sulfate anhydrous (MgSO4) (Mallinckrodt, catalog number: A31H10 )
  17. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662-2.5KG )
  18. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786-2.5KG )
  19. Cholesterol (Sigma-Aldrich, catalog number: C8667-25G )
  20. Ampicillin sodium salt (EMD Millipore, catalog number: 171254-25GM )
  21. Bacto agar (BD, BactoTM, catalog number: 214030 )
  22. Bacto peptone (BD, BactoTM, catalog number: 211677 )
  23. Luria-Bertani (LB) broth (Sigma, catalog number: L3022-1KG )
  24. 95% ethanol (EMD Millipore, catalog number: EX0280-3 )
  25. 5 mg/ml cholesterol (see Recipes)
  26. 100 mg/ml ampicillin stock (see Recipes)
  27. 1 M potassium phosphate buffer (pH 6) (see Recipes)
  28. 1 M CaCl2 (see Recipes)
  29. 1 M MgSO4 (see Recipes)
  30. LB (see Recipes)
  31. LB agar plates with and without ampicillin (see Recipes)
  32. Nematode growth medium (NGM) agar plates (see Recipes)
  33. RNAi plates (see Recipes)
  34. Slow killing (SK) assay plates (see Recipes)

Equipment

  1. 500 ml conical flask (VWR, catalog number: 89091-420 )
  2. 1,000 ml conical flask (VWR, catalog number: 29136-106 )
  3. 2,000 ml conical flask (VWR, catalog number: 89090-858 )
  4. Pipetman (Eppendorf, models: P20, P1000)
  5. 20 °C and 25 °C C. elegans incubators (ThermoForma, model: 3920 )
  6. Centrifuge (VWR, model: Clinical 50 )
  7. Platinum wire worm pick (made in the laboratory as described in Wollenberg et al., 2013) and alcohol burner (VWR, catalog number: 470199-936 )
  8. Orbital Shaker (Thermo Fisher Scientific, catalog number: SK4000 )
  9. Autoclave (Consolidated Sterilizer Systems, model: SR-24D )
  10. Stereomicroscope (Leica, model: MZ7.5 )

Software

  1. Microsoft Excel
  2. GraphPad Prism

Procedure

  1. P. aeruginosa Lawn Avoidance Assay

    Day 1
    1. Synchronization of C. elegans
      1. The assays are described for conditions where a gene is knocked down by RNAi (see Notes for details and assays with mutants). Transfer gravid adult N2 animals from E. coli OP50 plates to control RNAi plates (15-20 animals per RNAi plate), as well as RNAi plates for any gene of interest. Use one RNAi plate for each gene knockdown.
      2. Incubate the plates at room temperature (22 °C) for 2 h.
      3. Remove the gravid adults from the RNAi plates and incubate the plates at 20 °C for 72 h to obtain synchronized adult animals.

    Day 2
    1. Take out a vial of frozen P. aeruginosa PA14 from -80 °C and immediately streak on an LB agar plate. Incubate the plate at 37 °C for 12-14 h.

    Day 3
    1. Pick a single colony of P. aeruginosa PA14, inoculate in 2 ml of LB in a 14 ml polypropylene round-bottom tube, place vertically on a shaker, and grow it for 10-12 h at 250 rpm and 37 °C.
    2. Place 20 µl of inoculum on the center of 35 mm SK plates (Figure 1A) that are modified NGM (3.5% instead of 2.5% peptone) plates. Let the inoculum dry at room temperature (20-30 min). Invert the plates upside down (to prevent cracks in agar from drying out) and incubate at 37 °C for 12 h.


      Figure 1. Schematic representation of plates. A. Avoidance assay plates contain a spot of pathogenic bacteria in the center. B. The two-choice preference assay plates contain spots of the two types of bacteria placed diagonally opposite. The cross (×) in the center of the two-choice preference assay plate indicates the site of the transfer of C. elegans. C. The surface of a training plate is fully covered with the pathogenic bacteria.

    Day 4
    1. Cool the P. aeruginosa plates from 37 °C to room temperature for 30 min.
    2. Transfer 30 synchronized adult animals just outside P. aeruginosa lawn on SK plates. Use three P. aeruginosa plates per C. elegans strain/condition. Scrape off the residual E. coli from outside P. aeruginosa lawns on SK plates using platinum wire worm pick.
    3. Incubate the plates with animals at 25 °C.
    4. At various times of incubation (2, 4, 8, 12, and 24 h or as required), count the number of animals that are inside the lawn, outside the lawn, and have crawled on the sidewalls of plates.

  2. Naïve Two Choice Preference Assay
    Day 1
    1. Synchronize C. elegans as described for lawn avoidance assay (Procedure A, Day 1) and incubate at 20 °C for 72 h.

    Day 2
    1. Take out a vial of frozen P. aeruginosa PA14 from -80 °C and immediately streak on an LB agar plate. Incubate the plate at 37 °C for 12-14 h.
    2. Streak RNAi bacteria (E. coli HT115) from -80 °C on LB agar plates containing 100 µg/ml ampicillin. Incubate the plate at 37 °C for 12-14 h.

    Day 3
    1. Pick a single colony of P. aeruginosa PA14, inoculate in 2 ml of LB in a 14 ml polypropylene round-bottom tube, place vertically on a shaker, and grow it for 10-12 h at 250 rpm and 37 °C.
    2. Pick a single colony of RNAi bacteria (E. coli HT115), inoculate in 10 ml of LB with 100 µg/ml ampicillin in a 15 ml tube, place horizontally on a shaker, and grow it for 10-12 h on at 250 rpm and 37 °C.
    3. After growth, concentrate RNAi bacteria 10-20 fold by centrifuging at 5,000 rpm for 5 min at room temperature.
    4. Place 20 µl of each inoculum diagonally opposite onto 35 mm SK plates (Figure 1B). Let the inoculum dry at room temperature (20-30 min). Invert the plates upside down and incubate at 37 °C for 12 h.

    Day 4
    1. Cool the two-choice preference assay plates from 37 °C to room temperature for 30 min.
    2. Transfer 30 synchronized adult animals to the centers of two-choice preference plates equidistant from both the lawns (Figure 1B). Use 3 two-choice preference assay plates per C. elegans strain/condition.
    3. Scrape off residual E. coli from the center of the two-choice preference assay plates using a worm pick (as described in Procedure A, Day 4).
    4. Incubate the plates with animals at 25 °C.
    5. At various times of incubation (2, 4, 8, 12, and 24 h or as required), count the number of animals that are on the E. coli lawn, P. aeruginosa lawn, outside the lawns, and have crawled on the sidewalls of plates.

  3. Trained Two Choice Preference Assay
    Day 1
    1. Synchronize C. elegans as described for lawn avoidance assay (Procedure A, Day 1) and incubate at 20 °C for 54 h.
    2. Take out a vial of frozen P. aeruginosa PA14 from -80 °C and immediately streak on an LB agar plate. Incubate the plate at 37 °C for 12-14 h.

    Day 2
    1. Pick a single colony of P. aeruginosa PA14, inoculate in 2 ml of LB in a 14 ml polypropylene round-bottom tube, place vertically on a shaker, and grow it for 10-12 h at 250 rpm and 37 °C. Store the P. aeruginosa plate at 4 °C.
    2. Place 20 µl of inoculum on 35 mm SK plates and spread it on the entire surface of the plates to obtain full lawns of P. aeruginosa (Figure 1C). Invert the plates upside down and incubate at 37 °C for 12 h.
    3. Streak RNAi bacteria (E. coli HT115) from -80 °C on LB agar plates containing 100 µg/ml ampicillin. Incubate the plate at 37 °C for 12-14 h.

    Day 3
    1. Pick a single colony of P. aeruginosa from the plate stored at 4 °C on Day 2, inoculate in 2 ml of LB in a 14 ml polypropylene round-bottom tube, place vertically on a shaker, and grow it for 10-12 h at 250 rpm and 37 °C.
    2. Pick a single colony of RNAi bacteria (E. coli HT115), inoculate in 10 ml of LB with 100 µg/ml ampicillin in a 15 ml tube, place horizontally on a shaker, and grow it for 10-12 h on at 250 rpm and 37 °C.
    3. Cool the full lawn P. aeruginosa plates from 37 °C to room temperature for at least 30 min.
    4. Transfer 54 h old synchronized C. elegans grown at 20 °C (from Day 1) to full lawns of P. aeruginosa, and incubate at 25 °C for 18 h. This will constitute the trained group of animals.
    5. For control naïve animals, maintain the animals on their corresponding RNAi plates and incubate at 25 °C for 18 h along with the training groups.
    6. Prepare two-choice preference plates (Figure 1B) as described above in naïve two-choice preference assay (Procedure B, Day 3). Invert the plates upside down and incubate at 37 °C for 12 h.

    Day 4
    1. Cool the two-choice preference assay plates from 37 °C to room temperature for 30 min.
    2. Transfer 30 synchronized adult animals from the full lawn P. aeruginosa plates to the center of a two-choice preference assay plate equidistant from both the lawns. Use 3 two-choice preference assay plates per C. elegans strain/condition. This will be the trained group of animals.
    3. Likewise, transfer 30 synchronized adult animals from RNAi plates to the center of a two-choice preference assay plate equidistant from both the lawns. Use 3 two-choice preference assay plates per C. elegans strain/condition. This will be the control group of naïve animals.
    4. Scrape off the residual bacteria from the centers of two-choice preference assay plates using a worm pick (as described in Procedure A, Day 4).
    5. Incubate the plates with animals at 25 °C.
    6. After 1 h of incubation, count the number of animals that are on the E. coli lawn, P. aeruginosa lawn, outside the lawns, and have crawled on the sidewalls of plates.

Data analysis

Percent lawn occupancy calculation: Count the number of animals inside and outside the bacterial lawns. Calculate percent lawn occupancy in Microsoft Excel as:




Where, Non is the number of animals on the lawn, Noff is the number of animals outside the lawn. The animals that have crawled on the sidewalls of plates are excluded from calculations. Plot the time course of percent lawn occupancy using GraphPad Prism. The representative data in Figure 2A show that animals that have knockdown of the genes aex-5 and nol-6 have enhanced and slowed rates of avoidance of P. aeruginosa lawns, respectively.

Choice index calculation: Count the number of animals on the two types of bacterial lawns. Calculate P. aeruginosa choice index (P. aeruginosa CI) in Microsoft Excel as:





The P. aeruginosa CI measures the preference of animals for P. aeruginosa with values ranging from -1 to 1. The values 1, -1, and 0 indicate that all animals are on P. aeruginosa, all animals are away from P. aeruginosa, and the same number of animals are on P. aeruginosa and E. coli, respectively. The P. aeruginosa CI can be plotted for single time points (trained two-choice preference assay) or for a time course (naïve two-choice preference assay) using GraphPad Prism. The representative data in Figure 2B show that naïve animals that have knockdown of the genes aex-5 and nol-6 have faster and slower switch in preference from P. aeruginosa to E. coli, respectively.
  Use GraphPad Prism 8 for statistical analysis of data. Combine data from three independent experiments and calculate the mean and standard deviation (SD). Use multiple t-tests–one per time point and calculate p value for a gene knockdown with respect to the control sample. Judge the data to be statistically significant when P < 0.05.


Figure 2. The time course of pathogen avoidance and change in bacterial preference. A. Time course of the percent occupancy of animals on P. aeruginosa lawns. B. Time course of the P. aeruginosa CI of naïve animals in a two-choice preference assay containing one lawn of each P. aeruginosa and E. coli. See Singh and Aballay (2019b), for details on aex-5 and nol-6. Error bars denote SD from three independent experiments. t-test was used for each time point, and P values for a gene knockdown (aex-5 or nol-6) were calculated with respect to the control RNAi. ***P < 0.001, **P < 0.01, and *P < 0.05. n.s., non-significant.

Notes

  1. The assays are described for P. aeruginosa. However, these protocols can be used for other pathogens such as Serratia marcescens Db11.
  2. The assays are described for conditions where a gene is knocked down by RNAi. The assays can be modulated for mutant animals where RNAi is not used. For such assays, carry out the synchronization of animals on E. coli OP50 plates and prepare lawns of E. coli OP50 and P. aeruginosa PA14 for two-choice preference assays.
  3. The assays can be carried out at different levels of oxygen in a hypoxia chamber.
  4. The incubation time of plates seeded with P. aeruginosa before animal exposure is critical for the kinetics of lawn avoidance. Differences in incubation time lead to different kinetics of avoidance (Singh and Aballay, 2019b).
  5. There are several modifications of training protocol for two-choice preference assays. In some cases, training is carried out from L1 larval stage on plates that contain both E. coli and P. aeruginosa (Zhang et al., 2005).
  6. P. aeruginosa is an opportunistic pathogen, and the PA14 strain is a highly virulent clinical isolate from a human patient. Handling of P. aeruginosa PA14 should be carried out under biosafety level 2 (BSL-2) laboratory practices.

Recipes

  1. 100 mg/ml ampicillin stock
    1. Add 1 g ampicillin sodium salt into a 15 ml tube and fill dH2O up to 10 ml
    2. Stir until dissolved and filter-sterilize (0.22 μm)
    3. Store in 1 ml aliquots at -20 °C for up to 1 year
  2. 5 mg/ml cholesterol
    1. Add 500 mg cholesterol to 100 ml 95% ethanol
    2. Stir until fully dissolved and filter-sterilize (0.22 μm)
    3. Store the cholesterol solution at room temperature
  3. 1 M potassium phosphate buffer (pH 6)
    1. Add 23 g K2HPO4, 118 g KH2PO4 into a 1,000 ml graduated bottle and fill dH2O up to 1,000 ml
    2. Stir until fully dissolved
    3. Keep bottle lid loose and autoclave for 30 min at 121 °C
    4. Store at room temperature
  4. 1 M CaCl2
    1. Dissolve 11.1 g CaCl2 in 100 ml dH2O
    2. Autoclave for 30 min at 121 °C
    3. Store at room temperature
  5. 1 M MgSO4
    1. Dissolve 12.0 g MgSO4 in 100 ml dH2O
    2. Autoclave for 30 min at 121 °C
    3. Store at room temperature
  6. LB (100 ml)
    1. Add 2 g LB broth to 100 ml dH2O in a 500 ml conical flask
    2. Autoclave for 30 min at 121 °C and store at room temperature
  7. LB agar plates without ampicillin
    1. Add 10 g LB broth, 7.5 g agar to 500 ml dH2O in a 1,000 ml conical flask
    2. Autoclave for 30 min at 121 °C and cool to 55 °C
    3. Pour 25 ml each into 100 mm Petri dishes and incubate at room temperature for 2 days
    4. Store at 4 °C in a box and use for 3 months
  8. LB agar plates with ampicillin
    1. Add 10 g LB broth, 7.5 g agar to 500 ml dH2O in a 1,000 ml conical flask
    2. Autoclave for 30 min at 121 °C and cool to 55 °C
    3. Add 500 µl of 100 mg/ml ampicillin while stirring
    4. Pour 25 ml each into 100 mm Petri dishes and incubate at room temperature for 2 days
    5. Store at 4 °C in a box and use for 3 months
  9. Nematode growth medium (NGM) agar plates seeded with E. coli OP50
    1. Add 2.3 g Bacto peptone, 2.8 g NaCl, 20.4 g agar to 960 ml dH2O in a 2,000 ml conical flask
    2. Autoclave for 30 min at 121 °C and cool to 55 °C
    3. Add 25 ml of 1 M potassium phosphate buffer, 1 ml of 1 M CaCl2, 1 ml of 1 M MgSO4, and 1 ml of 5 mg/ml cholesterol while stirring
    4. Pour 8 ml each into 60 mm Petri dishes and incubate at room temperature for 3 days
    5. Inoculate a single colony of E. coli OP50 in 100 ml of LB broth in a 500 ml conical flask and incubate at 37 °C at 225 rpm shaking for 18-20 h
    6. Spot 400 µl of E. coli OP50 culture on the center of plates that were incubated at room temperature for 3 days (from step 9d)
    7. Grow E. coli OP50 on the plates for 3 days at room temperature
    8. Store the plates at 4 °C in a box and use for 3 months
  10. RNAi plates (NGM plates with 3 mM IPTG and 100 µg/ml ampicillin)
    1. Add 2.3 g Bacto peptone, 2.8 g NaCl, 20.4 g agar to 960 ml dH2O in a 2,000 ml conical flask
    2. Autoclave for 30 min at 121 °C
    3. Dissolve 715 mg IPTG in 5 ml dH2O
    4. Cool media to 55 °C while stirring
    5. Add 25 ml of 1 M potassium phosphate buffer, 1 ml of 1 M CaCl2, 1 ml of 1 M MgSO4, 1 ml of 5 mg/ml cholesterol, 5 ml of IPTG solution prepared above (from step 10c), and 1 ml of 100 mg/ml ampicillin while stirring
    6. Pour 8 ml each into 60 mm Petri dishes and incubate at room temperature for 3 days
    7. Store the plates at 4 °C in a box and use for 3 months
  11. Slow killing (SK) assay plates
    1. Add 3.2 g Bacto peptone, 3.0 g NaCl, 20 g agar to 960 ml dH2O in a 2,000 ml conical flask
    2. Autoclave for 30 min at 121 °C and cool to 55 °C
    3. Add 25 ml of 1 M potassium phosphate buffer, 1 ml of 1 M CaCl2, 1 ml of 1 M MgSO4, and 1 ml of 5 mg/ml cholesterol while stirring
    4. Pour 3.5 ml each into 35 mm Petri dishes and incubate at room temperature for 3 days
    5. Pack stacks of the plates in plastic bags, store at 4 °C in a box and use for 3 months

Acknowledgments

This work was supported by NIH grants GM0709077 and AI117911 (to A.A.). The wild type Bristol N2 strain used in this study was provided by the Caenorhabditis Genetics Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). The protocols have been adapted from previous work (Singh and Aballay, 2019b).

Competing interests

The authors declare that they have no conflicts of interest or competing interests.

References

  1. Kavaliers, M., Ossenkopp, K. P. and Choleris, E. (2019). Social neuroscience of disgust. Genes Brain Behav 18(1): e12508. 
  2. Liberati, N. T., Urbach, J. M., Miyata, S., Lee, D. G., Drenkard, E., Wu, G., Villanueva, J., Wei, T. and Ausubel, F. M. (2006). An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl Acad Sci U S A 103(8): 2833-2838.
  3. Medzhitov, R., Schneider, D. S. and Soares, M. P. (2012). Disease tolerance as a defense strategy. Science 335(6071): 936-941. 
  4. Melo, J. A. and Ruvkun, G. (2012). Inactivation of conserved C. elegans genes engages pathogen- and xenobiotic-associated defenses. Cell 149(2): 452-466. 
  5. Singh, J. and Aballay, A. (2019a). Microbial Colonization Activates an Immune Fight-and-Flight Response via Neuroendocrine Signaling. Dev Cell 49(1): 89-99 e84.
  6. Singh, J. and Aballay, A. (2019b). Intestinal infection regulates behavior and learning via neuroendocrine signaling. eLife 8: 50033. 
  7. Singh, J. and Aballay, A. (2020). Neural control of behavioral and molecular defenses in C. elegans. Curr Opin Neurobiol 62: 34-40.
  8. Tran, A., Tang, A., O'Loughlin, C. T., Balistreri, A., Chang, E., Coto Villa, D., Li, J., Varshney, A., Jimenez, V., Pyle, J., Tsujimoto, B., Wellbrook, C., Vargas, C., Duong, A., Ali, N., Matthews, S. Y., Levinson, S., Woldemariam, S., Khuri, S., Bremer, M., Eggers, D. K., L'Etoile, N., Miller Conrad, L. C. and VanHoven, M. K. (2017). C. elegans avoids toxin-producing Streptomyces using a seven transmembrane domain chemosensory receptor. eLife 6: e23770. 
  9. Wollenberg, A. C., Visvikis, O., Alves, A. F. and Irazoqui, J. E. (2013). Staphylococcus aureus killing assay of Caenorhabditis elegans. Bio-protocol 3(19): e916.
  10. Zhang, Y., Lu, H. and Bargmann, C. I. (2005). Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature 438(7065): 179-184.

简介

[摘要 ] 物理避免病原体是宿主减少病原体感染的关键防​​御策略。主持人展示了使用多种策略来感知和避免病原体,从嗅觉到感知由病原体感染引起的损害。了解避免病原体的各种机制有可能揭示保守的宿主防御反应,这些反应对病原体感染很重要。在这里,我们描述协议研究线虫秀丽隐杆线虫模型中的病原体避免草坪行为以及细菌偏好的变化的协议 。此外,我们描述了在病原菌上对动物进行训练后测量病原性和非病原性细菌偏好的协议。这些测定法可用于发现宿主学习的各种机制,从而避免病原体。

[背景 ] 一种宿主使用多个策略来抵御病原体感染(Medzhitov 等人,2012)。物理规避病原体是宿主使用的各种防御策略之一(Medzhitov 等,2012;Kavaliers 等,2019; Singh和Aballay ,2020)。在检测到细菌毒素后,不同的感觉机制(包括化学感受和伤害感受神经元引起的疼痛诱发)会导致回避行为。对导致病原体回避的机制的更深入了解,有可能揭示保守的宿主防御反应,这些反应对病原体感染很重要。

线虫秀丽隐杆线虫已被广泛用于理解病原体回避行为和相关学习。秀丽隐杆线虫似乎使用多种机制来了解导致逃避行为的诱因的病原体(Singh和Aballay ,2020年)。秀丽隐杆线虫可以感知细菌代谢产物(Tran 等,2017)以及核心细胞活动的扰动(Melo 和Ruvkun ,2012)。此外,肠道感染可以调节神经内分泌信号传导,从而引发逃避行为(Singh和Aballay ,2019a和2019b)。因此,研究秀丽隐杆线虫的病原体回避行为和相关学习可以帮助破译宿主在感测病原体中使用的各种策略。

关键字:病菌趋避, 学习, 细菌感染, 趋避行为, 防御反应

材料和试剂


 


1. 35毫米培养皿(Tritech Research,目录号:T3500)      


2. 60毫米培养皿(Tritech Research,目录号:T3315)      


3. 100毫米培养皿(Tritech Research,目录号:T3361)      


4. 14毫升聚丙烯圆底管(VWR,目录号:60819-761)      


5. 15毫升离心管(VWR,目录号:21008-216)      


6. 10毫升注射器(BD,Bacto TM ,目录号:309604)      


7. 0.2 微米的无菌针筒过滤器(VWR,目录号: 28145-501 )      


8. 大肠埃希菌OP50菌株(秀丽隐杆线虫遗传学中心(CGC))      


9. 含有对照RNAi质粒(L4440)的大肠杆菌HT115菌株(来源BioScience ,Ahringer C. elegans RNAi集合)      


10. 表达任何目的基因的dsRNA的大肠杆菌HT115菌株(来源:BioScience ,Ahringer C. elegans RNAi Collection)   


11. 铜绿假单胞菌PA14菌株(Liberati 等,2006 )。   


12. 秀丽隐杆线虫野生型布里斯托尔N2菌株和/或任何目的突变体(CGC)   


13. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Anatrace 产品,目录号:I1003 100 GM)   


14. 氯化钠(Fisher Bioreagents,目录号:BP358-1)   


15. 氯化钙(CaCl 2 )(Sigma-Aldrich,目录号:746495-500G )   


16. 无水硫酸镁(MgSO 4 )(Mallinckrodt,目录号:A31H10 )   


17. 磷酸二氢钾(KH 2 PO 4 )(Sigma-Aldrich,目录号:P0662-2.5KG)   


18. 磷酸氢二钾(K 2 HPO 4 )(Sigma-Aldrich,目录号:P3786-2.5KG)   


19. 胆固醇(Sigma-Aldrich,目录号:C8667-25G)   


20. 氨苄西林钠盐(EMD密理博,目录号:171254-25GM)   


21. Bacto 琼脂(BD,Bacto TM ,目录号:214030)   


22. Bacto 蛋白ept (BD,Bacto TM ,目录号:211677)   


23. Luria-Bertani(LB)肉汤(Sigma,目录号:L3022-1KG)   


24. 95 %乙醇(EMD密理博,目录号:EX0280-3)   


25. 5 mg / ml胆固醇(请参阅食谱)   


26. 氨苄西林原液100 mg / ml(请参阅食谱)   


27. 1 M磷酸钾缓冲液(pH 6)(请参阅食谱)   


28. 1 M CaCl 2 (请参阅食谱)   


29. 1 M MgSO 4 (请参阅食谱)   


30. LB(请参阅食谱)   


31. 含和不含氨苄青霉素的LB琼脂平板(请参阅食谱)   


32. 线虫生长培养基(NGM)琼脂板(请参阅食谱)   


33. RNAi板(请参阅食谱)   


34. 慢速杀灭(SK)检测板(请参阅食谱)   


设备


 


500 ml锥形烧瓶(VWR,目录号:89091-420)
1,000 ml锥形烧瓶(VWR,目录号:29136-106)
2,000 ml锥形烧瓶(VWR,目录号:89090-858)
移液器仪(Eppendorf ,型号小号:P20,P1000)
20℃和25 ℃下 Ç 。线虫培养箱(ThermoForma ,型号:3920)
离心机(VWR,型号:Clinical 50)
铂丝蜗杆镐(如Wollenberg 等人所述,在实验室制造,2013年)和酒精燃烧器(VWR,目录号:470199-936)
轨道振荡器(Thermo Fisher Scientific ,目录号:SK4000)
高压灭菌器(联合灭菌系统,型号:SR-24D)
立体显微镜(莱卡,米Odel等:MZ7.5)
 


软件


 


微软Ë Xcel公司
GraphPad棱镜
 


程序


 


铜绿假单胞菌草坪回避测定
 


第一天


线虫的同步
描述了针对基因被RNAi敲除的条件的测定(有关详细信息和突变体测定,请参见注释)。从大肠杆菌OP50平板转移妊娠成年N2动物到对照RNAi平板(每个RNAi平板15-20只动物)以及任何感兴趣基因的RNAi平板。每个基因敲低使用一个RNAi板。
在室温(22 °C )下孵育平板2小时。
从RNAi板上移走妊娠母,并在20 °C 下孵育板72 h,以获得同步的成年动物。
 


第二天


从-80 °C 取出一小瓶冷冻的铜绿假单胞菌PA14,然后立即在LB琼脂平板上划线。在37 °C下孵育平板12-14小时。
 


第三天


挑选一个铜绿假单胞菌PA14的菌落,接种在14 ml聚丙烯圆底试管中的2 ml LB中,垂直放在摇床上,并在250 rpm和37 °C 下生长10-12 h 。
将20 µl接种物放在改良的NGM(3.5%代替2.5%蛋白ept)平板的35 mm SK平板(图1A)的中心。让接种物在室温下干燥(20-30分钟)。将板倒置颠倒(以防止琼脂的裂纹变干),并在37 °C 下孵育12小时。
 


D:\ Reformatting \ 2020-3-2 \ 1405--2003089 Jogender Singh 847750 \ Figs jpg \图1.jpg


图1.板s的示意图。A.回避测定板的中央有一个病原菌斑点。B.二选偏好分析板包含对角相对放置的两种细菌的斑点。两项选择偏好分析板中间的叉号(× )表示秀丽隐杆线虫转移的位置。C.训练板的表面完全覆盖有病原细菌。


 


第四天


将铜绿假单胞菌板从37 °C 冷却到室温30分钟。
在SK板上的铜绿假单胞菌草坪外转移30只同步的成年动物。每个线虫菌株/条件使用三个铜绿假单胞菌板。使用铂金丝虫镐从SK板上的铜绿假单胞菌草坪外部刮去残留的大肠杆菌。
将板与动物在25 °C下孵育。
在不同的孵化时间(2、4、8、12和24 h或根据需要),计算草坪内,草坪外并在板的侧壁上爬行的动物的数量。
 


幼稚的两种选择偏好分析
第一天


按照避免草皮试验中所述同步线虫(步骤A,第1天),并在20 °C 下孵育72小时。
 


第二天


从-80 °C 取出一小瓶冷冻的铜绿假单胞菌PA14,然后立即在LB琼脂平板上划线。在37 °C下孵育平板12-14小时。
从-80 °C 在含100 µg / ml氨苄青霉素的LB琼脂平板上划线RNAi细菌(大肠杆菌HT115)。在37 °C下孵育平板12-14小时。
 


 


第三天


挑选一个铜绿假单胞菌PA14的菌落,接种在14 ml聚丙烯圆底试管中的2 ml LB中,垂直放在摇床上,并在250 rpm和37 °C 下生长10-12 h 。
挑选一个RNAi细菌菌落(大肠杆菌HT115),在15 ml管中的100 ml / ml氨苄青霉素中接种10 ml LB,水平放置在摇床上,并以250 rpm的速度培养10-12 h和37 ℃ 。
生长后,浓缩物的RNAi细菌10-20在5倍离心,000rpm下,在室温下5分钟。
将每对接种物20 µl对角地放在35 mm SK板上(图1B)。让接种物在室温下干燥(20-30分钟)。将板倒置,在37 °C 下孵育12小时。
 


第四天


将两选偏好分析板从37 °C 冷却至室温30分钟。
将30只同步成年动物转移到与两个草坪等距的两选偏好板的中心(图1B)。每个秀丽隐杆线虫菌株/条件使用3个两选偏好分析板。
使用蠕虫镐(如步骤A,第4天中所述)从两选偏好分析板的中央刮下残留的大肠杆菌。
将板与动物在25 °C下孵育。
在不同的孵化时间(2、4、8、12和24 h或根据需要),计算在大肠杆菌草坪,铜绿假单胞菌草坪,草坪外并在其上爬行的动物的数量。板的侧壁。
 


训练有素的两种选择偏好分析
第一天


按照避免草皮试验中所述同步线虫(步骤A,第1天),并在20 °C 下孵育54小时。
从-80 °C 取出一小瓶冷冻的铜绿假单胞菌PA14,然后立即在LB琼脂平板上划线。在37 °C下孵育平板12-14小时。
 


第二天


挑选一个铜绿假单胞菌PA14的菌落,接种在14 ml聚丙烯圆底试管中的2 ml LB中,垂直放在摇床上,并在250 rpm和37 °C 下生长10-12 h 。将铜绿假单胞菌板保存在4 °C下。
将20 µl接种物放在35 mm SK平板上,并将其铺在平板的整个表面上,以获得铜绿假单胞菌的完整草坪(图1C)。将板倒置,在37 °C 下孵育12小时。
从-80 °C 在含100 µg / ml氨苄青霉素的LB琼脂平板上划线RNAi细菌(大肠杆菌HT115)。在37 °C下孵育平板12-14小时。
 


第三天


在第2天从4 °C 的平板中挑选一个铜绿假单胞菌菌落,接种在14 ml聚丙烯圆底试管中的2 ml LB中,垂直放置在摇床上,使其生长10-12 h在250 rpm和37 °C下。
挑选一个RNAi细菌菌落(大肠杆菌HT115),在15 ml管中的100 ml / ml氨苄青霉素中接种10 ml LB,水平放置在摇床上,并以250 rpm的速度培养10-12 h和37 ℃ 。
冷却充分草坪绿脓杆菌p 从37拉泰什℃下至室温下至少30分钟。
将生长在20 °C的54 h老同步线虫(来自D ay 1)转移到铜绿假单胞菌的完整草坪上,并在25 °C 孵育18 h。这将构成训练有素的动物群。
对于未经处理的对照动物,将其放在相应的RNAi板上,并与训练组一起在25 °C 下孵育18 h。
如上述在纯朴的二选偏好分析(程序B,第3天)中所述,准备二选偏好板(图1B )。在VERT板倒置,并培育在37 ℃下12小时。
 


第四天


将两选偏好分析板从37 °C 冷却至室温30分钟。
将30只同步成年动物从完整的草坪绿脓杆菌板转移到与两个草坪等距的两选偏好分析板的中心。每个秀丽隐杆线虫菌株/条件使用3个两选偏好分析板。这将是训练有素的动物。
同样,将30只同步成年动物从RNAi平板转移到与两个草坪等距的两选偏好分析平板的中心。每个秀丽隐杆线虫菌株/ 条件使用3个两选偏好分析板。这将是幼稚动物的对照组。
使用蠕虫镐(如步骤A,第4天所述)从两选偏好分析板的中心刮下残留细菌。
将板与动物在25 °C下孵育。
孵育1小时后,计算在草坪外的大肠杆菌草坪,铜绿假单胞菌草坪上爬过板侧壁的动物数量。
 


数据分析


 


草坪占用率计算:计算细菌草坪内外的动物数量。计算Microsoft E xcel中的草坪占用百分比为:


 






 


其中,N on 是草坪上的动物数量,N off 是草坪外的动物数量。在计算的侧壁上爬行的动物不包括在内。使用GraphPad Prism绘制草坪占用百分比的时间过程。图2A中的代表性数据表明,具有基因aex-5 和nol -6的敲低的动物分别具有增强的和减缓的铜绿假单胞菌草坪的避免率。


 


选择指数计算:计算两种细菌性草坪上的动物数量。计算Microsoft E xcel中的铜绿假单胞菌选择指数(P. aeruginosa CI)为:


 






 


在铜绿假单胞菌CI测量动物的偏好铜绿假单胞菌具有值范围从- 1比1的值1,- 1,且0表示所有的动物都在铜绿假单胞菌,所有的动物都远离铜绿假单胞菌,并且铜绿假单胞菌和大肠杆菌上分别有相同数量的动物。在铜绿假单胞菌CI可以被绘制为单时间点(训练两选择偏好测定)或一段时间场(幼稚双选择偏好分析使用GraphPad Prism)。图2B中的代表性数据显示,已敲除aex-5 和nol -6 基因的幼稚动物分别具有从铜绿假单胞菌到大肠杆菌的优先切换,速度更快和更慢。


使用GraphPad Prism 8进行数据统计分析。合并来自三个独立实验的数据,并计算平均值和标准差(SD)。使用多个t 检验–每个时间点一个,并计算相对于对照样品的基因敲低的p值。当P <0.05 时,判断数据具有统计学意义。


 


D:\ Reformatting \ 2020-3-2 \ 1405--2003089 Jogender Singh 847750 \ Figs jpg \图2.jpg


图2.避免病原体和细菌偏好变化的时间过程。A. 铜绿假单胞菌草坪上动物的占有率的时程。B. 在两种选择偏爱分析中,幼稚动物的铜绿假单胞菌CI的时程,其中每个铜绿假单胞菌和大肠杆菌都带有一个草坪。有关aex-5 和nol -6的详细信息,请参见Singh和Aballay (2019b )。误差棒表示来自三个独立实验的SD。在每个时间点使用t 检验,并计算相对于对照RNAi 的基因敲低(aex-5 或nol -6 )的P 值。*** P <0.001,** P <0.01和* P <0.05。ns 。,无关紧要。


 


笔记


 


描述了对铜绿假单胞菌的测定。但是,这些协议可以用于其他病原体,例如粘质沙雷氏菌(Serratia marcescens)Db11。
描述了针对RNAi敲除基因的条件的测定。可以对不使用RNAi的突变动物的检测方法进行调整。对于此类检测,请在大肠杆菌OP50板上进行动物同步,并准备大肠杆菌OP50和铜绿假单胞菌PA14的草皮,以进行两项选择。
所述测定可以在低氧室中在不同的氧气水平下进行。
在动物接触之前,接种铜绿假单胞菌的平板的孵育时间对于避免草坪的动力学至关重要。潜伏时间的不同导致规避动力学的不同(Singh和Aballay ,2019b)。
两种选择偏好分析的训练方案有多种修改。在某些情况下,从L1幼虫阶段开始在含有大肠杆菌和铜绿假单胞菌的平板上进行训练(Zhang 等人,2005)。
铜绿假单胞菌是机会病原体,并且PA14菌株是来自人类患者的高毒性临床分离株。铜绿假单胞菌PA14的处理应按照2级生物安全实验室标准(BSL-2)进行。
 


菜谱


 


100 mg / ml氨苄青霉素原液
在15 ml试管中加入1 g氨苄西林钠盐,并充满dH 2 O至10 ml
搅拌直至溶解并过滤灭菌(0.22 微米)
在-20 °C下以1毫升等分试样保存长达1年
5 mg / ml胆固醇
将500毫克胆固醇添加到100毫升95%乙醇中
搅拌直至完全溶解和过滤灭菌(0.22 微米)
将胆固醇溶液储存在室温下
1 M磷酸钾缓冲液(pH 6)
添加23克ķ 2 HPO 4 ,118克KH 2 PO 4 为1 ,000毫升刻度瓶中并填充卫生署2 O最多1 ,000毫升
搅拌直至完全溶解
保持瓶盖松动并在121 °C下高压灭菌30分钟
室温保存
1 M氯化钙2
将11.1 g CaCl 2 溶于100 ml dH 2 O
在121 °C下高压灭菌30分钟
室温保存
1 M硫酸镁4
将12.0 g MgSO 4 溶于100 ml dH 2 O
在121 °C下高压灭菌30分钟
室温保存
磅(100毫升)
在500 ml锥形瓶中将2 g LB肉汤添加到100 ml dH 2 O中
在121 °C下高压灭菌30分钟,并在室温下保存
不含氨苄青霉素的LB琼脂平板
在1000毫升的锥形烧瓶中,将10克LB肉汤,7.5克琼脂添加到500毫升dH 2 O 中
在121 °C下高压灭菌30分钟,然后冷却至55 °C
将25毫升每个倒入100毫米培养皿中,并在室温下孵育2天
存放在4 °C 的盒子中,使用3个月
LB带有氨苄青霉素的琼脂平板
在1000毫升的锥形烧瓶中,将10克LB肉汤,7.5克琼脂添加到500毫升dH 2 O 中
在121 °C下高压灭菌30分钟,然后冷却至55 °C
在搅拌的同时添加500 µl 100 mg / ml氨苄西林
将25毫升每个倒入100毫米培养皿中,并在室温下孵育2天
存放在4 °C 的盒子中,使用3个月
线虫生长培养基(NGM)琼脂接种与板大肠杆菌OP50
在一个2,000 ml锥形烧瓶中,将2.3 g Bacto 蛋白ept,2.8 g NaCl,20.4 g琼脂添加到960 ml dH 2 O 中
在121 °C下高压灭菌30分钟,然后冷却至55 °C
在搅拌的同时添加25 ml 1 M磷酸钾缓冲液,1 ml 1 M CaCl 2,1 ml 1 M MgSO 4 和1 ml 5 mg / ml胆固醇
分别倒入8毫升60毫米培养皿中,并在室温下孵育3天
在500 ml锥形瓶中的100 ml LB肉汤中接种大肠杆菌OP50 的单个菌落,并在37 °C下以225 rpm的转速孵育18-20 h
点400微升大肠杆菌上在室温下温育,对于3天(从板的中心OP50培养小号TEP 9D)
生长的大肠杆菌OP50上在室温下将板3天
将板在4 °C 的盒子中存放,使用3个月
RNAi板(具有3 mM IPTG和100 µg / ml氨苄青霉素的NGM板)
在一个2,000 ml锥形烧瓶中,将2.3 g Bacto 蛋白ept,2.8 g NaCl,20.4 g琼脂添加到960 ml dH 2 O 中
在121 °C下高压灭菌30分钟
将715 mg IPTG溶于5 ml dH 2 O
搅拌下将介质冷却至55 °C
加入25毫升1M的磷酸钾缓冲液,1毫升的1M的CaCl 2 ,1毫升的1M硫酸镁4 ,1毫升5毫克/毫升胆固醇的,上面制备的5毫升的IPTG溶液(由小号TEP 10C),并1毫升100毫克/毫升氨苄青霉素,同时搅拌
分别倒入8毫升60毫米培养皿中,并在室温下孵育3天
将板在4 °C 的盒子中存放,使用3个月
慢速杀灭(SK)检测板
在一个2000毫升的锥形烧瓶中,将3.2克Bacto 蛋白ept,3.0克NaCl,20克琼脂添加到960毫升dH 2 O 中
在121 °C下高压灭菌30分钟,然后冷却至55 °C
在搅拌的同时添加25 ml 1 M磷酸钾缓冲液,1 ml 1 M CaCl 2,1 ml 1 M MgSO 4 和1 ml 5 mg / ml胆固醇
将每份3.5毫升倒入35毫米培养皿中,并在室温下孵育3天
将板叠用塑料袋包装,在4 °C 的盒子中存放并使用3个月
 


致谢


 


NIH授予GM0709077和AI117911(授予AA)支持这项工作。本研究中使用的野生型Bristol N2菌株由秀丽隐杆线虫遗传学中心(CGC)提供,该中心由NIH研究基础设施计划办公室(P40 OD010440)资助。该协议已根据先前的工作进行了改编(Singh和Aballay ,2019b)。


 


利益争夺


 


作者宣称他们没有利益冲突或利益冲突。


 


参考文献


 


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Copyright Singh and Aballay. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Singh, J. and Aballay, A. (2020). Bacterial Lawn Avoidance and Bacterial Two Choice Preference Assays in Caenorhabditis elegans. Bio-protocol 10(10): e3623. DOI: 10.21769/BioProtoc.3623.
  2. Singh, J. and Aballay, A. (2019b). Intestinal infection regulates behavior and learning via neuroendocrine signaling. eLife 8: 50033. 
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