TRPM channels mediate learned pathogen avoidance following intestinal distention
- Department of Molecular Microbiology & Immunology, Oregon Health & Science University, United States
Figures
Figure 1 with 1 supplement
E. faecalis elicits fast avoidance in C. elegans.
(A) Schematic of avoidance assays (top) and representative photomicrographs (bottom) of C. elegans on lawns of E. coli OP50, P. aeruginosa PA14, or E. faecalis OG1RF at 0 hr (left) and 4 hr (right) on BHI media. Individual animals are outlined in red. Scale bar, 5 mm. (B) Occupancy index of N2 animals after 4 hr of incubation on E. coli, P. aeruginosa, E. faecalis, E. faecium, or S. aureus. One-way ANOVA with subsequent comparison to E. coli as the control group was performed. Occupancy Index = (number of animals on bacterial lawn)/(total number of animals). (C) Individual animals were tracked on lawns of E. faecalis and the time that they first left the lawn after transfer was recorded. Wild-type (WT, N2 Bristol) animals showed an average first leave time of 19.20 min while tax-2(p671) animals showed an average first leave time of 61.98 min. An unpaired t-test between the groups was performed. (D) N2 animals on standard avoidance assays plates with E. faecalis were observed for 10 min during two different time windows (0–1 hr and 3–4 hr) and the number of times the animals left and entered the bacteria lawns was counted. One-way ANOVA with subsequent comparisons between all groups was performed. (E) Occupancy index of N2 animals after 24 hr incubation on P. aeruginosa PA14 lawns or E. coli OP50 lawns supplemented with isolated total RNA from E. coli (E.c. RNA), P. aeruginosa (P.a. RNA), or E. faecalis (E.f. RNA). One-way ANOVA with subsequent comparison to E. coli RNA as the control group was performed.
Figure 1—figure supplement 1
Avoidance of Gram-positive pathogens.
(A) Occupancy index time course of N2 animals on either E. faecalis, E. faecium, or S. aureus. N = 3 for each point. Points represent the mean. (B) Response index of N2 animals to dry drops of E. coli, E. faecalis, E. faecium, BHI, LB, or 0.6 mM SDS buffer. Response Index = number of responses/total number of drops.
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Figure 1—figure supplement 1—source data 1
Raw data for Figure 1—figure supplement 1A.
- https://cdn.elifesciences.org/articles/65935/elife-65935-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 2 supplements
Anterior intestinal distention elicits avoidance of E. faecalis.
(A) Representative photomicrographs of animals at 4 hr on BHI media with E. coli (left), P. aeruginosa (middle), or E. faecalis (right). Red arrows point to the borders of the anterior intestine while blue arrows point the borders of the medial intestine. (B) Quantification of the anterior (top) and medial (bottom) intestinal lumen diameter of animals on E. coli, P. aeruginosa, or E. faecalis at 1, 2, and 4 hr. Two-way ANOVA was performed with comparison to the E. coli group at each time point. For E. coli, N = 11, 13, and 17 at 1, 2, and 4 hr, respectively. For P. aeruginosa, N = 18, 14, and 18. For E. faecalis, N = 13, 18, and 16. Points are the mean of each group and error bars are standard deviation. (C) Representative photomicrographs with fluorescent and brightfield images merged of E. coli, P. aeruginosa, and E. faecalis expressing GFP in the intestinal lumen of animals at 4 hr. (D) Occupancy index on E. faecalis at 4 hr of WT animals compared to either RNAi-mediated knockdown of nol-6 or a loss-of-function mutation in nol-6. One-way ANOVA was performed with subsequent comparison to the WT group as the control. (E) A representative photomicrograph of a nol-6(ac1) animal after exposure to E. faecalis for 4 hr. Red arrows point to the borders of the anterior intestine, and blue arrows point to the borders of the medial intestine. (F) Quantification of anterior and medial intestinal lumen diameter of nol-6(ac1) animals at 0 and 4 hr on E. faecalis. Two-way ANOVA was performed with comparison to the 0 hr groups for both anterior and medial quantifications.
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Figure 2—source data 1
Raw data for Figure 2B.
- https://cdn.elifesciences.org/articles/65935/elife-65935-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Animals evacuating E. faecalis lawns show significant anterior intestinal distention.
(A) Representative photomicrographs of wild-type animals taken from inside (top) or outside (bottom) a lawn of E. faecalis. Outside animals were picked immediately after they left the lawn, with animals inside the lawn picked at the same time. Scale bar, 50 μm. (B) Quantification of anterior intestinal diameter of animals as represented in (A). An unpaired t-test was performed.
Figure 2—figure supplement 2
E. faecalis but not P. aeruginosa causes anterior intestinal distention in nol-6 animals.
(A) Quantification of anterior and medial intestinal lumen diameter of nol-6(ac1) animals after 0 and 24 hr on P. aeruginosa. (B) Occupancy index of WT and nol-6(ac1) animals on P. aeruginosa at 24 hr. An unpaired t-test was performed. (C) Quantification of anterior intestinal lumen diameter of nol-6 RNAi animals at 0 and 4 hr on E. faecalis. An unpaired t-test was performed. (D) Quantification of medial intestinal lumen diameter of nol-6 RNAi animals at 0 and 4 hr on E. faecalis. An unpaired t-test was performed.
Figure 3 with 1 supplement
Avoidance of E. faecalis is independent of virulence.
(A) Occupancy index of N2 animals on virulent E. faecalis OG1RF and avirulent E. faecalis OG1RF ΔfsrB at 4 hr. An unpaired t-test was performed. (B) Quantification of anterior intestinal diameter of N2 animals on E. faecalis strain OG1RF or OG1RF ΔfsrB at 4 hr. An unpaired t-test was performed. (C) Representative fluorescent micrographs of clec-60p::GFP (top) and ilys-3p::GFP (bottom) animals at 0, 4, and 24 hr on E. faecalis. Scale bar, 200 µm. (D) Quantification of clec-60::GFP at 0, 4, and 24 hr on E. faecalis. One-way ANOVA was performed with comparisons to the 0 hr group as the control. (E) Quantification of ilys-3p::GFP at 0, 4, and 24 hr on E. faecalis. One-way ANOVA was performed with comparisons to the 0 hr group as the control.
Figure 3—figure supplement 1
Loss of key immune genes does not affect avoidance of E. faecalis.
Occupancy index for wild-type, pmk-1(km25), fshr-1(ok778), and bar-1(ga80) on E. faecalis at 4 hr. One-way ANOVA with comparison to the WT group was performed.
Figure 4 with 1 supplement
TAX-2/4 pathways regulate avoidance of E. faecalis and P. aeruginosa.
(A) Occupancy index of WT animals compared to npr-1(ad609) and npr-1(ok1447) after 4 hr on E. faecalis. One-way ANOVA with subsequent comparison to WT animals as the control group was performed. (B) Occupancy index of WT, npr-1(ad609) and npr-1(ok1447) on lawns of E. faecalis in atmospheric oxygen or a chamber containing 8% oxygen. Two-way ANOVA with subsequent comparison to WT animals of each respective oxygen condition was performed. (C) Occupancy index of WT, npr-1(ad609), and npr-1(ok1447) in an 8% oxygen chamber at 1, 2, 3, and 4 hr, and 1 hr after removal from the chamber (vertical dashed line). Two-way ANOVA with subsequent comparison at each time point to WT animals was performed. N = 9 for all animals. Points are the mean of each group and error bars are standard deviation. (D) Occupancy index for WT and double loss-of-function mutants for gcy-35, tax-2, or tax-4, and npr-1 on E. faecalis for 4 hr and P. aeruginosa for 24 hr. Two-way ANOVA with subsequent comparisons to WT animals for each bacterium were performed. (E) Diagram of the neurons affected by tax-2(p694) and tax-4(p678). The latter allele covers all tax-2 expressing sensory neurons, while the former covers a subset. (F) Occupancy index for WT and single loss-of-function mutants for tax-2 or tax-4 on E. faecalis for 4 hr and P. aeruginosa for 24 hr. Two-way ANOVA as in with subsequent comparisons to WT animals for each bacterium were performed.
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Figure 4—source data 1
Raw data for Figure 4C.
- https://cdn.elifesciences.org/articles/65935/elife-65935-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
The TRPV subunits OCR-2 and OSM-9 slightly speed up avoidance of E. faecalis.
(A) Occupancy index of WT animals compared to ocr-2(ak47) and osm-9(yz6) animals on E. faecalis at 4 hr. One-way ANOVA with subsequent comparison to WT animals was performed. (B) Occupancy index time course for WT animals compared to ocr-2(ak47) and osm-9(yz6) on E. faecalis. Two-way ANOVA with comparison to WT animals at each time point was performed. N = 9 for each point. Points represent the mean and error bars are the standard deviation.
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Figure 4—figure supplement 1—source data 1
Raw data for Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/65935/elife-65935-fig4-figsupp1-data1-v2.xlsx
Figure 5
ASE, AWB, and AWC neurons mediate avoidance of E. faecalis.
(A) Occupancy index on E. faecalis at 4 hr for WT, tax-2(p694) and animals with tax-2 expression in ASE neurons (flp-6p::tax-2) or AQR, PQR, or URX neurons (gcy-32p::tax-2) in the tax-2(p694) background. One-way ANOVA with subsequent comparison to wild-type (WT) animals was performed. (B) Occupancy index of WT animals and animals with ablated neurons on E. faecalis at 4 hr and P. aeruginosa at 24 hr. Ablation of sensory neurons either by mutation (che-1(p680) = ASE(−)) or by caspase expression (sra-6p::mCasp-1=ASH(−); gpa-4p::TU#813 + gcy-27p::TU#814 = ASI(−); str-1p::mCasp-1 = AWB(−); ceh-36p::TU#813 + ceh-36p::TU#814 = AWC(−)). Two-way ANOVA with subsequent comparisons to WT groups for each respective bacterium were performed. (C) Occupancy index at 1 hr for WT and ASI(−) animals on P. aeruginosa lawns. Animals were trained on E. coli OP50 lawns supplemented with either E. coli or P. aeruginosa RNA for 24 hr. Two-way ANOVA with subsequent comparison with WT control groups was performed. (D) Choice index after 1 hr for WT and ASI(−) animals choosing between E. coli and P. aeruginosa lawns. Two-way ANOVA with subsequent comparison with WT control groups was performed. Choice Index = (number of animals on E. coli – number of animals on P. aeruginosa)/(number of animals on E. coli + number of animals on P. aeruginosa). (E) Model for avoidance of P. aeruginosa (top) and E. faecalis (bottom) Avoidance of P. aeruginosa depends on both an ASI neuron-mediated bacterial sRNA pathway along with intestine-wide distention. The latter requires the NPR-1-dependent hyperoxia avoidance pathway along with AWB and AWC olfactory neurons. Avoidance of E. faecalis also depends on intestinal distention, though this is confined to the anterior intestine. This anterior intestinal distention-induced avoidance also requires AWB and AWC olfactory neurons, but also requires ASE chemosensory neurons. NPR-1-dependent hyperoxia avoidance opposes E. faecalis avoidance and depends on functional TAX-2/4.
Figure 6
AWB and AWC neurons are necessary for aversive olfactory learning following ingestion of E. faecalis.
(A) Schematic of the choice assay (top) and quantification (bottom) of choice index for N2 animals choosing between E. coli and E. faecalis lawns and either free to move for 1 hr (Free to move) or paralyzed upon arrival at a bacterial lawn (Paralyzed). An unpaired t-test between the groups was performed. (B) Schematic of the lid choice assay (top) and quantification of naïve and trained choice index (bottom, left) and learning index (bottom, right) for WT, tax-4(p678), tax-2(p671), tax-2(p694), AWB(−), and AWC(−) animals. To train animals, young adult animals were placed on E. faecalis lawns for 4 hr before the choice assay was performed. For choice index, two-way ANOVA with subsequent comparisons to the naïve and trained WT groups as controls were performed. For learning index, one-way ANOVA with comparison to the WT group as control was performed. (C) Schematic of the paired olfactory choice assay. (D) Quantification of choice index for naïve and trained WT, AWC(−), and AWB(−) animals, choosing between 1:200 benzaldehyde and ethanol. Two-way ANOVA with subsequent comparisons between naïve and trained groups and between WT and neuron ablated animals was performed. (E) Quantification of the learning index for WT, AWC(−), and AWB(−) animals from (D). One-way ANOVA with comparison to the WT group as control was performed. Learning index = (naïve choice index) – (trained choice index).
Figure 7 with 2 supplements
The TRPM channels GON-2 and GTL-2 are required for distention-induced pathogen avoidance.
(A) Occupancy index for wild-type, gtl-2(n2618), gon-2(q362), gon-2(q388), and glp-1(e2141) animals on E. faecalis at 4 hr or P. aeruginosa at 24 hr. Animals were grown to the young adult stage at 15°C. Two-way ANOVA with comparisons to the respective WT group for each bacterium were performed. (B) Occupancy index for wild-type, gtl-2(n2618), gon-2(q362), gon-2(q388), and glp-1(e2141) animals on E. faecalis at 4 hr or P. aeruginosa at 24 hr. Animals were grown to the young adult stage at 25°C. Two-way ANOVA with comparisons to the respective WT group for each bacterium were performed. (C) Occupancy index for wild-type, gon-2(q388), self-promoter rescue (gon-2(q388);gon-2p::gon-2), and intestine specific rescue (gon-2(q388);ges-1p::gon-2) animals on E. faecalis at 4 hr. One-way ANOVA with comparison to the wild-type group as control was performed. (D) Occupancy index for wild-type, gtl-2(tm1463), self-promoter rescue (gtl-2(tm1463);gtl-2p::gtl-2), and excretory cell specific rescue (gtl-2(tm1463);sulp-4p::gtl-2) animals on E. faecalis at 4 hr. One-way ANOVA with comparison to the wild-type group as control was performed.
Figure 7—figure supplement 1
Screening TRP channels for effect on avoidance of E. faecalis.
Occupancy index of WT animals on E. faecalis at 4 hr compared to five different TRP channel mutants: gon-2(q362), gon-2(q388), gtl-2(n2618), and gtl-2(tm1643). One-way ANOVA with subsequent comparison to WT animals was performed.
Figure 7—figure supplement 2
Rescue of gon-2 and gtl-2 expression in mutant backgrounds.
Representative micrographs of each rescue strain are shown. Top left: gon-2(q388); gon-2p::gon-2(cDNA)::SL2::GFP. Bottom left: gon-2(388);ges-1p::gon-2(cDNA)::SL2::GFP. Top right: gtl-2(tm1463);gtl-2p::gtl-2(cDNA)::SL2::GFP. Bottom right: gtl-2(tm1463);sulp-4p::gtl-2(cDNA)::SL2::GFP. Scale bars in order: 50 μm, 50 μm, 50 μm, and 10 μm.
Figure 8 with 1 supplement
GON-2 and GTL-2 diminish olfactory aversive learning following E. faecalis exposure.
(A) Occupancy index for WT, gon-2(q388), and gtl-2(n2618) animals with either RNAi-mediated knockdown of aex-5 or an empty vector (EV) control on the E. coli HT115 lawns these animals were raised on. Two-way ANOVA with subsequent comparison between all groups was performed. (B) Occupancy index for WT, gon-2(q388), and gtl-2(n2618) animals with either RNAi-mediated knockdown of aex-5 or an EV control on E. faecalis lawns at 4 hr. Two-way ANOVA with subsequent comparison between all groups was performed. (C) Choice index for wild-type, gtl-2(n2618), gtl-2(tm1463), gon-2(q362), and gon-2(q388) animals between E. faecalis and E. coli using the lid choice assay. Two-way ANOVA with comparison to the naïve and trained WT group was performed. (D) Quantification of learning index for animals from (C). One-way ANOVA with comparison to the WT group was performed. (E) Model for avoidance of P. aeruginosa (top) and E. faecalis (bottom), as in Figure 5E, with the addition of a germline role in avoidance of P. aeruginosa, GON-2 and GTL-2 regulation of avoidance of E. faecalis, and the contribution of odor sensing pathways to avoidance of both bacteria.
Figure 8—figure supplement 1
Loss of GON-2 or GTL-2 function does not affect anterior intestinal distention on E. faecalis.
(A–C) Representative photomicrographs of the anterior intestines of gtl-2(n2618), gtl-2(tm1463), and gon-2(q388) animals after 4 hr on E. faecalis. Scale bar, 50 μm. (D) Quantification of the anterior intestinal diameters of animals represented in (A–C). One-way ANOVA with subsequent comparison to WT animals was performed.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | OP50 | Caenorhabditis Genetics Center (CGC) | OP50 | |
| Strain, strain background (E. coli) | OP50-GFP | CGC | OP50-GFP | |
| Strain, strain background (E. coli) | HT115 | Source BioScience | HT115 | |
| Strain, strain background (Pseudomonas aeruginosa) | PA14 | Frederick M. Ausubel laboratory | PA14 | |
| Strain, strain background (P. aeruginosa) | PA14-GFP | Frederick M. Ausubel laboratory | PA14-GFP | |
| Strain, strain background (Enterococcus faecalis) | OG1RF | Danielle A. Garsin laboratory | OG1RF | |
| Strain, strain background (E. faecalis) | OG1RF-GFP | Danielle A. Garsin laboratory | OG1RF-GFP | |
| Strain, strain background (E. faecalis) | OG1RF ΔfsrB | Danielle A. Garsin laboratory | OG1RF ΔfsrB | |
| Strain, strain background (E. faecium) | E007 | Danielle A. Garsin laboratory | E007 | |
| Strain, strain background (Staphylococcus aureus) | NCTC8325 | National Collection of Type Cultures | NCTC8325 | |
| Strain, strain background (Caenorhabditis elegans) | N2 Bristol | CGC | N2 | |
| Strain, strain background (C. elegans) | npr-1(ad609) | CGC | DA609 | |
| Strain, strain background (C. elegans) | npr-1(ok1447) | CGC | RB1330 | |
| Strain, strain background (C. elegans) | ocr-2(ak47) | CGC | CX4544 | |
| Strain, strain background (C. elegans) | osm-9(ky10) | CGC | CX10 | |
| Strain, strain background (C. elegans) | gcy-35(ok769); npr-1(ad609) | Aballay laboratory | ||
| Strain, strain background (C. elegans) | tax-2(p694); npr-1(ad609) | Aballay laboratory | ||
| Strain, strain background (C. elegans) | tax-4(p678); npr-1(ad609) | Aballay laboratory | ||
| Strain, strain background (C. elegans) | tax-2(p671) | CGC | PR671 | |
| Strain, strain background (C. elegans) | tax-2(p694) | CGC | PR694 | |
| Strain, strain background (C. elegans) | tax-4(p678) | CGC | PR678 | |
| Strain, strain background (C. elegans) | tax-2(p694); lin-15 and lin-15A(n765); dbEx723[gcy-32p::tax-2(cDNA)::SL2::GFP + lin-15(+)] | CGC | AX2159 | |
| Strain, strain background (C. elegans) | tax-2(p694); lin-15 and lin-15A(n765); dbEx724[flp-6p::tax-2(cDNA)::SL2::GFP + lin-15(+)] | CGC | AX2161 | |
| Strain, strain background (C. elegans) | che-1(p680) | CGC | PR680 | ASE(−) |
| Strain, strain background (C. elegans) | peIs1713 [sra-6p::mCasp-1+unc-122p::mCherry] | CGC | JN1713 | ASH(−) |
| Strain, strain background (C. elegans) | oyIs84 [gpa-4p::TU#813 + gcy-27p::TU#814 + gcy-27p::GFP + unc-122p::DsRed] | CGC | PY7505 | ASI(−) |
| Strain, strain background (C. elegans) | peIs1715 [str-1p::mCasp-1+unc-122p::GFP] | CGC | JN1715 | AWB(−) |
| Strain, strain background (C. elegans) | oyIs85 [ceh-36p::TU#813 + ceh-36p::TU#814 + srtx-1p::GFP + unc-122p::DsRed] | CGC | PY7502 | AWC(−) |
| Strain, strain background (C. elegans) | agIs26 [clec-60p::GFP + myo-2p::mCherry] | CGC | JIN810 | |
| Strain, strain background (C. elegans) | unc-119(ed3); eEx650 [ilys-3p::GFP + unc-119(+)] | CGC | CB6710 | |
| Strain, strain background (C. elegans) | pmk-1(km25) | CGC | KU25 | |
| Strain, strain background (C. elegans) | fshr-1(ok778) | CGC | RB911 | |
| Strain, strain background (C. elegans) | bar-1(ga80) | CGC | EW15 | |
| Strain, strain background (C. elegans) | nol-6(ac1) | CGC | AY1 | |
| Strain, strain background (C. elegans) | gtl-2(n2618) | CGC | CZ9957 | |
| Strain, strain background (C. elegans) | gon-2(q362) | CGC | EJ26 | |
| Strain, strain background (C. elegans) | gon-2(q388) | CGC | EJ1158 | |
| Strain, strain background (C. elegans) | glp-1(e2141) | CGC | CB4037 | |
| Strain, strain background (C. elegans) | gtl-1(ok375) | CGC | VC244 | |
| Strain, strain background (C. elegans) | gtl-2(tm463) | CGC | LH202 | |
| Strain, strain background (C. elegans) | trpa-1(ok999) | CGC | RB1052 | |
| Strain, strain background (C. elegans) | trpa-2(ok3189) | CGC | TQ233 | |
| Strain, strain background (C. elegans) | gon-2(q388); gon-2p::gon-2(cDNA)::SL2::GFP | This Study | AY157 | gon-2 expression, own promoter |
| Strain, strain background (C. elegans) | gon-2(q388); ges-1p::gon-2(cDNA)::SL2::GFP | This Study | AY158 | gon-2 expression, intestine specific promoter |
| Strain, strain background (C. elegans) | gtl-2(n2618); gtl-2p::gtl-2(cDNA)::SL2::GFP | This Study | AY159 | gtl-2 expression, own promoter |
| Strain, strain background (C. elegans) | gtl-2(n2618); sulp-4p::gtl-2(cDNA)::SL2:GFP | This Study | AY160 | gtl-2 expression excretory cell promoter |
| Software, algorithm | GraphPad Prism 8 | GraphPad Software | https://www.graphpad.com/scientific-software/prism/ | |
| Software, algorithm | ImageJ | NIH | https://imagej.nih.gov/ij/ | |
| Software, algorithm | Leica LAS v4.6 | Leica | https://www.leica-microsystems.com/ | |
| Other | Hypoxia chamber | STEMCELL Technologies | CAT# 27310 |
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TRPM channels mediate learned pathogen avoidance following intestinal distention
eLife 10:e65935.
https://doi.org/10.7554/eLife.65935
