Calcineurin knockdown enhances C. elegans susceptibility to Pseudomonas aeruginosa

(A) Representative survival plots of N2 and tax-6(p675) animals on P. aeruginosa PA14 at 25°C. The animals were grown on E. coli OP50 at 20°C until 1-day-old adults before transferring to P. aeruginosa PA14 at 25°C. p<0.001

(B) Representative survival plots of N2 animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control and tax-6 RNAi. p<0.001.

(C) Representative survival plots of tax-6(p675) animals on P. aeruginosa PA14 at 25°C after treatment with the EV control and tax-6 RNAi. n.s., nonsignificant.

(D) Representative survival plots of N2 animals grown on bacteria for RNAi against tax-6 along with the EV control at 20°C. Day 0 represents young adults. p<0.001.

(E) Representative survival plots of fer-1(b232) animals on P. aeruginosa PA14 at 25°C after treatment with the EV control and tax-6 RNAi at 25°C. p<0.001.

(F) Representative survival plots of fer-1(b232) animals grown on bacteria for RNAi against tax-6 along with the EV control at 25°C. The animals were developed at 25°C. Day 0 represents young adults. p<0.001.

For all panels, n = 3 biological replicates; animals per condition per replicate > 90.

Calcineurin is required for the C. elegans defecation motor program (DMP)

(A) Representative fluorescence (top) and the corresponding brightfield (bottom) images of N2 animals incubated on P. aeruginosa-GFP for 6 hours at 25°C after growth on the empty vector (EV) control and tax-6 RNAi bacteria.

(B) Colony-forming units (CFU) per animal of N2 worms incubated on P. aeruginosa-GFP for 6 hours at 25°C after growth on the EV control and tax-6 RNAi bacteria. **p < 0.01 via the t-test (n = 3 biological replicates).

(C) Representative fluorescence (top) and the corresponding brightfield (bottom) images of N2 and tax-6(p675) animals incubated on P. aeruginosa-GFP for 6 hours at 25°C after growth on E. coli OP50 at 20°C.

(D) CFU per animal of N2 and tax-6(p675) worms incubated on P. aeruginosa-GFP for 6 hours at 25°C after growth on E. coli OP50 at 20°C. *p < 0.05 via the t-test (n = 3 biological replicates).

(E) Representative photomicrographs of N2 animals grown on the EV control and tax-6 RNAi bacteria at 20°C until 1-day-old adults. Arrows point to the border of the intestinal lumen.

(F) Quantification of the diameter of the intestinal lumen of N2 animals grown on the EV control and tax-6 RNAi bacteria at 20°C until 1-day-old adults. ***p<0.001 via the t-test (n = 21 worms each).

(G) The number of expulsion events observed in 20 minutes in 1-day-old adult N2 animals grown on the EV control and tax-6 RNAi bacteria at 20°C. ***p<0.001 via the t-test (n = 6 worms each).

(H) Percent of 1-day-old adult worms having irregular DMP. ***p<0.001 via the t-test (n = 4 biological replicates).

Calcineurin knockdown enhances lifespan via DMP defects-mediated calorie restriction

(A) Representative photomicrographs of oil-red-O (ORO) stained 1-day-old adult N2 animals grown on the empty vector (EV) control and tax-6 RNAi bacteria at 20°C. Scale bar = 200 µm.

(B) Quantification of ORO intensity per animal of 1-day-old adult N2 animals grown on the EV control and tax-6 RNAi bacteria at 20°C. ***p<0.001 via the t-test (n = 10 worms each).

(C) Quantitative reverse transcription-PCR (qRT-PCR) for pha-4 mRNA levels in N2 animals grown on the EV control and tax-6 RNAi bacteria at 20°C until 1-day-old adults. ***p < 0.001 (n = 3 biological replicates).

(D) Representative survival plots of eat-2(ad465) animals grown on the bacteria for RNAi against tax-6 along with the EV control at 20°C. Day 0 represents young adults. n.s., nonsignificant (n = 3 biological replicates; animals per condition per replicate > 90).

(E-I) Representative survival plots of aak-2(ok524) (E), raga-1(ok386) (F), rsks-1(ok1255) (G), daf-16(mu86) (H), and nhr-49(nr2041) (I) animals grown on the bacteria for RNAi against tax-6 along with the EV control at 20°C. Day 0 represents young adults. p<0.001 for all the plots (n = 3 biological replicates; animals per condition per replicate > 80).

Calcineurin inhibition enhances lifespan via HLH-30 and NHR-8

(A) Representative survival plots of hlh-30(tm1978) animals grown on the bacteria for RNAi against tax-6 along with the empty vector (EV) control at 20°C. Day 0 represents young adults. n.s., nonsignificant (n = 3 biological replicates; animals per condition per replicate > 90).

(B) Representative photomicrographs of oil-red-O (ORO) stained 1-day-old adult hlh-30(tm1978) animals grown on the EV control and tax-6 RNAi bacteria at 20°C. Scale bar = 200 µm.

(C) Quantification of ORO intensity per animal of 1-day-old adult hlh-30(tm1978) animals grown on the EV control and tax-6 RNAi bacteria at 20°C. n.s., nonsignificant via the t-test (n = 10 worms each).

(D) Representative survival plots of nhr-8(ok186) animals grown on the bacteria for RNAi against tax-6 along with the EV control at 20°C. Day 0 represents young adults. n.s., nonsignificant (n = 3 biological replicates; animals per condition per replicate > 90).

(E) Representative survival plots of daf-9(rh50) animals grown on the bacteria for RNAi against tax-6 along with the EV control at 20°C. Day 0 represents young adults. p<0.001 (n = 3 biological replicates; animals per condition per replicate > 80).

(F) Representative photomicrographs of ORO stained 1-day-old adult nhr-8(ok186) animals grown on the EV control and tax-6 RNAi bacteria at 20°C. Scale bar = 200 µm.

(G) Quantification of ORO intensity per animal of 1-day-old adult nhr-8(ok186) animals grown on the EV control and tax-6 RNAi bacteria at 20°C. ***p<0.001 via the t-test (n = 10 worms each).

(H) Quantitative reverse transcription-PCR (qRT-PCR) for nhr-8 mRNA levels in N2 animals grown on the EV control, tax-6, flr-1, nhx-2, and pbo-1 RNAi bacteria at 20°C until 1-day-old adults. ***p < 0.001 via the t-test (n = 3 biological replicates).

(I) Model depicting the mechanism of increased lifespan and enhanced susceptibility to pathogen upon inhibition of calcineurin via the defects in the defecation motor program (DMP).

Calcineurin knockdown enhances C. elegans susceptibility to Pseudomonas aeruginosa

Representative survival plots of N2 animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control, tax-6, crtc-1, and crh-1 RNAi.. p<0.001 for tax-6 and p<0.01 for crtc-1 and crh-1 compared to EV (n = 3 biological replicates; animals per condition per replicate > 90).

Calcineurin inhibition enhances susceptibility to P. aeruginosa independent of known immunity pathways

(A-E) Representative survival plots of pmk-1(km25) (A), kgb-1(km21) (B), dbl-1(nk3) (C), zip-2(ok3730) (D), and daf-16(mu86) (E) animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control and tax-6 RNAi. p<0.001 for all the plots (n = 3 biological replicates; animals per condition per replicate > 90).

Calcineurin inhibition leads to defects in the defecation motor program (DMP) without affecting the pharyngeal pumping rate

(A) Pharyngeal pumps per 30 seconds of 1-day-old adult N2 animals grown on the empty vector (EV) control and tax-6 RNAi bacteria at 20°C. n.s., nonsignificant via the t-test (n = 20 worms each).

(B) The number of expulsion events observed in 20 minutes in 1-day-old adult N2 and regular and irregular tax-6(p675) animals grown on E. coli OP50 at 20°C. ***p<0.001, n.s., nonsignificant via the t-test (n = 6 worms each).

Calcineurin inhibition leads to defects in the defecation motor program (DMP) in hlh-30(tm1978) animals

(A) The number of expulsion events observed in 20 minutes in 1-day-old adult hlh-30(tm1978) animals grown on the empty vector (EV) control and tax-6 RNAi bacteria at 20°C. ***p<0.001 via the t-test (n = 5 worms each).

(B) Representative photomicrographs of hlh-30(tm1978) animals grown on the EV control and tax-6 RNAi bacteria at 20°C until 1-day-old adults. Arrows point to the border of the intestinal lumen.

(C) Quantification of the diameter of the intestinal lumen of hlh-30(tm1978) animals grown on the EV control and tax-6 RNAi bacteria at 20°C until 1-day-old adults. ***p<0.001 via the t-test (n = 13 worms each).

Primers used in the study