Calcineurin is a highly conserved calcium/calmodulin-dependent serine/threonine protein phosphatase with diverse functions. Inhibition of calcineurin is known to enhance Caenorhabditis elegans lifespan via multiple signaling pathways. Aiming to study the role of calcineurin in regulating innate immunity, we discover that calcineurin is required for the rhythmic defecation motor program (DMP) in C. elegans. Calcineurin inhibition leads to defects in the DMP, resulting in intestinal bloating, rapid colonization of the gut by bacteria, and increased susceptibility to bacterial infection. We demonstrate that intestinal bloating by calcineurin inhibition mimics calorie restriction that results in enhanced lifespan. The TFEB ortholog, HLH-30, is required for calcineurin inhibition-mediated lifespan enhancement by triggering lipolysis. Finally, we show that the nuclear hormone receptor, NHR-8, is upregulated by calcineurin inhibition and is required for increased lifespan. Our studies uncover a role for calcineurin in the C. elegans DMP and provide a new mechanism for calcineurin inhibition-mediated longevity extension.
This useful study has the potential to reveal insights into how calcineurin influences C. elegans lifespan through its role in controlling the defecation motor program. Currently, the evidence in support of the conclusions is still incomplete, largely due to concerns about partial gene inactivation by RNAi. The inclusion of experiments using a tax-6 null allele would mitigate these concerns.
Interventions that enhance lifespan also impart resistance to multiple stresses (Johnson et al., 2001). Indeed, the positive correlation between improved stress resistance and enhanced lifespan has been exploited to identify long-lived mutants (Castro et al., 2004; Denzel et al., 2014; Johnson et al., 2001; Muñoz and Riddle, 2003; Wang et al., 2004). Among stress responses, innate immunity appears to be a crucial factor for enhanced lifespan (Campos et al., 2021; Fabian et al., 2021; Soo et al., 2023; Xia et al., 2019). However, the correlation between innate immunity and lifespan is not always positive. Interventions that alter lifespan may not modulate innate immunity, and vice-versa (Labed et al., 2018; Naim et al., 2021; Otarigho and Aballay, 2020; Sun et al., 2011). Some signaling pathways also establish a tradeoff between innate immunity and lifespan. Mutants that have improved immunity but reduced lifespan have been identified (Amrit et al., 2019; Otarigho and Aballay, 2021; Ren and Ambros, 2015). Conversely, mutants with enhanced lifespans but declined immune responses have also been discovered (Kawli et al., 2010). Moreover, the genetic pathways for lifespan and immunity could be uncoupled in mutants exhibiting enhanced lifespan and improved immune responses (Alper et al., 2010; Guerrero et al., 2021). Therefore, the relationship between lifespan and innate immunity appears to be complex and remains to be fully understood.
Calcineurin, a conserved protein from yeast to humans, is a calcium/calmodulin-dependent serine/threonine protein phosphatase that is involved in diverse cellular processes and signal transduction pathways (Chen et al., 2022; Hogan et al., 2003; Schulz and Yutzey, 2004; Ulengin-Talkish and Cyert, 2023). Dephosphorylation of substrate proteins by calcineurin affects several cellular pathways, including transcriptional signaling programs (Ulengin-Talkish and Cyert, 2023). Calcineurin regulates the activity of the transcription factors of the nuclear factor of activated T cells (NFAT) family (Hogan et al., 2003). Dephosphorylation of NFATs by calcineurin triggers their nuclear localization and activates immune responses in vertebrates (Herbst et al., 2015; Hogan et al., 2003; Vandewalle et al., 2014). In the nematode Caenorhabditis elegans, calcineurin regulates thermotaxis, body size, fertility, and lifespan (Bandyopadhyay et al., 2002; Dong et al., 2007; Kuhara et al., 2002; Lee et al., 2013). Knockdown of the catalytic subunit of calcineurin, tax-6, is known to enhance C. elegans lifespan via multiple pathways, including autophagy and CREB-regulated transcriptional coactivators (CRTCs) (Dong et al., 2007; Dwivedi et al., 2009; Mair et al., 2011; Tao et al., 2013). However, the role of calcineurin in regulating C. elegans response to pathogen infections has not been studied. Because C. elegans lacks the NFAT transcription factors (Song et al., 2013), it will be intriguing to study how calcineurin inhibition impacts C. elegans innate immunity. These studies could also shed some light on the complex interplay between lifespan and immunity.
In this study, we examined the effect of calcineurin inhibition on C. elegans innate immunity. Surprisingly, we found that the knockdown of tax-6 enhanced the susceptibility of C. elegans to bacterial infection, despite enhancing lifespan. We discovered that tax-6 is required for the rhythmic defecation motor program (DMP). The knockdown of tax-6 resulted in intestinal bloating due to defects in the DMP which enhanced susceptibility to bacterial infection by increasing gut colonization by bacteria. Intestinal bloating resulted in calorie restriction-like phenotypes, including reduced lipid levels, and led to increased lifespan. We discovered that the TFEB ortholog, HLH-30, is required for calcineurin inhibition-mediated lifespan extension by triggering lipolysis. Finally, we found that the nuclear hormone receptor, NHR-8, is upregulated by calcineurin inhibition and is required for increased lifespan. Our studies uncover a new mechanism for calcineurin inhibition-mediated longevity extension.
Calcineurin knockdown enhances C. elegans susceptibility to Pseudomonas aeruginosa infection
To understand the role of calcineurin in C. elegans innate immune response, we studied the survival of a hypomorphic allele of tax-6, tax-6(p675), on the pathogenic bacterium P. aeruginosa PA14. Surprisingly, the tax-6(p675) animals had a drastically reduced survival on P. aeruginosa compared to wild-type N2 animals (Figure 1A). Similarly, the knockdown of tax-6 by RNA interference (RNAi) enhanced the susceptibility of animals to P. aeruginosa compared to that of control animals (Figure 1B). The effects of RNAi were specific to tax-6, as tax-6(p675) animals did not have a further increase in susceptibility to P. aeruginosa upon tax-6 RNAi (Figure 1C). As reported earlier (Dong et al., 2007; Dwivedi et al., 2009; Mair et al., 2011; Tao et al., 2013), we confirmed that tax-6 knockdown led to an increased lifespan on E. coli (Figure 1D). An earlier study demonstrated that calcineurin regulates cAMP response element-binding protein (CREB) and CRTCs to regulate lifespan in C. elegans (Mair et al., 2011). The knockdown of the CREB homolog-1 (crh-1) and crtc-1 enhanced lifespan similar to the tax-6 knockdown (Mair et al., 2011). We asked whether the knockdown of crh-1 and crtc-1 impacted C. elegans survival on P. aeruginosa comparable to the tax-6 knockdown. Interestingly, the knockdown of crh-1 and crtc-1 did not affect the survival on P. aeruginosa as adversely as the knockdown of tax-6 did (Figure S1). These results demonstrated that despite having an increased lifespan, tax-6 knockdown animals have drastically enhanced susceptibility to P. aeruginosa infection by a mechanism likely independent of crh-1 and crtc-1.
We observed enhanced matricidal hatching in tax-6 knockdown animals on P. aeruginosa. Therefore, we asked whether the enhanced susceptibility of tax-6 knockdown animals to P. aeruginosa was because of enhanced matricidal hatching. To this end, we studied the effects of tax-6 knockdown in fer-1(b232) temperature-sensitive mutants. When grown at 25°C, fer-1(b232) animals have unfertilized oocytes (Argon and Ward, 1980), and therefore, these animals will lack matricidal hatching. As shown in Figure 1E, fer-1(b232) animals with unfertilized oocytes also had reduced survival on P. aeruginosa upon tax-6 knockdown, indicating that the increased susceptibility upon tax-6 knockdown is not because of enhanced matricidal hatching. The fer-1(b232) animals also showed enhanced lifespan on E. coli upon knockdown of tax-6 (Figure 1F). These results suggested that the knockdown of tax-6 led to increased lifespan and enhanced susceptibility to pathogen infection.
Next, we asked whether the enhanced susceptibility to bacterial infection upon tax-6 knockdown was mediated by one or more of the established C. elegans innate immunity pathways, which include a MAP kinase pathway mediated by NSY-1/SEK-1/PMK-1 (Kim et al., 2002), the MLK-1/MEK-1/KGB-1 c-Jun kinase pathway (Kim et al., 2004), the TGF-β/DBL-1 pathway (Mallo et al., 2002), and the bZIP transcription factor ZIP-2 pathway (Estes et al., 2010). In mutants of all different immune pathways, the knockdown of tax-6 further enhanced susceptibility to P. aeruginosa (Figure S2A-D). The enhanced susceptibility of tax-6 knockdown animals to P. aeruginosa was also independent of the FOXO transcription factor DAF-16 (Figure S2E). Thus, the enhanced susceptibility to P. aeruginosa upon tax-6 knockdown appears to be independent of established C. elegans innate immunity pathways.
Calcineurin is required for the C. elegans defecation motor program (DMP)
To understand why tax-6 knockdown animals had enhanced susceptibility to P. aeruginosa infection, we studied the colonization of the intestine of tax-6 RNAi animals by P. aeruginosa. Knockdown of tax-6 led to enhanced intestinal colonization by P. aeruginosa compared to the control animals (Figure 2A, B). The tax-6(p675) animals also had increased intestinal colonization by P. aeruginosa (Figure 2C, D). Animals with bloated intestinal lumens exhibit enhanced bacterial colonization of the gut, an improved lifespan, and an increased susceptibility to pathogens (Kumar et al., 2019; Singh and Aballay, 2019a). Because tax-6 knockdown animals displayed all of these phenotypes, we asked whether tax-6 knockdown led to the bloating of the intestinal lumen. Indeed, we found that tax-6 knockdown resulted in bloated intestinal lumens (Figure 2E, F).
Intestinal bloating could be an outcome of either defects in the pharyngeal pumping (Kumar et al., 2019) or defects in the defecation motor program (DMP) (Singh and Aballay, 2019a). Knockdown of tax-6 did not affect the pharyngeal pumping rate (Figure S3A). We studied whether calcineurin was required for the DMP. The C. elegans DMP is a highly coordinated rhythmic behavior required for regular expulsion of the intestinal lumen contents with a frequency of about one cycle per minute. The DMP involves a series of muscle contractions involving posterior body muscle contraction, anterior body muscle contraction, and expulsion muscle contraction to release the lumen content (Thomas, 1990). We counted the number of expulsion events for 20 minutes per animal and found that tax-6 knockdown animals had drastically reduced expulsion events (Figure 2G), leading to irregular DMP. The DMP-defect phenotype had a low penetrance in the tax-6(p675) animals. The tax-6(p675) animals exhibited both regular and irregular DMPs (Figure S3B). About 36% of the tax-6(p675) animals had irregular and slowed DMP, while the rest had a regular DMP (Figure 2H), suggesting that tax-6(p675) is a weak allele. Taken together, these results showed that calcineurin is required for the DMP, and its inhibition enhances susceptibility to P. aeruginosa by impeding the DMP.
Calcineurin inhibition enhances lifespan via DMP defects-mediated calorie restriction
Defects in C. elegans DMP result in reduced absorption of nutrients leading to diminished intestinal fat (Sheng et al., 2015). We asked whether tax-6 knockdown resulted in reduced fat levels. To this end, we carried out oil-red-O (ORO) staining of tax-6 RNAi animals. Knockdown of tax-6 resulted in drastically reduced fat levels (Figure 3A, B). Next, we tested whether the reduced fat levels in tax-6 knockdown animals resulted in calorie restriction-like phenotypes. Calorie restriction is known to upregulate the expression of the FoxA transcription factor PHA-4 (Panowski et al., 2007). Knockdown of tax-6 also resulted in the upregulation of pha-4 expression levels (Figure 3C). Because calorie restriction enhances lifespan (Kaeberlein et al., 2006; Lakowski and Hekimi, 1998; Panowski et al., 2007), we asked whether calcineurin inhibition resulted in increased lifespan via calorie restriction. The knockdown of tax-6 in the genetic model of calorie restriction, eat-2(ad465) (Lakowski and Hekimi, 1998), did not increase the lifespan (Figure 3D). These results suggested that calcineurin inhibition enhances lifespan via calorie restriction.
The mechanisms of lifespan enhancement by dietary or calorie restriction are complex and depend on several different downstream pathways (Chamoli et al., 2014; Greer and Brunet, 2009; Hansen et al., 2008). Further, different pathways are operational under different dietary regimens (Greer and Brunet, 2009). Therefore, we tested the role of different pathways linked with calorie restriction-mediated lifespan enhancement in increasing lifespan upon calcineurin inhibition. We studied the interactions of the AMP-activated kinase, aak-2 (Greer and Brunet, 2009), the mTOR pathway, raga-1 (Robida-Stubbs et al., 2012; Zhang et al., 2019), ribosomal S6 kinase, rsks-1 (Chen et al., 2009; Selman et al., 2009; Zhang et al., 2019), the FOXO transcription factor, daf-16 (Greer and Brunet, 2009), and the nuclear hormone receptor, nhr-49 (Chamoli et al., 2014), with calcineurin inhibition in regulating lifespan. Mutants defective in aak-2 appeared to have only a partial increase in lifespan upon calcineurin inhibition (Figure 3E). On the other hand, loss-of-function mutants of raga-1, rsks-1, daf-16, and nhr-49 exhibited enhanced lifespan upon calcineurin inhibition (Figure 3F-I).
Calcineurin knockdown enhances lifespan via HLH-30 and NHR-8
To further explore longevity pathways regulated by calorie restriction that might enhance lifespan downstream of calcineurin inhibition, we studied the role of TFEB ortholog HLH-30. HLH-30 is required for starvation response and triggers lipid depletion under nutrient-deprived conditions (O’Rourke and Ruvkun, 2013). Knockdown of tax-6 did not enhance the lifespan of hlh-30(tm1978) animals (Figure 4A), indicating that HLH-30 is required for increased lifespan downstream of calcineurin inhibition. We studied whether HLH-30 affected fat depletion upon the knockdown of tax-6. While the ORO levels declined significantly in N2 animals upon knockdown of tax-6 (Figure 3A, B), the ORO levels did not change in hlh-30(tm1978) animals upon tax-6 knockdown (Figure 4B, C). Knockdown of tax-6 resulted in defects in the DMP and intestinal bloating in hlh-30(tm1978) animals (Figure S4A-C). Therefore, the lack of fat depletion in hlh-30(tm1978) animals upon tax-6 RNAi was not because of the absence of DMP defects. These results suggested that HLH-30-mediated lipid depletion enhanced lifespan upon calcineurin inhibition.
Steroid hormone signaling mediated by NHR-8 is known to regulate calorie restriction-mediated enhancement in lifespan (Thondamal et al., 2014). We tested whether NHR-8 was required for lifespan increment by calcineurin inhibition. Indeed, the knockdown of tax-6 did not enhance the lifespan of nhr-8(ok186) animals (Figure 4D). While the ligands of NHR-8 remain poorly defined (Magner et al., 2013), the steroid hormones produced by the cytochrome P450 enzyme DAF-9 are known to enhance lifespan via NHR-8 under calorie restriction (Thondamal et al., 2014). Therefore, we tested whether DAF-9 mediated the enhanced lifespan downstream of calcineurin inhibition. We found that tax-6 knockdown enhanced the lifespan of daf-9(rh50) animals (Figure 4E), indicating that DAF-9-produced steroid hormones were not required for lifespan increment by calcineurin inhibition. We asked whether NHR-8 affected the fat depletion of tax-6 knockdown animals. The nhr-8(ok186) animals had drastically reduced fat levels upon the knockdown of tax-6 (Figure 4F, G). These results indicated that NHR-8 worked downstream of or in parallel with fat depletion.
Because calorie restriction can also enhance the expression of genes that increase lifespan, such as pha-4 (Panowski et al., 2007), we tested whether tax-6 knockdown modulated the mRNA levels of nhr-8. Indeed, we observed that tax-6 knockdown increased nhr-8 mRNA levels significantly (Figure 4H). To test whether tax-6 knockdown resulted in the upregulation of nhr-8 because of intestinal bloating or independent of intestinal bloating, we studied nhr-8 mRNA levels in other gene knockdown animals that had bloated intestinal lumens. We knocked down genes flr-1, nhx-2, and pbo-1 as their inhibition is known to lead to defects in the DMP, resulting in intestinal bloating and enhanced lifespan (Singh and Aballay, 2019a). Knockdown of flr-1, nhx-2, and pbo-1 also resulted in increased nhr-8 mRNA levels (Figure 4H), indicating that intestinal bloating resulted in nhr-8 upregulation. Therefore, calcineurin inhibition likely enhanced lifespan via NHR-8 by upregulating its expression levels. Taken together, these results suggested that intestinal bloating caused by calcineurin inhibition enhances lifespan by lipolysis via HLH-30 and upregulating expression of NHR-8 (Figure 4I).
We discovered that calcineurin is required for the C. elegans DMP. Calcineurin activity is regulated by intracellular calcium levels. Increased amounts of calcium ions activate the phosphatase activity of calcineurin via the binding of the calcium-sensing protein calmodulin to calcineurin (Klee et al., 1998). The rhythmic DMP cycle in C. elegans is known to be regulated by rhythmic calcium waves (Santo et al., 1999; Teramoto and Iwasaki, 2006). The calcium waves are regulated by the endoplasmic reticulum calcium channel, ITR-1, and mutations in the itr-1 gene affect defecation by preventing cytoplasmic calcium release (Santo et al., 1999). It is likely that calcium waves regulate DMP via calcineurin activities. Indeed, calcineurin is expressed in the enteric muscles that are required for contractions for DMP (Lee et al., 2005). It is interesting to note that tax-6 gain-of-function mutants are also known to have DMP defects (Lee et al., 2005). Therefore, optimum calcineurin activity appears to be crucial for maintaining a rhythmic DMP.
Calcineurin inhibition has been shown to extend C. elegans lifespan via multiple mechanisms (Dong et al., 2007; Dwivedi et al., 2009; Mair et al., 2011; Tao et al., 2013). Activation of autophagy has been shown to be one of the mechanisms of extended lifespan upon calcineurin inhibition (Dwivedi et al., 2009). We showed that defects in the DMP caused by calcineurin inhibition reduce lipid levels and mimic calorie restriction. Calorie restriction is known to induce autophagy (Morselli et al., 2010), and autophagy is required for calorie restriction-mediated lifespan extension (Hansen et al., 2008; Jia and Levine, 2007). Therefore, it is likely that reduced lipid levels because of intestinal bloating result in the activation of autophagy upon calcineurin inhibition. Importantly, the TFEB ortholog, HLH-30, is required for lipolysis and autophagy under starvation conditions (Lapierre et al., 2013; O’Rourke and Ruvkun, 2013). HLH-30 is also required for lifespan enhancement via autophagy and multiple longevity pathways (Lapierre et al., 2013; O’Rourke and Ruvkun, 2013). We observed that HLH-30 is also required for the increase in lifespan upon calcineurin inhibition. Because autophagy may regulate lifespan via multiple longevity pathways (Hansen et al., 2008; Lapierre et al., 2013), this could explain why calcineurin inhibition enhances lifespan via multiple mechanisms.
Dietary restriction-mediated lifespan extensions are complex and context-dependent (Greer and Brunet, 2009). Multiple longevity pathways have been identified under different paradigms of dietary restriction (Chamoli et al., 2020, 2014; Chen et al., 2009; Greer and Brunet, 2009; Lapierre et al., 2013; Matai et al., 2019; Selman et al., 2009; Thondamal et al., 2014). We found that tax-6 knockdown enhanced lifespan independent of multiple dietary restriction pathways, including raga-1, rsks-1, daf-16, and nhr-49. An earlier study identified that tax-6 knockdown enhanced lifespan via NHR-49 (Burkewitz et al., 2015). We currently do not understand the reasons for this discrepancy. We identified that tax-6 knockdown resulted in the upregulation of nhr-8 mRNA levels, and nhr-8 was required for the increased lifespan in tax-6 knockdown animals. NHR-8 has been shown to mediate calorie restriction-dependent lifespan via the regulation of xenobiotic responses (Chamoli et al., 2014; Verma et al., 2018). Therefore, it is likely that NHR-8 increases lifespan upon calcineurin inhibition by activating xenobiotic response pathways.
Recent studies in different organisms have shown that gut bloating has profound effects on food-seeking behaviors, immunity, and lifespan (Duvall et al., 2019; Filipowicz et al., 2021; Kumar et al., 2019; Min et al., 2021; Singh and Aballay, 2019a, 2019b, 2019c). Several C. elegans mutants with defects in the DMP and bloated intestinal lumens are known to have dampened nutrient absorption, leading to reduced lipid deposition in the gut, mimicking calorie restriction (Sheng et al., 2015). It is possible that some of the effects of intestinal bloating on the host physiology are mediated by calorie restriction. Indeed, the neuropeptide Y receptors, which control a diverse set of behaviors, including appetite, are activated by gut bloating (Singh and Aballay, 2019c). Calorie restriction is known to induce neuropeptide Y, which might trigger feeding behaviors (Aveleira et al., 2015; de Rijke et al., 2005; Ferreira-Marques et al., 2016). The complete characterization of the physiological changes downstream of gut bloating may provide broad insights into the effects of gut physiology on behaviors, immunity, and lifespan.
Materials and Methods
The following bacterial strains were used: Escherichia coli OP50, E. coli HT115(DE3), Pseudomonas aeruginosa PA14, and P. aeruginosa PA14 expressing green fluorescent protein (P. aeruginosa PA14-GFP). The cultures for these bacteria were grown in Luria-Bertani (LB) broth at 37°C.
C. elegans strains and growth conditions
C. elegans hermaphrodites were maintained on nematode growth medium (NGM) plates seeded with E. coli OP50 at 20°C unless otherwise indicated. Bristol N2 hermaphrodites were used as the wild-type control unless otherwise indicated. The following strains were used in the study: PR675 tax-6(p675), HH142 fer-1(b232), DA465 eat-2(ad465), CF1038 daf-16(mu86), STE68 nhr-49(nr2041), RB754 aak-2(ok524), AE501 nhr-8(ok186), VC222 raga-1(ok386), RG1228 daf-9(rh50), RB1206 rsks-1(ok1255), KU25 pmk-1(km25), KU21 kgb-1(km21), NU3 dbl-1(nk3), JIN1375 hlh-30(tm1978), and VC3056 zip-2(ok3730). Some of the strains were obtained from the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN). The fer-1(b232) hermaphrodites were maintained on E. coli OP50 at 15°C and were grown at 25°C prior to P. aeruginosa killing assays and longevity assays to obtain animals with unfertilized oocytes.
RNA interference (RNAi)
RNAi was used to generate loss-of-function phenotypes by feeding nematodes E. coli strain HT115(DE3) expressing double-stranded RNA homologous to a target gene. RNAi was carried out as described previously (Gokul and Singh, 2022; Ravi et al., 2023). Briefly, E. coli with the appropriate vectors were grown in LB broth containing ampicillin (100 μg/mL) at 37°C overnight and plated onto NGM plates containing 100 μg/mL ampicillin and 3 mM isopropyl β-D-thiogalactoside (IPTG) (RNAi plates). RNAi-expressing bacteria were allowed to grow overnight at 37°C. Gravid adults were transferred to RNAi-expressing bacterial lawns and allowed to lay eggs for 2 hours. The gravid adults were removed, and the eggs were allowed to develop at 20°C to 1-day-old adults for subsequent assays. The RNAi clones were from the Ahringer RNAi library and were verified by sequencing.
C. elegans longevity assays
Lifespan assays were performed on RNAi plates containing E. coli HT115(DE3) with the empty vector control and tax-6 RNAi clone in the presence of 50 µg/mL of 5-fluorodeoxyuridine (FUdR). Animals were synchronized on RNAi plates without FUdR and incubated at 20°C. At the late L4 larval stage, the animals were transferred onto the corresponding RNAi plates containing 50 µg/mL of FUdR and incubated at 20°C. For fer-1(b232) lifespan assays, the animals were synchronized on RNAi plates without FUdR and incubated at 25°C. The fer-1(b232) lifespan assays were carried out at 25°C without FUdR. Animals were scored every other day as live, dead, or gone. Animals that failed to display touch-provoked movement were scored as dead. Animals that crawled off the plates were censored. Experimental groups contained more than 80 animals per condition per replicate. Young adult animals were considered as day 0 for the lifespan analysis. Three independent experiments were performed.
C. elegans killing assays on P. aeruginosa PA14
Bacterial cultures were prepared by inoculating individual bacterial colonies of P. aeruginosa into 2 mL of LB and growing them for 10-12 hours on a shaker at 37°C. Bacterial lawns were prepared by spreading 20 µL of the culture on the entire surface of 3.5-cm-diameter modified NGM agar plates (0.35% instead of 0.25% peptone). The plates were incubated at 37°C for 12-16 hours and then cooled to room temperature for at least 30 minutes before seeding with synchronized 1-day-old adult animals. The killing assays were performed at 25°C, and live animals were transferred daily to fresh plates. Animals were scored at times indicated and were considered dead when they failed to respond to touch.
P. aeruginosa-GFP colonization assay
Bacterial cultures were prepared by inoculating individual bacterial colonies of P. aeruginosa-GFP into 2 mL of LB and growing them for 10-12 hours on a shaker at 37°C. Bacterial lawns were prepared by spreading 20 µL of the culture on the entire surface of 3.5-cm-diameter modified NGM agar plates (0.35% instead of 0.25% peptone) containing 50 μg/mL of kanamycin. The plates were incubated at 37°C for 12 hours and then cooled to room temperature for at least 30 minutes before seeding with 1-day-old adult gravid adults. The assays were performed at 25°C. After 6 hours of incubation, the animals were transferred from P. aeruginosa-GFP plates to fresh E. coli OP50 plates and visualized within 5 minutes under a fluorescence microscope.
Quantification of intestinal bacterial loads
The intestinal bacterial loads were quantified by measuring colony-forming units (CFU) as described earlier (Singh and Aballay, 2019b, 2019a) with appropriate modifications. Briefly, P. aeruginosa-GFP lawns were prepared as described above. After 6 hours of exposure, the animals were transferred from P. aeruginosa-GFP plates to the center of fresh E. coli OP50 plates thrice for 10 minutes each to eliminate bacteria stuck to their body. Afterward, ten animals/condition were transferred into 50 μL of PBS containing 0.01% triton X-100 and ground using glass beads. Serial dilutions of the lysates (101, 102, 103, 104) were seeded onto LB plates containing 50 μg/mL of kanamycin to select for P. aeruginosa-GFP cells and grown overnight at 37°C. Single colonies were counted the next day and represented as the number of bacterial cells or CFU per animal. At least three independent experiments were performed for each condition.
Fluorescence imaging was carried out as described previously (Gokul and Singh, 2022; Ravi et al., 2023). Briefly, the animals were anesthetized using an M9 salt solution containing 50 mM sodium azide and mounted onto 2% agarose pads. The animals were then visualized using a Nikon SMZ-1000 fluorescence stereomicroscope. The fluorescence intensity was quantified using Image J software.
Quantification of intestinal lumen bloating
The 1-day-old adult animals grown on the empty vector control and tax-6 RNAi were anesthetized using an M9 salt solution containing 50 mM sodium azide, mounted onto 2% agarose pads, and imaged. The diameter of the intestinal lumen was measured using the Zeiss Zen Pro 2011 software. At least ten animals were used for each condition.
Measurement of defecation motor program (DMP) rate
Wild-type N2 and tax-6(p675) animals were synchronized and grown at 20°C on E. coli OP50 for 4 and 5 days, respectively, before measuring the DMP rate. For RNAi experiments, N2 worms were synchronized on EV and tax-6 RNAi plates and grown for 4 days at 20°C before measuring the DMP rate. The DMP cycle length was scored by assessing the time between expulsions (which are preceded by posterior and anterior body wall muscle contraction and the contraction of enteric muscles in a normal regular pattern) (Thomas, 1990). The number of expulsion events in 20 minutes was measured for each worm. The DMP rate was recorded for 5-6 worms/condition.
Pharyngeal pumping assay
For the pharyngeal pumping assay, 1-day-old adult animals grown on the empty vector control and tax-6 RNAi were used. The number of contractions of the terminal bulb of the pharynx was counted for 30 seconds per worm. The pumping rates for 20 worms were recorded for each condition.
The oil-red-O (ORO) staining was carried out as described earlier (Lynn et al., 2015) with appropriate modification. Briefly, wild-type N2, hlh-30(tm1978), and nhr-8(ok186) animals were synchronized on empty vector control and tax-6 RNAi bacteria and grown at 20°C for 4 days. About 300-400 synchronized gravid adult worms from experimental plates were washed three times with 1X PBS plus 0.01% triton X-100 (PBST). To permeabilize the cuticle, 600 µL of 40% isopropanol was added to 100 µL of worm pellet and was rocked for three minutes. The animals were spun down at 500 rpm for 30 seconds, and then 600 µL of supernatant was removed. Subsequently, 600 µL of ORO working stock was added to the worm pellet to stain the worms and incubated at room temperature for 1 hour on a shaker. ORO working stock was freshly prepared by diluting the stock 0.5% ORO in isopropanol to 60% in water and rocked at room temperature for 2 hours, followed by the removal of debris with a 0.22 µm filter. Afterward, worm samples were pelleted, 600 µL of supernatant was removed, 600 µL of PBST was added, and the samples were rocked for 30 minutes at room temperature. After that, the worms were mounted on a 2% agarose pad and imaged. At least ten worms/condition were imaged. Three independent biological replicates were performed. The ORO intensity was quantified using Image J software.
RNA isolation and quantitative reverse transcription-PCR (qRT-PCR)
Animals were synchronized by egg laying. Approximately 40 N2 gravid adult animals were transferred to 9-cm RNAi plates seeded with E. coli HT115 expressing the appropriate vectors and allowed to lay eggs for 4-5 hours. The gravid adults were then removed, and the eggs were allowed to develop at 20°C for 96 hours. Subsequently, the animals were collected, washed with M9 buffer, and frozen in TRIzol reagent (Life Technologies, Carlsbad, CA). Total RNA was extracted using the RNeasy Plus Universal Kit (Qiagen, Netherlands). Residual genomic DNA was removed using TURBO DNase (Life Technologies, Carlsbad, CA). A total of 6 μg of total RNA was reverse-transcribed with random primers using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).
qRT-PCR was conducted using the Applied Biosystems One-Step Real-time PCR protocol using SYBR Green fluorescence (Applied Biosystems) on an Applied Biosystems 7900HT real-time PCR machine in 96-well-plate format. Twenty-five-microliter reactions were analyzed as outlined by the manufacturer (Applied Biosystems). The relative fold-changes of the transcripts were calculated using the comparative CT(2-ΔΔCT) method and normalized to pan-actin (act-1, -3, -4) as described earlier (Singh and Aballay, 2019a, 2017). The cycle thresholds of the amplification were determined using StepOnePlus software (Applied Biosystems). All samples were run in triplicate. The primer sequences are shown in Table S1.
Quantification and statistical analysis
The statistical analysis was performed with Prism 8 (GraphPad). All error bars represent mean±standard deviation (SD). The two-sample t-test was used when needed, and the data were judged to be statistically significant when p < 0.05. In the figures, asterisks (*) denote statistical significance as follows: *, p<0.05, **, p<0.001, ***, p<0.0001, as compared with the appropriate controls. The Kaplan-Meier method was used to calculate the survival fractions, and statistical significance between survival curves was determined using the log-rank test. All experiments were performed in triplicate.
Some strains used in this study were provided by the Caenorhabditis Genetics Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).
This work was supported by IISER Mohali intramural funds, Science and Engineering Research Board (SERB) Startup Research Grant (Ref. No. SRG/2020/000022) awarded by DST, India, and Ramalingaswami Re-entry Fellowship (Ref. No. BT/RLF/Re-entry/50/2020) awarded by the Department of Biotechnology, India. P.D. was supported by a junior research fellowship from the Council of Scientific & Industrial Research (CSIR), India.
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