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. 2023 Nov 13;226(1):iyad197. doi: 10.1093/genetics/iyad197

Germline mitotic quiescence and cell death are induced in Caenorhabditis elegans by exposure to pathogenic Pseudomonas aeruginosa

Daniel P Bollen 1,2, Kirthi C Reddy 3,4, Laura I Lascarez-Lagunas 5, Dennis H Kim 6,✉, Monica P Colaiácovo 7,✉,3
Editor: J Engebrecht
PMCID: PMC10763535  PMID: 37956057

Abstract

The impact of exposure to microbial pathogens on animal reproductive capacity and germline physiology is not well understood. The nematode Caenorhabditis elegans is a bacterivore that encounters pathogenic microbes in its natural environment. How pathogenic bacteria affect host reproductive capacity of C. elegans is not well understood. Here, we show that exposure of C. elegans hermaphrodites to the Gram-negative pathogen Pseudomonas aeruginosa causes a marked reduction in brood size with concomitant reduction in the number of nuclei in the germline and gonad size. We define 2 processes that are induced that contribute to the decrease in the number of germ cell nuclei. First, we observe that infection with P. aeruginosa leads to the induction of germ cell apoptosis. Second, we observe that this exposure induces mitotic quiescence in the proliferative zone of the C. elegans gonad. Importantly, these processes appear to be reversible; when animals are removed from the presence of P. aeruginosa, germ cell apoptosis is abated, germ cell nuclei numbers increase, and brood sizes recover. The reversible germline dynamics during exposure to P. aeruginosa may represent an adaptive response to improve survival of progeny and may serve to facilitate resource allocation that promotes survival during pathogen infection.

Keywords: germline, C. elegans, P. aeruginosa, pathogen, germ cell apoptosis, meiosis

Introduction

Host survival and reproductive capacity during infection contribute to evolutionary fitness. How infection influences reproduction and germline physiology is not well understood. The bacterivorous nematode Caenorhabditis elegans encounters a wide range of pathogenic microbes in its native environment (Schulenburg and Félix 2017). Caenorhabditis elegans responds to infection through innate immune signaling (Kim and Ewbank 2018) and behavioral avoidance mechanisms (Kim and Flavell 2020).

Pseudomonas aeruginosa is an environmental bacterium that resides in soil and water and can cause devastating opportunistic infections in humans. Pseudomonas aeruginosa is capable of infecting an evolutionarily diverse range of host organisms (Campa et al. 1993; Rahme et al. 1995; Reynolds and Kollef 2021). Infection of C. elegans by P. aeruginosa has been studied for over 20 years, and P. aeruginosa has been shown to kill C. elegans under a range of experimental conditions (Mahajan-Miklos et al. 1999; Tan et al. 1999). Its long history of study and the existence of tools such as an ordered transposon insertion mutant library (Liberati et al. 2006) make it an attractive and tractable model pathogen to study in this context.

We sought to understand how reproduction of C. elegans hermaphrodites is affected by exposure to P. aeruginosa. It has been reported that pathogenic bacteria such as Shigella spp., Burkholderia spp., Microbacterium sp., Staphylococcus aureus, and Serratia marcescens reduce the number of progeny of C. elegans, but the underlying mechanisms are not understood (O’Quinn et al. 2001; Irazoqui et al. 2010; Kesika et al. 2015; Madhu et al. 2019; Le et al. 2022). Elucidation of these mechanisms may shed light on how animals sense and effectively utilize resources upon pathogen exposure.

Here, we study the effects of P. aeruginosa infection on the C. elegans germline. We show that exposure to P. aeruginosa induces a reduction in brood size in C. elegans. We find that exposure to P. aeruginosa induces germ cell apoptosis, as well as mitotic quiescence in the proliferative region of the gonad. Importantly, these phenotypes are seen to constitute a dynamic process; they are reversible and contingent on exposure to the Pseudomonas lawn milieu. These responses may represent protective processes taken to ensure progeny viability or the effects of shunting resources from reproduction to ensure host survival in the face of pathogenic stress.

Materials and methods

Caenorhabditis elegans strains

All C. elegans strains used in this study were maintained as previously described, with N2 Bristol worms used as the wild-type background (Brenner 1974). Animals were maintained and assays were performed at 20°C. The following strains were used in this study: MD701 (bcIs39 [lim-7p::ced-1::GFP + lin-15(+)]), ZD541 (pmk-1(km25); bcIs39), ZD2678 (cep-1(gk138); bcIs39), and ZD2691 (zip-2(ok3730); bcIs39).

Bacteria propagation

Plates for P. aeruginosa exposure assays were prepared as described (Meisel et al. 2014). Briefly, LB broth was inoculated with P. aeruginosa  PA14 glycerol stock. Cultures were incubated overnight shaking at 37°C to allow growth to stationary phase. Two hundred microliters of culture was seeded onto 3.5-cm prewarmed plates of slow-kill assay (SKA) media (Tan et al. 1999). Plates were tilted briefly to coat the entire agar surface with the bacterial culture. Plates were first incubated overnight at 37°C and then at room temperature for 24 h. The same procedure was followed to prepare Escherichia coli  OP50 exposure plates for use as a control.

Brood size assays

To measure the brood sizes of animals, age-synchronized hermaphrodite animals were grown to young adults (66 h old) and then moved to bacteria of interest. For assays where the number of progeny produced during the exposure window was counted, animals were singled onto SKA plates seeded with the bacteria of interest; otherwise, they were picked to a single shared plate for the duration of exposure.

At the end of the exposure period, animals were singled to nematode growth media (NGM) plates seeded with 100 µL E. coli  OP50 for recovery. Animals were moved every 24 h to a fresh plate, and progeny on the vacated plates was counted immediately. Progeny includes both eggs and larvae on the plate at the end of the exposure/recovery period. If animals were dead or missing at the end of the 24 h period, the plate was omitted from analysis.

Germline apoptosis assays

Germline cell death was assessed by using either a ced-1::gfp reporter strain or by visualizing “button-like” cell corpses using differential interference contrast (DIC) microscopy (Gumienny et al. 1999; Zhou et al. 2001). Young adult animals were exposed to bacteria of interest for 6 h unless otherwise stated. A total of 20–25 animals were then mounted under a coverslip with 7 μL of 3 mM sodium azide on either a 1.5% or 3% agar pad for fluorescence or DIC analysis, respectively. Germline nuclei enveloped with fluorescent CED-1 or highly refractive by DIC optics were observed in late pachytene and scored under ×40 magnification using a Zeiss Axio Imager Z1 microscope. All focal planes were searched to identify corpses. Partially obscured gonad arms were not scored.

Germline size reduction assays

To assay germline size reduction, young adult animals were exposed to bacteria of interest for 24 h. A total of 20–25 animals were dissected, DAPI-stained, and mounted as described previously (Colaiácovo et al. 2003). Z-stack images of one germline hemisphere were taken, and max projections of these z-stacks were stitched together with an ImageJ plugin (Preibisch et al. 2009). These composite images were used to count nuclei for each gonad arm sample, from the distal tip up to the end of late pachytene, omitting diplotene and diakinesis stage nuclei.

Egg-laying assays

To stage eggs laid after exposure to bacteria, young adult animals (66 h old) were exposed to bacteria of interest for 24 h. After this exposure, animals were moved to NGM plates seeded with E. coli  OP50, 5 per plate. Three plates per condition were assayed. After 1 h, animals were removed and the stage of the eggs laid was observed using a dissecting microscope. Stages were categorized according to a previously established rubric (Ringstad and Horvitz 2008). For statistical analysis, eggs were categorized as either “delayed” or “not delayed” according to previous reports on egg-laying by wild-type animals under standard laboratory conditions (Hirsh, Oppenheim, and Klass 1976). The resulting contingency table was analyzed using Fisher's exact test to determine the statistical significance of the number of delayed eggs laid.

Fluorescence immunostaining

Whole mount preparation of dissected gonads, fixation, and immunostainings were performed as described previously (Colaiácovo et al. 2003). An α-phospho-histone H3 (Ser10) primary rabbit antibody (Cell Signaling) was used at 1:2,000 dilution. The secondary antibody used was Alexa-488 goat α-rabbit (Jackson ImmunoResearch Laboratories) at a 1:500 dilution. Vectashield from Vector Laboratories (Burlingame, CA) was used as a mounting media and antifading agent.

Imaging

Images of whole-mounted gonads were collected at 0.5-µm z-intervals with an IX-70 microscope (Olympus) and a cooled charge-coupled device (CCD) camera (model CH350; Roper Scientific) controlled by the DeltaVision system (Applied Precision). Images were deconvolved using the SoftWorx 3.3.6 software (Applied Precision) and processed with Fiji ImageJ (Schindelin et al. 2012).

Statistical analysis

Statistical tests were carried out using GraphPad Prism. Tests used are described in figure legends. Prior to statistical comparisons, outliers were detected using robust regression and outlier removal (ROUT) (Q = 1%) and removed from analysis.

Results and discussion

Exposure to P. aeruginosa results in a reduction in C. elegans brood size

To quantify the effect of exposure to P. aeruginosa on progeny production, we exposed young adult animals to P. aeruginosa  PA14 for 24 h. All exposures in this study were done with agar plates completely seeded with the bacterial lawn to prevent the confounding effects of pathogen avoidance. We found that for the duration of the exposure, animals on the P. aeruginosa lawn laid few eggs when compared with animals on the standard laboratory food source, nonpathogenic E. coli  OP50 (Fig. 1a and Supplementary Data 1). These data were consistent with a previous report that C. elegans feeding on P. aeruginosa produced few progeny (Tan et al. 1999). The progeny count includes both eggs and larvae on the plate at the end of the exposure period. We saw no evidence of embryonic lethality in eggs laid on P. aeruginosa (Supplementary Fig. 1 and Data 1).

Fig. 1.

Fig. 1.

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Exposure to P. aeruginosa results in decreased brood size that is independent of egg retention in the uterus. a) Caenorhabditis elegans lays significantly fewer eggs on P. aeruginosa compared with E. coli. Young adult hermaphrodites were singled onto SKA plates seeded with E. coli  OP50 and P. aeruginosa  PA14 for 24 h. At the end of the exposure time, the number of progeny was counted. Each point represents a unique animal (n ≈ 20). Welch's t-test, 2 tailed, ****P < 0.0001. b) Caenorhabditis elegans does not retain eggs on P. aeruginosa in short timescales. Young adult hermaphrodites were exposed to P. aeruginosa and E. coli or were starved for 6 h. Animals were then moved to E. coli  OP50 to lay eggs for 1 h. Animals exposed to P. aeruginosa did not lay eggs at later stages compared with E. coli, though starved animals did. Average of 3 experiments with at least 50 eggs scored per condition per experiment from 15 animals for each condition. To determine statistical significance, eggs were categorized as either “delayed” or “not delayed.” Fisher's exact test of the resulting contingency table showed no significant difference (ns) between the E. coli and P. aeruginosa conditions. c) Animals exposed to P. aeruginosa do retain eggs after longer exposure times. Young adult hermaphrodites were exposed to P. aeruginosa or E. coli for 24 h. Animals were then moved to E. coli  OP50 to lay eggs for 1 h. Animals exposed to P. aeruginosa laid eggs at later stages. Average of 3 experiments with at least 25 eggs scored per condition per experiment from 25 animals for each condition. To determine statistical significance, eggs were categorized as either “delayed” or “not delayed.” Fisher's exact test of the resulting contingency table showed a significant difference between the E. coli and P. aeruginosa conditions, ****P < 0.0001. d) Egg-laying defect in response to P. aeruginosa does not result in a significant increase of eggs in the uterus. Young adult hermaphrodites were exposed to P. aeruginosa or E. coli for 24 h. Animals were anesthetized, and the number of eggs in the uterus of each animal was counted. Each point represents a unique animal (n ≈ 30). Student's t-test, 2-tailed, ns, not significant.

We next investigated whether defective egg-laying might be due to egg retention induced by exposure to P. aeruginosa. In hermaphrodite animals with defects in egg-laying, eggs are retained in the uterus and laid at later developmental stages (Trent et al. 1983). As such, egg retention can be measured by identifying the developmental stage of recently laid eggs (Ringstad and Horvitz 2008). To determine if the reduction in brood size was strictly due to a retention of eggs in the uterus, we exposed animals to P. aeruginosa and then assayed the developmental stage of eggs laid. We found that compared with the canonical egg retention induced by removal of food (Trent et al. 1983), exposure to P. aeruginosa caused no egg-laying deficit after 6 h (Fig. 1b and Supplementary Data 1). However, after a longer exposure time of 24 h, a remarkable egg-laying deficit appeared when compared with what we observed for hermaphrodites exposed to E. coli  OP50 (Fig. 1c and Supplementary Data 1). We also noted that the number of eggs in the uterus did not increase after a 24 h exposure to P. aeruginosa, suggesting that the missing progeny were not simply retained and that fewer eggs were produced overall (Fig. 1d and Supplementary Data 1). Whereas these data suggest that exposure to P. aeruginosa causes a defect in egg-laying that may contribute to decreased progeny production, our observations also suggest that there is a reduction in the number of fertilized oocytes during exposure to P. aeruginosa.

Exposure to P. aeruginosa induces germline shrinkage

We next assessed whether the reduction in progeny upon exposure to P. aeruginosa might be due to physiological changes in the germline. The C. elegans reproductive system consists of 2 gonad arms connected to a uterus (Fig. 2a). Nuclei are positioned in a spatiotemporal gradient along each gonad arm with a stem cell niche at the distal end giving rise to a population of mitotically proliferating nuclei (the premeiotic tip or proliferative zone) which then progress out of mitosis into meiosis (Kimble and Crittenden 2005).

Fig. 2.

Fig. 2.

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Caenorhabditis elegans germline undergoes shrinkage upon exposure to P. aeruginosa. a) Diagram of C. elegans reproductive system, with its 2 symmetrical gonad arms. The mitotic region is highlighted, as well as the “death zone” where nuclei can undergo apoptosis. Germline nuclei that do not undergo apoptosis complete oogenesis into mature oocytes and are fertilized, ultimately passing into the uterus where they will develop until laid. Diagram created with BioRender.com. b) Germlines of animals exposed to P. aeruginosa are reduced in size compared with those of animals exposed to E. coli and nonpathogenic P. aeruginosa mutant gacA. DAPI-stained germlines of animals exposed to bacteria of interest for 24 h. Scale bar, 25 µm. c) Quantification of the number of germline nuclei after 24 h exposure to bacteria of interest. There is a significant reduction for animals exposed to P. aeruginosa but not those exposed to the nonpathogenic P. aeruginosa mutant gacA. Welch's t-test, ****P < 0.0001, ns, not significant (n ≈ 10).

A dramatic reduction in the size of the gonads was observed for animals exposed to pathogen (Fig. 2b). Notably, this reduction was absent in animals exposed to the P. aeruginosa gacA mutant that is defective in virulence (Rahme et al. 1995). This decrease in size could be due to a reduction in either the cytoplasmic material in the gonadal syncytium or in the number of germline nuclei. To identify changes in the number of nuclei in the germline, animals exposed to P. aeruginosa  PA14 for 24 h were dissected, and germlines were isolated and DAPI-stained. Quantification of the number of DAPI-stained nuclei in the germline revealed a significant reduction in the number of nuclei in the gonad arms upon exposure to pathogenic P. aeruginosa but notably no significant decrease in animals exposed to the P. aeruginosa gacA mutant (Fig. 2c and Supplementary Data 1). This suggests that the pathogenicity of P. aeruginosa is responsible for the decrease in germline nuclei, as opposed to a difference in bacterial food quality between E. coli  OP50 and P. aeruginosa  PA14.

Exposure to P. aeruginosa induces germline cell death

Possible explanations for this reduction in germline nuclei include increased oocyte maturation rate, a reduction in mitotic proliferation, or an increase in germline apoptosis rates. Due to our observation of a reduction in the number of eggs produced during exposure, we considered an increased oocyte maturation rate unlikely. To determine if germline apoptosis plays a role in the reduction in germline size, we measured rates of germline cell death by either using a ced-1::gfp reporter strain or by scoring cellularized corpses by DIC microscopy. ced-1 encodes for a phagocytic corpse recognition engulfment protein essential in the apoptotic process. Nuclei engulfed during germ cell apoptosis become enveloped in CED-1 and are highly refractive by DIC optics acquiring a “button-like” appearance (Fig. 3a, Supplementary Fig. 2 and Data 1) (Gumienny et al. 1999; Zhou et al. 2001) There is a basal physiological rate of germline cell death, and this can be increased upon exposure to chemical, osmotic, or heat stress, as well as starvation (Gumienny et al. 1999; Salinas et al. 2006). We found that after 6 h of exposure to P. aeruginosa  PA14, a greater number of CED-1-positive nuclei appear compared with E. coli  OP50 (Fig. 3b and Supplementary Data 1), and this was further supported by elevated levels of germline cell corpses observed by DIC (Supplementary Fig. 2). Importantly, ΔgacA  PA14 did not induce germ cell apoptosis, again suggesting that these effects are due to pathogenic infection, in line with our observation for gonad size reduction (Fig. 3b, Supplementary Fig. 2 and Data 1). We found similar results for animals exposed to P. aeruginosa mutants defective in other virulence factors, such as rhlR and lasR (Fig. 3b and Supplementary Data 1) (Kariminik et al. 2017). We notably see attenuated but not ablated virulence in the lasR mutant, consistent with what has been previously reported (O'Loughlin et al. 2013; Mukherjee et al. 2017).

Fig. 3.

Fig. 3.

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Exposure to P. aeruginosa induces apoptosis in the C. elegans germline. a) Representative image of ced-1::gfp reporter strain MD701 on E. coli. Germline outline in yellow on DIC. Apoptotic nuclei can be seen in the death zone of the germline enveloped with CED-1::gfp, an example of which is marked with a red arrowhead. b) Quantification of apoptotic germ cell nuclei after 6 h exposure to bacteria of interest. Animals exposed to P. aeruginosa showed induction of germ cell apoptosis which was not observed in mutants in pathogenic regulators gacA, rhlR, and lasR. c) Mutants in the infection response genes pmk-1 and zip-2 show no greater magnitude of induction of germ cell apoptosis upon exposure to P. aeruginosa. Animals were exposed to bacteria of interest for 6 h. Combined data from 3 biological replicates, n ≍ 30 gonads per replicate. Mann–Whitney test used, ****P < 0.0001, ns, not significant. d) Mutants in infection response pmk-1 and zip-2 show more rapid induction of germline cell death upon exposure to P. aeruginosa. Animals were exposed to bacteria of interest for 1 h. e) p53 homolog CEP-1 is not necessary for induction of germline cell death upon exposure to P. aeruginosa. Welch's t-test used for statistical comparisons in d) and e), ****P < 0.0001, ***P < 0.001, ns, not significant (n ≍ 30 gonads per condition).

Exposure of C. elegans to P. aeruginosa  PA14 was previously reported to not cause increased germ cell apoptosis (Aballay and Ausubel 2001). This prior study relied solely upon the use of the SYTO 12 dye for staining of apoptotic corpses, and the authors noted technical concerns regarding dye uptake by animals in the presence of P. aeruginosa. We confirmed our observations of germ cell apoptosis induced by P. aeruginosa  PA14 by 2 independent methods—the ced-1::gfp reporter strain and by direct observation and scoring of cellularized corpses by DIC microscopy.

Germline cell death in response to infection by Salmonella enterica has been reported to be dependent on PMK-1 p38 mitogen-activated protein kinase (MAPK) (Aballay et al. 2003). In contrast, we observed that upon exposure to P. aeruginosa, PMK-1 was not required for induction of germ cell apoptosis (Fig. 3c and Supplementary Data 1). Because pmk-1 mutants are more susceptible to pathogen infection (Kim et al. 2002), we sought to assess the role that increased susceptibility to pathogenesis may have on germline cell death. To test this, we examined germ cell apoptosis in a zip-2 mutant. The bZIP transcription factor ZIP-2 regulates the innate immune response of C. elegans independently of pmk-1 (Estes et al. 2010). Mutants in zip-2 showed no greater induction of germ cell apoptosis than wild-type animals after a 6 h exposure, similar to what was observed for pmk-1 mutants (Fig. 3c and Supplementary Data 1). To assess if pathogen susceptibility had any effect on the kinetics of the induction of germline cell death, we examined induction of germ cell apoptosis at earlier time points. We found that within 1 h of exposure, both zip-2 and pmk-1 mutant animals showed induction of germ cell apoptosis in response to P. aeruginosa, while wild-type animals did not (Fig. 3d and Supplementary Data 1), indicative of accelerated kinetics of induction of germline cell death in mutants defective in the innate immune response. The differences in our observations here and prior reports of PMK-1-dependent germline cell death during Salmonella infection (Aballay et al. 2003) may be due to differences in how each pathogen induces germ cell apoptosis or differences in experimental conditions.

One possibility for this induction of germ cell apoptosis is increased DNA damage among meiotic nuclei in the germline. In C. elegans, the p53 homolog CEP-1 is necessary for induction of germ cell apoptosis in response to DNA double-strand breaks (DSBs) caused by ionizing radiation and in mutants with either elevated programmed meiotic DSBs or impaired DSB repair (Schumacher et al. 2001). Our data shows that cep-1 mutant animals exhibited no defect in the induction of germ cell apoptosis in response to P. aeruginosa (Fig. 3e and Supplementary Data 1). This suggests that the increased germline cell death following exposure to P. aeruginosa is not in response to DNA damage and must occur by some other mechanism.

Exposure to P. aeruginosa induces mitotic quiescence in the proliferative zone of the germline

Another possible process by which the number of germline nuclei may be reduced is a cessation of mitosis in the proliferative zone of the gonad arm. Starvation of C. elegans was previously observed to result in the induction of mitotic quiescence, as detected by a loss of nuclei positive for histone H3 phosphorylation, a marker indicative of M phase (Seidel and Kimble 2015).

To determine if mitotic quiescence was occurring in response to P. aeruginosa exposure, young adult animals were exposed to P. aeruginosa  PA14 for 6 h, and their whole-mounted gonads were immunostained for phospho-histone H3. We found that phospho-histone H3–positive nuclei were almost completely absent from P. aeruginosa–exposed animals, though not in animals exposed to E. coli  OP50 or ΔgacA P. aeruginosa  PA14 (Fig. 4a and b and Supplementary Data 1). This was coupled with a reduction in the total number of nuclei in the proliferative zone of the germline (Supplementary Fig. 3 and Data 1) and suggests that exposure to pathogen induces mitotic quiescence in C. elegans.

Fig. 4.

Fig. 4.

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Exposure to P. aeruginosa induces mitotic quiescence in the C. elegans germline. a) Representative high-resolution images of nuclei in the premeiotic region of the germline immunostained for phospho-histone H3 (Ser10) (pH3). Scale bar, 25 µm. b) Quantification of pH3 immunostaining shows loss of mitotically dividing nuclei upon exposure to P. aeruginosa but not in the nonpathogenic gacA mutant. Mann–Whitney test, ****P < 0.0001, ns, not significant (n ≈ 20 gonads scored for each).

Pathogen-induced germline changes are reversible upon cessation of exposure to P. aeruginosa

To determine if the changes in the germline that we observed during exposure to P. aeruginosa were reversible, we exposed animals to P. aeruginosa  PA14 and then returned them to E. coli  OP50. After 24 h of exposure to P. aeruginosa, while animals showed reduced egg-laying capacity during the next 24 h period, animals transferred back to E. coli  OP50 recovered and ultimately laid a similar number of eggs to animals exposed only to E. coli  OP50 (Fig. 5a and Supplementary Data 1). In addition, animals transferred from P. aeruginosa to E. coli  OP50 after a 6 h exposure exhibited phospho-histone H3–positive nuclei within 2 h (Fig. 5b and Supplementary Data 1). This 2-h timescale is consistent with the recovery time sufficient for recovery from starvation-induced mitotic quiescence (Seidel and Kimble 2015). We also observed that a 2 h recovery on E. coli  OP50 after transfer from P. aeruginosa was sufficient to reduce the number of apoptotic nuclei in the gonad arms (Fig. 5c and Supplementary Data 1). The relatively rapid recovery in the levels of germline cell death induction suggests that exposure to the pathogenic lawn, and not necessarily the longer-term effects of damage from infection and a subsequent innate immune response, contributes to the induction of germ cell apoptosis. We further observed that animals removed from P. aeruginosa and placed on E. coli  OP50 exhibited recovery of the germline, with nuclei numbers increasing over several days (Fig. 5d and Supplementary Data 1).

Fig. 5.

Fig. 5.

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Germline changes in response to P. aeruginosa are reversible upon removal of P. aeruginosa. a) Young adult animals exposed to P. aeruginosa show sustained but reversible reduction in brood size. Animals were exposed to bacteria of interest for 24 h and then moved to E. coli NGM plates to recover. Animals were moved every 24 h to a fresh plate, and the number of progeny produced was counted. Points represent the average of 25 animals. Error bars represent the standard deviation. b) Mitotic quiescence is abated within 2 h of recovery on E. coli. Animals were exposed to bacteria of interest for 6 h. A subset of animals exposed to P. aeruginosa were allowed to recover on E. coli for 2 h; then, phospho-histone H3–positive nuclei were assayed. Mann–Whitney test was used for nonparametric comparisons, and Student's t-test was used to compare parametric data, ****P < 0.0001, ns, not significant (n ≈ 15 gonads per condition). c) Germline cell death induction is abated within 2 h of recovery on E. coli. ced-1::gfp animals were exposed to bacteria of interest for 6 h, assessed for the number of apoptotic nuclei per gonad arm, and then assessed again 2 h later, with a subset of animals exposed to P. aeruginosa allowed to recover on E. coli for 2 h. ****P < 0.0001, Welch's t-test (n ≍ 30 gonads per condition). d) Gonad size recovers in animals removed from P. aeruginosa. Animals were exposed to bacteria of interest for 24 h, and then, the number of DAPI-stained nuclei in each dissected gonad was scored. Animals were then moved to E. coli  OP50 to recover and were dissected 24 and 48 h later. Each point represents the average of n ≈ 15 animals. Error bars represent the standard deviation.

These observations show that exposure to pathogenic bacteria results in a reduced number of progeny laid, with a concomitant increase in germline cell death and induction of mitotic quiescence in the germline. These observations are accompanied by a reduction in the number of nuclei in the germline, and these phenomena appear to be reversible once the animals are in the presence of nonpathogenic bacteria. We speculate that the combination of germline cell death and mitotic quiescence in response to exposure to P. aeruginosa may represent a protective program to maintain brood viability in the presence of a pathogenic microbial environment, with a reduction in the number of progeny laid in an unfavorable microbial environment to preserve progeny viability and promote robust brood sizes when there has been a transition to a more beneficial microbial environment.

This suite of phenotypes is also known to occur in response to starvation (Salinas et al. 2006; Angelo and Van Gilst 2009 ; Seidel and Kimble 2015). Furthermore, reports have shown that exposure to P. aeruginosa reduces lipid stores in C. elegans (Nhan et al. 2019) and leads to downregulation of the acyl-CoA dehydrogenase acdh-1 (Fletcher et al. 2019) which has previously been identified as a marker of the starvation response (Van Gilst et al. 2005). As such, these phenotypes bare some resemblance to adult reproductive diapause (ARD). However, the lethal nature of P. aeruginosa exposure does not allow for the same long-term reproductive quiescence reported in ARD (Angelo and Van Gilst 2009; Seidel and Kimble 2011; Gerisch et al. 2020). While our experiments with nonpathogenic P. aeruginosa mutants shows that the causative factors in these changes are not due to an underlying nutritive difference between 2 bacterial species, the similarities in the germline phenotypes induced by these distinct stressors suggest that there may be commonalities in underlying host responses to trigger reversible germline changes. Recent research into immunometabolic crosstalk has uncovered relationships between metabolic stress and immune activation (Penkov et al. 2019; Anderson and Pukkila-Worley 2020; Ayres 2020; Peterson et al. 2022). The similarities between ARD and our here described germline pathogen response may arise from this immunometabolic crosstalk.

While here we describe a set of reversible germline phenotypes accompanying a reduction in progeny number upon exposure to P. aeruginosa, the molecular factors involved remain elusive. Further study of the pathogen and host-associated factors necessary for this germline response may yield insight into how animals sense and effectively respond to pathogens.

Supplementary Material

iyad197_Supplementary_Data
iyad197_supplementary_data.zip (903KB, zip)

Acknowledgments

Some strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We also thank members of the Kim and Colaiácovo labs for helpful conversations and feedback on this work.

Contributor Information

Daniel P Bollen, Division of Infectious Diseases and Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Kirthi C Reddy, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

Laura I Lascarez-Lagunas, Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.

Dennis H Kim, Division of Infectious Diseases and Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.

Monica P Colaiácovo, Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.

Data availability

Strains and reagents are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.

Supplemental material available at GENETICS online.

Funding

This work was supported by National Institutes of Health grants R35GM141794 to D.H.K. and R01GM072551 to M.P.C.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

iyad197_Supplementary_Data
iyad197_supplementary_data.zip (903KB, zip)

Data Availability Statement

Strains and reagents are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.

Supplemental material available at GENETICS online.

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