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Modulation of sensory perception by hydrogen peroxide enables Caenorhabditis elegans to find a niche that provides both food and protection from hydrogen peroxide

['Jodie A. Schiffer', 'Biology Department', 'Northeastern University', 'Boston', 'Massachusetts', 'United States Of America', 'Stephanie V. Stumbur', 'Maedeh Seyedolmohadesin', 'Physics Department', 'Yuyan Xu']

Date: 2022-02 

Hydrogen peroxide (H 2 O 2 ) is the most common chemical threat that organisms face. Here, we show that H 2 O 2 alters the bacterial food preference of Caenorhabditis elegans, enabling the nematodes to find a safe environment with food. H 2 O 2 induces the nematodes to leave food patches of laboratory and microbiome bacteria when those bacterial communities have insufficient H 2 O 2 -degrading capacity. The nematode’s behavior is directed by H 2 O 2 -sensing neurons that promote escape from H 2 O 2 and by bacteria-sensing neurons that promote attraction to bacteria. However, the input for H 2 O 2 -sensing neurons is removed by bacterial H 2 O 2 -degrading enzymes and the bacteria-sensing neurons’ perception of bacteria is prevented by H 2 O 2 . The resulting cross-attenuation provides a general mechanism that ensures the nematode’s behavior is faithful to the lethal threat of hydrogen peroxide, increasing the nematode’s chances of finding a niche that provides both food and protection from hydrogen peroxide.

One of the most common lethal threats that nematodes encounter is hydrogen peroxide, which is produced by a wide variety of microorganisms. In this microbial battlefield, how do nematodes find a niche that provides the food and safety necessary for growth and reproduction? In the present study, we developed a model ecosystem to study the behavioral mechanisms that enable the nematode C. elegans to find those niches. We found that C. elegans adjust their behavior to find bacterial communities that provide protection from hydrogen peroxide. Hydrogen peroxide and bacteria had opposing effects on the activity of sensory neurons that modulate the nematode’s locomotion towards bacteria and away from hydrogen peroxide. The diminished perception of bacteria unable to degrade hydrogen peroxide in the environment represents a general mechanism enabling nematodes to leave environments where the bacterial community does not provide them and their future progeny with sufficient protection from hydrogen peroxide.

Funding: This work was funded by a National Science Foundation CAREER grant #1750065 to J.A., a Burroughs Wellcome Fund award to V.V., an American Federation for Aging Research award to V.V., a National Institutes of Health grant DP2DK116645 to B.S.S., a Department of Energy Joint Genome Institute grant CSP503338 to B.S.S., a National Science Foundation Research Experiences for Undergraduates Award #1757443 to O.B., and a Northeastern University Tier 1 award to V.V. and J.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we show that hydrogen peroxide alters the bacterial food preference of C. elegans, enabling the nematodes to find food patches that provide hydrogen peroxide protection, where they can grow and reproduce. When H 2 O 2 is present in the environment, the nematodes are more likely to leave food patches of laboratory and microbiome bacteria if those bacteria lack enzymes necessary for the degradation of environmental H 2 O 2 . This change in nematode behavior occurs because when bacterial communities have insufficient H 2 O 2 -degrading capacity, environmental H 2 O 2 can excite the ASJ sensory neurons that promote escape from H 2 O 2 and can prevent the response to bacteria of multiple classes of sensory neurons that promote locomotion towards bacteria. Thus, the modulation of C. elegans’ sensory perception by the interplay of hydrogen peroxide and bacteria adjusts the nematode’s behavior to improve the nematode’s chances of finding a niche that provides both food and protection from hydrogen peroxide.

C. elegans is ideally suited for studying how bacteria shape the evolution of behaviors that enable animals to find food and H 2 O 2 protection because of C. elegans’ small size, well-described anatomy [ 10 , 11 ], and tractable microbiome [ 12 – 16 ]. C. elegans associates with a bacterial microbiome recruited from the surrounding environment [ 12 – 14 ] that includes bacteria in genera that degrade or produce H 2 O 2 [ 17 ]. Hydrogen peroxide produced by a bacterium from the C. elegans microbiome, Neorhizobium sp., causes DNA damage to the nematodes [ 18 ]. Many bacteria—including Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus oralis, and Enterococcus faecium—kill C. elegans by producing millimolar concentrations of H 2 O 2 [ 19 – 21 ]. In the complex and variable habitat where C. elegans lives, deciding whether to leave or stay in a bacterial food patch when there is hydrogen peroxide in the environment is critical for survival.

Hydrogen peroxide (H 2 O 2 ) is the most common chemical threat in the microbial battlefield [ 4 ]. Bacteria, fungi, plants, and animal cells have long been known to excrete H 2 O 2 to attack prey and pathogens [ 5 , 6 ]. H 2 O 2 is also a byproduct of aerobic respiration [ 7 ]. Prevention and repair of the damage that hydrogen peroxide inflicts on macromolecules are critical for cellular health and survival [ 7 ]. To avoid damage from H 2 O 2 , cells rely on conserved physiological defenses, including H 2 O 2 -degrading catalases [ 4 ]. We recently found that C. elegans represses their own H 2 O 2 defenses in response to sensory perception of Escherichia coli, the nematode’s food source, because E. coli can deplete H 2 O 2 from the local environment and thereby protect the nematodes [ 8 ]. Thus, the E. coli self-defense mechanisms create a public good [ 9 ], an environment safe from the threat of H 2 O 2 , that benefits C. elegans [ 8 ]. Whether similar interactions between nematodes and bacteria shaped the evolution of behavioral responses protecting C. elegans from H 2 O 2 remains poorly understood.

To grow and reproduce in an ever-changing natural environment, animals must adjust their behavior to find both food and safety. Animals co-evolved in close association with complex bacterial communities that can remodel both the animals’ behavior and their environment [ 1 , 2 ]. Because of this complexity, our understanding of the evolution of the mechanisms that adjust animal behavior to enable them to find food and safety in variable environments remains limited [ 3 ]. In the present study, we developed a model ecosystem to determine how the environment-dependent sensory perception of the natural bacterial community enables Caenorhabditis elegans nematodes to adjust their behavior and find a niche that provides both food and protection from hydrogen peroxide.

Results

Hydrogen peroxide alters the bacterial food preference of C. elegans The bacterium E. coli, the food source of C. elegans under standard laboratory conditions, degrades environmental H 2 O 2 primarily by expressing two catalases, KatG and KatE. These enzymes account for over 95% of E. coli’s H 2 O 2 -degrading capacity. The peroxiredoxin, AhpCF, plays a minor role [22]. Previously, we found that E. coli JI377, a katG katE ahpCF triple null mutant strain which cannot degrade H 2 O 2 in the environment [22], did not protect C. elegans adults from 1 mM H 2 O 2 killing, whereas the E. coli MG1655 parental wild-type strain was protective [8]. We observed a similar pattern when we quantified the development of C. elegans embryos in the presence or absence of 1 mM H 2 O 2 in the environment: when no H 2 O 2 was present, most embryos cultured on petri plates with either E. coli MG1655 or E. coli JI377 lawns developed into fertile adults (S1 Fig). Embryos on plates with E. coli MG1655 and H 2 O 2 also developed into fertile adults (S1 Fig); however, those on plates with E. coli JI377 and H 2 O 2 did not develop into adulthood and instead died as first stage (L1) larvae, similar to embryos that hatched on H 2 O 2 plates without food (S1 Fig). These findings showed that H 2 O 2 -degrading enzymes from E. coli created an environment where C. elegans was safe from the threat of H 2 O 2 , enabling the nematode’s development and subsequent reproduction. Given the threat of H 2 O 2 to C. elegans development and reproduction, we set out to determine the extent to which C. elegans would modulate their behavior to find an environment with both food and safety from the threat of H 2 O 2 . To determine whether C. elegans preferred E. coli strains that could degrade H 2 O 2 , we quantified the migration of populations of adult nematodes in a binary choice assay towards MG1655 and JI377 lawns (108 bacterial cells each) on opposite sides of a petri plate (Fig 1A). In this assay, a choice index of 1 indicated complete preference for MG1655, a choice index of -1 indicated complete preference for JI377, and a choice index of 0 indicated no preference [23]. In the absence of added H 2 O 2 in the environment, the nematodes moved toward both MG1655 and JI377 (Fig 1A). The fraction of nematodes on each lawn approximated a steady state after 30 minutes (Fig 1B). The nematodes exhibited a slight preference for MG1655: a choice index of 0.20 at the end of the two-hour assay (Fig 1C). In contrast, in the presence of 1 mM H 2 O 2 in the environment, the nematodes showed a strong preference for MG1655 (a final choice index of 0.63, Fig 1C). We conclude that H 2 O 2 altered the E. coli preference of C. elegans, increasing the nematode’s chances of finding an E. coli lawn that degrades H 2 O 2 . PPT PowerPoint slide

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TIFF original image Download: Fig 1. Hydrogen peroxide alters the bacterial food preference of C. elegans. (A) Diagram summarizing experimental strategy (top) and series of pictures of the E. coli MG1655 and JI377 lawns at the specified timepoints from representative food-choice assays (bottom) without added H 2 O 2 (left) and with 1 mM H 2 O 2 (right). (B) The proportion of nematodes on the E. coli MG1655 and JI377 lawns in assays without added H 2 O 2 (left) and with 1 mM H 2 O 2 (right) is plotted against time. P < 0.001 for times other than zero (ANOVA). n ≥ 15 assays per condition. (C) The food-choice indices for the assays shown in (B) are plotted against time. H 2 O 2 induced an increase in food-choice index. Groups labeled with different letters exhibited significant differences (P < 0.05, Tukey HSD test) otherwise (P > 0.05). (D) The H 2 O 2 -dependent increase in the proportion of nematodes on the E. coli MG1655 lawns compared to JI377 lawns in two-hour food-choice assays was absent in choice-trap assays, in which the paralytic agent sodium azide was added to the bacterial lawns. ** indicates P < 0.002 and “ns” indicates P > 0.05 (standard least-squares regression). (E) The H 2 O 2 -induced increase in food-choice index was absent in choice-trap assays, for the assays shown in (D). Groups labeled with different letters exhibited significant differences (P < 0.01, Tukey HSD test) otherwise (P > 0.05). Data are represented as mean ± s.e.m. https://doi.org/10.1371/journal.ppat.1010112.g001 The increased nematode preference for E. coli MG1655 in the presence of H 2 O 2 appeared to be in part due to an H 2 O 2 -induced change in nematode behavior after reaching the E. coli JI377 lawn. Instead of staying on the JI377 lawn as they did on assays without H 2 O 2 , in the presence of H 2 O 2 a large proportion of nematodes left the JI377 lawn (Fig 1A); as a result, the fraction of nematodes on the JI377 lawn peaked 15 minutes after the start of the assay and decreased 2.4-fold thereafter, while the fraction of nematodes on the MG1655 lawn continued to increase (Fig 1B). To test whether the increased preference for MG1655 in the presence of H 2 O 2 was due to an H 2 O 2 -dependent increase in the proportion of nematodes that left the JI377 lawn after reaching it, we used the paralytic agent sodium azide to prevent nematodes from leaving the bacterial lawns that they reached. Under these conditions, environmental H 2 O 2 no longer increased the nematodes’ preference for MG1655 (Fig 1D and 1E). These findings suggested that environmental H 2 O 2 increased C. elegans’ preference for E. coli lawns that degraded H 2 O 2 primarily by increasing the chances that nematodes would leave lawns that did not degrade environmental H 2 O 2 .

Production of serotonin inhibits H 2 O 2 -induced food leaving Because food levels affected the H 2 O 2 -induced food leaving behavior of C. elegans, we speculated that this behavior may be regulated by the neurotransmitter serotonin. Expression of the serotonin biosynthetic tryptophan hydroxylase gene tph-1 increases with food [30–35] and serotonin regulates many food-related behaviors [30–32,36]. We found that tph-1(mg280) null mutants, which specifically lack serotonin [30], were more likely than wild-type animals to leave a lawn of E. coli JI377 when 1 mM H 2 O 2 was added to the environment (Fig 3A). The tph-1(mg280) mutation did not affect either the proportion of nematodes leaving a lawn of E. coli JI377 when no H 2 O 2 was added, or the proportion of nematodes leaving a lawn of E. coli MG1655 even when H 2 O 2 was added (Fig 3A). A second, independently derived, tph-1(n4622) null deletion allele [37], exhibited the same behavior as tph-1(mg280) (Fig 3B). Therefore, serotonin biosynthesis inhibits C. elegans’ decision to leave a food lawn that does not provide H 2 O 2 protection. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Production of serotonin inhibits H 2 O 2 -induced food leaving. (A-B) tph-1 null mutations increase the proportion of nematodes that leave an E. coli JI377 food patch in the presence of H 2 O 2 . (C) A mod-5 null mutation decreases the proportion of nematodes that leave an E. coli JI377 food patch in the presence of H 2 O 2 . Data are represented as mean ± s.e.m. Groups labeled with different letters exhibited significant differences (P < 0.05, Tukey HSD test) otherwise (P > 0.05). https://doi.org/10.1371/journal.ppat.1010112.g003 Since lowering serotonin levels increased food-leaving when the E. coli lawn did not degrade environmental H 2 O 2 , we determined whether increasing serotonin levels would be sufficient to lower food leaving. Nematodes with a serotonin reuptake transporter gene mod-5(n822) null mutation have higher presynaptic serotonin levels [38]. We found that mod-5(n822) mutants were less likely to leave an E. coli JI377 lawn than wild-type animals (Fig 3C). We conclude that serotonin functions in a dose-dependent manner to inhibit the nematode’s H 2 O 2 -induced food leaving behavior.

Hydrogen peroxide and bacteria have opposing effects on the activity of sensory neurons How did C. elegans overcome its strong attraction towards E. coli to specifically leave lawns that did not degrade hydrogen peroxide? C. elegans relies on sensory perception of bacterially-derived cues to efficiently find the bacteria it feeds on [39]. Most sensory functions in C. elegans hermaphrodites are performed by 60 ciliated and 12 non-ciliated neurons [40]. Twelve pairs of those ciliated neurons make up the nematode’s major sensory organs, the two amphids, responsible for smell, taste, and temperature sensation [41]. To assess the extent to which E. coli and H 2 O 2 modulated the function of amphid sensory neurons, we examined their activity in response to combinations of E. coli and H 2 O 2 . The activity of C. elegans sensory neurons is strongly correlated with their calcium responses [42]. We presented nematodes expressing the genetically encoded calcium indicator GCaMP6 in sensory neurons with six combinations of stimuli consisting of suspensions of E. coli MG1655, E. coli JI377, or water, pre-mixed with or without 1 mM H 2 O 2 for 20 hours (to give the bacteria an opportunity to break down the H 2 O 2 ). We used a custom-built microfluidic device to deliver these stimuli to the amphids of each L4 stage nematode in a randomized order, in 15 second intervals, preceded and followed by 45 second intervals without stimuli, while recording with single-cell resolution the activity of 26 sensory neurons via fluorescence microscopy (Fig 4A–4D). Our imaging studies covered 11 of the 12 pairs of amphid neurons (ADF, ADL, ASE, ASG, ASH, ASI, ASJ, ASK, AWA, AWB, and AWC), and 2 pairs of non-amphid neurons (BAG and URX). The six combinations of stimuli induced two major patterns of neuronal modulation, described below, with each pattern affecting the activity of multiple sensory neurons: one pattern was induced by 1 mM H 2 O 2 and E. coli JI377 with 1 mM H 2 O 2 , and the other pattern was induced by E. coli MG1655, E. coli JI377, and E. coli MG1655 with 1 mM H 2 O 2 (Fig 4E). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Hydrogen peroxide and bacteria have opposing effects on the activity of sensory neurons. (A) Schematic of the microfluidic setup for controlled delivery of sensory stimuli and calcium imaging of nematodes. (B) Multi-channel microfluidic device. The black arrow marks the inlet channel for loading the nematode, the purple arrow marks the outlet channel for fluid waste, the black asterisks mark the stimuli delivery channels that were used, and the purple asterisk marks the buffer delivery channel. (C) Magnified view of the channel where the sensory endings in the head of the immobilized nematode are stimulated with mixtures of bacteria and H 2 O 2 . (D) Relative fluorescence of the GCaMP6 genetically encoded calcium indicator expressed by the animal. 26 sensory neurons in the nematode’s head (13 shown) were imaged with single cell resolution. (E) Clustering of the mean changes in GCaMP6 fluorescence of 26 sensory neurons in response to six stimuli consisting of suspensions of E. coli MG1655, E. coli JI377, or water, with or without adding 1 mM H 2 O 2 . (F-K) Average GCaMP6 fluorescence traces of (F) ASJ, (G) ADF, (H) AWA, (I) BAG, (J) ASK, and (K) ASH neuronal classes in response to six different stimuli (left sub-panels) and average changes in fluorescence in response to those stimuli (right sub-panels). The stimulus delivery interval is indicated by a shaded box. Data are represented as mean ± s.e.m. The number of neurons imaged was 28 ADF, 28 ADL, 14 ASEL, 14 ASER, 28 ASG, 28 ASH, 27 ASI, 28 ASJ, 28 ASK, 28 AWA, 13 AWB, 24 AWC, 18 BAG, and 28 URX. Groups labeled with different letters exhibited significant differences (P < 0.05, Tukey HSD test) otherwise (P > 0.05). Traces for the URX, ADL, ASEL, ASER, ASG, ASI, AWB, and AWC neuronal classes are shown in S5 Fig. https://doi.org/10.1371/journal.ppat.1010112.g004 H 2 O 2 strongly excited (increased [Ca2+]) the ASJ neuron pair (Fig 4F) which, later in this manuscript, we show is required for H 2 O 2 avoidance. In the ASK and URX neuron pairs, H 2 O 2 also increased the GCaMP6 signal relative to the water-only control, albeit more weakly than in the ASJ neuron pair (Figs 4J and S5A). These H 2 O 2 -induced increases in neuronal activity were abolished when H 2 O 2 was combined with E. coli MG1655 (which degrades H 2 O 2 ) but persisted when H 2 O 2 was combined with E. coli JI377 (which does not degrade H 2 O 2 ) (Figs 4F, 4J and S5A). Therefore, E. coli’s H 2 O 2 -degrading enzymes prevented the excitation of ASJ, ASK, and URX neurons by environmental H 2 O 2 . E. coli MG1655 and JI377 modulated the activity of mostly overlapping but distinct sets of sensory neurons. Both E. coli strains excited the ADF, AWA, AWB, and BAG neuron pairs (Figs 4G–4I and S5G) and inhibited (decreased [Ca2+]) the ASK pair (Fig 4J). MG1655 also excited ASEL (S5C Fig) and inhibited the ASH pair (Fig 4K), while JI377 excited the ADL pair (S5B Fig). When combined with H 2 O 2 , MG1655 elicited the same response pattern in ADF, ASK, ASH, and BAG as it did without H 2 O 2 (Fig 4G and 4I–4K). In contrast, when combined with H 2 O 2 , JI377 no longer elicited a significant response in ADF, ADL, ASK, AWA, and BAG (Figs 4G–4K and S5B). Therefore, E. coli’s H 2 O 2 -degrading enzymes prevented H 2 O 2 from blocking the specific excitation or inhibition of most of the sensory neurons that were modulated by E. coli. Most of the neuronal classes excited by E. coli MG1655 mediate locomotion towards attractive cues. ADF and ASEL sense water-soluble attractants [43,44], AWA detect attractive volatile odorants [45], and BAG sense oxygen and carbon dioxide [46,47]. The neuronal classes inhibited by E. coli MG1655 mediate locomotion away from repulsive cues; ASK and ASH sense various repellents [48,49]. We propose that, when E. coli cannot degrade environmental H 2 O 2 , the strong attraction of C. elegans to E. coli is weakened because H 2 O 2 prevents the modulation of those classes of neurons by E. coli, increasing the chances that the nematode would leave the E. coli lawn.

The H 2 O 2 -sensing ASJ neurons are required for H 2 O 2 avoidance Because the ASJ neuronal pair was strongly excited by H 2 O 2 (Fig 4F), we speculated these neurons may mediate an aversive locomotory response to H 2 O 2 , in line with the role of ASJ in triggering an aversive response when excited by cues from the C. elegans predator Pristionchus pacificus [50] and from the bacterial pathogen Pseudomonas aeruginosa [51,52]. To quantify H 2 O 2 avoidance, we exposed nematodes to a drop of 1 mM H 2 O 2 or water and recorded the proportion of avoidance responses (a reversal followed by an omega bend, Fig 5A) in response to these stimuli [48,50,53]. H 2 O 2 elicited a significant increase in the proportion of avoidance responses relative to the water control (Fig 5B). In animals in which the ASJ neurons were genetically ablated via ASJ-specific caspase expression [54], that increase was absent (Fig 5C). Therefore, the H 2 O 2 -sensing ASJ neurons were required for H 2 O 2 avoidance. This ASJ-dependent aversive response to H 2 O 2 enables C. elegans to escape environments with lethal H 2 O 2 levels. PPT PowerPoint slide

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TIFF original image Download: Fig 5. The H 2 O 2 -sensing ASJ neurons are required for H 2 O 2 avoidance. (A) Schematic overview of hydrogen peroxide drop avoidance assay, with worm body shapes extracted from tracking data [97]: upon sensing the small volume of 1 mM H 2 O 2 on its path (i), the nematode initiates an avoidance response consisting of a reversal phase (ii), an omega turn (iii-iv), and the resumption of locomotion (v). (B) H 2 O 2 induces an increase in avoidance responses. ** indicates P < 0.002 (t-test). (C) Ablation of the ASJ neurons suppresses the increase in avoidance responses induced by H 2 O 2 . Groups labeled with different letters exhibited significant differences (P < 0.01, Tukey HSD test) otherwise (P > 0.05). Data are represented as mean ± s.e.m. of the average avoidance response of each animal per condition. https://doi.org/10.1371/journal.ppat.1010112.g005

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010112

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