Caenorhabditis elegans and its cognate bacterial diet comprise a reliable, widespread model to study diet and microbiota effects on host physiology. Nonetheless, how diet influences the rate at which neurons die remains largely unknown. A number of models have been used in C. elegans as surrogates for neurodegeneration. One of these is a C. elegans strain expressing a neurotoxic allele of the mechanosensory abnormality protein 4 (MEC-4d) degenerin/epithelial Na + (DEG/ENaC) channel, which causes the progressive degeneration of the touch receptor neurons (TRNs). Using this model, our study evaluated the effect of various dietary bacteria on neurodegeneration dynamics. Although degeneration of TRNs was steady and completed at adulthood in the strain routinely used for C. elegans maintenance (Escherichia coli OP50), it was significantly reduced in environmental and other laboratory bacterial strains. Strikingly, neuroprotection reached more than 40% in the E. coli HT115 strain. HT115 protection was long lasting well into old age of animals and was not restricted to the TRNs. Small amounts of HT115 on OP50 bacteria as well as UV-killed HT115 were still sufficient to produce neuroprotection. Early growth of worms in HT115 protected neurons from degeneration during later growth in OP50. HT115 diet promoted the nuclear translocation of DAF-16 (ortholog of the FOXO family of transcription factors), a phenomenon previously reported to underlie neuroprotection caused by down-regulation of the insulin receptor in this system. Moreover, a daf-16 loss-of-function mutation abolishes HT115-driven neuroprotection. Comparative genomics, transcriptomics, and metabolomics approaches pinpointed the neurotransmitter γ-aminobutyric acid (GABA) and lactate as metabolites differentially produced between E. coli HT115 and OP50. HT115 mutant lacking glutamate decarboxylase enzyme genes (gad), which catalyze the conversion of GABA from glutamate, lost the ability to produce GABA and also to stop neurodegeneration. Moreover, in situ GABA supplementation or heterologous expression of glutamate decarboxylase in E. coli OP50 conferred neuroprotective activity to this strain. Specific C. elegans GABA transporters and receptors were required for full HT115-mediated neuroprotection. Additionally, lactate supplementation also increased anterior ventral microtubule (AVM) neuron survival in OP50. Together, these results demonstrate that bacterially produced GABA and other metabolites exert an effect of neuroprotection in the host, highlighting the role of neuroactive compounds of the diet in nervous system homeostasis.

Funding: Millennium Scientific Initiative of the Chilean Ministry of Economy, Development, and Tourism (P029-022-F) to AC, Proyecto Apoyo Redes Formacion de Centros (REDES180138) to AC, and CYTED grant P918PTE 3 to AC. MC received funding from Fondecyt 1181089 to AJM and AC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2020 Urrutia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Genetically encoded prodegenerative stimuli, such as a dominant gain-of-function mutation on the mechanosensory channel gene mec-4 (mec-4d), encoding the MEC-4d degenerin, have proven effective in deciphering common molecular players of neuronal degeneration in invertebrates and mammals [ 16 , 17 , 18 ]. The touch receptor neurons (TRNs) of C. elegans respond to mechanical stimuli by causing an inward Na + current through the MEC-4 channel, a member of the degenerin/epithelial Na + (DEG/ENaC) family. Mutations near the second transmembrane (TM) helix (A713), termed mec-4d, cause the constitutive opening of the channel and the degeneration of the TRN, rendering animals insensitive to touch [ 19 ]. Necrosis of the TRNs in mec-4d worms is presumably due to unregulated Na + and Ca 2+ entry as well as reactive oxygen species (ROS) imbalance [ 18 , 20 , 21 ]. The degeneration of the TRN is a stochastic process that begins with the fragmentation of the axon followed by the swelling of the soma. Notably, the use of this model has allowed for interventions that can delay neurodegeneration, such as caloric restriction, antioxidant treatment, and mitochondria blockage [ 16 ], as well as diapause entry [ 22 ]. In this study, we evaluated the rate of degeneration of C. elegans neurons in different dietary bacteria and found that specific dietary bacteria promote protection from neuronal degeneration. Combining systems biology approaches coupled to genetics, we discovered that γ-aminobutyric acid (GABA) produced by bacteria is protective for C. elegans neurons undergoing degeneration. Moreover, further characterization indicated that GABA was not the sole metabolite involved in neuroprotection, and lactate was also identified.

Intestinal microbes regulate many aspects of host physiology [ 1 ], including immune system maturation [ 2 , 3 , 4 ], neurodevelopment, and behavior [ 5 , 6 , 7 ], among others. Recent reports show that in mood disorders and neurodegenerative diseases, the microbiome composition and abundance is altered, and this has provided a glimpse at the role of specific bacterial metabolites with neuroactive potential in the prevention of such disorders [ 8 , 9 , 10 , 11 ]. However, whether bacterial metabolites directly influence neuronal degeneration and their mechanisms of action are largely unknown. The bacterivore nematode Caenorhabditis elegans continues to provide an excellent model to study the relationship between bacteria and host [ 12 ]. Both the animal and its bacterial diet are genetically tractable, making them suitable for individual gene and large-scale mutation analysis. This system has been instrumental in deciphering specific metabolites from gut bacteria that influence developmental rate, fertility and aging [ 13 ], and host factors mediating germline maintenance in response to a variety of bacterial diets [ 14 ] as well as defensive behavioral strategies against pathogens [ 15 ].

Results

Bacterial diet influences the rate at which neurons degenerate We measured the effect of different dietary bacteria on the progression of genetically induced neuronal degeneration of the TRNs in a C. elegans mec-4d strain expressing a mutant mechanosensory channel, MEC-4d [19]. We previously showed that mec-4d-expressing anterior ventral microtubule (AVM) touch neuron dies in a stereotyped fashion and defined the window of time when animals feed on the standard laboratory E. coli OP50 diet [16]. Right after hatching, mec-4d mutant animals were fed different bacteria, and the AVM neuronal integrity was quantified in adulthood (72 hours later). The pertinence of the use of each of these strains is explained in the Materials and methods section. The dietary bacteria used were E. coli OP50, E. coli B, E. coli HT115, E. coli K-12, Comamonas aquatica, Comamonas testosteroni, Bacillus megaterium, and the mildly pathogenic Pseudomonas aeruginosa PAO1. As a soil nematode, C. elegans feeds on a large range of bacteria in its natural environment [23]. We also selected three bacterial species previously coisolated with wild C. elegans from soil by our group, namely Pseudochrobactrum kiredjianiae, Stenotrophomonas humi, and B. pumilus. In accordance with our previous reports, neurodegeneration steadily occurred when feeding with E. coli OP50: only a very low percentage of worms (1%–3%) maintained AVM axons after 3 days (Fig 1A). Notably, although neurodegeneration occurred with E. coli B, C. testosteroni, B. megaterium, and P. kiredjianiae similarly to when feeding with E. coli OP50, the bacteria E. coli HT115, E. coli K-12, C. aquatica, P. aeruginosa, S. humi, and B. pumilus gave significant protection (Fig 1B). E. coli HT115 was the most protective, with over 40% of wild-type axons 72 hours after hatching, compared with less than 6% in E. coli OP50 (Fig 1B and 1D). The broad difference on neuronal integrity in mec-4d worm populations feeding on E. coli OP50 or HT115 can be observed in Fig 1C. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Dietary bacteria determine the rate of neuronal degeneration. (A) TRNs expressing GFP in wild-type and mec-4d worms. The latter shows the stereotypical progression of AVM degeneration of mec-4d mutants and constitutes the axonal categories assessed during the experiment shown in (B). Scale bars represent 20 μm. (B) Percentage of all morphological axonal categories in mec-4d worms after 72 hours of growth in different bacterial strains. (C) Fluorescence microscopy fields of GFP-expressing mec-4d worms raised in the indicated E. coli strains comparing the presence of AVM axons on each preparation at 10× magnification. (D) Percentage of wild-type axons in the experiment shown in (B). (E) Percentage of touch responsiveness of animals after growth in the different dietary bacteria. ****P < 0.0001, ***P < 0.001, **P < 0.005, *P < 0.05, ns. The underlying numerical data and statistical analysis for each figure panel can be found in S1 and S2 Datasets, respectively. AVM, anterior ventral microtubule; Axϕ, degenerated axon; AxT, truncated axon; AxW, wild-type axon; GFP, green fluorescent protein; mec-4d, mechanosensory abnormality protein 4; ns, not significant; TRN, touch receptor neuron. https://doi.org/10.1371/journal.pbio.3000638.g001 TRNs are neurons expressing receptors of gentle mechanical stimuli [24]. Hence, we determined the response to gentle touch in worms fed the different strains to test whether morphological protection shown in Fig 1B translates into functional responses. Fig 1E shows that the number of responses in worms correlates with the morphological categories AxW and AxL, the two axonal categories defined as functional in previous work [22].

Bacterial components promote neuroprotection Phenotypical outcomes mediated by intestinal bacteria can be a result of either a modulation of host physiology by interspecies live interactions (i.e. bacterial colonization) or by the exposure of the host to a bacterial metabolite. The first one requires bacteria to be alive in the intestine, whereas the second does not. To distinguish between these two possibilities, we fed mec-4d animals with UV-killed HT115 bacteria, the most protective among those tested, and scored the AVM integrity at 72 hours. Additionally, we also evaluated UV-killed P. aeruginosa PAO1, a mild pathogen that needs to be alive to colonize and induce long-term defensive responses in the animal [15]. In both cases, dead bacteria protected to the same extent as live bacteria (Fig 2A). This indicates that protective molecules of E. coli HT115 bacteria are produced prior to exposure to the animals, and thus neuronal protection is independent of the induction of bacterial responses by the interaction with the host. Furthermore, worms raised in dead HT115 bacteria cultivated to different optical densities (ODs) displayed the same levels of neuroprotection, suggesting that the protective factors are present in the bacteria during all phases of the growth curve (S1A and S1B Fig). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Bacterial components have neuroprotective activity. (A) Axonal categories in worms raised in the indicated live or UV-killed bacterial strains. (B–C) All axonal categories (B) or wild-type (C) axons in worms rose in different proportions of UV-killed E. coli HT115 on UV-killed OP50. (D) Wild-type axons of mec-4d animals fed with E. coli HT115 or OP50 bacterial pellet supplemented with supernatant of OP50 or HT115. ****P < 0.0001, ***P < 0.001, **P < 0.005, *P <0.05; ns. “a” and “b” are used to indicate statistically significant differences. The underlying numerical data and statistical analysis for each figure panel can be found in S1 and S2 Datasets, respectively. Axϕ, degenerated axon; AxT, truncated axon; AxW, wild-type axon; LB, Luria-Bertani; mec-4d, mechanosensory abnormality protein 4; NGM, nematode growth media; ns, not significant. https://doi.org/10.1371/journal.pbio.3000638.g002 The large difference in neuroprotection between E. coli OP50 and HT115 strains raises two possibilities: (1) E. coli OP50 actively promotes the degeneration of the neuron, and (2) HT115 has a protective effect. To discern this matter, we fed worms with a mix of UV-killed E. coli HT115 and OP50 in different proportions (Fig 2B and 2C). A 1/100 (1%) dilution of E. coli HT115 in OP50 was sufficient to protect AVM neurons significantly more than pure OP50. This strongly suggested that E. coli HT115 produces a neuroprotective compound that is needed in small amounts. To test whether the neuroprotective molecules are being secreted by the bacteria, we separated the supernatant of both bacterial strains from their pellets by centrifugation and mixed the supernatant of E. coli HT115 with OP50 pellet and vice versa (see Materials and methods for details). E. coli HT115 supernatant was not capable of providing protective activity when mixed with E. coli OP50 pellet (Fig 2D). This suggests either that the protective factor is not secreted or that the amounts contained in the supernatant are not sufficient for protection. As expected, E. coli OP50 supernatant did not alter the protection pattern of E. coli HT115.

E. coli HT115 diet promotes long-term protection of mechanoreceptors and interneurons of the touch receptor circuit E. coli HT115 was shown to be neuroprotective throughout the development of the animal and into young adulthood (Fig 1B). We explored whether AVM neurons are still protected after worms reached maturity. To that end, we fed newly hatched mec-4d animals with E. coli HT115 and scored their neuronal integrity every 24 hours for 168 hours. While on E. coli OP50, all animals had degenerated neurons at the final time point, and on HT115 food, 25% of animals had wild-type AVM axons (Fig 3A and 3C), confirming the ability of HT115 to significantly protect at later life stages. Notably, between 12 and 24 hours after hatching in HT115, there was a statistically significant rise in wild-type axons (AxW, Fig 3C), suggesting that neurons could be growing after an initial truncation. To assess this, we followed individual animals in a longitudinal fashion on E. coli HT115 and scored the neuronal integrity of each nematode every 24 hours for 3 days. We scored axons separately according to their initial and final morphology and classified axonal outcome as “protection” when the morphology of axons did not change from truncated or wild type and “degeneration” when axonal morphology changed from truncated axon (AxT) to degenerated axon (Axϕ) or was maintained as Axϕ. Finally, “regeneration” refers to axon growth from truncations to wild type. Although the most prevalent category is protection (40%), 30% of axons regenerated between 24 and 72 hours after hatching on E. coli HT115 (Fig 3D). This suggests that under HT115 protective conditions, a portion of neurons can repair broken axons. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Neuroprotection induced by dietary E. coli HT115 is long lasting and extends to other neuronal types. Time course of axonal categories of worms fed with E. coli OP50 (A) or HT115 (B) for 168 hours. (C) Percentage of wild-type axons in (A) and (B). (D) Proportion of axonal categories in longitudinal assays. Protection (green) indicates axons that were not degenerated over time regardless of the initial category, with the exception of Axϕ. Regeneration (gray) accounts for axons that grew in size over time. Degeneration (red) accounts for axons that degenerated over time. Determination of wild-type axons in AVM (E), ALM (F), PLM (G), and PVC (H) neurons of worms fed E. coli OP50 or HT115. ****P < 0.0001, ***P < 0.001, ns. The underlying numerical data and statistical analysis for each figure panel can be found in S1 and S2 Datasets, respectively. ALM, anterior lateral microtubule; AVM, anterior ventral microtubule; Axϕ, degenerated axon; AxT, truncated axon; AxW, wild-type axon; ns, not significant; PLM, posterior lateral microtubule; PVC, ventral cord interneuron; PVM, posterior ventral microtubule. https://doi.org/10.1371/journal.pbio.3000638.g003 Next, we explored whether other neurons of the touch circuit are protected from degeneration in E. coli HT115 diet. It has been already reported that at hatching, four embryonic TRN, two anterior lateral microtubule (ALMs), and two posterior lateral microtubule (PLM) neurons have already degenerated when growing at 20 °C in this model [16,25]. At 25 °C, however, degeneration proceeds at a slower rate [16,22]. To analyze the degeneration rate of ALM and PLM neurons, animals in the fourth larval stage (L4) were grown at 25 °C, and their progenies were synchronized at birth. The neuronal integrity of ALM, PLM, and AVM cells was assessed at 12, 24, and every 24 hours after birth until 168 hours at 25 °C. The percentage of wild-type neurons in E. coli HT115 diet is significantly higher than that in the OP50 diet throughout the temporal course for all three neurons (Fig 3E and 3G, full morphological characterization is shown in S2A and S2F Fig). We tested next whether E. coli HT115 was capable of protecting the ventral cord interneuron (PVC) expressing the degenerin-1 (deg-1) prodegenerative stimulus. deg-1(u38) animals progressively lose the ability to respond to posterior touch due to the time-dependent degeneration of the PVC interneuron [26]. We tested the posterior touch response of deg-1 animals during development feeding on E. coli OP50 and HT115. E. coli HT115 promotes a larger functional response than E. coli OP50, suggesting that this neuron is also protected (Fig 3H). Taken together, these results demonstrate that HT115 diet is protective over different neuronal types undergoing degeneration. Neurodegeneration of the TRNs is directly related to the expression of a neurotoxic form of the mechanosensory channel. Therefore, it is formally possible that a decrease in the expression of the channel would diminish the prodegenerative stimulus and promote protection. We sought to evaluate whether HT115 diet changes the expression of the MEC-4d channel in the membrane. We constructed a double mutant of mec-4d expressing MEC-4::green fluorescent protein (GFP) and quantified the number of channels of PLMs in HT115-fed animals compared with OP50. S3A and S3B Fig show that channel number remains constant in both diets, ruling out that protection conferred by HT115 affects expression of MEC-4d channel in the membrane.

Early exposure of animals to E. coli HT115 is sufficient for neuronal protection Ad libitum feeding on E. coli HT115 protected mec-4d-expressing neurons from degeneration for long periods of time. We sought to investigate whether a constant stimulus provided by the HT115 metabolite is required to achieve neuroprotection or an early, discrete time-lapse exposure to the diet is sufficient. We fed animals for the first 6 hours after hatching (previous to the birth of the AVM) and for 12 hours after hatching (at birth of the neuron) with UV-killed E. coli HT115 and immediately switched to E. coli OP50. We scored the neuronal morphology 12, 24, 48, and 72 hours posthatching (S4A and S4D Fig). In parallel, both diets were fed ad libitum as controls. Strikingly, animals that ingested E. coli HT115 for only 6 hours showed a significantly larger number of wild-type neurons at 72 hours (14.3%) than animals continuously fed OP50 (3.6%, Fig 4A) and had more axons in the other categories (S4A and S4C Fig). Feeding HT115 to mec-4d animals for the first 12 hours after hatching conferred a significantly larger protection than feeding HT115 for only 6 hours (Fig 4A). These results show that although early short exposures are not equally protective as a permanent HT115 diet, they do have a long-lasting effect in neurons compared with an uninterrupted diet of E. coli OP50. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Diet of E. coli HT115 at early stages of life is necessary and sufficient to confer neuroprotection. (A and B) Percentage of wild-type axons of animals fed for 6 and 12 hours with (A) E. coli HT115 or (B) E. coli OP50 and then changed to OP50 or HT115 diet, respectively. (C) Percentage of wild-type axons of animals feeding on either E. coli OP50 or HT115 whose parents were fed on either diet. The underlying numerical data and statistical analysis for each figure panel can be found in S1 and S2 Datasets, respectively. F, filial generation; P, parental generation. https://doi.org/10.1371/journal.pbio.3000638.g004 We then tested the effect of early exposure to nonprotective bacteria. We fed mec-4d animals for 6 and 12 hours with UV-killed E. coli OP50 and then changed them to HT115. The morphology of AVM neurons was scored at 12, 24, 48, and 72 hours posthatching. Six hours of E. coli OP50 exposure did not prevent HT115 from protecting AVM neurons later in adulthood (Fig 4B and S4E Fig). Exposure for 12 hours, however, precludes protection of the AVM (Fig 4B and S4F Fig). This suggests that the time between the first 6 and 12 hours of development is crucial for the protective effect to take place. In C. elegans, some dietary bacteria–induced traits show heritable properties [15]. Therefore, we tested whether neuronal protection could be inherited. Animals were fed either E. coli OP50 or HT115 from birth, and their F1 progeny transferred to OP50 or HT115. Neuronal integrity of descendants was tested in a time course fashion. One generation of parental feeding on HT115 did not improve neuronal protection in the progeny feeding on OP50, nor did E. coli food preclude protection of filial generation (F) 1 feeding on HT115 (Fig 4C). This result indicates that the protective effect of E. coli HT115 is not transmitted intergenerationally.

Identification of uniquely expressed genes on neuroprotective bacteria To identify the bacterial molecule(s) conferring neuroprotection, we looked for differences in the genomes and transcriptomes of the two E. coli strains. We reasoned that genes important for neuronal protection would be uniquely expressed or up-regulated in E. coli HT115 compared with E. coli OP50. We first sequenced the genomes of E. coli HT115 and OP50 using the Illumina MiSeq platform. Notably, we found that E. coli OP50 has a deletion of 23 Kbp, containing genes for the regulators of capsular system (rcsDB, rcsC) required for envelope stress response, genes for short-chain polyhydroxybutyrate synthesis (atoSC, atoDAEB), and the pseudogene yfaATSQP [27]. Thus, RcsB is a unique E. coli HT115 gene that codes a main transcription factor required for the activation of the glutamate decarboxylase enzyme gene (gad) operons, alone or coupled to other regulators by positive feedback as illustrated in Fig 5A. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Enzymes of the GAD enzyme operon are uniquely and highly expressed in E. coli HT115. (A) Scheme of regulation of the gad operon. (B) Log2 Fold Change of genes coding for enzymes of GABA metabolism. The underlying numerical data and statistical analysis for each figure panel can be found in S1 Dataset and S2 File, respectively. GABA, γ-aminobutyric acid; GAD, glutamate decarboxylase; gad, glutamate decarboxylase enzyme gene; rcs, regulator of capsular system. https://doi.org/10.1371/journal.pbio.3000638.g005 Transcriptomic analysis determined that the genes involved in resistance to acidic environments were highly up-regulated in E. coli HT115 (Fig 5B). The glutamate decarboxylase operons gadAX and gadBC include the genes gadA and gadB, both encoding for the enzyme glutamate decarboxylase (GAD), which converts glutamate to GABA, whereas gadC encodes a glutamate/GABA antiporter. Other overexpressed genes in HT115 include those for the periplasmic acid stress chaperones hdeA and hdeB. Importantly, this overexpression occurs while global regulators also involved in the acidic response remain equally expressed in both strains (CRP-AMPc, H-NS, or Fis). Therefore, rcsB deletion seems to induce a specific metabolic difference between both bacteria, i.e., the abolition of GABA production in E. coli OP50. To endure this deficit, genes related to sodium/glutamate and glutamate/aspartate transport were up-regulated in E. coli OP50 as shown in Fig 5B (gltS Log 2 Fold Change (LgFC) = −4.69, gltK LgFC = −2.08, respectively). Additionally, other genes related to GABA metabolism (the transaminase gabT LgFC = 2.67) and membrane permeability (permease gabP LgFC = 2.74) were also up-regulated in E. coli HT115 from non-rcsB-dependent operons. Interestingly, no other metabolic enzyme-related genes were up-regulated in either bacterium. This suggests that enzymes and metabolites involved in the pathway of GABA production and utilization are good candidate neuroprotective players.

GAD and its product GABA are required for E. coli HT115 neuroprotection To test the role of GAD and its product GABA in neuroprotection, we first generated a gad null mutant of E. coli HT115 by homologous recombination (HT115Δgad, details in Materials and methods). To corroborate that HT115Δgad lacked GAD activity, we used a colorimetric assay based on pH elevation given by the conversion of glutamate to GABA [28,29]. As expected, wild-type E. coli HT115 raised the pH of the solution, whereas neither HT115Δgad nor OP50 were able to do so. To confirm that a rise in pH is due to the expression of GAD, we transformed E. coli OP50 with a plasmid expressing gadA (pGgadA). E. coli OP50 pGgadA supplemented with glutamate showed potent enzymatic activity, raising the pH of the solution above HT115 levels (Fig 6A). Secondly, we fed mec-4d animals with E. coli HT115Δgad and scored its protective potential at 72 hours. HT115Δgad was not able to protect degenerating AVM neurons, showing a significant reduction of wild-type axons compared with wild-type strains (Fig 6B). This shows that GAD activity plays a pivotal role in the protection conferred by HT115 bacteria. Moreover, plasmid pGgadA was able to rescue protective potential in null mutant HT115Δgad. Additionally, dietary supplementation of UV-killed HT115Δgad with 2 mM of GABA was sufficient to provide neuroprotection (Fig 6B and S5A Fig). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. Bacterial GABA is crucial for neuroprotection. (A) Measurements of GAD enzyme activity normalized as a percentage of HT115 GAD activity in wild-type, mutant, and transformed bacterial strains. (B and C) Percentage of wild-type axons in wild-type and Δgad mutant HT115 strain (B) and wild-type OP50 strain (C) supplemented with GABA and genetically transformed with the pGgadA plasmid. ****P < 0.0001, ***P < 0.001, ns. The underlying numerical data and statistical analysis for each figure panel can be found in S1 and S2 Datasets, respectively. GABA, γ-aminobutyric acid; GAD, glutamate decarboxylase; gad, glutamate decarboxylase enzyme gene; ns, not significant. https://doi.org/10.1371/journal.pbio.3000638.g006 Finally, we fed mec-4d with E. coli OP50 pGgadA to test whether gadA is sufficient to provide protective activity in the presence or absence of glutamic acid, the substrate for GAD. Whereas E. coli OP50 pGgadA alone was not sufficient to increase wild-type axons incidence, glutamate addition to the bacterial culture significantly increased the presence of wild-type axons compared with E. coli OP50 pGgadA and E. coli OP50 wild type (Fig 6C and S5B Fig). This is coherent with the increased GAD activity of E. coli OP50 supplemented with pGgadA shown in Fig 6A. Furthermore, supplementation of HT115Δgad with 2 mM GABA was sufficient to provide neuroprotection (Fig 6C). Importantly, addition of 2 mM GABA to UV-killed E. coli OP50 lawn protected mec-4d neurons significantly more than OP50 alone, even though it did not reach HT115 levels (Fig 6C). Taken together, these results show that GAD and its product GABA play an important role in E. coli HT115–mediated neuroprotection.

GAD activity and GABA levels correlate with neuroprotection conferred by other bacteria The amount of a neuroprotective metabolite present in a given bacterium could be an indicator of that bacteria’s ability to confer neuronal protection. Bacteria tested by us in Fig 1B gave different degrees of protection to mec-4d animals. We evaluated GAD activity in all strains and normalized it against E. coli HT115 (Fig 8A). All strains except E. coli K-12 exhibited less GAD activity than E. coli HT115. Additionally, we directly measured GABA production using the GABA‐aminotransferase plus succinic semialdehyde dehydrogenase (GABase test [30] and S9 Fig). E. coli HT115 pellet had the highest GABA levels, whereas OP50 and HT115Δgad were indistinguishable from each other (Fig 8B). This demonstrates that GABA is being produced in E. coli HT115 and not in OP50 or the HT115 Δgad strain. P. aeruginosa PAO1 and B. pumilus had less GABA than HT115 but significantly more than most strains (Fig 8B). To understand whether there was a correlation between GAD and GABA levels with neuroprotection conferred by these bacteria, we performed a Pearson correlation test. Fig 8C shows that GAD expression and GABA levels are correlated with neuroprotective activity in all strains. Importantly, GABA concentration was a better indicator (r = 0.88) than GAD activity (r = 0.67). These results support the previous evidence that bacterial GAD enzyme and its product GABA are key for neuroprotection. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 8. GAD activity and GABA levels correlate with neuroprotection conferred by other bacteria. Measurements of GAD enzyme activity normalized as a percentage of HT115 GAD activity (A) and GABA concentration found in pellets (B) in all bacteria used. (C) Correlation between GAD activity and GABA concentration in bacteria diet and percentage of wild-type axons in C. elegans. ****P < 0.0001, **P < 0.005; ns. “a,” “b,” “c,” and “d” are used to indicate statistically significant differences. The underlying numerical data and statistical analysis for each figure panel can be found in S1 and S2 Datasets, respectively. GABA, γ-aminobutyric acid; GAD, glutamate decarboxylase; ns, not significant. https://doi.org/10.1371/journal.pbio.3000638.g008