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Colonization of the Caenorhabditis elegans gut with human enteric bacterial pathogens leads to proteostasis disruption that is rescued by butyrate

['Alyssa C. Walker', 'Department Of Microbiology', 'Cell Science', 'University Of Florida', 'Gainesville', 'Florida', 'United States Of America', 'Rohan Bhargava', 'Alfonso S. Vaziriyan-Sani', 'Christine Pourciau']

Date: 2022-02 

Protein conformational diseases are characterized by misfolding and toxic aggregation of metastable proteins, often culminating in neurodegeneration. Enteric bacteria influence the pathogenesis of neurodegenerative diseases; however, the complexity of the human microbiome hinders our understanding of how individual microbes influence these diseases. Disruption of host protein homeostasis, or proteostasis, affects the onset and progression of these diseases. To investigate the effect of bacteria on host proteostasis, we used Caenorhabditis elegans expressing tissue-specific polyglutamine reporters that detect changes in the protein folding environment. We found that colonization of the C. elegans gut with enteric bacterial pathogens disrupted proteostasis in the intestine, muscle, neurons, and the gonad, while the presence of bacteria that conditionally synthesize butyrate, a molecule previously shown to be beneficial in neurodegenerative disease models, suppressed aggregation and the associated proteotoxicity. Co-colonization with this butyrogenic strain suppressed bacteria-induced protein aggregation, emphasizing the importance of microbial interaction and its impact on host proteostasis. Further experiments demonstrated that the beneficial effect of butyrate depended on the bacteria that colonized the gut and that this protective effect required SKN-1/Nrf2 and DAF-16/FOXO transcription factors. We also found that bacteria-derived protein aggregates contribute to the observed disruption of host proteostasis. Together, these results reveal the significance of enteric infection and gut dysbiosis on the pathogenesis of protein conformational diseases and demonstrate the potential of using butyrate-producing microbes as a preventative and treatment strategy for neurodegenerative disease.

Protein conformational diseases are one of the leading causes of geriatric death and disability, worldwide. Individuals suffering from these ailments are limited to palliative care, as there are no cures or effective treatments. Correlational evidence suggests that the human gut microbiota is a culprit, but the effect of individual bacteria remains elusive, in part, due to the complexity of the microbiome. A single-bacterium approach can help to deconvolute the complexity of the microbiome and reveal the effect of individual bacterial species on organismal proteostasis. As such, we utilized the intestine of C. elegans as a “test tube” to identify the effect of bacteria on the host using tissue-specific polyglutamine repeats as protein folding sensors. We found that colonization of the C. elegans intestine with pathogenic gram-negative bacteria disrupted proteostasis in the intestine, muscle, neurons, and gonads. Furthermore, we demonstrated that butyrogenic bacteria enhanced proteostasis, which was evidenced by a decrease in polyglutamine aggregation and suppression of aggregate-dependent toxicity. Further experiments revealed that co-colonization with butyrogenic bacteria inhibited protein aggregation in C. elegans and the butyrate-mediated suppression of aggregation is dependent on SKN-1/Nrf2 and DAF-16/FOXO–two transcription factors involved in the regulation of oxidative stress responses. While the mechanism of bacteria-mediated induction of protein aggregation remains elusive, our results suggest that bacterial aggregates, in addition to the contribution of oxidative stress, are the contributing factor. These results are intriguing as they suggest that enteric bacteria directly contribute to the pathogenicity of protein conformational diseases.

Here, we show that colonization of the C. elegans gut with gram-negative enteric bacterial pathogens disrupts host proteostasis, leading to aggregation and proteotoxicity of polyglutamine (polyQ) tracts across multiple tissues, including intestine, muscle, neurons, and the gonads. Such tissue non-autonomous effects of bacteria on the host may explain the impact of the gut microbiota on the onset and progression of PCDs. While pathogenic bacteria contribute to aggregation, commensal strains suppress it. Additionally, we show that butyrate and butyrogenic bacteria enhance host proteostasis and lead to suppression of bacteria-mediated polyQ aggregation and proteotoxicity. Oxidative stress and bacteria-derived protein aggregates seem to contribute to the observed disruption of proteostasis. Collectively, our results suggest that dysbiosis between enteric pathogens and commensal butyrogenic bacteria contributes to the pathogenicity of PCDs.

The human gut microbiota (HGM) is a complex assembly of microorganisms capable of synthesizing molecules that can impact the host’s physiology [ 14 ]. A healthy commensal relationship between a balanced microbiome and the host is fostered by their cross-talk [ 15 ]. Conversely, gut dysbiosis results from the loss of beneficial bacteria accompanied by the overgrowth of opportunistic pathogens, a condition often induced by antibiotics [ 16 , 17 ]. Recent evidence has established a link between the HGM and PCDs, whereby gut dysbiosis or direct enteric infection exacerbate the disease [ 18 – 20 ]. Commensal residents of the human microbiota, specifically those involved in the synthesis of short-chain fatty acids (SCFAs) such as butyrate, were shown to be beneficial to the host [ 21 ]. Unfortunately, environmental factors and the complexity of the human microbiome often hinder the consistency of results from experiments that pertain to PCDs [ 22 ]. To eliminate such complexity, we employed Caenorhabditis elegans as a model to study the effect of enteric pathogens on host proteostasis and examined the benefits provided by butyrogenic bacteria.

Neurodegenerative protein conformational diseases (PCDs), including amyotrophic lateral sclerosis (ALS), Alzheimer’s, Huntington’s, and Parkinson’s disease, are characterized by the misfolding and aggregation of metastable proteins that reside within the proteome, often resulting in loss of tissue function that manifests in disease progression [ 1 ]. Despite the high prevalence and enormous financial and social burdens imposed on afflicted individuals and their families [ 2 ], no effective treatment or cure has been found; moreover, the etiology of these diseases remains largely unknown [ 3 ]. Factors such as age, diet, stress, trauma, toxins, infections, or antibiotics, have been shown to increase the risk of PCDs [ 4 – 10 ]. Notably, these triggers are also associated with changes in the microbiome [ 11 – 13 ], suggesting that bacteria may contribute to the pathogenesis of PCDs, which may explain their sporadic onset. Nonetheless, the relationship between the microbiome and disease progression remains poorly defined.

The human gut microbiota is a polymicrobial community and the effect of the interactions between individual strains and the host is hindered by the complexity and wealth of its composition. We co-colonized the C. elegans gut with pathogenic and butyrogenic bacteria as a strategy to begin deconvoluting complex polymicrobial interactions within the human microbiome and their effect on the host. We fed worms cultures of E. coli Bt with either E. coli OP50 as a control or P. aeruginosa PAO1, which showed the most robust enhancement of aggregation. In the presence of L-arabinose, we found a significant decrease in aggregation of intestinal polyQs in co-cultures with both strains ( Fig 10A ). E. coli Bt suppressed E. coli OP50- and P. aeruginosa PAO1-mediated aggregation by approximately 4- and 2-fold, respectively. These results indicate that endogenous butyrate synthesized by butyrogenic bacteria can enhance host proteostasis and suppress protein misfolding. Furthermore, our experiments demonstrate the potential benefits of butyrate and butyrogenic bacteria in the suppression of bacteria-induced proteotoxicity associated with metastable proteins and also emphasize the importance of having a balance between commensal butyrogenic and enteropathogenic microbes ( Fig 10B ).

We expanded our investigation to animals carrying a neuronal polyQ40 reporter by measuring toxicity using the TOP phenotypic readout. We employed this method to assess the effect of endogenous butyrate on aggregate-dependent toxicity. The availability of L-arabinose to control E. coli strains (E. coli OP50 and E. coli WT ) did not improve motility. Still, the butyrate produced by E. coli Bt significantly enhanced motility in animals carrying the neuronal polyQ reporter ( Fig 9D ), but not in N2 control worms ( Fig 9E ). While further investigation is needed to elucidate the basis of this response, the result could be key to understanding the mechanism(s) by which butyrogenic bacteria affect host proteostasis.

A) The effect of butyrogenic bacteria (E. coli Bt ) on the aggregation profile of worms expressing muscle-specific polyQ35. Data are represented as the average number of aggregates of muscle-specific polyQ35 (AM140) per worm normalized to the control (no arabinose). Animals were fed four different strains of bacteria: non-butyrogenic controls (E. coli OP50, E. coli WT , E. coli Δ ) and conditional butyrogenic E. coli (E. coli Bt ). B) The effect of butyrogenic bacteria on the motility of animals expressing muscle-specific polyQ35 and C) control N2 worms. Data are represented as the average number of body bends per worm normalized to control (no arabinose). Each bar is an average of three independent experiments with a total of 45 animals. D) The effect of butyrogenic bacteria on the motility of animals expressing neuronal polyQ40 and on E) control N2 worms. Data are represented as the average TOP seconds per worm normalized to control animals (no arabinose). Error bars represent SEM. Statistical significance between each pair was calculated using Student’s t-test (*p<0.05, **p<0.01, ***p<0.0005 ****p<0.0001).

We further investigated whether the effect of endogenous butyrate produced by E. coli Bt strain was limited to the intestine or was ubiquitous across tissues, as seen when the animals were exposed to exogenous butyrate. To accomplish this, we colonized the intestines of animals expressing muscle and neuronal polyQ with E. coli Bt . While supplementation of 3% L-arabinose into NGM plates did not affect intestinal polyQ aggregation in animals colonized by control E. coli strains ( Fig 8A ), it did enhance aggregation in animals expressing muscle-specific polyQs ( Fig 9A ). Intriguingly, this enhancement of aggregation was significantly reduced in the presence of L-arabinose in animals colonized by E. coli Bt , suggesting that endogenously produced butyrate suppresses protein aggregation in the muscle tissue as well ( Fig 9A ). We then assessed the effects of this strain on aggregate-dependent toxicity in this model utilizing the body bend assay. Our results show that worms expressing muscle-specific polyQ have 2.5x more body bends when E. coli Bt is endogenously producing butyrate, compared to worms colonized with non-butyrogenic bacteria or butyrogenic bacteria in the absence of the sugar substrate ( Fig 9B ). The observed beneficial effect of endogenous butyrate is dependent on the presence of polyQ since endogenous butyrate did not increase the number of body bends in control wild-type worms ( Fig 9C ). In fact, the presence of endogenous butyrate was slight, but significantly detrimental to wild-type C. elegans. These results support the beneficial effect of butyrate in the suppression of aggregation and the associated proteotoxicity in the muscle tissues.

Animals were fed four different strains of bacteria: non-butyrogenic controls (E. coli OP50, E. coli WT , E. coli Δ ) and conditional butyrogenic E. coli (E. coli Bt ). A) The graphs represent the average number of intestinal polyQ44 aggregates per worm normalized to the control (no arabinose). Each bar is an average of three independent experiments with a total of 100 animals. B) Intestinal aggregate-dependent toxicity normalized to the control (no arabinose) assessed with the TOP phenotype. The left panel represents roller worms (Control), the middle panel represents polyQ33 worms, and the right panel represents worms expressing polyQ44. Each bar is an average of three independent experiments with a total of 60 animals. Error bars represent SEM. Statistical significance between each pair was calculated using Student’s t-test (*p<0.05, ****p<0.0001).

SCFA-producing bacteria are common residents of the human gut microbiota. Many of these bacteria, such as those belonging to the Firmicutes phylum, are known to produce butyrate [ 42 ]. Butyrate has been demonstrated to provide numerous health benefits against a variety of ailments, and neurodegenerative diseases are no exception [ 32 ]. To determine the effect that butyrogenic bacteria have on organismal proteostasis, we used an E. coli strain, LW393 (E. coli Bt ), engineered to conditionally overproduce butyrate [ 43 ]. Colonizing C. elegans intestines with this strain allows us to model simplified physiological, yet controlled, conditions. The strain was derived from E. coli W (E. coli WT ), a non-pathogenic strain closely related to commensal bacteria by first creating a quadruple deletion mutant (ldhA, frdABCD, ackA, and adhE), E. coli BEM9 (E. coli Δ ) strain, followed by overexpression of enzymes that channel intermediates into the pathway and which are essential for butyrate synthesis [ 43 ]. Therefore, we used E. coli WT and, in some instances, E. coli Δ , as controls. E. coli Bt synthesizes butyrate in the presence of 5- and 6-carbon sugar substrates to levels up to three times higher than any other available commercial strain [ 43 ]. We first used this strain to colonize the intestine of C. elegans expressing the intestinal polyQ44 reporter over a period of four days. We demonstrated that, on days three and four, C. elegans intestines are colonized by viable bacteria ( S11 Fig ). While glucose is a suitable substrate for butyrate synthesis, we found that it affects polyQ aggregation and thus cannot be utilized in our experiments ( S12 Fig ); however, L-arabinose is another butyrate synthesis substrate and does not affect aggregation. To confirm that L-arabinose from the NGM plates is available to intestinal bacteria, we colonized C. elegans intestines with bacteria that express an arabinose-inducible fluorescent reporter and cultured them in the absence of the sugar. We then transferred these animals onto plates containing E. coli OP50 and 3% L-arabinose and detected a strong induction of fluorescence, indicating that L-arabinose is accessible to the intestinal bacteria ( S13 Fig ). We supplemented NGM plates with 3% L-arabinose and quantified the number of aggregates after four days feeding polyQ-expressing animals the three control (E. coli OP50, E. coli WT , and E. coli Δ ) and the butyrate-producing (E. coli Bt ) strains. We did not detect any effect on polyQ aggregation in worms colonized with each of the control E. coli strains; however, supplementation of L-arabinose did result in significant suppression of polyQ aggregation in animals colonized by E. coli Bt ( Fig 8A ). To determine whether the aggregate-suppressing effect of E. coli Bt would also rescue polyQ-associated motility defects, we measured the TOP phenotype in worms expressing intestinal polyQ44. In agreement with the observed suppression of aggregation, supplementation of L-arabinose to animals colonized by E. coli Bt also improved motility, indicating that bacteria-derived butyrate can rescue aggregation and aggregate-associate toxicity ( Fig 8B ).

A) The effect of hsf-1, skn-1, and daf-16 knockdown on intestine-specific polyQ44 aggregation in the presence of exogenous butyrate supplementation, compared to empty vector control (L4440). Data are represented as the average number of aggregates per C. elegans intestine. Each data point is an average of a minimum of three independent experiments with a total of at least 85 animals. Error bars represent SEM. Statistical significance was calculated using one-way ANOVA followed by multiple comparison Dunnett’s post-hoc test (*p<0.05, ****p<0.0001). B) Fluorescent and Nomarski images of worms expressing reporter constructs regulated by HSF-1, SKN-1, and DAF-16. Worms were fed E. coli expressing either the control empty vector (EV) shown in the left panels or specific RNAi shown in the right panels. To activate the reporters, animals expressing hsp70p::GFP and gcs-1p::GFP were either heat shocked (HS) or exposed to 5 mM acrylamide (AA), respectively. Functional knockdowns were confirmed by the attenuated GFP expression in animals fed RNAi versus EV. Scale bar = 200 μm.

To gain a mechanistic understanding of how butyrate suppresses bacteria-mediated aggregation, we investigated the dependence of the observed response on three major evolutionary-conserved transcription factors, HSF-1, SKN-1/Nrf2, and DAF-16/FOXO. Each of these transcription factors was previously shown to provide protection against proteotoxicity [ 38 – 40 ]. Because butyrate suppressed aggregation upon colonization with multiple strains of bacteria, including E. coli OP50 ( Fig 4 ), we investigated the dependence of butyrate-mediated suppression of aggregation on the abovementioned transcription factors by knocking-down each candidate using RNAi. C. elegans carrying intestinal polyQ44 that were fed E. coli HT115 (DE3) expressing empty vector control (L4440) RNAi, exhibited a significant suppression of aggregation in the presence of butyrate (10–100 mM) ( Fig 7A ). While butyrate-mediated suppression of aggregation was not affected when HSF-1 was knocked-down, the beneficial effect of butyrate was abolished upon downregulation of SKN-1/Nrf2 and DAF-16/FOXO transcription factors. As expected, HSF-1 knockdown enhanced aggregation in the absence of butyrate when compared to control animals ( Fig 7A ) [ 41 ]. To confirm that the knockdown is functional, we used fluorescent reporters to assess the expression of downstream target genes of HSF-1 (hsp70p::GFP also known as C12C8.1p::GFP) and SKN-1 (gcs-1p::GFP), and we used DAF-16 (DAF-16::GFP) fluorescent fusion ( Fig 7B ). The expression of all fluorescent reporters was attenuated upon knockdown of the respective target genes.

To determine whether exogenous butyrate would also rescue the aggregate-dependent motility defect observed in the intestinal polyQ44 model, we measured the TOP phenotype in animals colonized by E. coli OP50, K. pneumoniae KP182, and P. aeruginosa PAO1 in the absence or presence of 100 mM butyrate. While little to no changes in TOP were observed in the roller controls, exogenous butyrate supplementation (100 mM) rescued motility defects in intestinal polyQ44 worms ( Figs 2C and 5 ). We further explored the ability of butyrate to suppress aggregation in other tissues by repeating the above experiment using the muscle polyQ model and assessed the effect of butyrate on bacteria-induced protein aggregation at concentrations ranging from 0–50 mM. Unlike the intestinal polyQ44 model, worms expressing muscle polyQ35 did not experience a significant enhancement of aggregation at lower butyrate concentrations and exhibited a dose-response or near dose-response suppression of aggregation across all bacterial strains except for P. mirabilis and P. aeruginosa PAO1, wherein the only suppressive concentrations of butyrate were 25 and 50 mM, respectively ( Fig 6 ). It is worth noting that the trends of butyrate-suppressed aggregation in worms expressing muscle polyQ parallel those seen in worms expressing intestinal polyQs ( Fig 4 ). For example, animals cultured on plates containing 0–50 mM butyrate exhibited a significant dose-dependent decrease in intestinal and muscle aggregation when colonized by E. coli OP50 or Klebsiella spp. On the other hand, lower concentrations of butyrate did not significantly suppress aggregation in either tissue when animals were fed P. aeruginosa PAO1, S. enterica 12023, and P. mirabilis, suggesting that the beneficial effect of butyrate is, at least in part, facilitated by bacteria ( Figs 4 and 6 ).

A trade-off between organismal resources available to maintain either proteostasis or fecundity has been proposed [ 37 ]. As such, we asked whether butyrate affects fecundity in our experiments, which may explain the suppression of aggregation. Indeed, we found that butyrate decreases the number of progeny per worm, but the effect seems to depend on the presence of polyQ ( S10A and S10B Fig ). Moreover, 25 mM butyrate significantly decreased the number of progeny in worms colonized by P. aeruginosa ( S10C Fig ). Yet, we found that under the same conditions, butyrate increased polyQ aggregation in animals colonized by P. aeruginosa ( Figs 4 and S2C ). These results indicate that a decrease in fecundity alone does not explain the observed suppression of polyQ aggregation.

Furthermore, to rule out the possibility that butyrate decreased bacteria-mediated protein aggregation by merely killing bacteria, we tested the effect of butyrate on bacterial growth and viability in liquid medium and on solid NGM plates, respectively, at concentrations up to 100 mM. While butyrate did affect the growth and viability of some bacteria, all of the strains tested remained viable. We found that most overnight cultures supplemented with butyrate at concentrations that inhibited aggregation reached optical densities comparable to unsupplemented controls ( S6 Fig ). We also tested the effect of butyrate on bacteria cultured on solid NGM media and found that, while some strains were affected, all were viable across concentrations ( S7 Fig ). Additionally, feeding C. elegans dead bacteria resulted in higher aggregation profiles that are distinct from those treated with 100 mM butyrate, further suggesting that butyrate does not suppress bacteria-mediated aggregation by solely affecting bacterial viability ( S8 Fig ). Next, we asked whether butyrate could affect colonization of the intestine. We found that at 25 mM, butyrate significantly enhanced intestinal colonization by E. coli OP50, but exhibited no effect on colonization at 100 mM ( S9 Fig ). These results indicate that it is unlikely that the butyrate’s effect on bacterial colonization affects aggregation.

The depletion of butyrogenic bacteria in the gut establishes an environment in which pathogenic bacteria, particularly Enterobacteriaceae, can flourish [ 31 ]. In addition, butyrate itself has been found to suppress the growth of pathogenic bacteria and was shown to protect against neurodegeneration [ 32 – 34 ]. To assess the effect of exogenous butyrate on the observed bacteria-induced aggregation, we first tested its effect on intestinal polyQ aggregation in C. elegans. We used all bacterial genera that induced protein aggregation by three-fold or more ( Fig 1B ). The physiological concentration of human colonic butyrate is estimated to be 10–20 mM, and a therapeutic dose of 150 mM butyrate was safely delivered via enema directly into the human large intestine [ 35 , 36 ]. Therefore, we tested the effect of butyrate on bacteria-induced protein aggregation at concentrations ranging from 10–100 mM. Unexpectedly, we found that lower concentrations of butyrate differentially affected host proteostasis where enhancement of aggregation was dependent on the type of bacteria colonizing the intestine: 10 mM: S. enterica 12023, P. mirabilis; 25 mM: P. mirabilis, P. aeruginosa PAO1; 50 mM: P. aeruginosa PAO1. However, at higher butyrate concentrations (>25 mM), bacteria-induced aggregation was suppressed in a dose-dependent manner in worms colonized with all bacteria except P. aeruginosa. Supplementation with 100 mM butyrate suppressed bacteria-induced aggregation across all strains. ( Fig 4 ). These results are unexpected, as they suggest that at low physiological concentrations (<25 mM), butyrate may contribute to the bacteria-mediated disruption of host proteostasis, as demonstrated by differentially enhanced polyQ aggregation; however, with the exception of P. aeruginosa PAO1, higher concentrations (50–100 mM) of butyrate suppressed aggregation induced by all other bacterial strains, which suggests therapeutic potential. We employed western blot analysis of polyQ44::YFP levels to eliminate the possibility that these results are due to changes in protein level ( S1C and S1D Fig ), and we confirmed these results by assessing insoluble fractions ( S2B and S2C Fig ).

Thus far, we demonstrated that colonization of the C. elegans gut with enteric bacterial pathogens leads to proteostasis imbalance and proteotoxicity in proximal tissues, including muscle and neurons. To further examine the extent to which gut bacteria influence host proteostasis, we asked whether colonization of the C. elegans intestine had any effect on the gonad. If the colonization of the intestine affects the muscle and neurons, we reasoned that bacteria might also affect proteostasis in the germline—if this is the case, then we should be able to detect changes in polyQ aggregation in the progeny of parents that were colonized by pathogenic bacteria. To test the effect of bacteria on the next generation, we fed C. elegans either control E. coli OP50, K. pneumoniae KP182, or P. aeruginosa PAO1 for three days, followed by “bleaching” adults, and age-synchronizing the progeny. C. elegans synchronization involves dissolving gravid adults by hypochlorite treatment leaving intact and sterile embryos which hatch into L1 larvae. Synchronized F1 animals were placed on fresh plates containing a lawn of control E. coli OP50 and intestinal aggregates were enumerated after 92 hours (h). The F2 generation was isolated and assessed using the same method ( Fig 3H ). Strikingly, we found that colonization of the parental intestines with the select bacteria enhanced polyQ aggregation in the F1 progeny, despite the fact that they never encountered the bacteria that were fed to the parental generation (with the exception of E. coli OP50) ( S5 Fig ). The extent of the enhancement in the number of polyQ aggregates per worm correlated with previously assessed phenotypes where P. aeruginosa showed the highest induction of aggregation ( Fig 3I ). Since P. aeruginosa induced the strongest effect, we looked at the F2 progeny from a parental generation fed these bacteria. We saw no enhancement of aggregation compared to control E. coli, indicating that only F1 generation is affected ( Fig 3I ). Using western blotting, we ruled out the possibility that these results were due to changes in polyQ44::YFP expression level ( S1E Fig ) and we confirmed an increase of the insoluble polyQ44::YFP ( S2D Fig ). Collectively, these results are intriguing as they demonstrate that colonization of the parental intestines affects proteostasis in the offspring.

To determine whether proteostasis in neuronal tissue is also affected by bacterial colonization of the intestine, we utilized worms carrying neuronal polyQ40 (F25B3.3p::Q40::YFP) [ 30 ]. The small size and high concentration of neurons preclude us from accurately quantifying the exact number of aggregates in this tissue; as such, we measured toxicity by assessing TOP motility. Animals carrying this reporter exhibit an age-dependent decrease in motility compared to control N2 worms ( Fig 3E ). We found that colonization of the intestine with K. pneumoniae and P. aeruginosa led to a significant defect in motility when compared to the control E. coli OP50 ( Fig 3F and 3G ).

A) The effect of select bacteria on protein folding in the muscle. Data are represented as the average number of aggregates per worm normalized to E. coli OP50 control strain. Each bar is an average of three independent experiments with a total of 100 animals. B) Age-dependent decline in motility assessed by enumerating body bends per 30 seconds in muscle-specific polyQ35 and N2 control worms. The data are represented as the average number of body bends quantified on day 3–5 post-hatching in a total of 15 animals per each day. The effect of bacteria on the motility of C) control animals and D) animals expressing muscle-specific polyQ35. Data are represented as the average number of body bends per worm normalized to worms fed E. coli OP50 control strain. Each bar is an average of three independent experiments with a total of 45 animals. E) Age-dependent decline in motility assessed by increased TOP. The effect of bacteria on the motility of F) control animals and G) animals expressing neuron-specific polyQ40. Data are represented as the average TOP per worm normalized to animals fed E. coli OP50 control strain. Each bar is an average of three independent experiments with a total of 60 animals. H) A cartoon depicting experiments which demonstrated that bacterial colonization of the C. elegans intestine affects the F1 generation. I) Quantification of intestinal aggregates in the F1 and F2 generations from parental animals that were fed select test and control bacteria. Each bar represents the average number of aggregates per intestine normalized to the control (E. coli OP50). Data are representative of three (F1) and one (F2) independent experiments with a total of 100 and 30 animals, respectively. Error bars represent SEM. Statistical significance was calculated using one-way ANOVA followed by multiple comparison Dunnett’s post-hoc test (*p<0.05, **p<0.01, ****p<0.0001).

Because bacteria-induced polyQ aggregation in the intestine influenced C. elegans motility, an effect that likely involves distal tissues, we sought to determine whether the influence of bacteria on protein aggregation is confined to the intestine or if bacteria can impact protein folding across other tissues. We employed nematodes expressing muscle-specific polyQ35 (unc-54p::Q35::YFP) as a proxy to detect changes in proteostasis in that tissue. Different tissues have different thresholds for polyQ aggregation; therefore, while polyQ33 did not aggregate in the intestine, polyQ35 aggregates in the muscle. This reporter exhibits age-dependent and muscle-specific aggregation and the associated motility defect [ 29 ]. We colonized the intestines of these worms with two strains that enhanced intestinal polyQ aggregation: K. pneumoniae KP182 and P. aeruginosa PAO1. Animals expressing muscle polyQs exhibit a more robust aggregation profile compared to the intestinal polyQ model; thus, we had to quantify the aggregates on day three instead of day four. The results paralleled the trend seen in the aggregation profile of the intestinal polyQ model, where K. pneumoniae enhanced aggregation compared to control E. coli OP50 and P. aeruginosa PAO1 showed the most robust enhancement ( Fig 3A ). To determine whether the bacteria-induced proteostasis imbalance detected by the polyQ aggregation in the muscle also leads to tissue dysfunction, we employed a previously characterized motility readout to enumerate the body bends of polyQ-expressing worms fed these two bacterial strains. The animals expressing muscle polyQ exhibit a sharp and age-dependent decrease in body bends ( Fig 3B ). We found that colonization of the C. elegans intestine with enteric bacterial pathogens led to a significant defect in motility, particularly in worms fed the most potent enhancer of aggregation, P. aeruginosa, which decreased the number of body bends 2-fold when compared to the control E. coli OP50; a larger and more significant defect than what was observed in control wild-type animals ( Fig 3C and 3D ).

Tissue non-autonomous effects between C. elegans neurons and other somatic cells (i.e., intestine and epithelium) have been recently described where perturbation of proteostasis in one tissue impacts another [ 26 – 28 ]. As such, we postulated that bacteria-induced disruption of proteostasis in the intestine could affect other tissues. To begin assessing the impact of bacteria on muscle, we examined motility. The nematodes that express intestinal polyQ have an integrated rol-6 marker, which results in a roller phenotype and renders animals incompatible with known motility assays, such as thrashing. Thus, we developed a novel motility readout that entails sliding an eyebrow hair under the mid-section of the worm and counting the number of seconds until the worm completely crawls off—we refer to this phenotype as t ime- o ff- p ick (TOP) ( Fig 2A ). We found that TOP increases with age and the decrease in motility is dependent on polyQ when compared to control roller worms that do not express polyQs, as well as those that express non-aggregating polyQs ( Fig 2B ). To ensure that the TOP method measures polyQ-specific defects and not an undesired effect related to the roller phenotype or bacterial pathogenicity, we used non-roller strains that express muscle-specific polyQ35 (unc-54p::Q35::YFP, AM140) and polyQ40 (unc-54p::Q40::YFP, AM141). Both of these strains are known to exhibit polyQ-length and age-dependent motility defects [ 29 ]. In support of the functionality of our assay, we found that worms expressing these constructs also exhibited a significant polyQ-length and age-dependent TOP motility defect that correlated with the enumeration of body bends, which is a well-established assessment of motility in C. elegans ( S3A Fig ). Additionally, colonization of these polyQ-expressing strains with P. aeruginosa PAO1 inhibited motility in both assays yet had little to no effect in wild-type N2 animals. These findings indicate that polyQ toxicity, and not bacterial pathogenicity, is the factor that is measured by TOP ( S3B Fig ). Therefore, we further used the TOP method to determine whether colonization of the C. elegans intestine with bacteria that induced polyQ aggregation aggravates this phenotype. We chose two bacterial strains: K. pneumoniae KP182 (medium inducer); P. aeruginosa PAO1 (high inducer) and found that the “time-off-pick” was significantly increased by both strains ( Fig 2C ), supporting the trend seen in their respective aggregation profiles ( Fig 1B ). We did not detect any effect on the TOP phenotype in roller controls or in roller nematodes expressing a non-aggregating and intestine-specific polyQ33, confirming that this motility defect was the result of bacteria-induced polyQ-dependent toxicity rather than general pathogenicity from a bacterial infection ( Fig 2C ). While it is known that worms expressing polyQ33 do not exhibit age-dependent aggregation, we wanted to ensure that aggregation does not occur when these animals are colonized by bacteria that induce polyQ44 aggregation. We did not detect polyQ33 aggregates in animals colonized with the strongest inducer, P. aeruginosa PAO1 ( S4 Fig ). Based on our results, we conclude that bacterial colonization of the C. elegans intestine affects protein folding in the immediate environment and that such localized intestinal protein aggregation influences motility, likely by acting on muscle or neuronal tissues.

To begin understanding how bacteria could contribute to the disruption of host proteostasis, we used E. coli defective in the production of curli, which are aggregation-prone amyloids that are specific to Enterobacteriaceae [ 24 ]. We generated two mutant E. coli strains: one deficient in csgA, which encodes an amyloid structural subunit of curli fimbriae, and one deficient in csgD, a transcription factor that positively regulates the curli operon ( Fig 1D ). While staining with congo red confirmed a deficiency in curli production ( Fig 1E ), staining for bacterial protein aggregates using ProteoStat, which provides superior sensitivity in aggregate detection [ 25 ], revealed that only csgD mutant cells have significantly fewer aggregates ( Fig 1F ). In agreement with these results, we found that colonization of C. elegans only with the ΔcsgD E. coli strain led to a significant decrease in the number of polyQ aggregates per intestine ( Fig 1G ). These results suggest that bacteria-derived aggregates significantly contribute to the disruption of host proteostasis.

A) Age-dependent aggregation of intestinal polyQs (polyQ44) colonized by E. coli OP50. Data are represented as the number of aggregates per 20–30 intestines quantified in animals at day 1–5 post-hatching. B) The effect of select enteric bacteria on polyQ aggregation in the C. elegans intestine. Data are represented as the average number of aggregates per C. elegans intestine normalized to E. coli OP50 control strain (lower dotted line). Each data point is an average of a minimum of three independent experiments with a total of at least 100 animals. Black circles represent control bacteria and bacteria that did not have any significant effect on polyglutamine aggregation upon colonization of the C. elegans intestine. Red solid circles represent bacteria that significantly enhanced aggregation by <3-fold. Red open circles represent bacteria that significantly enhanced aggregation by >3-fold (arbitrary threshold). Arrows represent bacteria that were chosen for follow-up experiments. C) The effect of select commensal bacteria on polyQ aggregation in the C. elegans intestine. Data are represented as the average number of aggregates per C. elegans intestine normalized to E. coli OP50 control strain. Each data point is an average of a minimum of three independent experiments with a total of at least 100 animals. D) PCR confirmation of E. coli MG1655 curli mutant strains. WT, csgA::kan (left two bands) amplified with primers flanking the ΔcsgA locus. WT, csgD::kan (right two bans) amplified with primers flanking the ΔcsgD locus. E) Phenotypic confirmation of the curli-deficient ΔcsgA and ΔcsgD mutant strains using Congo Red plate assay. F) ProteoStat staining of total aggregates produced by E. coli MG1655 wild-type (WT), MG1655 ΔcsgA, and MG1655 ΔcsgD. Data are represented as the average fluorescent signal per bacterial strain stained with ProteoStat. Each data point is an average of two independent experiments with three replicates per run. G) The effect of E. coli MG1655 curli mutants on polyQ (polyQ44) aggregation in the C. elegans intestine. Data are represented as the average number of aggregates per C. elegans intestine. Each data point is an average of three independent experiments with a total of 90 animals. Error bars represent standard error of the mean (SEM). Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by multiple comparison Dunnett’s post-hoc test (****p< 0.0001).

We tested the ability of select bacterial species from the Enterobacteriaceae family to affect the protein folding environment in C. elegans upon intestinal colonization. Select bacterial species were from the following genera: Escherichia, Klebsiella, Proteus, Citrobacter, Shigella, and Salmonella, as well as additional pathogenic bacteria that are associated with gut microbiota; these include gram-negative Pseudomonas and Acinetobacter. Although the physical manifestations of human proteopathies involve aggregate-induced dysfunction of neuronal and musculoskeletal tissue, we concluded that the intestinal tissue was best suited for our initial experiments because it is the immediate environment of bacterial colonization, presumably resulting in the most robust effect. As such, to assess the effect of these candidate strains on the protein folding environment in the C. elegans gut, we used animals that constitutively express intestine-specific polyQs fused to yellow fluorescent protein (polyQ44::YFP) [ 23 ]. Nematodes carrying this reporter exhibit age-dependent aggregation of intestinal polyQs ( Fig 1A ). These aggregates present as quantifiable fluorescent foci and were previously characterized as a proxy to assess the influence of bacteria on aggregation [ 23 ]. To determine the age at which nematodes harbor an intermediate range of aggregation for identifying bacteria that suppress or enhance aggregation, we quantified aggregates between days 1–5, post-hatching. Worms were grown at room temperature (~23°C) on the control bacteria, E. coli OP50. Because a high number of aggregates can introduce counting error, an aggregation threshold was set at 40 aggregates per intestine to ensure the reliability of counting. Aggregates increased each day, becoming visible after three days, and reaching our set threshold after five days ( Fig 1A ). Hence, to identify bacteria that either enhance or suppress aggregation, we chose to grow C. elegans on test bacteria for four days before assessing the ability of each strain to influence protein aggregation in the intestine. Out of 19 strains tested, one did not have any effect on aggregation, six strains significantly enhanced aggregation two- to three-fold, and 12 strains enhanced aggregation more than three-fold with respect to E. coli OP50 ( Fig 1B ). We found that the strongest enhancers were P. aeruginosa, K. pneumoniae, C. freundii, S. Typhimurium, K. aerogenes, and P. mirabilis. The only strain that did not significantly induce aggregation relative to E. coli OP50 was E. coli HB101, a non-pathogenic strain derived from E. coli K-12. To eliminate the possibility that bacteria change polyQ expression levels, using an antibody specific to YFP/GFP ( S1A Fig ), we confirmed that polyQ44:YFP soluble protein levels do not change between E. coli OP50 and P. aeruginosa PAO1 –two bacterial strains that elicited the lowest and highest intestinal aggregation, respectively ( S1B Fig ). Moreover, western blot analysis of the insoluble polyQ44::YFP fraction confirmed bacteria-mediated increase in aggregation ( S2A Fig ), as it parallels the aggregation profile of worms fed E. coli OP50 and P. aeruginosa PAO1 ( Fig 1B ). These results are intriguing as they demonstrate that common human enteric pathogens significantly affect polyQ aggregation, and therefore host proteostasis. We next asked whether gram-negative bacteria that are associated with commensal microflora would also enhance aggregation. We tested three strains known to be part of the commensal microbiome: K. oxytoca HM-624, Prevotella disiens HM-1171, and Prevotella corporis HM-1294. While K. oxytoca enhanced aggregation, as we have seen with the other Klebsiella spp., both Prevotella spp. suppressed aggregation relative to E. coli OP50 ( Fig 1C ). In fact, P. corporis almost completely suppressed aggregation.

Discussion

We used a simple metazoan model, C. elegans, to study the effect of bacterial gut colonization on organismal proteostasis. We found that the colonization of the C. elegans intestine with enteric bacterial pathogens disrupts organismal proteostasis and enhances proteotoxicity associated with polyQ. Importantly, our results demonstrate that bacteria alone do not cause major toxicity in control animals that do not express polyQ, which would hinder the interpretation of our data (Figs 2B, 2C, 3C, and 3F). It is not surprising that the detrimental effect of bacteria on polyQ aggregation is observed in the most proximal tissue, the intestine, as similar results were previously observed [23]. However, to our knowledge, this is the first report of bacteria having detrimental, tissue non-autonomous effects on protein folding. Recently, Bacillus subtilis, a probiotic strain that is also part of the human commensal microbiota, was shown to inhibit α-synuclein aggregation in C. elegans muscle [44]. The fact that certain bacteria disrupt host proteostasis while others can enhance it emphasizes the importance of microbes in the pathogenesis of PCDs. Previous research suggests that increased presence of bacterial species such as K. pneumoniae and P. aeruginosa in the human gut is associated with an increased prevalence of PCDs [45,46]. Interestingly, among all of the strains that we tested, Klebsiella spp. and P. aeruginosa were the most potent inducers of polyQ aggregation–this was the case even for a commensal strain, K. oxytoca (Fig 1B and 1C). Both of these species are ubiquitous in the environment, and multidrug-resistant strains of these bacteria are often associated with nosocomial and opportunistic infections [47]. Due to their rapidly increasing resistance to antibiotics, they are classified by the Centers for Disease Control and Prevention as serious threats [48]. While these strains are often associated with infections, their enteric presence is also evident in healthy individuals and may possibly support their contribution to neurodegenerative diseases [49,50]. In fact, both of these microbes have recently been associated with neurodegenerative diseases in humans [45,46], a correlation that is in agreement with our results. The increasing prevalence of antibiotic resistance among Klebsiella spp. and P. aeruginosa strains may specifically enrich for their growth within the human microbiome upon an antibiotic treatment while decreasing the abundance of beneficial bacteria. In agreement with this hypothesis, population-based studies of patients with Parkinson’s and ALS revealed that a history of antibiotic treatment is associated with an elevated risk for these diseases [7,8].

Another notable strain that induced aggregation by more than three-fold is P. mirabilis. This gram-negative bacterium is part of human commensal microbiota but can also become an opportunistic pathogen, most often leading to urinary tract infections, and eventually, bacteremia [51]. Interestingly, in one study, blood cultures of PD septic patients had a five-fold higher prevalence of P. mirabilis compared to non-PD septic patients [52]. Recently, it was demonstrated in a mouse model that P. mirabilis contributes to PD pathogenesis, including the induction of α-synuclein in the mouse brain [53]. Furthermore, colonization of the gut with P. mirabilis and Enterobacteriaceae, in particular, K. pneumoniae, was shown to be accelerated by antibiotic treatment, leading to detrimental dysbiosis in mouse microbiota [54]. On the other side of the aggregation spectrum, the non-pathogenic E. coli HB101 did not significantly enhance aggregation in our C. elegans polyQ model. The E. coli HB101 strain is also a common laboratory food for C. elegans; hence, we did not expect any enhancement with these bacteria. However, both P. disiens HM-1171 and P. corporis HM-1294 suppressed aggregation relative to E. coli OP50 (Fig 1C). The mechanism by which these Prevotella spp. suppress aggregation does not depend on butyrate as both strains were deficient in its production [55,56]. Interestingly, Prevotella was the only bacterial genus whose abundance significantly decreased in patients suffering from constipation, a condition that is a hallmark in Parkinson’s disease and is associated with up to 80% of cases [57–59]. Further evidence that these two species could be beneficial to host proteostasis comes from a recent study that demonstrated a negative correlation between the abundance of Prevotella in the gut of Parkinson’s disease patients and the severity of symptoms [60]. A negative correlation between the abundance of Prevotella and pathogenesis of Alzheimer’s disease mouse model has also been observed [61]. Collectively, these results suggest that the members of the Prevotella genus could play a more direct role in enhancing host proteostasis, and that the mechanisms by which bacteria affect host proteostasis in our C. elegans model may be conserved in higher organisms.

Although P. aeruginosa was previously found to induce neurodegeneration in C. elegans [62], the results of decreased polyQ-dependent motility of animals colonized with P. aeruginosa and K. pneumoniae were unexpected and suggest that either bacteria, or host responses to these bacteria, affect the function of distal tissues (i.e., muscle or neurons), but only in animals that express metastable aggregation-prone proteins such as polyQs (Figs 2B, 2C, and 3A–3G). Recent studies show that colonization of the C. elegans intestine with pathogenic bacteria affects bacterial avoidance behavior [63] and consequently increases lifespan [64]. While it is possible that feeding behavior might have been affected in our experiments, it is unlikely that avoidance would contribute to an increase in polyQ aggregation and the associated motility defects. In fact, if the animals in our experiments experienced food avoidance behavior, we would expect to see enhanced proteostasis, as dietary restriction was previously shown to suppress polyQ aggregation [65].

It is known that bacterial colonization of the C. elegans intestine increases the production of reactive oxygen species (ROS), which in turn enhances aggregation in that tissue [23]. Oxidative stress is known to affect overall animal physiology, including motility [66]. Since our results show the bacteria-mediated motility defects only in the presence of aggregation-prone polyQ44, but not in non-aggregating polyQ33 or non-polyQ controls (Fig 2C), it is possible that ROS generated by the worm in response to bacterial colonization enhanced polyQ aggregation, which further affected the function of other tissues involved in motility. Such a mechanism likely contributes to the observed bacteria-mediated disruption of host proteostasis which is supported by our results demonstrating that butyrate rescued polyQ aggregation in a DAF-16 and SKN-1-dependent manner (Fig 7A). However, ROS is likely not the sole contributor since our results also indicate that bacterial aggregates affect host proteostasis and consequently increase aggregation of host polyQ (Fig 1E–1G).

Tissue non-autonomous regulation has been noted in C. elegans under various conditions [27,67]. Our results demonstrating that bacterial colonization of the C. elegans intestine affects proteostasis across tissues are intriguing, as they suggest that bacteria impose tissue non-autonomous effects on the host. In human and mouse studies, the effect of bacterial gut colonization on host proteostasis has been primarily attributed to non-specific mechanisms, such as systemic inflammation, which broadly affects many tissues [68]; however, C. elegans lacks an inflammatory response, indicating that other and possibly more direct mechanisms may be involved. Bacterial amyloids have also been shown to specifically contribute to pathogenesis by affecting metastable aggregation-prone proteins [69]. The introduction of metastable proteins is known to affect the stability and folding of other proteins within the proteome [70,71]. As such, it is likely that bacteria may be secreting factors that translocate from the intestine across other tissues and affect proteostasis. A cross-seeding between disease-associated proteins (i.e., Aβ) with bacterial amyloids, including those from E. coli and P. aeruginosa, was previously observed [72,73]. Also, an enhancement of alpha-synuclein aggregation mediated by bacterial curli was shown in mouse and C. elegans Parkinson’s disease models [74]. While our results indicate that E. coli curli fimbriae were not responsible for the enhancement of polyQ aggregation, E. coli aggregates did play a contributing factor (Fig 1G). It is possible that alpha-synuclein and polyQ are affected differently by bacterial amyloids. In addition to regulating the csg operon, CsgD also functions as a global transcriptional regulator and its target gene(s), other than csgA, could affect host proteostasis [75]. It is intriguing to speculate that the transmission of aggregation-prone proteins between tissues [67], may underlie how bacterial colonization of parental intestines affects protein aggregation in the progeny (Fig 3I).

A symbiosis between commensal and pathogenic bacteria can be disrupted by many factors, including infections, diet, and antibiotics [76]. While dysbiosis of the human gut microbiota has been associated with the pathogenesis of PCDs, the mechanisms that contribute to the disruption of host proteostasis and disease progression remain poorly understood. Our results demonstrate that butyrate, a common metabolite produced by commensal microbiota, can suppress aggregation and the associated toxicity when supplied exogenously (Figs 4 and 6) or produced by intestinal bacteria (Figs 8 and 9). We also showed that butyrate did not simply inhibit intestinal colonization (S9 Fig). In agreement with our results, the protective effects of butyrate have been previously demonstrated in a mouse model and in humans [21,32]. Our data indicate that the benefits of butyrate depend on SKN-1/Nrf2 and DAF-16/FOXO transcription factors, suggesting that butyrate may inhibit bacteria-induced aggregation by activating protective stress responses, in particular, oxidative stress responses. Indeed, bacteria are known to trigger oxidative stress, which can contribute to polyQ aggregation [23]. It is possible that various bacteria introduce different levels of oxidative stress and the strongest contributors affect host proteostasis, consequently leading to misfolding and aggregation of metastable proteins present within the proteome. In fact, oxidative stress is one of the major contributors to PCDs [77]. Recently, it was found that butyrate and β-hydroxybutyrate provide Nrf2- and FOXO-dependent protection against oxidative stress, respectively [78,79]. In addition, both SKN-1 and DAF-16 were shown to be involved in β-hydroxybutyrate-mediated lifespan extension [80]. These results, along with our data, collectively suggest that butyrate activates SKN-1/Nrf2 and DAF-16/FOXO, which attenuate bacteria-induced oxidative stress. The effect of butyrate on polyQ aggregation also seems to depend on the type of pathogenic bacteria present, suggesting that butyrate may contribute to host proteostasis by acting on bacteria in addition to the host. These results are consistent throughout the intestinal and muscle-specific polyQ models. Additionally, the co-colonization experiment further supports that the extent of the beneficial effect of butyrate depends on the bacteria present (Fig 10A). The fact that bacteria are viable in the presence of butyrate at concentrations that suppress aggregation (S6 and S7 Figs) and that dead bacteria exhibit a distinct effect on aggregation (S8 Fig) suggest that microbial processes which affect the host may be targeted by butyrate. Whether butyrate affects specific bacterial signals that contribute to PCDs remains to be determined and further studies are underway to identify bacterial factors that enhance aggregation of host metastable proteins. Whatever the mechanism may be, our results suggest a therapeutic potential of butyrate in the prevention or treatment of PCDs.

High metabolic rates of bacteria can lead to depletion of available oxygen and introduce a hypoxic environment on the plates. Low oxygen levels are known to disrupt cellular processes leading to increased polyQ aggregation [81]. One of the possible explanations regarding how butyrate may inhibit bacteria-mediated polyQ aggregation is by suppressing bacterial metabolism, which restores the normoxic environment. However, if this were true, we would expect to have seen the restoration of a normoxic environment when we fed worms heat-killed bacteria, which would result in polyQ aggregation similar to 100 mM butyrate (S8 Fig). Therefore, it is likely that butyrate could have some additional effects on bacteria in addition to the host. In fact, it is known that butyrate can affect bacterial pathogenicity [34,82,83]. Further research is underway to determine what changes butyrate might exert on bacteria and how that influences the host.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009510

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