Multitier regulation of the E. coli extreme acid stress response by CsrA.

CsrA is an RNA-binding protein that regulates processes critical for growth and survival, including central carbon metabolism, motility, biofilm formation, stress responses, and expression of virulence factors in pathogens. Transcriptomics studies in Escherichia coli suggested that CsrA repressed genes involved in surviving extremely acidic conditions. Here, we examine the effects of disrupting CsrA-dependent regulation on the expression of genes and circuitry for acid stress survival and demonstrate CsrA-mediated repression at multiple levels. We show that this repression is critical for managing the trade-off between growth and survival; overexpression of acid stress genes caused by csrA disruption enhances survival under extreme acidity but is detrimental for growth under mildly acidic conditions. In vitro studies confirmed that CsrA binds specifically to mRNAs of structural and regulatory genes for acid stress survival, causing translational repression. We also found that translation of the top-tier acid stress regulator, evgA, is coupled to that of a small leader peptide, evgL, which is repressed by CsrA. Unlike dedicated acid stress response genes, csrA and its sRNA antagonists, csrB and csrC, did not exhibit a substantial response to acid shock. Furthermore, disruption of CsrA regulation of acid stress genes impacted host-microbe interactions in Caenorhabditis elegans, alleviating GABA deficiencies. This study expands the known regulon of CsrA to genes of the extreme acid stress response of E. coli and highlights a new facet of the global role played by CsrA in balancing the opposing physiological demands of stress resistance with the capacity for growth and modulating host interactions.IMPORTANCETo colonize/infect the mammalian intestinal tract, bacteria must survive exposure to the extreme acidity of the stomach. E. coli does this by expressing proteins that neutralize cytoplasmic acidity and cope with molecular damage caused by low pH. Because of the metabolic cost of these processes, genes for surviving acid stress are tightly regulated. Here, we show that CsrA negatively regulates the cascade of expression responsible for the acid stress response. Increased expression of acid response genes due to csrA disruption improved survival at extremely low pH but inhibited growth under mildly acidic conditions. Our findings define a new layer of regulation in the acid stress response of E. coli and a novel physiological function for CsrA.

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

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.

Bacteria-Derived Protein Aggregates Contribute to the Disruption of Host Proteostasis.

Neurodegenerative protein conformational diseases are characterized by the misfolding and aggregation of metastable proteins encoded within the host genome. The host is also home to thousands of proteins encoded within exogenous genomes harbored by bacteria, fungi, and viruses. Yet, their contributions to host protein-folding homeostasis, or proteostasis, remain elusive. Recent studies, including our previous work, suggest that bacterial products contribute to the toxic aggregation of endogenous host proteins. We refer to these products as bacteria-derived protein aggregates (BDPAs). Furthermore, antibiotics were recently associated with an increased risk for neurodegenerative diseases, including Parkinson's disease and amyotrophic lateral sclerosis-possibly by virtue of altering the composition of the human gut microbiota. Other studies have shown a negative correlation between disease progression and antibiotic administration, supporting their protective effect against neurodegenerative diseases. These contradicting studies emphasize the complexity of the human gut microbiota, the gut-brain axis, and the effect of antibiotics. Here, we further our understanding of bacteria's effect on host protein folding using the model Caenorhabditis elegans. We employed genetic and chemical methods to demonstrate that the proteotoxic effect of bacteria on host protein folding correlates with the presence of BDPAs. Furthermore, the abundance and proteotoxicity of BDPAs are influenced by gentamicin, an aminoglycoside antibiotic that induces protein misfolding, and by butyrate, a short-chain fatty acid that we previously found to affect host protein aggregation and the associated toxicity. Collectively, these results increase our understanding of host-bacteria interactions in the context of protein conformational diseases.

Identification of proteotoxic and proteoprotective bacteria that non-specifically affect proteins associated with neurodegenerative diseases.

There are no cures for neurodegenerative protein conformational diseases (PCDs), such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Emerging evidence suggests the gut microbiota plays a role in their pathogenesis, though the influences of specific bacteria on disease-associated proteins remain elusive. Here, we reveal the effects of 229 human bacterial isolates on the aggregation and toxicity of Aß1-42, α-synuclein, and polyglutamine tracts in Caenorhabditis elegans expressing these culprit proteins. Our findings demonstrate that bacterial effects on host protein aggregation are consistent across different culprit proteins, suggesting that microbes affect protein stability by modulating host proteostasis rather than selectively targeting disease-associated proteins. Furthermore, we found that feeding C. elegans proteoprotective Prevotella corporis activates the heat shock response, revealing an unexpected discovery of a microbial influence on host proteostasis. Insight into how individual bacteria affect PCD proteins could open new strategies for prevention and treatment by altering the abundance of microbes.

Identification of proteotoxic and proteoprotective bacteria that non-specifically affect proteins associated with neurodegenerative diseases.

Neurodegenerative protein conformational diseases (PCDs), such as Alzheimer's, Parkinson's, and Huntington's, are a leading cause of death and disability worldwide and have no known cures or effective treatments. Emerging evidence suggests a role for the gut microbiota in the pathogenesis of neurodegenerative PCDs; however, the influence of specific bacteria on the culprit proteins associated with each of these diseases remains elusive, primarily due to the complexity of the microbiota. In the present study, we employed a single-strain screening approach to identify human bacterial isolates that enhance or suppress the aggregation of culprit proteins and the associated toxicity in Caenorhabditis elegans expressing Aß1-42, α-synuclein, and polyglutamine tracts. Here, we reveal the first comprehensive analysis of the human microbiome for its effect on proteins associated with neurodegenerative diseases. Our results suggest that bacteria affect the aggregation of metastable proteins by modulating host proteostasis rather than selectively targeting specific disease-associated proteins. These results reveal bacteria that potentially influence the pathogenesis of PCDs and open new promising prevention and treatment opportunities by altering the abundance of beneficial and detrimental microbes.

Time-off-pick Assay to Measure Caenorhabditis elegans Motility.

Caenorhabditis elegans is a simple metazoan that is often used as a model organism to study various human ailments with impaired motility phenotypes, including protein conformational diseases. Numerous motility assays that measure neuro-muscular function have been employed using C. elegans . Here, we describe "time-off-pick" (TOP), a novel assay for assessing motility in C. elegans . TOP is conducted by sliding an eyebrow hair under the mid-section of the worm and counting the number of seconds it takes for the worm to crawl completely off. The time it takes for the worm to crawl off the eyebrow hair is proportional to the severity of its motility defect. Other readouts of motility include crawling or swimming phenotypes, and although widely established, have some limitations. For example, worms that are roller mutants are less suitable for crawling or swimming assays. We demonstrated that our novel TOP assay is sensitive to age-dependent changes in motility, thus, providing another more inclusive method to assess motor function in C. elegans . Graphical abstract: Conceptual overview of the "time-off-pick" (TOP) assay. Various C. elegans models exhibit age-dependent defects in motility. The time it takes for a worm to crawl off of an eyebrow pick that is slid under its mid-section is measured in TOP seconds. A greater TOP is indicative of a greater motility defect. Eventually, worms with phenotypes that lead to paralysis will not be able to leave the pick.

Quantification of Bacterial Loads in Caenorhabditis elegans.

Caenorhabditis elegans is a ubiquitous free-living nematode that feeds on bacteria. The organism was introduced into a laboratory setting in the 1970s and has since gained popularity as a model to study host-bacteria interactions. One advantage of using C. elegans is that its intestine can be colonized by the bacteria on which it feeds. Quantifying the bacterial load within C. elegans is an important and easily obtainable metric when investigating host-bacteria interactions. Although quantification of bacteria harbored in C. elegans via whole-worm lysis is not a novel assay, there is great variation between existing methods. To lyse C. elegans, many protocols rely on the use of a hand-held homogenizer, which could introduce systematic error and subsequent variation between researchers performing the same experiment. Here, we describe a method of lysing the intestines of C. elegans to quantify the bacterial load within the intestine. Our method has been optimized for removing exogenous bacteria while maintaining worm paralysis, to ensure no bactericidal agents are swallowed, which could kill bacteria within the intestine and affect results. We utilize and compare the efficiency of two different homogenization tools: a battery-powered hand-held homogenizer, and a benchtop electric homogenizer, where the latter minimizes variability. Thus, our protocol has been optimized to reduce systematic error and decrease the potential for variability among experimenters. Graphic abstract: Simplified overview of the procedure used to quantify the bacterial load within C. elegans. The two different methods are herein described for worm lysis: "Option 1" is a hand-held homogenizer, and "Option 2" is a benchtop homogenizer.

Automating Aggregate Quantification in Caenorhabditis elegans.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.