Recent research suggests that people with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS), exhibit changes in the bacterial composition of their gut microbiome. However, identifying which bacteria may be associated with neurodegeneration is like finding the proverbial needle in a haystack.

The results of studies by University of Florida (UF) scientists have now for the first time made the link between specific bacterial species and the physical manifestations of neurodegenerative diseases. The research team, headed by Daniel Czyz, PhD, an assistant professor at the University of Florida (UF)/Institute of Food and Agricultural Sciences (IFAS) department of microbiology and cell science, and Alyssa Walker, a microbiology and cell science doctoral candidate, used the intestine of the roundworm model organism Caenorhabditis elegans, as a form of “test tube,” to investigate the effects of different human gut bacteria on host tissues.

Their results point to the potential to use specific types of bacteria as a preventative and treatment strategy for neurodegenerative disorders. “Looking at the microbiome is a relatively new approach to investigating what causes neurodegenerative diseases,” said Czyz. “In this study, we were able to show that specific species of bacteria play a role in the development of these conditions.”

Czyz is senior author of the team’s published study in PLOS Pathogens, which is titled, “Colonization of the Caenorhabditis elegans gut with human enteric bacterial pathogens leads to proteostasis disruption that is rescued by butyrate.”

Scientists still don’t understand exactly what causes neurodegenerative disorders, and this gap in knowledge represents a significant roadblock to developing effective treatments or preventive approaches. All neurodegenerative diseases can be traced to problems with the way proteins are handled in the body. If proteins are misfolded, they build up and accumulate in tissues. These protein aggregates interfere with cell functioning and lead to neurodegenerative disorders that are known as protein conformational diseases (PCDs). “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,” the scientists noted.

Factors such as age, diet, stress, trauma, toxins, infections, or antibiotics, have been shown to increase the risk of PCDs, and these triggers are also associated with changes in the microbiome. “This suggests that bacteria may contribute to the pathogenesis of PCDs, which may explain their sporadic onset,” the team noted. But despite these pointers, the relationship between the microbiome and disease progression remains poorly defined. And as the team noted, “the effect of individual bacteria remains elusive, in part, due to the complexity of the microbiome.”

Czyz and his co-authors set out to investigate whether introducing certain bacteria into the C. elegans worms would be followed by protein aggregation in the organisms’ tissues. Recent studies had established a link between human gut microbiota (HGM) and PCDs, and indicated that gut dysbiosys, or direct infection could exacerbate disease. Research had also indicated that some species resident in the human gut microbiome, and specifically those involved in synthesizing short-chain fatty acids (SCFAs) such as butyrate, were beneficial. However, identifying individual species that may be implicated in PCDs has been hampered by the complexity of the human microbiome, coupled with the impact of environmental factors. So, the team noted, “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.”

“Studies like ours are possible because these worms normally feed on bacteria,” Czyz said. “They are also transparent and have a simple body plan … The worms are only one millimeter long, and they each have exactly 959 cells,” Czyz explained. “But in many ways, they are a lot like us humans—they have intestines and muscles and nerves, but instead of being composed of billions of cells, each organ is just a handful of cells. They are like living test tubes. Their small size allows us to do experiments in a much more controlled way and answer important questions we can apply in future experiments with higher organisms and, eventually, people.

Worms colonized by a non-pathogenic control E. coli [University of Florida]

The team tested whether colonization of the C. elegans gut by specific bacterial species from the Enterobacteriaceae family could impact on protein folding environment in the organisms’ tissues. “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,” the authors explained. “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.”

Any resulting protein aggregation could be visually identified. “We have a way of marking the aggregates so they glow green under the microscope,” Czyz said. “We saw that worms colonized by certain bacteria species were lit up with aggregates that were toxic to tissues, while those colonized by the control bacteria were not. This occurred not just in the intestinal tissues, where the bacteria are, but all over the worms’ bodies, in their muscles, nerves and even reproductive organs.”

The worms colonized by pathogenic bacteria have more protein aggregates linked to neurodegenerative diseases. [University of Florida]

Czyz claims the findings demonstrate that specific species of bacteria play a role in the development of neurodegenerative conditions. “We also showed that some other bacteria produce compounds that counteract these ‘bad’ bacteria. Recent studies have shown that patients with Parkinson’s and Alzheimer’s disease are deficient in these ‘good’ bacteria, so our findings may help explain that connection and open up an area of future study,” he added.

So while the results showed that colonization of the C. elegans intestine with pathogenic gram-negative bacteria effectively disrupted proteostasis in the intestine, muscle, neurons, and gonads, colonization with butyrogenic bacteria enhanced proteostasis. “Further experiments revealed that co-colonization with butyrogenic bacteria inhibited protein aggregation in C. elegans …” the scientists wrote. “ … these results are intriguing as they suggest that enteric bacteria directly contribute to the pathogenicity of protein conformational diseases … While pathogenic bacteria contribute to aggregation, commensal strains suppress it.” They pointed out that their work demonstrates the potential benefits of butyrate and butyrogenic bacteria in suppressing the proteotoxic effects of detrimental bacteria and also emphasizes the importance of having a balance between commensal butyrogenic and enteropathogenic microbes.

Surprisingly, the offspring of affected worms also showed increased protein aggregation – even though they had never encountered the bacteria originally associated with the condition. “This is very interesting because it suggests that these bacteria generate some sort of a signal that can be passed along to the next generation,” Czyz said.

And worms colonized by the “bad” bacteria also lost mobility, a common symptom of neurodegenerative diseases. “A healthy worm moves around by rolling and thrashing,” Walker noted. “When you pick up a healthy worm, it will roll off the pick, a simple device that we use to handle these tiny animals. But worms with the bad bacteria couldn’t do that because of the appearance of toxic protein aggregates.”

Czyz added, “You could compare the pick to an obstacle course: just as a person with a neurodegenerative disease will have trouble getting across, the same is true with these worms, just at a much smaller scale … The worms are very delicate, so you need a tool that won’t damage them.”

Currently the Czyz lab is testing hundreds of strains of bacteria found in the human gut to see how they affect protein aggregation in C. elegans. The group is also investigating how bacteria associated with neurodegeneration cause protein misfolding at the molecular level.

Czyz is also interested in possible connections between antibiotic-resistant bacteria and protein misfolding. “Almost all of the bacteria we found associated with protein misfolding are also associated with antibiotic-resistant infections in people. However, it will take many more years of research before we can understand what, if any, connection there is between antibiotic resistance and neurodegenerative diseases,” he acknowledged.

The authors concluded, “Collectively, our results suggest that dysbiosis between enteric pathogens and commensal butyrogenic bacteria contributes to the pathogenicity of PCDs … 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.”

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