Maintaining balance in the microverse: investigating microbial impacts on host gut inflammation

Abstract

Enteric microbes are known for impacting digestion, maintaining the immune system, and have implications for overall gastrointestinal health. Microbial mechanisms driving and alleviating gut inflammation have been established, however, are challenging to decipher specific microbial mechanism as well as how these fit in a complex community like the colon. Using multi-omics from a systematic to individual microbial populations level, we investigate two microbial pathways, one that was responsible for driving persistent gut inflammation, as well as one potential microbial product that might alleviate inflammation and promote colon recovery.

My first study investigates Enterobacteriaceae, which are known to promote gut inflammation and blooms during dysbiosis, with little speculation on how they drive inflammation. Through a holistic host-microbe approach in a dysbiotic induced colitis murine model, we showed that Enterobacteriaceae members increase in abundance in an inherited dysbiosis model. Through comparison of the host gene response and microbial abundances I found a positive correlation with Enterobacteriaceae abundance and host genes associated with the uptake of an amino acid L-cysteine. Suggesting the bloom of Enterobacteriaceae may a result of microbial use for L-cysteine for growth. Cultivation of Enterobacteriaceae isolates from murine fecal samples, and a nutrient dependency assay using L-cysteine revealed that Enterobacteriaceae can utilize L-cysteine for increased growth, fueling the growth needed to drive inflammation. This amino acid is important for both microbial and host functions, as it allows for redox reactions in microbes with the sulfur being utilized for energy and growth. For humans, L-cysteine is essential in maintaining and repairing the gut epithelium, and is an established biomarker of inflammatory bowel disease, with patients often exhibiting low serum levels. Our findings suggest that Enterobacteriaceae may compete with the host for this essential gastrointestinal amino acid L-cysteine, to bloom and drive inflammation.

In my second study, the main objective is to investigate further the impact of Klebsiella pneumoniae on driving colon epithelial inflammation and to mitigate the effects using a molecular target. I identified the cultured Enterobacteriaceae of interest as Klebsiella pneumoniae and demonstrated that when cultured with colon epithelial cells it displayed an increased gene expression of outer membrane proteins, and host-attachment proteins. Interestingly, one of the highest expressed genes was outer membrane protein A (OmpA). OmpA is an essential component of the membrane of K. pneumoniae and has virulence qualities including adhesion to the host, biofilm formation, and antimicrobial resistance. With this knowledge and OmpA chosen as a molecular target of K. pneumoniae, we synthesized a peptide that binds and inactivates OmpA. We found that this peptide AOA-2 successfully limited attachment of K. pneumoniae to the host, thereby limiting a proliferative and inflammatory response revealing a potential therapeutic alternative to antibiotics. Additionally, we show that treating K. pneumoniae with AOA-2 also lowers the sensitivity to several antimicrobials, thus highlighting a therapeutic option of AOA-2 alongside antimicrobials to work more effectively against resistant K. pneumoniae.

Finally, in the third study, we observed an increase in the microbial family Lachnospiraceae in dysbiotic mice that showed similar host inflammation levels as control mice. We utilized 5 commercially available strains of Lachnospiraceae to probe their functional potential that could contribute to this recovery of dysbiosis induced colitis mice. Using metabolomics, we investigated products that may aid in oxidative stress and inflammation alleviation. One strain, Eubacterium rectale, produced significantly more reduced glutathione, a potent antioxidant, in addition to several amino acids that are essential for gut repair. We also showed that microbial spent media significantly lowered oxidative stress through ROS, and inflammatory outputs in addition to lowering colon cell gene expression of host repair and pro-inflammatory genes. Overall, these findings help us to better understand the gut microbial landscape and the contributions of specific microbes and subpopulations in maintaining the balance of the gut microbiota and gut health.