Buck, Amy

Pedersen, Amy

Du, Xiaochen

2024-06-04T09:45:37Z

2024-06-04T09:45:37Z

2024-06-04

The human gastrointestinal tract hosts a complex and diverse population of microorganisms collectively known as the gut microbiota. The gut microbiota plays pivotal roles in maintaining human health. Research links an imbalance in gut microbiota to several human diseases like inflammatory bowel diseases, obesity, diabetes mellitus, and gastrointestinal cancers. Consequently, modulating the gut microbiota is increasingly recognized as vital for treating these conditions, and several approaches, including probiotics, prebiotics, and faecal microbiota transplantation, are being explored for this purpose. However, strategies are lacking for precisely manipulating the composition of the gut microbiota. One emerging strategy is the use of microRNAs (miRNAs) which may be involved in the specific regulation of bacterial genes. miRNAs are a class of small, single-stranded RNA, typically 18–25 nucleotides in length. These small RNAs primarily regulate gene expression by either facilitating the degradation of messenger RNA (mRNA) or repressing mRNA translation in mammals. Over the last 15 years, extensive literature has demonstrated the presence of miRNAs in a cell-free form including serum, saliva, urine, and faeces. Recent reports suggest that mammalian miRNAs can directly influence the composition and activity of gut bacteria by entering bacteria and interacting with bacterial genes. However, many questions remain regarding how miRNA-bacteria interactions occur under physiological conditions. This thesis aims to investigate the specificity and mechanisms underlying small RNA trafficking from mouse intestinal cells to bacteria within the gut environment. To explore the miRNA-bacteria interactions, a crucial first step is to identify the miRNAs that are naturally transferred to the gut microbiota. We developed a purification method to isolate pure gut microbiota from mouse gut contents and sequenced small RNAs in the purified gut microbiota (PGM). Our data suggest host miRNAs are naturally present within gut microbiota and show that the miRNA composition within PGM is distinct to that found in total gut contents, implying specificity. We then employed miRNA-FISH (Fluorescence In Situ Hybridization) to visualize the presence of host miRNAs within gut bacterial cells and to detect miR-21a-5p, which is the most abundant miRNA in PGM. Moreover, co-staining experiments conducted in PGM using probes for miR-21a-5p and Lactobacillus demonstrated co-localization, suggesting the uptake of miR-21a-5p by Lactobacillus. Our work provides a promising foundation on which to discover additional interactions between miRNA and bacteria within the gut. Next, we investigated the transport mechanism of host miRNAs into bacteria. In mammals, miRNAs usually function with an Argonaute protein and it has been observed that miRNA-Ago2 (Argonaute 2) complexes are secreted by mouse intestinal epithelial cells. We hypothesized that miRNAs move with Ago2 protein and the miRNA-Ago2 complexes regulate gene expression within the gut microbiota. To test this hypothesis, we investigated whether the mouse Ago2 protein is detectable within gut microbiota and adapted a method to identify bacterial genes directly targeted by miRNA-Ago2 complexes. The detection of Ago2 was performed in PGM using western blot analysis and immunofluorescence imaging and our results suggest that there is no consistent Ago2 signal in PGM. However, PGM does not encompass the entire spectrum of bacterial species within the mouse gut. To account for possibility that Ago2 signal could be lost in the PGM purification, we employed another method to capture Ago2 and isolate associated RNAs from the intact gut tissue: CLEAR-CLIP (covalent ligation of endogenous Argonaute-bound RNAs–Crosslinking and immunoprecipitation). We identified high-confidence bacterial targets co-purifying with Ago2 in samples obtained from different segments of the gut, suggesting the transfer of host miRNAs with Ago2 protein and their potential functional role in gut bacteria. Nevertheless, further bioinformatic analysis and experimental validation are required to confirm these as genuine targets. In order to account for other transport methods beyond Ago2, we then investigated extracellular vesicles (EVs). These lipid-bound vesicles are secreted by cells into the extracellular space and play important roles in both intercellular and inter-organismal communication. We isolated EVs from a mouse intestinal epithelial cell line (Mode-K) and profiled the miRNAs content of Mode-K EVs. Our results show that miRNAs from the let-7 family are abundant and enriched in Mode-K EVs. To explore potential specificity in the uptake of EVs by bacteria, we conducted a comparison using fluorescently labelled EVs obtained from Mode-K cells and from the gastrointestinal parasite Heligmosomoides bakeri. We show that Salmonella Typhimurium SL1344 internalize Mode-K EVs but not parasite EVs when cultured in M9 medium and the uptake of mouse or parasite EVs is not observed in E. coli W3110. These data demonstrate specificity in the interaction between mammalian EVs and SL1344. Additionally, our data indicated that Mode-K EVs exerted a stimulatory effect on the growth of SL1344 and this growth-promoting impact is dependent on the dosage of Mode-K EVs, as higher concentrations correlated with increased growth. Co-culture experiments using Triton X-100-treated Mode-K EVs confirmed the necessity of intact EVs for the observed growth promotion in SL1344. We also show that mouse miRNAs are detected in S. Typhimurium after co-culturing with Mode-K EVs. In conclusion, Mode-K EVs have the capability to deliver RNA cargo to bacteria and affect bacterial growth. However, whether this promotion effect of Mode-K EVs on SL1344 is induced by mouse miRNAs requires further investigation. Finally, we used a genetic reporter system (Cre-loxP system) to investigate whether Mode-K EVs can deliver functional cargo into S. Typhimurium. This technique involves the donor cells secreting EVs containing Cre mRNA, which are then internalized by the reporter cells. If the Cre mRNA is translated after internalization, Cre recombinase can remove the loxP-flanked transcription terminator, leading to the production of fluorescent molecules in the reporter cells. Our co-culture experiments of mouse Cre-expressing EVs with reporter S. Typhimurium demonstrated the functional transmission of cargo from mouse to bacteria via EVs. In summary, this thesis demonstrates that host miRNAs are present within gut microbiota under physiological conditions and provides an approach to identify interactions between miRNA and bacteria within the gut. We also demonstrate that EVs derived from intestinal epithelial cells can act as a transport mechanism for host miRNAs into specific bacteria and impact bacteria growth. These findings enhance our understanding of the specificity and mechanism of RNA trafficking from mouse to bacteria within the gut environment which could hold promising implications for modulating the gut microbiota to treat associated diseases in the future.

https://hdl.handle.net/1842/41835

http://dx.doi.org/10.7488/era/4558

en

The University of Edinburgh

Du, Xiaochen, Ruth Ley, and Amy H. Buck. "MicroRNAs and extracellular vesicles in the gut: new host modulators of the microbiome?" Microlife 2 (2021): uqab010.

gut microbiota

microRNAs

miRNAs

miR-21a-5p

Ago2 protein

extracellular vesicles

Mode-K EVs

Specificity and mechanism of RNA trafficking from mouse to bacteria in the gut

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy