Periodic Reporting for period 1 - ExtractABact (Deciphering host-gut microbiota spatio-functional plasticity in inflammation)
Periodo di rendicontazione: 2023-02-01 al 2025-07-31
Over the past two decades, gut bacteria have emerged as major regulators of human health. The focus in the field thus far has been on bacterial taxonomy, with their spatial organization and functionality largely overlooked. Crohn’s disease (CD) vividly portrays this spatiofunctional dimension, as it features patches of gut inflammation (skip lesions) surrounded by uninflamed regions—in the same host—with a clear demarcation but unknown cause. Our preliminary data demonstrate host-microbe functional feedback loops in which bacterial strains can adapt and modify their immunomodulatory functions in response to the host. Moreover, we find functional alterations in gut bacteria of IBD patients. Thus, we hypothesize that bacterial spatio-functionality can be largely affected by host physiology, and, in turn, modulate the pathophysiological state, creating a functional feedback loop.
We propose to study bacterial spatiofunctional plasticity and mechanisms of host-microbe interactions in CD, and their potential causal effects on inflammation, combining microbiology, immunology and systems biology approaches. We intend to focus on three independent yet complementary aims: (1) Characterize host-microbe spatiotemporal functional alterations in skip lesions and assess their potential causal effects on inflammation; (2) Decipher the functional and molecular mechanisms of host-microbe feedback loops in healthy and inflamed intestines; and (3) Develop a toolbox for high-resolution functional analyses of gut bacteria directly in their natural environments.
This study will address, for the first time, bacterial functional plasticity in response to host inflammation, unveiling the phenomena, its mechanism of action, and potential causal effects on gut inflammation. Such understanding can shift our perception on microbiota-host interactions, may explain contradictions in the field, point to novel contributing factors to CD and related disorders, and guide future translational studies.
We propose to study bacterial spatiofunctional plasticity and mechanisms of host-microbe interactions in CD, and their potential causal effects on inflammation, combining microbiology, immunology and systems biology approaches. We intend to focus on three independent yet complementary aims: (1) Characterize host-microbe spatiotemporal functional alterations in skip lesions and assess their potential causal effects on inflammation; (2) Decipher the functional and molecular mechanisms of host-microbe feedback loops in healthy and inflamed intestines; and (3) Develop a toolbox for high-resolution functional analyses of gut bacteria directly in their natural environments.
This study will address, for the first time, bacterial functional plasticity in response to host inflammation, unveiling the phenomena, its mechanism of action, and potential causal effects on gut inflammation. Such understanding can shift our perception on microbiota-host interactions, may explain contradictions in the field, point to novel contributing factors to CD and related disorders, and guide future translational studies.
(Aim 1) To study spatio-functional alterations in inflamed gut, we are developing a methodology that combines protocols for colon clarity and detection of bacteria using combination of fluorophores and antibodies. Integration of these methodologies allows us to spatially analyse bacteria within the gut tissue, using light-sheet microscopy for a three-dimension (3D) imaging in the context of host cells. To this day, we have adapted and optimised protocols (iDISCO and CLARITY) to enhance tissue transparency and bacterial detection. Currently, we are troubleshooting tissue clarity steps without losing bacteria during the process. In addition, we developed and published a protocol for the real-time detection and monitoring of live communities of anaerobic gut bacteria in their natural environment (Hajjo, et al. 2023).
Furthermore, we aim to analyse bacterial functionality by monitoring structural genomic variations between inflamed and healthy tissue. We demonstrated and published that phase variation correlates to bacterial transcriptomics and gut inflammation in IBD patients (Carraso, et al. 2024). To study bacterial causality to inflammation, in the gut tissue, we are in the process of engineering bacteria to express one variant of the genomic phases “locked”, that were found to correlate to inflammation. These engineered bacteria will be tested in mice models for gut inflammation.
(Aim 2) Host-bacteria feedback loop is a process where bacteria are adapted to the gut ever changing environment, and in turn, they are affecting the environment including the host. Here, we aimed to study the bilateral crosstalk between the host (i.e. immune system) and gut resident bacteria. To do so, we focused on two known immunomodulatory human gut bacteria; Bifidobacterium longum (B. longum), a gram-positive, which suppresses oxidative stress an reduce inflammation, and Bacteroides thetaiotaomicron (B. theta), a gram-negative bacteria, also known to induce anti-inflammatory effect by induction of regulatory T cells. To this day, we have demonstrated and published that B. longum secrete vesicles that mediate immunomodulation (Mandelbaum, et al. 2023). In addition, we are in the process of studying immunological factors that affect bacterial functionality. We are using Knock-Out (KO) Germ-Free (GF) mice to exclude specific immunological pathways (e.g. Rag -/-).
(Aim 3) The isolation of specific bacterial strains from complex microbial communities can provide insight into bacterial diversity and functionality within various environments, which could be utilized for a variety of applications. In this study, we demonstrate a new method for the specific isolation of live bacteria strains from their natural environment. Our approach relies on metagenomic analysis and combines Fluorescent In-Situ hybridisation (FISH)-sorting followed by antibody-based sorting.
To demonstrate the technology feasibility, we isolated Bacteroides fragilis (B. fragilis) from human stool samples. We designed and tested the specificity of FISH B. fragilis FISH probe through flow cytometry analysis. We screened for potential probes and focused on a probe that successfully targeted B. fragilis, and differentiated it from non-relevant bacteria, within a stool spiked with B. fragilis. The probed B. fragilis was later used as an immunogen to vaccinate laying chickens to elicit antibodies against B. fragilis. Then, the antibodies were purified from egg yolk and tested for their specificity against B. fragilis. Following absorption of the antibodies against non-relevant bacteria (negative stool sample), we improved antibodies specificity with reduced background. We demonstrated that the antibodies are specific to B. fragilis and can be used to sort live bacteria from stool containing B. fragilis. We demonstrated that the sorted bacteria are viable and can be further grown in anaerobic conditions or analysed by sequencing. Currently we are in the process of improving antibodies specificity and validations of our new anaerobic sorter which was purchased by the generous funding of this ERC grant.
Furthermore, we aim to analyse bacterial functionality by monitoring structural genomic variations between inflamed and healthy tissue. We demonstrated and published that phase variation correlates to bacterial transcriptomics and gut inflammation in IBD patients (Carraso, et al. 2024). To study bacterial causality to inflammation, in the gut tissue, we are in the process of engineering bacteria to express one variant of the genomic phases “locked”, that were found to correlate to inflammation. These engineered bacteria will be tested in mice models for gut inflammation.
(Aim 2) Host-bacteria feedback loop is a process where bacteria are adapted to the gut ever changing environment, and in turn, they are affecting the environment including the host. Here, we aimed to study the bilateral crosstalk between the host (i.e. immune system) and gut resident bacteria. To do so, we focused on two known immunomodulatory human gut bacteria; Bifidobacterium longum (B. longum), a gram-positive, which suppresses oxidative stress an reduce inflammation, and Bacteroides thetaiotaomicron (B. theta), a gram-negative bacteria, also known to induce anti-inflammatory effect by induction of regulatory T cells. To this day, we have demonstrated and published that B. longum secrete vesicles that mediate immunomodulation (Mandelbaum, et al. 2023). In addition, we are in the process of studying immunological factors that affect bacterial functionality. We are using Knock-Out (KO) Germ-Free (GF) mice to exclude specific immunological pathways (e.g. Rag -/-).
(Aim 3) The isolation of specific bacterial strains from complex microbial communities can provide insight into bacterial diversity and functionality within various environments, which could be utilized for a variety of applications. In this study, we demonstrate a new method for the specific isolation of live bacteria strains from their natural environment. Our approach relies on metagenomic analysis and combines Fluorescent In-Situ hybridisation (FISH)-sorting followed by antibody-based sorting.
To demonstrate the technology feasibility, we isolated Bacteroides fragilis (B. fragilis) from human stool samples. We designed and tested the specificity of FISH B. fragilis FISH probe through flow cytometry analysis. We screened for potential probes and focused on a probe that successfully targeted B. fragilis, and differentiated it from non-relevant bacteria, within a stool spiked with B. fragilis. The probed B. fragilis was later used as an immunogen to vaccinate laying chickens to elicit antibodies against B. fragilis. Then, the antibodies were purified from egg yolk and tested for their specificity against B. fragilis. Following absorption of the antibodies against non-relevant bacteria (negative stool sample), we improved antibodies specificity with reduced background. We demonstrated that the antibodies are specific to B. fragilis and can be used to sort live bacteria from stool containing B. fragilis. We demonstrated that the sorted bacteria are viable and can be further grown in anaerobic conditions or analysed by sequencing. Currently we are in the process of improving antibodies specificity and validations of our new anaerobic sorter which was purchased by the generous funding of this ERC grant.
Monitoring bacterial functionality on the genomic level is beyond the state-of-the-art, as it allows to add another layer of information, on top of a widely used existing sequencing technology. Currently, shotgun sequencing is used to study bacterial abondance and potential genes. Here we demonstrate that for the same data (generated by other or by us), we can apply additional phase variation analysis, which is in high correlation to expression for the genes that under phase variation control.
Developing a method to isolate a specific bacteria from a complex environment is beyond the state-of-the-art. This technique can be applied to a wide range of bacteriological studies. To the best of our knowledge, there is no available methodology to enrich or deplete a live bacteria from different natural samples (excluding culturing techniques). By isolating specific strains, we can gain insights into their functional roles, develop personalised therapies and explore innovative solutions for gut dysbiosis.
Developing a method to isolate a specific bacteria from a complex environment is beyond the state-of-the-art. This technique can be applied to a wide range of bacteriological studies. To the best of our knowledge, there is no available methodology to enrich or deplete a live bacteria from different natural samples (excluding culturing techniques). By isolating specific strains, we can gain insights into their functional roles, develop personalised therapies and explore innovative solutions for gut dysbiosis.