Lachnospiraceae in the gut microbiome and their role in disease

Despite the gut microbiome being linked to numerous diseases, fundamental questions remain regarding how it influences mammalian physiology. Many studies show correlation between gut bacteria and specific diseases, but with little indication of the mechanism by which they may affect disease initiation or progression.

Applying the BBSRC approach of Integrative Microbiome Research we have generated exceptional preliminary data by carrying out research that combines the skills, methodologies and expertise from a range of disciplines from within the biosciences and beyond. This has enabled us to begin to understand a unique mechanism by which the gut microbiome can directly influence mammalian health. Our recent work shows how a unique family of bacteria within the mammalian gut, the Lachnospiraceae, produce two molecules (3M-4-TMAB and 4-TMAP) that were unknown until our recent discovery. These molecules were found in every organ in a mouse, even crossing into white matter. Their significance however lies in their structural mimicry of carnitine, a molecule critical to mammalian energy production. Carnitine acts as a carrier molecule, transporting fatty acids into mitochondria where they are burned for energy. However the bacterial molecules we discovered inhibit this process, reducing the amount of energy cells can produce when they are present. This is incredibly significant as the process of energy production in the mitochondria is known to be affected in a number of human diseases including type 2 diabetes and regressive/non-syndromic autism.

While we propose an unprecedented input for the gut microbiome into mammalian disease, our recent breakthrough work (in press at Science Advances) and the preliminary data we present here, provide solid evidence for both the systemic presence and inhibitory potential of these molecules. Also the presence of the Lachnospiraceae bacteria that produce these molecules at significantly increased levels in both type 2 diabetes patients and those with regressive autism is well known. We even determined that others have identified the presence of 3M-4-TMAB and 4-TMAP in prior studies of type 2 diabetes and autism, although at the time they were unaware of what these molecules were or their significance.

Given our findings we believe that understanding the role of these molecules in mammalian physiology is imperative. We intend in this proposal to;

- Firstly, elucidate the means by which these molecules interfere with energy production in the mitochondria
This will entail detailed studies of their interactions with enzymes linked to this process as well as wider effects these molecules may have on mammalian physiology. We will also check to see if, when they are produced in the intestine, they lead to a reduction in carnitine levels as it may act as a precursor molecule for making 3M-4-TMAB and 4-TMAP. Such a reduction in carnitine levels (as seen in T2D and ASD) would further contribute to a drop in energy production by mammalian cells. We will also test inhibitors against the bacteria to see if we can stop production of 3M-4-TMAB and 4-TMAP, an approach that could lead to future therapeutic interventions in these diseases.

- Secondly,we will determine the effects of these molecules on health and disease in mammalian cells and animal models
We will use specific cells to see if presence of these molecules induces specific signs of disease. For type 2 diabetes this would be any indication cells are no longer responding to insulin, leading to glucose build up in the blood. In obesity, we would see fat build up in cells as energy generation is blocked, while in regressive ASD stem cells in the brain that require fatty acid breakdown to generate energy would now begin to proliferate rapidly. Our work will then use animals, some lacking a gut microbiome, to test the ability of these molecules to alter mammalian physiology with respect to each disease.

The gut microbiome is an area of rapidly expanding research interest as scientists have shown, or hypothesised, its input across a wide range of diseases. Here we will build on our recent discovery of a unique pair of structurally similar metabolites (3M-4-TMAB and 4-TMAP) that originate from the gut microbiome and disseminate to every organ in a mouse model, including the brain. Given we now know these molecules affect metabolism and disrupt mitochondrial function, our focus will be on diseases where metabolic and mitochondrial dysfunction have been described; obesity, type 2 diabetes and regressive autism. Each disease has been described in the literature as having specific fatty acid oxidation related defects while carnitine has been used in each to alleviate disease symptoms.

Gut microbiome input into each disease has also been strongly supported in the literature and the family that produce these metabolites are significantly increased in affected patients. Additionally molecule(s) of identical mass to charge ratio (m/z) and molecular fingerint as 3M-4-TMAB and 4-TMAP have been detected and have been highlighted as significantly changed across all these diseases. Our recent publication (Science Advances - in press) has demonstrated these molecules at m/z 160.133 are highly likely to be 3M-4-TMAB and 4-TMAP, their single mass (usual indicative of a single molecule) alongside different structures rendering structural elucidation incredibly demanding.

Here we will determine the mechanism of action, effects at the cellular level, and consequences in vivo of the presence of 3M-4-TMAB and 4-TMAP. This proposal will capitalize on our unique knowledge of these molecules and our preliminary data indicating that they can recapitulate many of the before mentioned disease phenotypes in vitro (see Case for Support). This Integrated Microbiome Research proposal aims to finally define a fundamental mechanistic role for unique bacterial metabolites in human disease.

Due to the novelty of our findings, and their application in this proposal to diseases of significant impact and increasing incidence, we expect this proposal to have a considerable impact as outlined below. We have identified stakeholders and detail below how each will be impacted in the short and longer term.

As outlined in detail in the Academic Beneficiaries section we expect significant impact across academic research from our findings. Those that work on microbiome research and on the diseases to be studied in this proposal will be most impacted in the short term. We expect our findings will also be applicable more widely outside the diseases we study and expect in the longer term general impact across the study of neurological conditions and metabolic diseases where the microbiome has known or suspected input.

Patients, and those in the third sector that represent them, will be impacted through increased knowledge of the potential cause/microbiome input into disease in the short term. In the longer term there may be significant opportunities for intervention strategies to be introduced to counteract the presence of these metabolites or reduce their presence. These may improve disease outcomes or even prevent disease occurrence. The early stages for development of these intervention strategies are built into the proposal through screening of available inhibitors from the Schofield lab at Oxford University, but we also envisage future effective intervention strategies being developed that target the Lachnospiraceae identified in this proposal through the use of probiotics or targeted antimicrobials.

The general public will be impacted through the increased knowledge of a fundamental mechanism of action of the microbiota in disease. While presently most microbiome disease data is correlative, a detailed explanation of a mechanism of input for the gut microbiome in disease would impact how the general public views microbiome research and its impact on their health. This increased knowledge will also help the public make informed decisions about lifestyle choices, especially in the cases of metabolic diseases which have to date been of unknown cause. An understanding of the mechanisms underlying disease can help the public understand what leads to disease development (e.g. high fat diets leading to increased Lachnospiraceae colonisation of the gut, which may lead to type-2-diabetes).

Industry will gain increased knowledge of how the microbiome communicate with the host, of particular interest to companies already working on microbiome-based intervention strategies in disease (e.g. Microbiotica (UK)). Industry working on intervention strategies on the diseases to be studied here will also gain new insight into potential novel/microbiome inputs into the diseases they work on. We envisage that these impacts will be both short term, in terms of strategy changes in response to our findings, and longer term, as companies focus attention on exploiting our findings for therapeutic intervention.

Our findings will also impact clinicians in the short term as they factor in the influence of the gut microbiome on diseases of previously unknown etiology. Having increased explanatory knowledge of the potential impact of the microbiome on patient health will also be a tool for education to show that lifestyle choices can have long term consequences. In the longer term therapeutic (or preventative) interventions to target the gut microbiome rather than the disease itself (BBSRC strategy of Health through Stealth) may become the norm as clinicians and the NHS target metabolic diseases of increasing frequency.

Substantial impact will also be felt by those working on the project. Increased knowledge through an integrative approach to addressing the influence of the microbiome in mammalian disease will offer significantly increased knowledge for the PIs and knowledge, training and development opportunities for the PDRAs.