GABA를 생산하는 장내미생물, 그리고 우울증과의 관계

[현준]

Strandwitz P, Kim KH, Terekhova D, Liu JK, Sharma A, Levering J, McDonald D, Dietrich D, Ramadhar TR, Lekbua A, Mroue N, Liston C, Stewart EJ, Dubin MJ, Zengler K, Knight R, Gilbert JA, Clardy J, Lewis K. GABA-modulating bacteria of the human gut microbiota. Nat Microbiol. 2019 Mar;4(3):396-403. doi: 10.1038/s41564-018-0307-3. Epub 2018 Dec 10. PMID: 30531975; PMCID: PMC6384127.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384127/

The gut microbiota affects many important host functions, including the immune response and the nervous system1. However, while substantial progress has been made in growing diverse microorganisms of the microbiota2, 23–65% of species residing in the human gut remain uncultured3,4, which is an obstacle for understanding their biological roles. A likely reason for this unculturability is the absence of key growth factors in artificial media, which are provided by neighboring bacteria in situ5,6. In the present study, we used co-culture to isolate KLE1738, which required the presence of Bacteroides fragilis to grow. Bio-assay driven purification of B. fragilis supernatant led to the isolation of the growth factor, which, surprisingly, is the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). GABA was the only tested nutrient that supported growth of KLE1738, and genome analysis supported a GABA-dependent metabolism. Using growth of KLE1738 as an indicator, we isolated a variety of GABA-producing bacteria, and found that Bacteroides ssp. produced large quantities of GABA. Genome based metabolic modeling of the human gut microbiota revealed multiple genera with the predicted capability to produce or consume GABA. Transcriptome analysis of human stool from healthy individuals showed that GABA producing pathways are actively expressed by Bacteroides, Parabacteroides, and Escherichia species. By coupling 16S rRNA sequencing with fMRI imaging in patients with Major Depressive Disorder (MDD), a disease associated with an altered GABAergic response, we found that relative abundance levels of fecal Bacteroides are negatively correlated with brain signatures associated with depression.

장내 미생물은 면역반응과 신경계 활동을 비롯하여 사람의 많은 기능에 영향을 끼친다

하지만 아직 많은 장내미생물들이 생물학적 역할에 대해서 명확하게 밝혀지지 않았다.

왜냐하면, 장내미생물에 대해서 연구하기 위해선 체외에서 즉 인공배지에서 배양하여 많은 수를 만든다음에 연구하여야 하는데 인공배지에는 장내미생물들이 잘 자라기 위한 성장인자가 부족하기 때문이다.

보통 이러한 성장인자는 주변에 있는 다른 미생물이 만들어주는 경우가 많다.

이러한 이유로 장내미생물의 23~65%정도가 아직 그 기능과 역할이 밝혀지지 않았다.

이 논문에서는 KLE 1738 미생물의 주요 성장인자가 신경전달물질이기도 한 GABA인것을 밝혀낸 후 이러한 성질을 이용하여 사람의 대변내 미생물을 분석한 결과 장내미생물중 GABA를 만들어내는 미생물로

Bacteroides, Parabacteroides, Escherichia 가 있음을 알아내었다.

-> 장내미생물이 GABA를 만들어내고 있다 !

그런데 이렇게 생산된 GABA가 체내에서 신경전달물질로 사용이 될까?

이후 우울증 환자들의 뇌MRI와 대변을 분석하여 우울증의 정도와 대변내 GABA를 생성할 수 있는 미생물의 사이의 상관관계를 밝혀내었다.

-> 장내에 GABA를 만들어내는 미생물이 많아질수록 우울증의 심한정도가 감소한다 !

우울증 환자는 GABA를 만들어 낼 수 있는 미생물을 보충해주어야 한다 (본인생각)

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The unique GABA-dependence of KLE1738 was exploited to identify additional GABA producers in a co-culture assay. Stool samples were mixed with molten FAAy agar, allowed to solidify, and KLE1738 was then spread on top of the medium. By looking for zones of KLE1738 growth, we were able to identify gut bacteria that produce GABA (Fig. 2A, B). In addition to Bacteroides species, representatives from the Parabacteroides, Eubacterium, and Bifidobacterium genera were identified as GABA producers in this assay (Fig. 2B). Of those, Bacteroides ssp. (and to a lesser extent Parabacteroides sp.) were found to produce GABA within the pH range of the human large intestines (Fig. 2B).

GABA에 의존적인 KLE1738 미생물의 특성을 이용하여 GABA를 생산해내는 미생물을 찾기 위한 실험을 설계하였음

대변샘플에 KLE1738를 넣은후 인공배지에 넣고 배양하였음

배양후 KLE1738이 많이 성장한 구역을 찾아서 이 구역에 많이 있는 장내미생물이 GABA를 만들어냈을 것으로 추정하였음.

이 결과 Bacteroides, Parabacteroides, Eubacterium, Bifidobacterium에 속한 미생물들이 GABA를 만들어냈음을 알 수 있었음.

Fig. 2.
In vitro and in silico identification of GABA modulating bacteria.
(A) To screen for GABA-producing bacteria, homogenized human stool sample was diluted and mixed with molten FAAy, with or without pH 7.0 MOPS buffer. KLE1738 was then spread on top of the agar and plates were incubated anaerobically for a week. Colonies that KLE1738 grew around were GABA producers (Inset). (B) Identified GABA producers were grown in liquid medium buffered at a pH of 5.0 and 7.0, and GABA levels of the spent medium was quantified using LC/MS and final pH of the medium was tested with pH strips. N=2 independent samples, and error is based on standard error. (C) 1,159 available gut genomes (consisting of 919 species) were analyzed for the genetic potential to produce and/or consume GABA (pathways associated with production or consumption highlighted in Supplemental Information Table 5). (D). The biochemical potential of 533 available gut-related metabolic models in KBase were examined for the capability to produce GABA or consume GABA. Shown are genera that represent at least 0.5% of the 533 models and contain either GABA producers or consumers.

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Accordingly, we sought to explore whether Bacteroides, perhaps the major bacterial producers of GABA in the human gut, were associated with clinically diagnosed Major Depressive Disorder (MDD). To do this, stool from 23 patients with MDD was collected and analyzed by 16S rRNA sequencing using the American Gut protocols. Resting state fMRI was acquired within 3 days of stool sample collection and scans were co-registered to MNI-space. To investigate the relationship between neural circuitry important in depression and fecal Bacteroides abundance we focused, a priori, on the left dorsolateral prefrontal cortex (DLPFC) and default mode network (DMN). Our choice of the left DLPFC was guided by the highly replicated finding that the left DLPFC is hypoactive in depression28. Similarly, the DMN is involved in self-referential processing29 and negative rumination in depression30. Functional connectivity is elevated both within the DMN and with other networks in depression31 and normalizes with treatment response32. We found an expansive region of the default mode network spanning the left anterior medial frontal cortex in which functional connectivity with the left DLPFC was inversely correlated with the relative abundance of fecal Bacteroides (Fig. 3A,B). This region of significance overlapped extensively with both the left medial prefrontal and left frontopolar cortex, regions highly interconnected to the limbic system and thought to be important in emotional reappraisal and reward processing33,34. This cluster was unique, and we found no clusters in which functional connectivity was positively correlated with the relative abundance of Bacteroides. We found no associations with KLE1738, perhaps due to its low abundance.

Fig. 3.
Fecal Bacteroides relative abundance inversely correlates with functional connectivity between left DLPFC and DMN structures in patients with Major Depressive Disorder (MDD).
(A) 3-Dimensional plots of the medial surface of the left and right hemispheres in patients with MDD (n=23). Significant cluster of 387 voxels in which fecal Bacteroides relative abundance correlated inversely with functional connectivity between this cluster and the left DLPFC. Colorbar shows Z scores of beta weights of the Bacteroides relative abundance covariate of a multiple linear regression with functional connectivity as the dependent variable. (B) Scatter plot of the average functional connectivity (Z score) over a sphere of radius 5mm centered at the voxel of peak significance (+12, −57, 0 in MNI coordinates) and abundance of fecal Bacteroides (Pearson r = −0.67, p=.0005). n=23.

장내미생물중 GABA의 주요 생산자인 Bacteroides 미생물이 우울증(Major Depressive Disorder MDD)과 어떤 관계가 있는지 확인하고자 실험을 하였음

우울증 환자 23명의 대변을 채집하고, 뇌의 fMRI를 촬영하여 비교하였음

결과적으로 대변샘플내에 Bacterioides가 풍부하게 들어있을 수록 fMRI 영상상 우울증과 관련이 있는 영역의 크기가 작았음

장내미생물총에 GABA를 만들어낼 수 있는 Bacteroides가 풍부하게 있을수록

뇌가 건강하고 우울증의 심한정도가 감소한다 !

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결론

우울증환자, GABA가 필요한 증상을 겪는 환자는

장내미생물총을 건강하고 다양하게, 그리고 특정 미생물을 보충하여

장내 GABA생산을 원활하게 만들면 증상을 개선할 수 있다 !

(본인생각)