Multi-layer analysis of gut metagenomic functional potential in major depressive disorder

Major depressive disorder (MDD) is associated with gut microbiota dysbiosis, but previous studies have mostly focused on species composition and are subject to confounding factors such as age and diet. Robust differences at the functional pathway level remain unclear.

We included 98 indigenous residents of Xinjiang, China, whose families had resided there for three or more generations (49 MDD patients and 49 healthy controls) and performed shotgun metagenomic sequencing. Functional profiles were annotated with HUMAnN3 across seven levels: GBM, GMM, KO, EC, Module, Pathway, and PathAbundance. Features were transformed using the centered log-ratio (CLR) method. After controlling for 10 covariates (age, sex, BMI, bowel movement frequency, diet, gastrointestinal symptoms, years of smoking, alcohol consumption, physical activity, and sleep), OLS regression was performed with Benjamini-Hochberg FDR correction (q < 0.05). Complementary analysis was conducted using MaAsLin2, together with age caliper matching (52 participants), recurrent/first-episode subgroup analyses, and sensitivity analyses for treatment history. Overall functional profiles were evaluated by PERMANOVA based on Aitchison distance.

After covariate adjustment, overall functional profiles did not differ significantly between groups (PERMANOVA R²=1.9%, p = 0.071; PERMDISP p = 0.98). After FDR correction, 33 significant features were detected across five levels: EC, Pathway, Module, GMM, and PathAbundance (no significant signals at the GBM or KO levels). Key biologically meaningful signals included: propionate CoA-transferase (EC 2.8.3.1, β=-1.42, q = 0.022)↓, teichoic acid biosynthesis (map00552, β=-0.34, q = 0.045)↓, vancomycin resistance module (M00651, β=-1.28, q = 0.014)↓, galacturonate degradation II (MF0023, β=+1.82, q = 0.048)↑, and heme b biosynthesis I (PathAbundance, β=+0.93, q = 0.033)↑. MaAsLin2 effect sizes were correlated with the main OLS results at the Pathway level (R²=0.84) with consistent direction. GRSA independently confirmed downregulation of the phosphotransferase system (map02060, padj<0.001) and teichoic acid biosynthesis. The propionate CoA-transferase and teichoic acid biosynthesis signals were directionally consistent in the main model and recurrent subgroup, but did not survive correction in the matched subset; the first-episode subgroup (n = 11) was underpowered and its results are reported as exploratory.

Gut microbiota functional potential in MDD patients shows a mild but directionally consistent shift after controlling for confounders, involving short-chain fatty acid production, Gram-positive bacterial cell wall metabolism, and cofactor metabolism. This study measures functional potential and requires validation by metabolomics.