IDDF2024-ABS-0357 Veillonella parvula reduces the lactylation of SMAD3 in CTLA4+ TIGIT+ treg cells to promote colorectal cancer

Background

Evidence implicates the role of gut microbiota in colorectal cancer (CRC). Veillonella parvula (V. parvula), an anaerobic bacterium that metabolizes lactic acid, is positively related to the development of CRC. Previous studies demonstrated that V. parvula might promote tumorigenesis by modulating the tumor immune microenvironment (TIME), but its causative role and molecular mechanism remain unclear.

Methods

We performed single-cell sequencing and signaling pathway enrichment of blood, stool and tissue samples from the clinical cohort. We did flow cytometry and mass spectrometry combined with proteomics analyses to identify CTLA4+TIGIT+Treg cells enriched in the mucosa of CRC patients. The NCBI (National Center for Biotechnology Information) database and AlphaFold2 (www.alphafold.ebi.ac.uk), an artificial intelligence protein structure prediction system developed by DeepMind, were used to predict the protein structure and lactylation sites of small mother against decapentaplegic 3 (SMAD3) based on ColabFold (www.github.com). We did further in vitro and in vivo tests to explore the mechanism among V. parvula, Treg cells and CRC.

Results

We found that the peripheral blood CTLA4+TIGIT+Treg cells, one of the Treg cell subsets, was significantly positively correlated with the relative abundance of V. parvula in the stools and Treg cell-related signaling pathways were significantly enriched in the mucosa of CRC patients. We further identified the down-regulated lysine lactylation (K-Lac) level at the K378 site of SMAD3, a key protein of Treg cell differentiation. Spleen cells from wild-type mice were collected, and purified naïve T cells were obtained using the CD4 T cell isolation kit. The differentiation of Treg cells was inhibited by lactic acid while promoted by V. parvula, and only live V. parvula could rescue the inhibition of lactic acid. (IDDF2024-ABS-0357 Figure 1)

Conclusions

To sum up, we showed that V. parvula down-regulated K-Lac by depleting lactic acid in the tumor environment and competitively up-regulated lysine acetylation (K-Ac) at the K378 site of SMAD3. This, in turn, activated the transcription of SMAD3 downstream genes, promoting the differentiation from primitive T cells into CTLA4+ TIGIT+ Treg cells and leading to CRC immunosuppression and tumor progression.