Structural and functional analysis of colorectal cancer-relevant Fusobacterium nucleatum autotransporter adhesins

• Applicant: Dr. Daniel Roderer
• Subject Area: Structural Biology ; Biochemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 548509121

The human intestinal microbiome (IM) is decisive for the host’s health. A pathological modulation, i.e., dysbiosis, can cause and promote the progression of colorectal cancer (CRC). Fusobacterium nucleatum (Fn) is often overrepresented microbes in the IM of CRC patients and can initiate the emergence of tumors and downregulate the host’s immune defense. Its presence correlates with metastasis and bad prognosis. A key molecule for Fn-driven CRC progression is Fap2, a 400 kDa bifunctional adhesin. Fap2 mediates tumor colonization of Fn by interaction with the glycan Gal-GalNAc, the receptor on cancer cells, and natural killer cell deactivation through interaction with TIGIT, the receptor on immune cells. This makes Fap2 a prime target for treating microbe-driven cancer. However, mechanistic details of Fap2-receptor interaction remain open questions due to the lack of high-resolution structural data, a hurdle for structure-based anticancer drug design. We propose to unravel molecular details of Fap2 interaction with its two receptors Gal-GalNAc and TIGIT at hitherto unachieved resolution and completeness using and combining cryo electron microscopy (cryo-EM), site-directed mutagenesis, protein-protein interaction assays, mass spectrometry, and cryo tomography (cryo-ET). For this, we will apply our recently developed system to recombinantly produce the extracellular domain (ECD) of Fap2 and truncated versions thereof. These will then be mixed with the receptors, and the resulting complexes will be subjected to cryo-EM and single particle analysis to achieve near-atomic resolution insights into mechanism interaction mechanisms (work packages WP1.1 & 1.2). Mutagenesis of the Fap2-receptor interaction sites and binding assays (surface plasmon resonance for TIGIT and cell-based fluorescence assays for Gal-GalNAc) will complement the structural data (WP1.3). To gain insight into the abundance and distribution of Fap2 on the Fn surface, we will compare the expression levels of Fap2 and other autotransporter adhesins under varying culture conditions, e.g., in the presence and absence of other bacteria or immune cells, using surface-shaving proteomics (WP2.1). The surface of Fn grown under high Fap2 expression conditions will then be analyzed by cryo-ET and subtomogram averaging to display individual Fap2 molecules in situ. Hence, we aim to understand how several Fap2 adhesins might act together in coordinative receptor binding (WP2.2). Our proposal is based on strong preliminary work where we have established production of the Fap2-ECD, TIGIT-ECD, solved the structure of the Fap2-ECD by cryo-EM, showed interaction of Fap2 with receptors in vitro, identified their interaction sites, and visualized adhesins on the Fn surface by cryo-ET. Here, we will provide groundbreaking molecular insights into Fap2-mediated tumor colonization and immune evasion of Fn, paving the way for the development of therapeutics to target the underlying protein-protein interactions.

• DFG Programme: Research Grants
• Co-Investigators: Dr. Christoph Andreas Diebolder; Professorin Dr. Fan Liu; Professorin Dr. Annette Moter