Immunodominant Proteins of Fusobacterium nucleatum: Comparative Analyses in Colorectal Cancer Patients, Healthy Individuals, and Vaccinated Mice Models

Abstract

Fusobacterium nucleatum (Fn) is an anaerobic oral bacterium that has been strongly linked to colorectal cancer (CRC) through a growing body of experimental and clinical evidence. Fn has been shown to colonize tumors, invade host cells, and promote immune evasion through surface-exposed adhesins. Although Fn tumor colonization has been linked to poor clinical outcomes, the bacterial proteins most strongly targeted by the host humoral immune response remain poorly defined. I hypothesized that antibody recognition of Fn would preferentially target specific outer membrane proteins, and that immunodominant antigen profiles would differ depending on host immune status, including vaccination history in mice, and antibody response strength, tumor-associated bacterial load, and disease state in human patients. To test this hypothesis, whole-cell Fn lysates were incubated with plasma from mice vaccinated with O₂-killed Fn, unvaccinated controls, or no-serum controls, followed by immunoprecipitation (IP) and mass spectrometry (MS) to identify antibody-bound bacterial antigens. Quantitative analysis based on unique spectral counts of Fn-originating peptide fragments revealed that the most enriched targets were outer membrane-localized proteins. Vaccinated mouse sera selectively enriched a defined subset of Fn proteins, including the highly enriched outer membrane adhesins RadD and Fap2 and the abundant porin FomA. These findings demonstrate that vaccination induces robust antibody responses against specific Fn surface proteins in mice and establishes IP-MS as a powerful approach for defining humorally immunodominant bacterial antigens. In future work, I will compare antibody recognition patterns across CRC patients and healthy individuals to identify candidate targets for diagnostic or immunotherapeutic strategies.