Fusobacterium nucleatum is implicated in accelerating colorectal cancer (CRC) and is found within metastatic CRC cells in patient biopsies. Here, we found that bacterial invasion of CRC cells and cocultured immune cells induced a differential cytokine secretion that may contribute to CRC metastasis. We used a modified galactose kinase markerless gene deletion approach and found that F. nucleatum invaded cultured HCT116 CRC cells through the bacterial surface adhesin Fap2. In turn, Fap2-dependent invasion induced the secretion of the proinflammatory cytokines IL-8 and CXCL1, which are associated with CRC progression and promoted HCT116 cell migration. Conditioned medium from F. nucleatum–infected HCT116 cells caused naïve cells to migrate, which was blocked by depleting CXCL1 and IL-8 from the conditioned medium. Cytokine secretion from HCT116 cells and cellular migration were attenuated by inhibiting F. nucleatum host-cell binding and entry using galactose sugars, l-arginine, neutralizing membrane protein antibodies, or fap2 deletion. F. nucleatum also induces the mobilization of immune cells in the tumor microenvironment. However, in neutrophils and macrophages, the bacterial-induced secretion of cytokines was Fap2 independent. Thus, our findings show that F. nucleatum both directly and indirectly modulates immune and cancer cell signaling and migration. Because increased IL-8 and CXCL1 production in tumors is associated with increased metastatic potential and cell seeding, poor prognosis, and enhanced recruitment of tumor-associated macrophages and fibroblasts, we propose that inhibition of host-cell binding and invasion, potentially through vaccination or novel galactoside compounds, could be an effective strategy for reducing F. nucleatum–associated CRC metastasis.

Fusobacterium nucleatum is implicated in the acceleration of colorectal cancer (CRC), y et the mechanisms by which this bacterium modulates the tumor microenvironment remain understudied. Here we show that binding and cellular invasion of CRC cells selectively induces the secretion of the pro-inflammatory and metastatic cytokines IL-8 and CXCL1, which we then show induces robust migration of HCT116 cancer cells. Next, we demonstrate that cytokine signaling by cancer cells is largely driven by invasion coordinated by the surface adhesin Fap2. By contrast, we show that F. nucleatum induced secretion of CCL3, CXCL2, and TNFα cytokines from neutrophils and macrophages is Fap2 independent. Finally, we show that inhibiting F. nucleatum host-cell binding and entry using galactose sugars, neutralizing membrane antibodies, and deletion of the fap2 gene, lead to attenuated cytokine secretion and cellular migration. As elevated IL-8 and CXCL1 levels in cancer have been associated with increased metastatic potential and cell seeding, poor prognosis, and enhanced recruitment of tumor-associated macrophages and fibroblasts within tumor microenvironments, these data show that F. nucleatum directly and indirectly modulates immune and cancer cell signaling and migration. In conclusion, as viable F. nucleatum were previously shown to migrate within metastatic CRC cells, we propose that inhibition of host cell binding and invasion, potentially through vaccination or novel galactoside compounds, could be an effective strategy for reducing F. nucleatum -induced signaling that drives metastasis and cancer cell seeding. ________________________________________________________________________________________ microbe-accelerated cancers. Two recent studies reported that F. nucleatum directly induces cancer cell metastasis through NF-κB increased expression of Keratin 7 (KRT7) ( 11 ), as well as increased expression of caspase activation and recruitment domain 3 (CARD3), and downregulation of E-cadherin ( 12 ). Herein we add to the mechanisms used by F. nucleatum to induce cellular migration. We show that direct binding and invasion of host cancer and immune cells by F. nucleatum induces the secretion of the proinflammatory and prometastatic cytokines IL-8 and CXCL1, and that conditioned media from F. nucleatum infected HCT116 CRC cells causes non-Fusobacterium exposed cells to migrate towards this cytokine rich media.Chemokines/cytokines play a crucial role in tumor initiation, progression, and metastasis ( 13 ). Initially discovered as chemotactic mediators of leukocytes, they are now known to be secreted by several cell types and can be expressed constitutively or induced by inflammatory stimuli, including bacterial infections, and function in a variety of roles including cell survival, proliferation, angiogenesis, and cell migration. In cancer, chemokines mainly function in regulating angiogenesis, activating tumor-specific immune responses, and directly stimulating the tumor through autocrine or paracrine mechanisms ( 13 ).The cytoki...

Fusobacterium spp. are Gram-negative, anaerobic, opportunistic pathogens involved in multiple diseases, including a link between the oral pathogen Fusobacterium nucleatum and the progression and severity of colorectal cancer. The identification and characterization of virulence factors in the genus Fusobacterium has been greatly hindered by a lack of properly assembled and annotated genomes. Using newly completed genomes from nine strains and seven species of Fusobacterium, we report the identification and corrected annotation of verified and potential virulence factors from the type 5 secreted autotransporter, FadA, and MORN2 protein families, with a focus on the genetically tractable strain F. nucleatum subsp. nucleatum ATCC 23726 and type strain F. nucleatum subsp. nucleatum ATCC 25586. Within the autotransporters, we used sequence similarity networks to identify protein subsets and show a clear differentiation between the prediction of outer membrane adhesins, serine proteases, and proteins with unknown function. These data have identified unique subsets of type 5a autotransporters, which are key proteins associated with virulence in F. nucleatum. However, we coupled our bioinformatic data with bacterial binding assays to show that a predicted weakly invasive strain of F. necrophorum that lacks a Fap2 autotransporter adhesin strongly binds human colonocytes. These analyses confirm a gap in our understanding of how autotransporters, MORN2 domain proteins, and FadA adhesins contribute to host interactions and invasion. In summary, we identify candidate virulence genes in Fusobacterium, and caution that experimental validation of host-microbe interactions should complement bioinformatic predictions to increase our understanding of virulence protein contributions in Fusobacterium infections and disease. IMPORTANCE Fusobacterium spp. are emerging pathogens that contribute to mammalian and human diseases, including colorectal cancer. Despite a validated connection with disease, few proteins have been characterized that define a direct molecular mechanism for Fusobacterium pathogenesis. We report a comprehensive examination of virulence-associated protein families in multiple Fusobacterium species and show that complete genomes facilitate the correction and identification of multiple, large type 5a secreted autotransporter genes in previously misannotated or fragmented genomes. In addition, we use protein sequence similarity networks and human cell interaction experiments to show that previously predicted noninvasive strains can indeed bind to and potentially invade human cells and that this could be due to the expansion of specific virulence proteins that drive Fusobacterium infections and disease.

In this report, we describe a hybrid MinION whole-genome sequencing pipeline and the genomic characteristics of the first eight Fusobacterium strains deposited in the FusoPortal database. This collection of highly accurate and complete genomes drastically improves upon previous multicontig assemblies by correcting and newly identifying a significant number of open reading frames. We believe that the availability of this resource will result in the discovery of proteins and molecular mechanisms used by an oral pathogen, with the potential to further our understanding of how Fusobacterium nucleatum contributes to a repertoire of diseases, including periodontitis, preterm birth, and colorectal cancer.

Fusobacterium nucleatum is an oral opportunistic pathogen associated with diseases that include cancer and preterm birth. Our understanding of how this bacterium modulates human disease has been hindered by a lack of genetic systems.