Type IV pili facilitated natural competence in Fusobacterium nucleatum.

OBJECTIVES: Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV pili facilitated natural competence in Fusobacterium nucleatum, which is a Gram-negative, anaerobic bacterium that participates in a range of infections and diseases including periodontitis, preterm birth, and cancer. METHODS: Here we used bioinformatics on multiple Fusobacterium species, as well as molecular genetics to characterize natural competence in strain F. nucleatum subsp. nucleatum ATCC 23726. RESULTS: We bioinformatically identified components of the Type IV conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type IV pili in natural competence in F. nucleatum ATCC 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. CONCLUSIONS: In summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.

Enhanced Fusobacterium nucleatum Genetics Using Host DNA Methyltransferases To Bypass Restriction-Modification Systems.

Bacterial restriction-modification (R-M) systems are a first-line immune defense against foreign DNA from viruses and other bacteria. While R-M systems are critical in maintaining genome integrity, R-M nucleases unfortunately present significant barriers to targeted genetic modification. Bacteria of the genus Fusobacterium are oral, Gram-negative, anaerobic, opportunistic pathogens that are implicated in the progression and severity of multiple cancers and tissue infections, yet our understanding of their direct roles in disease have been severely hindered by their genetic recalcitrance. Here, we demonstrate a path to overcome these barriers in Fusobacterium by using native DNA methylation as a host mimicry strategy to bypass R-M system cleavage of transformed plasmid DNA. We report the identification, characterization, and successful use of Fusobacterium nucleatum type II and III DNA methyltransferase (MTase) enzymes to produce a multifold increase in gene knockout efficiency in the strain Fusobacterium nucleatum subsp. nucleatum 23726, as well as the first system for efficient gene knockouts and complementations in F. nucleatum subsp. nucleatum 25586. We show plasmid protection can be accomplished in vitro with purified enzymes, as well as in vivo in an Escherichia coli host that constitutively expresses F. nucleatum subsp. nucleatum MTase enzymes. In summary, this proof-of-concept study characterizes specific MTases that are critical for bypassing R-M systems and has enhanced our understanding of enzyme combinations that could be used to genetically modify clinical isolates of Fusobacterium that have thus far been inaccessible to molecular characterization. IMPORTANCE 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. Here, we show that F. nucleatum DNA methyltransferase-modified plasmid DNA overcomes the transformation barrier and has allowed the development of a genetic system in a previously inaccessible strain. We present a strategy that could potentially be expanded to enable the genetic modification of highly recalcitrant strains, thereby fostering investigational studies to uncover novel host-pathogen interactions in Fusobacterium.

Utilizing Whole Fusobacterium Genomes To Identify, Correct, and Characterize Potential Virulence Protein Families.

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.IMPORTANCEFusobacterium 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.

Fusobacterium nucleatum induces proliferation and migration in pancreatic cancer cells through host autocrine and paracrine signaling.

The tumor microbiome is increasingly implicated in cancer progression and resistance to chemotherapy. In pancreatic ductal adenocarcinoma (PDAC), high intratumoral loads of Fusobacterium nucleatum correlate with shorter survival in patients. Here, we investigated the potential mechanisms underlying this association. We found that F. nucleatum infection induced both normal pancreatic epithelial cells and PDAC cells to secrete increased amounts of the cytokines GM-CSF, CXCL1, IL-8, and MIP-3α. These cytokines increased proliferation, migration, and invasive cell motility in both infected and noninfected PDAC cells but not in noncancerous pancreatic epithelial cells, suggesting autocrine and paracrine signaling to PDAC cells. This phenomenon occurred in response to Fusobacterium infection regardless of the strain and in the absence of immune and other stromal cells. Blocking GM-CSF signaling markedly limited proliferative gains after infection. Thus, F. nucleatum infection in the pancreas elicits cytokine secretion from both normal and cancerous cells that promotes phenotypes in PDAC cells associated with tumor progression. The findings support the importance of exploring host-microbe interactions in pancreatic cancer to guide future therapeutic interventions.

Fusobacterium nucleatum CbpF Mediates Inhibition of T Cell Function Through CEACAM1 Activation.

F. nucleatum is an anaerobic bacterium that is associated with several tumor entities and promotes tumorigenesis. Recent evidence suggests that F. nucleatum binds the inhibitory receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) via the trimeric autotransporter adhesin CbpF. However, whether this binding is functional or whether other fusobacterial trimeric autotransporter adhesins are involved in CEACAM1 activation is unknown. In this study, using F. nucleatum mutants lacking the type 5c trimeric autotransporter adhesins fvcA (CbpF), fvcB, fvcC, and fvcD, we show that F. nucleatum CbpF binds and activates CEACAM1 and also binds carcinoembryonic antigen (CEA), a tumor-associated protein. We further find that CEACAM antibodies directed against the CEACAM N-terminal domain block the CbpF-CEACAM1 interaction. In functional assays, we demonstrate CbpF-dependent inhibition of CD4+ T cell response. Thus, we characterize an immune evasion mechanism in which F. nucleatum uses its surface protein CbpF to inhibit T cell function by activating CEACAM1.

Fusobacterium nucleatum host-cell binding and invasion induces IL-8 and CXCL1 secretion that drives colorectal cancer cell migration.

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.

Complete Genome Sequence of Fusobacterium necrophorum subsp. necrophorum ATCC 25286.

Fusobacterium necrophorum is a pathogenic Gram-negative, anaerobic bacterium. In this study, we present the first complete genome sequence of Fusobacterium necrophorum subsp. necrophorum ATCC 25286. These data provide a critical advancement in our understanding of virulence factors that could contribute to F. necrophorum pathogenesis in both human and livestock infections.

FusoPortal: an Interactive Repository of Hybrid MinION-Sequenced Fusobacterium Genomes Improves Gene Identification and Characterization.

Here we present FusoPortal, an interactive repository of Fusobacterium genomes that were sequenced using a hybrid MinION long-read sequencing pipeline, followed by assembly and annotation using a diverse portfolio of predominantly open-source software. Significant efforts were made to provide genomic and bioinformatic data as downloadable files, including raw sequencing reads, genome maps, gene annotations, protein functional analysis and classifications, and a custom BLAST server for FusoPortal genomes. FusoPortal has been initiated with eight complete genomes, of which seven were previously only drafts that ranged from 24 to 67 contigs. We have showcased that the genomes in FusoPortal provide accurate open reading frame annotations and have corrected a number of large (>3-kb) genes that were previously misannotated due to contig boundaries. In summary, FusoPortal (http://fusoportal.org) is the first database of MinION-sequenced and completely assembled Fusobacterium genomes, and this central Fusobacterium genomic and bioinformatic resource will aid the scientific community in developing a deeper understanding of how this human pathogen contributes to an array of diseases, including periodontitis and colorectal cancer.IMPORTANCE 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.