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

Abstract

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.

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

“…While FadA has been shown to drive interactions with cancer cells, we note that these studies were done in the strain F. nucleatum 12230 ( 31 , 37 , 38 ). Upon bioinformatic analysis, we found that F. nucleatum 23726 contains four fadA ortholog genes ( fadA2, fadA3a, fadA3b, fadA3c ), and F. nucleatum 12230 encodes for only one orthologue ( 23 ). This could mean that a single deletion of the fadA gene in F. nucleatum 23726 remains adhesive, and that multiple fadA family proteins are contributing to binding and invasion.…”

“…We recently bioinformatically identified five Type 5c adhesins in the strain F. nucleatum 23726, with four of these (CbpF, FvcB, FvcC, FvcD) containing all of the classic domains that make up a complete adhesin. The fifth protein (FvcE, not characterized) lacks a 'head' domain that is predicted to coordinate adhesion ( 23 ). To study these proteins, we made the first complete, single gene knockouts of these six adhesin genes ( fadA, fap2, cbpF, fvcB, fvcC, fvcD) in F. nucleatum 23726, as well as multiple gene deletions per strain ( Table S1-3 ) using a new version of a galactose kinase (GalK) genetic system previously used in F. nucleatum ( 35 ), as well as many classical studies in Clostridium ( 36 ).…”

Role of FAD-I in Fusobacterial Interspecies Interaction and Biofilm Formation

“…The genes encoded by homologs of FN1529 and FN1528 were previously unnamed and will be referred to in this study as rapA (radD-associated proteins A) and rapB, respectively. Based on the sequence similarity of RapA to the previously characterized FadA adhesin [18], rapA was also suggested to be named fadA2 by a very recent bioinformatic study [23]. All the four genes of the operon are predicted to be associated with membrane with Predict Protein (www.predictprotein.com accessed on 10 October 2019).…”

“…No function has been identified for the two other proteins encoded by the additional genes in the operon, which we denominated as RapA and RapB (RadD-associated proteins). While a recent study highlights the similarity of RapA to the fusobacterial adhesin FadA and suggests it to be FadA2, functional characterization is still missing [23].…”

A Computational Framework to Quantify Host-Microbiome Interactions in Clostridioides Difficile Infection

“…Consistent with the findings from previous reports 59,60 , Epulopiscium was significantly enriched in the CDI group and played an important role in differentiating this comparison. Among those features selected from the classification of CDI and Non-CDI groups, Enterobacter and Fusobacterium have been considered as opportunistic pathogens involved in multiple diseases 61,62 .…”