Comparative Genomic Analysis Of Fusobacterium Nucleatum

ObjectivesFusobacterium,a gram-negative anaerobic bacterium,is commonly detected in the human mouth and throat,but in recent years has also been found in other sites,such as the gastrointestinal and genitourinary tracts.Fusobacterium can cause local infection and a variety of invasive diseases,such as tonsillitis,paratonsillar abscess,dental sepsis,sepsis,brain abscesses,Lemierre syndrome,etc.,of which gingivitis and periodontitis are the most common infections.Fusobacterium nucleatum(F.nucleatum)is one of the most common pathogenic bacteria in Fusobacterium,which can promote early and late bacterial colonization and play an important role in the formation and maturation of dental plaque biofilm.Based on the DNA-DNA hybridization technique,glutamine dehydrogenase,2oxoglutarate reductase electrophoretic patterns,and genetic similarity analysis,F.nucleatum was divided into four subspecies:Fusobacterium nucleatum subspecies nucleatum(subsp.nucleatum),Fusobacterium nucleatum subspecies polymorphum(subsp.polymorphum),Fusobacterium nucleatum subspecies vincentii(subsp.vincentii)/Fusobacterium nucleatum subspecies fusiforme(subsp.fusiforme),Fusobacterium nucleatum subspecies animalis(subsp.animalis).Our knowledge of the genomic characteristics and pathogenic effectors of different F.nucleatum strains is limited.This study mainly conducted a comprehensive comparative genome study on F.nucleatum strains and predicted the existence of functional genes,virulence genes,resistance genes,unique genes,etc.,which can help us gain a deeper understanding of the relationship between different subspecies/strains genetic,functional and pathogenic mechanisms differences.MethodsIn this study,the whole genome DNA of four strains of F.nucleatum(FNV,FNU,FNA,FNP)was sequenced through the PacBio Sequel platform and Illumina NovaSeq PE150,and a complete genome map of the whole sequence was obtained.In addition to the 4 newly sequenced strains,the genomes of all F.nucleatum strains available in the NCBI database were also downloaded.Then,all downloaded genomic nucleic acid sequences were annotated with Prokka software.OrthoFinder was used to construct an evolutionary tree of single-copy orthologous genes for phylogenetic analysis.RAxML-NG software was used to construct a maximum likelihood(ML)phylogenetic tree,the optimal amino acid substitution model was used.Finally,FigTree software was used to visualize the optimal ML tree results and analyze the phylogenetic relationships between subspecies and strains of F.nucleatum.To further assess the boundaries between different subspecies of F.nucleatum,average nucleotide identity(ANI)analysis was performed using FastANI software with default parameters and the results were visualized as a heat map.This study performed a comprehensive phylogenetic and comparative genomic analysis of 30 F.nucleatum strains with complete genome sequences.Pan-genome analysis of 30 F.nucleatum strains was performed using Roary software,and their genomes were analyzed using the Gene Ontology database(GO)and the Kyoto Encyclopedia of Genes and Genomes database(KEGG).Virulence factors database(VFDB),Comprehensive antibiotic resistance database(CARD),and KEGG database were used to predict virulence genes,antibiotic resistance genes,and protein secretion systems.At the same time,the genomic islands(GIs),clustered regularly interspaced short palindromic repeats(CRISPR),secondary metabolite biosynthetic gene clusters,and the distribution of prophage and gene families were predicted.Finally,the adhesion protein FadA of different subspecies of F.nucleatum was studied.In addition,genomic characteristics of F.nucleatum were analyzed at the subspecies and strain levels,providing a new basis for further understanding of its pathogenic effects.Results1.We have completed the whole genome sequencing of 4 F.nucleatum strains(FNV,FNU,FNA,FNP),obtained the whole genome sequence of these 4 strains,obtained the complete circle map of the bacterial genome.Phylogenetic analysis showed that the strains of the same subspecies clustered together,but FNU did not cluster into any of the subtype subspecies of F.nucleatum.At the same time,the annotation was completed on 40 F.nucleatum strains that had not been previously annotated.2.The genetic composition analysis of 30 F.nucleatum strains showed that there were 517 "core/soft-core genes" with housekeeping functions,which were widely distributed in almost all strains.Each subspecies had unique gene clusters shared by strains within the subspecies.Analysis of the virulence factors revealed that many virulence factors were widely distributed across all strains,while some virulence genes showed no consistent occurrence rule at the subspecies level and exhibited strain-specific distribution.The resistance genes,genomic islands,and the type and number of prophage sequences mainly revealed strainspecific characteristics instead of subspecies-level consistency.Clustered regularly interspaced short palindromic repeats(CRISPR)types and secondary metabolite biosynthetic gene clusters were identically distributed in strains from the same subspecies.We also studied the polymorphism of F.nucleatum at the single gene level and found that the variation of amino acid sites in adhesion protein FadA of F.nucleatum did not affect the monomer and dimer 3D structure,while they may affect the binding and stability to host receptors.Conclusions1.Phylogenetic analysis showed that strains of the same subspecies clustered together,F.nucleatum was divided into 4 main subspecies,and a new subspecies of F.nucleatum——FNU.At the same time,the other three sequenced strains FNV,FNA,and FNP were found to belong to subsp.vincentii,animalis,and polymorphum,respectively.2.Based on the phylogenetic and comparative genomic analysis of the genomes of F.nucleatum strains,it was confirmed that FNU belongs to F.nucleatum,but it is different from other existing subspecies of F.nucleatum and is likely to be a new subspecies.Comparing FNU to other subspecies of F.nucleatum helps to better understand the biology of this unique strain.Furthermore,genomic analysis of F.nucleatum at the subspecies and strain levels revealed its differences and diversity at the genome level.When studying the pathogenesis,drug resistance,and clinical treatment of F.nucleatum,special consideration should be given to genomic features at the subspecies and strain levels.At the same time,this study also demonstrates the importance and application value of genomics technology in microbiology research and provides new methods and ideas for future microbiology research.Future research can combine transcriptome,proteome,and metabolome analysis methods to deeply explore the pathogenic mechanism and biological characteristics of F.nucleatum,and provide a deeper understanding and guidance for the prevention and treatment of related diseases.