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A survey of Fusobacterium nucleatum genes modulated by host cell infection.

ABSTRACT: Here, we report comprehensive transcriptomic profiles from Fusobacterium nucleatum under conditions that mimic the first stages of bacterial infection in a highly differentiated adenocarcinoma epithelial cell line. Our transcriptomic in vitro adenocarcinoma approach allows us to measure the expression dynamics and regulation of bacterial virulence and response factors in real time, and is a novel strategy for clarifying the role of F. nucleatum infection in colorectal cancer (CRC) progression. Our data show that: (i) infection alters metabolic and functional pathways in F. nucleatum, allowing the bacterium to adapt to the host-imposed milieu; (ii) infection also stimulates the expression of genes required to help induce and promote a hypoxic and inflammatory microenvironment in the host; and (iii) F. nucleatum invasion occurs by a haematogenous route of infection. Our study identifies novel gene targets from F. nucleatum that are activated during invasion and which may aid in determining how this species invades and promotes disease within the human gastrointestinal tract. These invasion-specific genes may be useful as biomarkers for CRC progression in a host and could also assist in the development of new diagnostic tools and treatments (such as vaccines or small molecule drug targets), which will be able to combat infection and inflammation in the host while circumventing the potential problem of F. nucleatum tolerization.

SUBMITTER: Cochrane K

PROVIDER: S-EPMC7067209 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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A survey of Fusobacterium nucleatum genes modulated by host cell infection.

Cochrane Kyla K Robinson Avery V AV Holt Robert A RA Allen-Vercoe Emma E

Microbial genomics 20191029 2

Here, we report comprehensive transcriptomic profiles from Fusobacterium nucleatum under conditions that mimic the first stages of bacterial infection in a highly differentiated adenocarcinoma epithelial cell line. Our transcriptomic in vitro adenocarcinoma approach allows us to measure the expression dynamics and regulation of bacterial virulence and response factors in real time, and is a novel strategy for clarifying the role of F. nucleatum infection in colorectal cancer ...[more]

PMID: 31661053

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Project description:Fusobacterium nucleatum is a ubiquitous opportunistic pathogen with an emerging role as an oncomicrobe in colorectal cancer and other cancer settings. F. nucleatum can adhere to and invade host cells in a manner that varies across F. nucleatum strains and host cell phenotypes. Here, we performed pairwise cocultures between three F. nucleatum strains and two immortalized primary host cell types (human colonic epithelial [HCE] cells and human carotid artery endothelial [HCAE] cells) followed by transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to investigate transcriptional and epigenetic host cell responses. We observed that F. nucleatum-induced host cell transcriptional modulation involves strong upregulation of genes related to immune migration and inflammatory processes, such as TNF, CXCL8, CXCL1, and CCL20. Furthermore, we identified genes strongly upregulated in a cell line-specific manner. In HCE cells, overexpressed genes included UBD and DUOX2/DUOXA2, associated with p53 degradation-mediated proliferation and intestinal reactive oxygen species (ROS) production, respectively. In HCAE cells, overexpressed genes included EFNA1 and LIF, two genes commonly upregulated in colorectal cancer and associated with poor patient outcomes, and PTGS2 (COX2), a gene associated with the protective effect of aspirin in the colorectal cancer setting. Interestingly, we also observed downregulation of numerous histone modification genes upon F. nucleatum exposure. We used the ChIP-seq data to annotate chromatin states genome wide and found significant chromatin remodeling following F. nucleatum exposure in HCAE cells, with increased frequencies of active enhancer and low-signal/quiescent states. Thus, our results highlight increased inflammation and chemokine gene expression as conserved host cell responses to F. nucleatum exposure and extensive host cell epigenomic changes specific to host cell type. IMPORTANCE Fusobacterium nucleatum is a bacterium normally found in the healthy oral cavity but also has an emerging role in colorectal cancer and other cancer settings. The host-microbe interactions of F. nucleatum and its involvement in tumor initiation, progression, and treatment resistance are not fully understood. We explored host cell changes that occur in response to F. nucleatum. We identified key genes differentially expressed in response to various conditions of F. nucleatum exposure and determined that the conserved host cell response to F. nucleatum was dominated by increased inflammation and chemokine gene expression. Additionally, we found extensive host cell epigenomic changes as a novel aspect of host modulation associated with F. nucleatum exposure. These results extend our understanding of F. nucleatum as an emerging pathogen and highlight the importance of considering strain heterogeneity and host cell phenotypic variation when exploring pathogenic mechanisms of F. nucleatum.

Project description: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.

Dataset Information

A survey of Fusobacterium nucleatum genes modulated by host cell infection.

Publications

A survey of <i>Fusobacterium nucleatum</i> genes modulated by host cell infection.

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