Biology subjects

Slade, D. J.

Publications and source records attributed to Slade, D. J..

12 recordsLinked to original sources

bioRxiv2023

Type 4 pili mediated natural competence in Fusobacterium nucleatum

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 4 pili mediated natural competence in Fusobacterium nucleatum, which are Gram-negative, anaerobic bacteria that participate in a range of infections and diseases including periodontitis, preterm birth, and cancer. We bioinformatically identified components of the Type 4 conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type 4 pili in natural competence in F. nucleatum strain 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. 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.

microbiology↗

bioRxiv2022

DNA methyltransferase enhanced Fusobacterium nucleatum genetics

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 user introduced plasmid DNA. We report the identification, characterization, and successful use of Fusobacterium nucleatum (Fn) Type II and III DNA methyltransferase (DMTase) enzymes to produce a multi-fold increase in gene knockout efficiency in the strain Fusobacterium nucleatum subsp. nucleatum 23726 (Fnn 23726), as well as the first efficient gene knockouts and complementations in Fnn 25586. We show plasmid protection can be accomplished in vitro with purified enzymes, as well as in vivo in an E. coli host that constitutively expresses Fnn DMTase enzymes. By characterizing specific DMTases that are critical for bypassing R-M systems, we have enhanced our understanding of potential enzyme combinations, with the goal of expanding these studies to genetically modify clinical isolates of Fusobacterium that have thus far been inaccessible to molecular characterization. This proof-of-concept study provides a roadmap to guide molecular microbiology efforts of the scientific community to facilitate the discovery of new Fusobacterium virulence genes, thereby leading to a new era of characterizing how an oral opportunistic pathogen contributes to an array of human infections and diseases. IMPORTANCEFusobacterium nucleatum is an oral opportunistic pathogen associated with diseases including 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 allows the development of genetic systems in previously inaccessible strains. We present a strategy that can be expanded to enable the genetic modification of clinical isolates, thereby fostering investigational studies to uncover novel host-pathogen interactions in Fusobacterium.

microbiology↗

bioRxiv2022

Mucosal microbiomes and Fusobacterium genomics in Vietnamese colorectal cancer patients

Perturbations in the gut microbiome have been linked to the promotion and prognosis of colorectal cancer (CRC), with the colonic overabundance of Fusobacterium nucleatum shown as the most consistent marker. Despite the increasing health burden inflicted by CRC in low- and middle-income countries like Vietnam, the CRC-specific microbiome in these populations remains underexplored. Here we conducted a study in Vietnam to enrol 43 CRC patients (cases) and 25 patients with non-cancerous colorectal polyps (controls) between December 2018 and January 2020. Our study investigated the mucosal microbiome signature and genomic diversity of Fusobacterium in Vietnamese CRC patients, using a combination of 16S rRNA gene profiling, anaerobic microbiology, and whole genome sequencing. We found that several oral bacteria, including F. nucleatum and Leptotrichia, were significantly more abundant in the tumour mucosa, and these two bacteria were also more enriched in tumours of advanced CRC stages (III-IV). We obtained 53 Fusobacterium genomes from the saliva, tumour and non-tumour mucosa of six CRC patients. Isolates from the gut mucosa belonged to diverse F. nucleatum subspecies (nucleatum, animalis, vincentii, polymorphum) and a potential new subspecies of F. periodonticum. The Fusobacterium population within each individual was distinct and in many cases diverse, with minimal intra-clonal variation. Phylogenetic analyses showed that within each individual, tumour-associated Fusobacterium were clonal to those isolated from non-tumour mucosa, but distantly related to those isolated from saliva. Genes encoding major virulence factors (Fap2 and RadD) showed variability in length and evidence of horizontal gene transfer. Our work provides a framework to understand the genomic diversity of Fusobacterium within the CRC patients, which can be exploited for the development of CRC diagnostic and therapeutic options targeting this oncobacterium.

microbiology↗

bioRxiv2022

CRISPR-Cas systems in Fusobacterium: An untapped genetic frontier

Fusobacterium nucleatum has recently received significant attention for its strong connection with the acceleration and gravity of multiple cancers (e.g., colorectal, pancreatic, esophageal). However, our understanding of the molecular mechanisms that drive infection by this oncomicrobe have been hindered by a lack of universal genetic tools. Herein we report a global bioinformatic identification and characterization of multiple Fusobacterium CRISPR-Cas adaptive immune systems including Cas13c, and detailed report of the proteins, spacer/repeat loci, trans- activating CRISPR RNA (tracrRNA), and CRISPR RNA (cRNA) from a Type II-A CRISPR-Cas9 system. Since most Fusobacterium are currently genetically intractable, this CRISPR-Cas bioinformatic roadmap could be used to build new genome editing and transcriptional tuning tools to characterize an increasingly important genus of human opportunistic-pathogens connected to the onset, progression, and severity of cancer.

microbiology↗

bioRxiv2021

Fusobacterium nucleatum infection induces pancreatic cancer cell proliferation and migration through regulation of host cytokine signaling

Pancreatic ductal adenocarcinoma (PDAC) harbors a complex tumor microbiome that has been implicated in cancer progression and resistance to chemotherapy. Recent clinical investigations uncovered a correlation between high loads of intratumor Fusobacterium nucleatum and decreased patient survival. Here we show that healthy and cancerous pancreatic cell lines harboring intracellular F. nucleatum secrete increased levels of cancer-associated cytokines including GM-CSF, CXCL1, IL-8, and MIP-3. We report that GM-CSF (granulocyte-macrophage colony stimulating factor) secretion directly increases the proliferation and migration of pancreatic cancer cells via an autocrine mechanism, notably in the absence of immune cell participation. Furthermore, we show that non-cancerous pancreatic epithelial cells do not exhibit increased proliferation or migration in response to these cytokines, but nevertheless, their secreted cytokines stimulate these responses in cancerous cell lines through paracrine signaling. Our results provide evidence that intratumor F. nucleatum in the pancreas elicits an infection-specific cytokine secretion profile from both normal and cancerous cells that adversely contributes to cancer progression through autocrine and paracrine mechanisms. Therefore, these results support the importance of investigating the contributions of both microbiome and host driven processes in pancreatic cancer to guide future therapeutic interventions.

cancer biology↗

bioRxiv2021

A Shigella type 3 effector protein co-opts host inositol pyrophosphates for activity

The type III secretion system is required for virulence of many pathogenic bacteria. Bacterial effector proteins delivered into target host cells by this system modulate host signaling pathways and processes in a manner that promotes infection. Here, we define the activity of the effector protein OspB of the human pathogen Shigella spp., the etiological agent of shigellosis and dysenteric disease. Using the yeast Saccharomyces cerevisiae as a model organism, we show that OspB sensitizes cells to inhibition of TORC1, the central regulator of growth and metabolism. In silico analyses reveal that OspB bears structural homology to bacterial cysteine proteases, and we define a conserved cysteine-histidine catalytic dyad required for OspB function. Using yeast genetic screens, we identify a crucial role for the arginine N-degron pathway in the growth inhibition phenotype and show that inositol hexakisphosphate is an OspB cofactor. We find that a yeast substrate for OspB is the TORC1 component Tco89p, proteolytic cleavage of which generates a C-terminal fragment that is targeted for degradation via the arginine N-degron pathway; processing and degradation of Tco89p is required for the OspB phenotype. In all, we demonstrate that the Shigella T3SS effector OspB is a cysteine protease and decipher its interplay with eukaryotic cell processes. ImportanceShigella spp. are important human pathogens and one of the leading causes of diarrheal mortality worldwide, especially in children. Virulence depends on the Shigella type III secretion system (T3SS), and definition of the roles of the bacterial effector proteins secreted by the T3SS is key to understanding Shigella pathogenesis. The effector protein OspB has been shown to contribute to a range of phenotypes during infection, yet the mechanism of action is unknown. Here, we show that OspB possesses cysteine protease activity in both yeast and mammalian cells, and that enzymatic activity of OspB depends on a conserved cysteine-histidine catalytic dyad. We determine how its protease activity sensitizes cells to TORC1 inhibition in yeast, finding that OspB cleaves a component of yeast TORC1, and that the degradation of the C-terminal cleavage product is responsible for OspB mediated hypersensitivity to TORC1 inhibitors. Thus, OspB is a cysteine protease that depends on a conserved cysteine-histidine catalytic dyad.

microbiology↗

bioRxiv2020

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

Fusobacterium nucleatum is implicated in the acceleration of colorectal cancer (CRC), yet 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 TNF 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.

cancer biology↗

bioRxiv2019

Reevaluating the Fusobacterium Virulence Factor Landscape

Fusobacterium are Gram-negative, anaerobic, opportunistic pathogens involved in multiple diseases, including the oral pathogen Fusobacterium nucleatum being linked to the progression and severity of colorectal cancer. The global identification of virulence factors in 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 correction of virulence factors from the Type 5 secreted autotransporter and FadA protein families, with a focus on the genetically tractable strain F. nucleatum subsp. nucleatum ATCC 23726 and the classic typed strain F. nucleatum subsp. nucleatum ATCC 25586. Within the autotransporters, we employed protein sequence similarity networks to identify subsets of virulence factors, and show a clear differentiation between the prediction of outer membrane adhesins, serine proteases, and proteins with unknown function. These data have defined protein subsets within the Type 5a effectors that are present in predicted invasive strains but are broadly lacking in passively invading strains; a key phenotype associated with Fusobacterium virulence. However, our data shows that prior bioinformatic analysis that predicted species of Fusobacterium to be non-{inverted exclamation}nvasive can indeed invade human cells, and that pure phylogenetic analysis to determine the virulence within this bacterial genus should be used cautiously and subsequently paired with experiments to validate these hypotheses. In addition, we provide data that show a complex interplay between autotransporters, MORN2 domain containing proteins, and FadA adhesins that we hypothesize synergistically contribute to host cell interactions and invasion. In summary, we report that accurate open reading frame annotations using complete Fusobacterium genomes, in combination with experimental validation of invasion, redefines the repertoire of virulence factors that could be contributing to the species specific pathology of multiple Fusobacterium induced infections and diseases. IMPORTANCEFusobacterium are emerging pathogens that contribute to the progression and severity of multiple mammalian and human infectious diseases, including colorectal cancer. Despite a validated connection with disease, a limited number of proteins have been characterized that define a direct molecular mechanism for pathogenesis in a diverse range of host tissue infections. 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 invasion experiments to show that previously predicted non-invasive strains can indeed enter human cells, and that this is likely due to the expansion of specific virulence proteins that drive F. nucleatum infections and disease.

microbiology↗

bioRxiv2018

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 varied from 24-67 contigs. We showcase that genomes in FusoPortal provide accurate open reading frame annotations, and have corrected a number of large genes (>3 kb) 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.\n\nImportanceIn this study, we report a hybrid MinION whole genome sequencing pipeline, and describe the genomic characteristics of the first eight strains deposited in the FusoPortal database. This collection of highly accurate and complete genomes drastically improves upon previous multi-contig assemblies by correcting and newly identifying a significant number of open reading frames. We believe 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 F. nucleatum contributes to a repertoire of diseases including periodontitis, pre-term birth, and colorectal cancer

microbiology↗

bioRxiv2018

Fusobacterium genomics using MinION and Illumina sequencing enables genome completion and correction

Understanding the virulence mechanisms of human pathogens from the genus Fusobacterium has been hindered by a lack of properly assembled and annotated genomes. Here we report the first complete genomes for seven Fusobacterium strains, as well as resequencing of the reference strain F. nucleatum subsp. nucleatum ATCC 25586 (seven total species, eight total genomes). A highly efficient and cost-effective sequencing pipeline was achieved using sample multiplexing for short-read Illumina (150 bp) and long-read Oxford Nanopore MinION (>80 kbp) platforms, coupled with genome assembly using the open-source software Unicycler. When compared to currently available draft assemblies (previously 24-67 contigs), these genomes are highly accurate and consist of only one complete chromosome. We present the complete genome sequence of F. nucleatum 23726, a genetically tractable and biomedically important strain, and in addition, reveal that the previous F. nucleatum 25586 genome assembly contains a 452 kb genomic inversion that has been corrected using our sequencing and assembly pipeline. To enable the scientific community, we concurrently use these genomes to launch FusoPortal, a repository of interactive and downloadable genomic data, genome maps, gene annotations, and protein functional analysis and classification. In summary, this study provides detailed methods for accurately sequencing, assembling, and annotating Fusobacterium genomes, which will enhance efforts to properly identify virulence proteins that may contribute to a repertoire of diseases including periodontitis, pre-term birth, and colorectal cancer.

microbiology↗

bioRxiv2017

FplA From Fusobacterium nucleatum Is A Type Vd Autotransporter Phospholipase With A Proposed Role In Altered Host Signaling And Evasion Of Autophagy

Fusobacterium nucleatum is a pathogenic oral bacterium that is linked to multiple human infections and colorectal cancer. While most Gram-negative pathogens utilize secretion systems for cellular invasion and infection, F. nucleatum lacks Type I, II, III, IV, and VI secretion. By contrast, F. nucleatum strains are enriched in Type V secreted autotransporters, which are Gram-negative bacterial virulence factors critical for binding and entry into host cells. Here we present the first biochemical characterization of a F. nucleatum Type Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename Fusobacterium phospholipase autotransporter (FplA). FplA is expressed as a full-length 85 kDa outer membrane embedded protein, or as a truncated phospholipase domain that remains associated with the outer membrane. Using multiple FplA constructs we characterized lipid substrate specificity, potent inhibitors, and chemical probes to detect and track this enzyme family. While the role of FplA is undetermined in F. nucleatum virulence, homologous phospholipases from intracellular pathogens are critical for vacuole escape, altered host signaling, and intracellular survival. We hypothesize that upon intracellular invasion of the host, FplA could play a role in phagosomal escape, subversion of autophagy, or eicosanoid-mediated inflammatory signaling, as we show that FplA binds with high affinity to host phosphoinositide signaling lipids critical to these processes. Our identification of substrates, inhibitors, and chemical probes for FplA, in combination with an fplA gene deletion strain, encompass a powerful set of tools for the future analysis of FplA in vivo. In addition, these studies will guide the biochemical characterization of additional Type Vd autotransporter phospholipases.\n\nIMPORTANCEF. nucleatum is an emerging pathogen that is linked to the pathogenesis of colorectal cancer, yet there is a critical knowledge gap in the mechanisms used by this bacterium to elicit changes in the host for intracellular entry and survival. As phospholipases are critical virulence factors for intracellular bacteria to initiate vacuole lysis, cell-to-cell spread, and evasion of autophagy, we set out to characterize a unique Type Vd secreted phospholipase A1 enzyme from F. nucleatum. Our results show a potential role for modulating host signaling pathways through cleavage of phosphoinositide dependent signaling lipids. These studies open the door for further characterization of this unique enzyme family in bacterial virulence, host-pathogen interactions, and for F. nucleatum, in colorectal carcinogenesis.

microbiology↗

bioRxiv2017

N-(3-Oxododecanoyl)-L-Homoserine Lactone Interactions In The Breast Tumor Microenvironment: Implications For Breast Cancer Viability And Proliferation In Vitro

It is well documented that the tumor microenvironment profoundly impacts the etiology and progression of breast cancer, yet the contribution of the resident microbiome within breast tissue remains poorly understood. Tumor microenvironmental conditions, such as hypoxia and dense tumor stroma, predispose progressive phenotypes and therapy resistance, however the role of bacteria in this interplay remains uncharacterized. We hypothesized that the effect of individual bacterial secreted molecules on breast cancer viability and proliferation would be modulated by these tumor-relevant stressors differentially for cells at varying stages of progression. To test this, we incubated human breast adenocarcinoma cells (MDA-MB-231, MCF-DCIS.com) and non-malignant breast epithelial cells (MCF-10A) with N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), a quorum-sensing molecule from Pseudomonas aeruginosa that regulates bacterial stress responses. This molecule was selected because Pseudomonas was recently characterized as a significant fraction of the breast tissue microbiome and OdDHL is documented to impact mammalian cell viability. After OdDHL treatment, we demonstrated the greatest decrease in viability with the more malignant MDAMB-231 cells and an intermediate MCF-DCIS.com (ductal carcinoma in situ) response. The responses were also culture condition (i.e. microenvironment) dependent. These results contrast the MCF-10A response, which demonstrated no change in viability in any culture condition. We further determined that the observed trends in breast cancer viability were due to modulation of proliferation for both cell types, as well as the induction of necrosis for MDA-MB-231 cells in all conditions. Our results provide evidence that bacterial quorum-sensing molecules interact with the host tissue environment to modulate breast cancer viability and proliferation, and that the effect of OdDHL is dependent on both cell type as well as microenvironment. Understanding the interactions between bacterial signaling molecules and the host tissue environment will allow for future studies that determine the contribution of bacteria to the onset, progression, and therapy response of breast cancer.

cancer biology↗