Fusobacterium nucleatum infection modulates the transcriptome and epigenome of HCT116 colorectal cancer cells in an oxygen-dependent manner.

Fusobacterium nucleatum, a gram-negative oral bacterium, has been consistently validated as a strong contributor to the progression of several types of cancer, including colorectal (CRC) and pancreatic cancer. While previous in vitro studies have shown that intracellular F. nucleatum enhances malignant phenotypes such as cell migration, the dependence of this regulation on features of the tumor microenvironment (TME) such as oxygen levels are wholly uncharacterized. Here we examine the influence of hypoxia in facilitating F. nucleatum invasion and its effects on host responses focusing on changes in the global epigenome and transcriptome. Using a multiomic approach, we analyze epigenomic alterations of H3K27ac and global transcriptomic alterations sustained within a hypoxia and normoxia conditioned CRC cell line HCT116 at 24 h following initial infection with F. nucleatum. Our findings reveal that intracellular F. nucleatum activates signaling pathways and biological processes in host cells similar to those induced upon hypoxia conditioning in the absence of infection. Furthermore, we show that a hypoxic TME favors F. nucleatum invasion and persistence and therefore infection under hypoxia may amplify malignant transformation by exacerbating the effects induced by hypoxia alone. These results motivate future studies to investigate host-microbe interactions in tumor tissue relevant conditions that more accurately define parameters for targeted cancer therapies.

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.

Smooth Muscle Organization and Nerves in the Rat Vagina: A First Look Using Tissue Clearing and Immunolabeling.

Smooth muscle fibers within the vagina, as well as the nerve fibers that contribute to their control mechanisms, are important for the maintenance and alteration of vaginal length and tone. Vaginal smooth muscle (VaSM) is typically described as being arranged into two distinct concentric layers: an inner circular muscular layer and an outer longitudinal muscular layer. However, the distribution of VaSM oriented in the longitudinal direction (LD) and circumferential direction (CD) has never been quantified. In this study, tissue clearing and immunohistochemistry were performed so that the VaSM, and surrounding nerves, within whole rat vaginas ([Formula: see text]) could be imaged without tissue sectioning, preserving the three-dimensional architecture of the organs. Using these methods, the vagina was viewed through the full thickness of the muscularis layer, from the distal to the proximal regions. The VaSM orientation in the proximal and distal regions and the VaSM content along the LD and CD were quantified. Additionally, a qualitative assessment of vaginal nerves was performed. When compared using a permuted version of the Watson [Formula: see text] test, the orientation of VaSM in the proximal and distal regions were found to be significantly different in 4 of the 6 imaged rat vaginas ([Formula: see text]). While the distal vagina contained a similar amount of VaSM oriented within [Formula: see text] of the LD and within [Formula: see text] of the CD, the proximal vagina contained significantly more VaSM oriented towards the LD than towards the CD. Nerve fibers were found to be wavy, running both parallel and perpendicular to vascular and non-vascular smooth muscle within the vagina. Micro-structural analyses, like the one conducted here, are necessary to understand the physiological function and pathological changes of the vagina.

Comparative Analysis of Colon Cancer-Derived Fusobacterium nucleatum Subspecies: Inflammation and Colon Tumorigenesis in Murine Models.

Fusobacteria are commonly associated with human colorectal cancer (CRC), but investigations are hampered by the absence of a stably colonized murine model. Further, Fusobacterium nucleatum subspecies isolated from human CRC have not been investigated. While F. nucleatum subspecies are commonly associated with CRC, their ability to induce tumorigenesis and contributions to human CRC pathogenesis are uncertain. We sought to establish a stably colonized murine model and to understand the inflammatory potential and virulence genes of human CRC F. nucleatum, representing the 4 subspecies, animalis, nucleatum, polymorphum, and vincentii. Five human CRC-derived and two non-CRC derived F. nucleatum strains were tested for colonization, tumorigenesis, and cytokine induction in specific-pathogen-free (SPF) and/or germfree (GF) wild-type and ApcMin/+ mice, as well as in vitro assays and whole-genome sequencing (WGS). SPF wild-type and ApcMin/+ mice did not achieve stable colonization with F. nucleatum, whereas certain subspecies stably colonized some GF mice but without inducing colon tumorigenesis. F. nucleatum subspecies did not form in vivo biofilms or associate with the mucosa in mice. In vivo inflammation was inconsistent across subspecies, whereas F. nucleatum induced greater cytokine responses in a human colorectal cell line, HCT116. While F. nucleatum subspecies displayed genomic variability, no distinct virulence genes associated with human CRC strains were identified that could reliably distinguish these strains from non-CRC clinical isolates. We hypothesize that the lack of F. nucleatum-induced tumorigenesis in our model reflects differences in human and murine biology and/or a synergistic role for F. nucleatum in concert with other bacteria to promote carcinogenesis. IMPORTANCE Colon cancer is a leading cause of cancer morbidity and mortality, and it is hypothesized that dysbiosis in the gut microbiota contributes to colon tumorigenesis. Fusobacterium nucleatum, a member of the oropharyngeal microbiome, is enriched in a subset of human colon tumors. However, it is unclear whether this genetically varied species directly promotes tumor formation, modulates mucosal immune responses, or merely colonizes the tumor microenvironment. Mechanistic studies to address these questions have been stymied by the lack of an animal model that does not rely on daily orogastric gavage. Using multiple murine models, in vitro assays with a human colon cancer cell line, and whole-genome sequencing analysis, we investigated the proinflammatory and tumorigenic potential of several F. nucleatum clinical isolates. The significance of this research is development of a stable colonization model of F. nucleatum that does not require daily oral gavages in which we demonstrate that a diverse library of clinical isolates do not promote tumorigenesis.

Harnessing Tissue Engineering Tools to Interrogate Host-Microbiota Crosstalk in Cancer.

Recent studies have begun to highlight the diverse and tumor-specific microbiomes across multiple cancer types. We believe this work raises the important question of whether the classical "Hallmarks of Cancer" should be expanded to include tumor microbiomes. To answer this question, the causal relationships and co-evolution of these microbiotic tumor ecosystems must be better understood. Because host-microbe interactions should be studied in a physiologically relevant context, animal models have been preferred. Yet these models are often poor mimics of human tumors and are difficult to interrogate at high spatiotemporal resolution. We believe that in vitro tissue engineered platforms could provide a powerful alternative approach that combines the high-resolution of in vitro studies with a high degree of physiological relevance. This review will focus on tissue engineered approaches to study host-microbe interactions and to establish their role as an emerging hallmark of cancer with potential as a therapeutic target.

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.

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.

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser³59 Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity.

The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr(313)-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ's Tyr(313)-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ's enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser(359). We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser(359.) In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser(359) phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr(313) docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity.

DNA-mediated cooperativity facilitates the co-selection of cryptic enhancer sequences by SOX2 and PAX6 transcription factors.

Sox2 and Pax6 are transcription factors that direct cell fate decision during neurogenesis, yet the mechanism behind how they cooperate on enhancer DNA elements and regulate gene expression is unclear. By systematically interrogating Sox2 and Pax6 interaction on minimal enhancer elements, we found that cooperative DNA recognition relies on combinatorial nucleotide switches and precisely spaced, but cryptic composite DNA motifs. Surprisingly, all tested Sox and Pax paralogs have the capacity to cooperate on such enhancer elements. NMR and molecular modeling reveal very few direct protein-protein interactions between Sox2 and Pax6, suggesting that cooperative binding is mediated by allosteric interactions propagating through DNA structure. Furthermore, we detected and validated several novel sites in the human genome targeted cooperatively by Sox2 and Pax6. Collectively, we demonstrate that Sox-Pax partnerships have the potential to substantially alter DNA target specificities and likely enable the pleiotropic and context-specific action of these cell-lineage specifiers.

Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element.

Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6-DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Šresolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = ß = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit.