Biology subjects

Casasanta, M. A.

Publications and source records attributed to Casasanta, M. A..

4 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↗

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↗

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↗