Transcriptomic analysis of Fusobacterium nucleatum-stimulated Human gingival fibroblasts
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE118691 | GEO | 2019/09/12
REPOSITORIES: GEO
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Project description:Fusobacterium nucleatum is a Gram-negative oral bacterial species associated with periodontal disease progression. As periodontal disease progresses, it is known F. nucleatum coaggregated with blood is frequently detected in the gingival crevice. However, it is largely unknown whether these interactions between F. nucleatum and blood induce a particular genetic response. We tested the cultures of F. nucleatum ATCC 25586 with or without blood in a semi-defined growth medium by microarray analysis and found 14 genes that were affected after only about 4hr. of adhered blood. Then, we selected 7 genes that changed significantly and tested these genes via real-time RT-PCR to confirm the validity of the microarray results. As a result, one amino sugar-binding protein on the membrane of F. nucleatum was especially expressed high via both microarray and real-time RT-PCR. Based upon these data, it appears that the protein on the F. nucleatum membrane binds and transfers the amino sugar only under blood conditions. This study aims to determine the effect of blood on the gene expression profiling of F. nucreatum. The study contains 2 separate experiments that both measure the cultures without or with blood. The samples with blood contain 9% and 33% blood.
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Project description:High throughput RNA sequencing For RNA sequencing, F. nucleatum was incubated with 1 mM or 5 mM metformin for 7 hours, when the bacterium were under logarithmic phase. Total RNA of F. nucleatum was stabilized with RNA protect Bacteria Reagent (QIAGEN, Germany) and extracted using a QIAGEN RNeasy kit (QIAGEN, Germany) following the manufacturer’s instructions.
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Project description:Periodontal disease is caused by dental plaque biofilms. Fusobacterium nucleatum is an important periodontal pathogen involved in the development of bacterial complexity in dental plaque biofilms. Human gingival fibroblasts (GFs) act as the first line of defense against oral microorganisms and locally orchestrate immune responses by triggering the production of reactive oxygen species and pro-inflammatory cytokines (IL-6 and IL-8). The frequency and severity of periodontal diseases is known to increase in elderly subjects. However, despite several studies exploring the effects of aging in periodontal disease, the underlying mechanisms through which aging affects the interaction between F. nucleatum and human GFs remain unclear. To identify genes affected by infection, aging, or both, we performed an RNA-Seq analysis using GFs isolated from a single healthy donor that were passaged for a short period of time (P4) 'young GFs' or for longer period of time (P22) 'old GFs', and infected or not with F. nucleatum. Comparing F. nucleatum-infected and uninfected GF(P4) cells the differentially expressed genes (DEGs) were involved in host defense mechanisms (i.e., immune responses and defense responses), whereas comparing F. nucleatum-infected and uninfected GF(P22) cells the DEGs were involved in cell maintenance (i.e., TGF-? signaling, skeletal development). Most DEGs in F. nucleatum-infected GF(P22) cells were downregulated (85%) and were significantly associated with host defense responses such as inflammatory responses, when compared to the DEGs in F. nucleatum-infected GF(P4) cells. Five genes (GADD45b, KLF10, CSRNP1, ID1, and TM4SF1) were upregulated in response to F. nucleatum infection; however, this effect was only seen in GF(P22) cells. The genes identified here appear to interact with each other in a network associated with free radical scavenging, cell cycle, and cancer; therefore, they could be potential candidates involved in the aged GF's response to F. nucleatum infection. Further studies are needed to confirm these observations.
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Project description:Fusobacterium nucleatum is a Gram negative oral bacterial species associated with periodontal disease progression. This species is perhaps best known for its ability to adhere to a vast array of other bacteria and eukaryotic cells. Numerous studies of F. nucleatum have examined various coaggregation partners and inhibitors, but it is largely unknown whether these interactions induce a particular genetic response. We tested coaggregation between F. nucleatum ATCC strain 25586 and various species of Streptococcus in the presence of a semi-defined growth medium containing saliva. We found that this condition could support efficient coaggregation, but surprisingly also stimulated a similar degree of autoaggregation. We further characterized the autoaggregation response, since few reports have examined this in F. nucleatum. After screening several common coaggregation inhibitors, we identified L-lysine as a competitive inhibitor of autoaggregation. We performed a microarray analysis of the planktonic vs. autoaggregated cells and found nearly 100 genes that were affected after only about 60 min. of aggregation. We tested a subset of these genes via real-time RT-PCR and confirmed the validity of the microarray results. Some of these genes were also found to be inducible in cell pellets created by centrifugation. Based upon these data, it appears that autoaggregation activates a genetic program that may be utilized for growth in a high cell density environment, such as the oral biofilm. The study aims to determine the effect of autoaggregation upon the transcriptome. The study contains 2 separate experiments that both measure dispersed (i.e. non-aggregated) vs. aggregated cells and each experiment was performed in duplicate. Samples with no added components other than medium were dispersed, samples containing 25% saliva were aggregated, and samples containing 25% saliva + 50mM L-lysine remained dispersed.
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Project description:To investigate the effect of the Zn ionophore, PBT2, on the transcriptomic response of Fusobacterium nucleatum ATCC 25586, RNA was extracted from bacterial samples which had been treated as follows: Untreated (0.006% DMSO v/v), DMSO-Zn treated (0.006% DMSO v/v + 200 uM ZnSO4), or PBT2-Zn treated (0.125 ug/mL PBT2 + 200 uM ZnSO4) (DMSO was the vehicle control). RNA samples were collected at 0h, 0.5h, and 1h post challenge in biological triplicate and sequenced using Illumina HiSeq platform. We mapped sequences to the reference genome F. nucleatum subsp. nucleatum ATCC 25586 and performed DEseq2 analysis to determine differentially expressed genes across time and treatment.
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Project description:Fusobacterium nucleatum is a Gram-negative oral bacterial species associated with periodontal disease progression. As periodontal disease progresses, it is known F. nucleatum coaggregated with blood is frequently detected in the gingival crevice. However, it is largely unknown whether these interactions between F. nucleatum and blood induce a particular genetic response. We tested the cultures of F. nucleatum ATCC 25586 with or without blood in a semi-defined growth medium by microarray analysis and found 14 genes that were affected after only about 4hr. of adhered blood. Then, we selected 7 genes that changed significantly and tested these genes via real-time RT-PCR to confirm the validity of the microarray results. As a result, one amino sugar-binding protein on the membrane of F. nucleatum was especially expressed high via both microarray and real-time RT-PCR. Based upon these data, it appears that the protein on the F. nucleatum membrane binds and transfers the amino sugar only under blood conditions.
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