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Sample GSM4674705

Query DataSets for GSM4674705

Status

Public on Sep 16, 2020

Title

Porphyromonas gingivalis WT from injected G.mellonela Wax18

Sample type

SRA

Source name

ATCC 33277

Organism

Porphyromonas gingivalis

Characteristics

treatment: Porphyromonas gingivalis WT from injected G.mellonela
genotype/variation: WT

Treatment protocol

Bacterial infection experiments. P. gingivalis wild-type and Δcas3 mutant were harvested from the broth culture by centrifugation, bacteria were washed 3x in RPMI-1640 medium and adjusted to OD660 1.0 (approximately 1x109 CFU/ml) and added to PMA-activated THP-1 cells at a multiplicity of infection of 100, in the antibiotic-free medium, and after 2h of infection, THP-1 cells were washed to remove non-adherent bacteria. After 2hr incubation, THP-1 cells were treated for 1 hr with metronidazole (200 μg/ml)/gentamicin (300 μg/ml) to kill extracellular bacteria (94), were washed to remove antibiotics, then were lysed with sterile water, followed by addition of an equal volume of 2x PBS, and serial 10-fold dilutions of each lysate were plated on blood agar plates. CFU/ml of P. gingivalis was calculated. In separate experiments, THP-1 cells were cultured with P. gingivalis wild-type or the mutant, and both cell culture supernatant fluids, and RNA were harvested for measurement of cytokine expression and dual transcription, respectively.
Galleria mellonella infection model. Seven groups of 15 larvae, ranging from 200 to 300 mg, and with no signs of melanization, were randomly chosen and used for subsequent infection. A 25-μl Hamilton syringe was used to inject 5-μl aliquots of bacterial inoculum into each larva's hemocoel via the last left proleg (96). Two groups received wild-type P.gingivalis (7.7x108, and 7.7x107 CFUs per larvae), two groups received the cas3 mutant (4.3x108 and 4.3x107 CFUs per larvae). Three control groups included THSB medium alone, THSB + P. gingivalis wild-type heat-killed (10 min at 75oC), and THSB + Δcas3 mutant heat-killed (10 min at 75oC). After injection, larvae were incubated at 37°C, and the appearance of melanization and survival were recorded at 0.5, 1, 1.5, 2, 3, 3.75, 4.25, 6.25, 22, 24, 28 and 42 hours. After injection, larvae were incubated at 37°C, and appearance (signs of melanization) and survival were recorded at selected intervals. Larvae were scored as dead when they displayed no movement in response to touch. Kaplan-Meier killing curves were plotted, and estimations of differences in survival were compared using a log-rank test. A P value of ≤0.05 was considered significant. All data were analyzed with GraphPad Prism,

Growth protocol

Bacterial growth conditions. Porphyromonas gingivalis ATCC 33277 was cultured anaerobically at 37°C. Cells maintained on BHI-Blood agar plates supplemented with 5 μg/ml hemin, and 1 μg/ml menadione (vitamin K). Two different liquid growth media were used in the experiments. For the construction of the mutant and the infection experiments, we used TSB broth supplemented with 1 mg/ml yeast extract, 5 μg/ml hemin, and 1 μg/ml menadione. For the transcriptome experiments, we grew P. gingivalis in 20% heat-inactivated human serum (Sigma-Aldrich H3667-20ML) supplemented with 5 μg/ml hemin as describe in Grenier et al.
THP-1 cells culture. The human monocyte/macrophage cell line THP-1 (ATCC®, TIB-202) was cultured at 37°C in a 5% CO2 incubator in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (2 mM), penicillin/streptomycin (100 U/100 µg/ml), HEPES (10 mM), sodium pyruvate (1 mM), glucose (4.5 mg/ml), sodium bicarbonate (1.5 mg/ml) and 2-mercaptoethanol (0.05 mM; Sigma-Aldrich, St. Louis, MO). Cells were adjusted to 5x105 viable cells/ml and, in order to induce differentiation into a macrophage-like state, THP-1 cells were placed into fresh medium containing 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO) and 1 ml of THP-1 cells were added to each well of 24-well cell culture plates. After 48 hrs of incubation, the cell culture medium was replaced by antibiotic-free medium, and cells were used in challenge studies.
Galleria mellonella: G. mellonella larvae were purchased from SWorm Ltd., South Korea, and were used within five days of receipt. For the G. mellonella killing assays, insects in the final instar larval stage were purchased from Vanderhorst, Inc. (St. Marys, OH), stored at 4°C in the dark.

Extracted molecule

total RNA

Extraction protocol

RNA Extraction. Total RNA was extracted from those samples using the mirVana RNA Isolation Kit (Life Technologies, Grand Island, NY). Samples were bead-beaten for 1 min at maximum speed with 300 μl of 0.1-mm zirconia-silica beads (BioSpec Products, Bartlesville, Okla.) in the mirVana lysis buffer. Bacterial ribosomal RNA was depleted using Ribo-Zero rRNA Removal Kits (Bacteria) (Epicentre, Madison, WI) following the manufacturer's protocol. For RNA processing for eukaryotic analysis, we used Dynabeads mRNA Purification Kit to isolate eukaryotic mRNA for transcriptome analysis.
RNA libraries were prepared for sequencing using standard Illumina protocols. For RNA sequencing, Illumina adapter-specific primers were used to amplify and selectively enrich for the cDNA generated from enriched mRNA. Quantified libraries were pooled and sequenced using a HiSeq 2500 machine, 2x100 Flow-cell (Illumina). The Nextera XT kit was used to generate libraries from amplified DNA.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Matrix_genes_counts_Pg_in_G.mellonella_WT.csv

Data processing

We used the PATRIC annotation for genome ID 431947.7 of P. gingivalis sequences 33277 for our analysis. Low-quality sequences were removed from the query files using Trimmomatic .
Cleaned files were aligned against the P. gingivalis ATCC 33277 genome database using bowtie2 .
Eukaryotic sequences, human and G. mellonella, were aligned against genome release 33 (GRCh38.p13) in GenCode (https://www.gencodegenes.org/) and RefSeq assembly accession GCF_003640425.2, respectively. Alignment for eukaryotic sequences was performed using STAR . Read counts from the BAM files were obtained using featureCounts.
In the case of THP-1 infection experiments, differential expression analysis was performed using NOISeqBio (102). After exploratory analysis with NOISeqBio, we selected RPKM normalization for THP-1 cells data, tmm normalization with length correction for P. gingivalis intracellular transcriptome, and tmm normalization without length correction for was P. gingivalis planktonic transcriptome. Only with significant differential expression and log changes > 2 were used in posterior analyses.
Genome_build: human: release 33 (GRCh38.p13), Porphyromonas gingivalis genome ID 431947.7 of P. gingivalis sequences 33277 , galleria mellonella: GCF_003640425.2.
Supplementary_files_format_and_content: tab-delimited text files
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

Submission date

Jul 16, 2020

Last update date

Sep 16, 2020

Contact name

Jose Solbiati

E-mail(s)

josesolbiati@ufl.edu

Organization name

University of Florida

Department

Oral biology