Journal: bioRxiv

Article Title: DNA glycosylase NEIL2 prevents Fusobacterium -mediated inflammation and DNA damage in colonic epithelial cells

doi: 10.1101/2020.06.11.147454

Figure Lengend Snippet: (A-D) Human colonic EDMs were infected with Fn at moi 100 for 24 h. The RNA from EDMs was used for qRT-PCR to determine the expression of genes involved in base excision repair, mismatch repair and for non-homologous end joining (NHEJ). (A) Schematic showing the experimental design. (B) The level of BER transcripts, NEIL1, NEIL2, NTH1, OGG1, (C) The level of MMR transcripts, MLH1, MLH3, MSH2, MSH6, PMS2, (D) The transcript level of NHEJ marker Ku70 were determined by qRT-PCR. (E-F) Human colonic EDMs were infected with commensal E. coli -K12 strain (E), or pathogenic IBD-associated adherent invasive E. coli LF-82 (F) to determine the expression level of NEIL2 following infection. In (B-F), the expression level of the transcripts was normalized to the housekeeping gene (18srRNA), and the normalized expression value was compared with the respective uninfected control cells. Data represent the mean ± SEM of three separate experiments. * indicates p≤0.05, and ** indicates p≤0.01 as calculated by the unpaired two-tailed student’s t-test.

Article Snippet: Escherichia coli K12 strain DH10B , (ATCC-PTA¬5105), was cultured on L.B. agar and L.B. broth and used to infect EDM at moi of 100.

Techniques: Infection, Quantitative RT-PCR, Expressing, Non-Homologous End Joining, Marker, Control, Two Tailed Test

Journal: bioRxiv

Article Title: DNA glycosylase NEIL2 prevents Fusobacterium -mediated inflammation and DNA damage in colonic epithelial cells

doi: 10.1101/2020.06.11.147454

Figure Lengend Snippet: (A) APC Min /+ EDMs derived from the uninvolved region of the colon were infected with different microbes; commensal E. coli K12, IBD-associated adherent-invasive E.coli LF82 and colon cancer-associated pathogens (NC101, H. pylori and Fn ). The supernatants were collected from the uninfected and infected EDMs done in the same experiments and assessed for oxidative DNA damage (right). Data represent the mean ± SEM of three separate experiments. * indicates p≤0.05, ** indicates p≤0.01 as assayed by student’s t-test. (B) The relative level of the oxidized bases produced by each microbe was compared with uninfected cells, which is considered as 1. The relative production of the oxidized base was compared between different microbes

Article Snippet: Escherichia coli K12 strain DH10B , (ATCC-PTA¬5105), was cultured on L.B. agar and L.B. broth and used to infect EDM at moi of 100.

Techniques: Derivative Assay, Infection, Produced

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: A-to-I mRNA editing can affect protein sequence and function in bacteria as evidenced by the case of HokB. ( A ) Adenosine is deaminated to inosine, which is similar to guanosine in its base-pair properties. ( B ) A-to-I mRNA editing by TadA in E. coli occurs in 80%–90% of endogenously expressed hokB transcripts at the logarithmic phase, and is assumed to recode a tyrosine to a cysteine codon at position 29 of HokB. ( C ) Growth analysis of E. coli (Top10-DH10B) co-expressing mCherry (control, black) or mCherry-HokB (green), with either GFP-TadA (left panel) or GFP (right panel). The mean and standard error of three biological replicates conducted on different days ( N = 3), each with 21 technical replicates, are shown. The expression of mCherry and HokB was induced from the beginning of the experiment (time point “0”) with 0.2% arabinose from a pBAD vector. Expression of GFP-TadA or GFP was induced with 1 mM IPTG from a pME6032 vector. ( D ) A-to-I mRNA editing identification by next-generation sequencing (NGS; Illumina; amplicon-seq) of plasmid-borne hokB RNA (cDNA) co-overexpressed with GFP-TadA or with GFP alone. Minimum observed reads coverage per sample that passed our quality filters ≥17 515 391. Statistical analysis was conducted using Student’s t -test; P -value <.0001 (****). ( E ) A-to-I mRNA editing validation by Sanger sequencing of plasmid-borne DNA and RNA (cDNA) of hokB when co-overexpressed with GFP-TadA or with GFP. The sequence above the chromatograms represents the gene (DNA) sequence. A black arrow marks the double peak of A and G(I) that was observed only in the cDNA (complementary DNA) samples. Note that the G(I) peak is higher than the A peak when overexpressing GFP-TadA and vice versa when overexpressing only GFP. ( F ) MS/MS spectrum of non-edited (Y29; top) and edited (C29; bottom) HokB peptides found in strains co-overexpressing HokB with GFP (top) or GFP-TadA (bottom). Black arrows mark identified peptides and their mass in the MS/MS spectra that show a mass shift corresponding to tyrosine or cysteine at the edited site. The gray arrow marks an example of a peptide and its mass in the MS/MS spectra that does not include the edited site (same mass). All peptides were discovered with false discovery rate (FDR) ≤ 0.01. The peaks weight, font size, and axis were adjusted from the original figure for better visualization and comparison. A comprehensive mass distribution and the original MS/MS spectra and data can be found in – .

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: Sequencing, Bacteria, Expressing, Control, Plasmid Preparation, Next-Generation Sequencing, Amplification, Biomarker Discovery, Tandem Mass Spectroscopy, Comparison

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: DNA-encoded cysteine residues are essential for the toxicity of edited HokB. ( A ) The protein sequence of non-edited and edited HokB according to their respective transcript. DNA-coded cysteines are shown in bold. ( B ) A description of the different plasmids containing different versions of HokB used in the growth assay is presented. ( C ) Growth analysis of E. coli (Top10-DH10B) WT strain expressing the HokB (Y29#, green), non-edited HokB (Y29, blue), and edited HokB (C29, red) fused to mCherry reporter protein (N-terminus) from the plasmid shown in panel (B). As a reference control, a plasmid harboring only mCherry was used (black). As previously reported , when highly expressed, edited HokB (C29) induces the highest level of toxicity. ( D ) Growth analysis as in panel (C), with all three versions of HokB having the C9S substitution. ( E ) Growth analysis as in panel (C), with all three versions of HokB having the C14S substitution. ( F ) Growth analysis as in panel (C), with all three versions of HokB having the C46S substitution. In all growth experiments, protein expression was induced from the beginning of the experiment (time point “0”) with 0.2% arabinose from a pBAD vector. The mean and standard error of three biological replicates conducted on different days ( N = 3), each with 21 technical replicates, are shown.

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: Sequencing, Growth Assay, Expressing, Plasmid Preparation, Control

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: In vivo disulfide bond formation is essential for the toxicity of the edited HokB. ( A ) Growth analysis of an E. coli Δ dsbA strain that expresses one of three versions of HokB, fused to mCherry from an inducible plasmid. As a reference control, we used a plasmid encoding only mCherry. ( B ) Growth analysis as in panel (A), but with overexpressing DsbA from a second plasmid (pME6032). ( C ) Growth analysis, as in panel (B), using an empty plasmid (pME6032 with no dsbA insert).

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: In Vivo, Plasmid Preparation, Control

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: Western blot analysis supports that A-to-I mRNA editing mediates an intramolecular disulfide bond between C29 and C46 in HokB. ( A ) Western blot of membrane enriched protein fraction of E. coli (Top10-DH10B) WT strain expressing either mCherry only (control; black) or the HokB (Y29#, green), non-edited HokB (Y29, blue), and edited HokB (C29, red) fused to mCherry (N-terminus) from the plasmid shown in Fig. . ( B ) Same as panel (A) but with the C9S substitution in the different expressed HokB versions. ( C ) Same as panel (A) but with the C14S substitution in the different expressed HokB versions. ( D ) Same as panel (A) but with the C46S substitution in the different expressed HokB versions.

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: Western Blot, Membrane, Expressing, Control, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: Lower levels of edited HokB induce early entrance to the stationary phase. ( A ) Growth analysis of WT E. coli as described in Fig. with 1:1000 lower arabinose concentration. The expression of mCherry and HokB was induced from the beginning of the experiment (time point “0”) with 0.0002% arabinose from a pBAD vector. Black and white triangles correspond to sampling times for panel (B). ( B ) CFU counts at 5 and 6 h of the beginning of growth. Notice that there are fewer CFUs when edited HokB is expressed, with similar numbers at 5 and 6 h after growth. The mean and standard error of four biological replicates conducted on different days ( N = 4) are shown. Statistical analysis was conducted using Student’s paired t -test followed by Benjamini–Hochberg FDR correction: P -value ≤.05 (*).

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: Concentration Assay, Expressing, Plasmid Preparation, Sampling

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: A-to-I mRNA editing of hokB is conserved in pathogenic E. coli and Shigella strains. Sanger sequencing of the endogenous hokB gene and its mRNA from the same sample of non-pathogenic E. coli (used throughout this work), enterohemorrhagic E. coli , enteropathogenic E. coli , uropathogenic E. coli , and Shigella sonnei . A black arrow marks the double peak of A and G(I) observed only in the cDNA samples. Note that the G(I) peak (black) is higher than the A peak (green) in most samples. Sequences were aligned to the E. coli reference genome ( NC_000913.3 ) and positions 1491982–1491990 are shown. See for exact genomic coordinates of the full-length hokB gene in each species.

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: Sequencing