Journal: Cancer Science

Article Title: Fusobacterium nucleatum induces excess methyltransferase‐like 3‐mediated microRNA‐4717‐3p maturation to promote colorectal cancer cell proliferation

doi: 10.1111/cas.15536

Figure Lengend Snippet: MicroRNA‐4717‐3p (miR‐4717) induces colorectal cancer (CRC) cell proliferation and is closely related to Fusobacterium nucleatum ‐positive ( Fn +) CRC. (A) miR‐4717 levels were analyzed by quantitative PCR (qPCR) in 50 pairs of CRC patient tumors and paracancerous tissue samples from individuals with or without evidence of F. nucleatum infection. * p < 0.05, ** p < 0.01, by Student's t test. Data are means with SD. (B) Correlations between F. nucleatum abundance and miR‐4717 expression levels in F. nucleatum ‐infected CRC tissues were assessed ( n = 25; Spearman correlation analysis). (C) Receiver operating characteristic (ROC) curves were generated based on miR‐4717 expression levels in patients with CRC that did or did not exhibit F. nucleatum infection. AUC, area under the ROC curve. (D) miR‐4717 expression in HCT‐116 and LoVo cells following F. nucleatum infection, Bacteroides fragilis ( Bf ) infection, or PBS treatment for 24 h by qPCR. (E) Expression of miR‐4717 was detected by RT‐qPCR in CRC cells (SW480, HT29, LoVo, HCT‐116, and NCM460) at 24 h after F. nucleatum infection. (F) Expression of miR‐4717 was detected by RT‐qPCR in CRC cells (LoVo or HCT‐116) after F. nucleatum infection at different MOI. (G–I) HCT‐116 cell proliferation was examined following F. nucleatum and/or miR‐4717 inhibitor treatment by EdU (G), cell cycle analysis (H), and CCK‐8 (I) assays. Scale bar, 125 μm. Data are means ± SD from triplicate experiments. * p < 0.05. ns, not significant. (J) Representative images from tumors in different groups, including mice treated with F. nucleatum and/or miR‐4717 antagomir. Images of xenograft (J, left panel) was generated, with average xenograft tumor weight (J, right panel) calculated, and Ki‐67 staining (J, middle panel) for xenograft shown. Scale bar, 125 μm.

Article Snippet: The customized F. nucleatum (NusG) primers used herein were synthesized by Sangon Biotech and are listed in Table .

Techniques: Real-time Polymerase Chain Reaction, Infection, Expressing, Generated, Quantitative RT-PCR, Cell Cycle Assay, CCK-8 Assay, Staining

Journal: Cancer Science

Article Title: Fusobacterium nucleatum induces excess methyltransferase‐like 3‐mediated microRNA‐4717‐3p maturation to promote colorectal cancer cell proliferation

doi: 10.1111/cas.15536

Figure Lengend Snippet: MicroRNA‐4717‐3p (miR‐4717) suppresses mitogen‐activated protein kinase kinase 4 (MAP2K4) expression in the context of Fusobacterium nucleatum ( Fn ) infection. (A) Following predictive bioinformatics analyses and consultation of the gene annotation, MAP2K4 was identified as a putative miR‐4717 target gene. (B) Predicted miR‐4717 binding sites within the MAP2K4 mRNA 3′‐UTR with corresponding WT and mutated (MT) sequences. (C) Impact of miR‐4717 mimic or inhibitor treatment on pMIR‐MAP2K4‐WT and pMIR‐MAP2K4‐MT reporter activity in HEK293 cells as assessed by luciferase reporter gene assay. * p < 0.05. (D, E) MAP2K4 mRNA and protein levels following miR‐4717 mimic or inhibitor treatment. (F) MAP2K4 mRNA levels were assessed by quantitative PCR in paired tumor and paracancerous tissue samples from colorectal cancer (CRC) patients with or without F. nucleatum infection. * p < 0.05, ** p < 0.01. (G) Representative western blot images illustrating MAP2K4 expression in patient CRC tissue samples that were positive or negative for F. nucleatum infection. (H) Correlations between F. nucleatum abundance and MAP2K4 expression levels in F. nucleatum ‐positive CRC tissues ( n = 25; Spearman's correlation analysis). (I, J) MAP2K4 mRNA and protein levels were assessed in HCT‐116 and LoVo cells following F. nucleatum infection, Bacteroides fragilis ( Bf ) infection, or PBS treatment for 24 h. (K) After miR‐4717 mimic or inhibitor treatment, F. nucleatum was used to infect HCT‐116 cells for 24 h, with MAP2K4 protein levels analyzed by western blotting. (L–N) miR‐4717 mimic/inhibitor, siRNA MAP2K4, or MAP2K4 overexpression plasmids were transfected into HCT‐116 cells for 24 h, with MAP2K4 protein levels evaluated by western blotting.

Article Snippet: The customized F. nucleatum (NusG) primers used herein were synthesized by Sangon Biotech and are listed in Table .

Techniques: Expressing, Infection, Binding Assay, Activity Assay, Luciferase, Reporter Gene Assay, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Transfection

Journal: Cancer Science

Article Title: Fusobacterium nucleatum induces excess methyltransferase‐like 3‐mediated microRNA‐4717‐3p maturation to promote colorectal cancer cell proliferation

doi: 10.1111/cas.15536

Figure Lengend Snippet: Methyltransferase‐like 3 (METTL3)‐dependent m 6 A methylation promotes microRNA‐4717‐3p (miR‐4717) upregulation in colorectal cancer cells. (A) Primary (pri)‐miR‐4717 levels were assessed in HCT‐116 cells following Fusobacterium nucleatum ( Fn ) infection or PBS treatment for 24 h. (B) Potential m 6 A modification sites were identified within pri‐miR‐4717, of which three were very high confidence positions as predicted using SRAMP. (C) m 6 A modification of pri‐miR‐471 was detected. The percentage of the input is shown. Data are presented as means ± SD. (D, E) Pri‐miR‐4717 or mature miR‐4717 levels were detected by quantitative PCR (qPCR) in HEK293T cell line upon transfected with WT or mutant‐type (MT) pri‐miR‐4717 plasmids. (F) m 6 A modification level of pri‐miR‐4717 was detected by qPCR between WT and MT plasmids. (G) m 6 A modification‐related gene expression levels in HCT‐116 cells following PBS or F. nucleatum treatment. (H) Following PBS treatment or F. nucleatum infection, METTL3 protein levels were quantified by western blotting in HCT‐116 and LoVo cells. (I) Mature miR‐4717 or pri‐miR‐4717 levels were measured by qPCR following METTL3 knockdown or overexpression. (J) m 6 A modified levels in siMETTL3 or control HCT‐116 cells as measured by qPCR to analyze the m 6 A modification of pri‐miR‐4717. * p < 0.05, *** p < 0.001.

Article Snippet: The customized F. nucleatum (NusG) primers used herein were synthesized by Sangon Biotech and are listed in Table .

Techniques: Methylation, Infection, Modification, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Gene Expression, Western Blot, Knockdown, Over Expression, Control

Journal: Cancer Science

Article Title: Fusobacterium nucleatum induces excess methyltransferase‐like 3‐mediated microRNA‐4717‐3p maturation to promote colorectal cancer cell proliferation

doi: 10.1111/cas.15536

Figure Lengend Snippet: Methyltransferase‐like 3 (METTL3) induces the proliferation of Fusobacterium nucleatum ( Fn )‐associated colorectal cancer in vitro and in vivo. (A–C) Ability of HCT‐116 cells to proliferate following F. nucleatum and/or si‐METTL3 treatment was assessed by (A) EdU, (B) cell cycle analyses, and (C) CCK‐8. (D) Xenograft tumors models were generated, with representative xenograft tumors (D, left panel) being shown. Ki‐67 staining of these xenografts (D, middle panel) were also carried out, and average tumor weights for xenograft tumors (D, right panel) were calculated. Data are means ± SD from triplicate experiments. * p < 0.05. Scale bar, 125 μm.

Article Snippet: The customized F. nucleatum (NusG) primers used herein were synthesized by Sangon Biotech and are listed in Table .

Techniques: In Vitro, In Vivo, CCK-8 Assay, Generated, Staining

Journal: Cancer Science

Article Title: Fusobacterium nucleatum induces excess methyltransferase‐like 3‐mediated microRNA‐4717‐3p maturation to promote colorectal cancer cell proliferation

doi: 10.1111/cas.15536

Figure Lengend Snippet: Fusobacterium nucleatum ( Fn ) drives the progression of colorectal cancer (CRC) through the methyltransferase‐like 3 (METTL3)/microRNA‐4717‐3p (miR‐4717)/mitogen‐activated protein kinase kinase 4 (MAP2K4) axis. (A) METTL3 mRNA levels were measured by quantitative PCR in paired tumor and normal tissue samples from CRC patients with or without F. nucleatum infection. (B) Correlations between METTL3 and MAP2K4 expression levels in F. nucleatum ‐infected CRC tissues ( n = 25; Spearman correlation analysis). (C) Representative images showing that the abundance of invasive F. nucleatum in CRC tissues is associated with high expression of METTL3 and low expression of MAP2K4. (D) HCT‐116 cells were transfected with METTL3 overexpression or siMETTL3 constructs for 24 h, after which MAP2K4 levels were assessed by western blotting. (E, F) Prior to F. nucleatum infection, miR‐4717 mimic or miR‐4717 inhibitor and METTL3 overexpression or siMETTL3 constructs were cotransfected into HCT‐116 cells, with MAP2K4 levels then examined by western blotting. (G, H) CCK‐8 and colony formation assays were carried out for HCT‐116 cells in which METTL3 had been knocked down or overexpressed following the transfection of MAP2K3 overexpression plasmids or MAP2K4‐specific siRNA constructs. * p < 0.05, ** p < 0.01.

Article Snippet: The customized F. nucleatum (NusG) primers used herein were synthesized by Sangon Biotech and are listed in Table .

Techniques: Real-time Polymerase Chain Reaction, Infection, Expressing, Transfection, Over Expression, Construct, Western Blot, CCK-8 Assay

Journal: Cancer Science

Article Title: Fusobacterium nucleatum induces excess methyltransferase‐like 3‐mediated microRNA‐4717‐3p maturation to promote colorectal cancer cell proliferation

doi: 10.1111/cas.15536

Figure Lengend Snippet: Schematic overview of Fusobacterium nucleatum ‐induced m 6 A modification‐dependent microRNA‐4717‐3p (miR‐4717) overexpression as a driver of colorectal cancer cell proliferation. MAP2K4, mitogen‐activated protein kinase kinase 4; METTL3, methyltransferase‐like 3; pri‐miRNA‐4717, primary miR‐4717.

Article Snippet: The customized F. nucleatum (NusG) primers used herein were synthesized by Sangon Biotech and are listed in Table .

Techniques: Modification, Over Expression

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A) Heat map showing percent identity of NanA homologs in sequenced F. nucleatum strains to the sialic acid lyase of E. coli MG1655. Homologs showed high similarity to amino acid sequence of lyase from F. nucleatum 23726 . (B) Amount of sialic acid remaining in the growth medium 24-48 hours post inoculation of different F. nucleatum strains (as indicated), relative to un-inoculated media control. All bacteria were grown in medium supplemented with free sialic acid (Neu5Ac, 100 μM). Error bars shown are standard deviation of the mean, from three or more experiments.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Sequencing, Control, Bacteria, Standard Deviation

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A-B) E. coli MG1655 ΔnanA was complemented with empty vector, E. coli nanA or putative nanA from F. nucleatum 23726. F.n. = F. nucleatum ; E.c. = E. coli . (A) Growth was assessed in minimal media with sialic acid by measuring absorbance at 600 nm. (B) Lysates of E. coli nanA mutant with empty vector or the complemented strain were incubated with Neu5Ac and its disappearance was monitored over time by DMB-HPLC. (C) Concentration of sialic acid (Neu5Ac) remaining in the medium 24 h post inoculation. Sialic acid consumption by F. nucleatum 23726 wild-type (WT) and ΩsiaT was studied in complete growth medium supplemented with free sialic acid. Data shown is representative of two independent experiments. Error bars show standard deviation from the mean value. (D) Growth of F. nucleatum ( F. nuc.) WT and ΩsiaT in low carbohydrate (carb) medium with or without supplementation with the indicated carbohydrates. Data shown is representative of two independent experiments. (E) Cell associated sialidase activity of anaerobically cultured G. vaginalis JCP8151B and F. nucleatum ATCC23726 strains was analyzed using fluorogenic 4MU-Neu5Ac substrate. (F) F. nucleatum was grown anaerobically in media that was either untreated or exposed to purified sialidase from Arthrobacter ureafaciens ( A.u.) or G. vaginalis ( G.v.). Total and free sialic acid content was measured at 0 and 48 h post inoculation. Bound sialic acids (Bound = Total - Free) are inaccessible to F. nucleatum , except in the presence of exogenous sialidase. Data shown are representative of at least two technical replicates per condition performed in two or more independent experiments. Error bars represent standard deviation from the mean value. See also , and .

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Plasmid Preparation, Mutagenesis, Incubation, Concentration Assay, Standard Deviation, Activity Assay, Cell Culture, Purification

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: Sialidase-producers in the vaginal microbial community release free sialic acids (red diamonds) from host glyco-conjugates, which may be accessed by F. nucleatum , which does not produce sialidase. SiaT = sialic acid transporter; NanA = N -acetylneuraminate lyase; More information about F. nucleatum genes shown in sialic acid catabolic gene cluster, and the enzymes they encode, can be found in .

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques:

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A) Schematic showing integration of plasmid containing siaT insert (pLR25) into the F. nucleatum chromosome. Positions of forward (Fwd) and reverse (Rev) primers used for confirming integration of plasmid are also indicated. (B) Agarose gel image with the expected PCR product confirming integration of plasmid into the siaT locus.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Plasmid Preparation, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: Relative amount of bound (bound = total-free) and free sialic acid remaining in the medium at 0 and 48 hours post inoculation. F. nucleatum was grown anaerobically in media that was either untreated or exposed to purified sialidase from Arthrobacter ureafaciens ( A.u. ) or P. bivia (P.b.) ATCC 29303. Bound sialic acids are inaccessible to F. nucleatum except in the presence of exogenous sialidase. Error bars represent standard deviation of the mean value.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Purification, Standard Deviation

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: Mouse vaginal microbial communities-collection and amplification. Step 1 - *Collection of mouse vaginal washes. Washes were pooled from mice co-housed in the same cage. A portion of vaginal wash pools was cultured overnight anaerobically (referred to as “microbiota pools”) to amplify the vaginal bacteria and frozen for subsequent use. Remaining pooled material was stored at −20°C. Step 2 - On the day of the experiment, frozen microbiota pools were used to recover mouse vaginal bacteria by streaking out on Columbia blood plates in anaerobic chamber and incubating for 24 h at 37°C. Colonies from these plates were re-suspended in liquid media either (a) for DNA extraction, or (b) for co-culture experiments with F. nucleatum . *Publication of this animal image was approved by IACUC.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Amplification, Cell Culture, Bacteria, DNA Extraction, Co-Culture Assay

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: Heat map showing the log10 abundance of different OTUs in the microbiota pools of Jackson (Jax, 1-4) mice and Envigo (Envi, 5-8) collected before estrogenization (BE), after estrogenization (AE), and at 1 day post inoculation (dpi) with F. nucleatum . Microbiome analysis was done on uncultured and cultured vaginal washes pooled from 5 mice in the same cage. Each column represents one microbiota pool = 1 cage = 5 mice, total = 4 microbiota pools per vendor per condition. OTUs were assigned using the UNOISE algorithm. Data was clustered using hierarchical Euclidean clustering in the R project for statistical computing.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A) Heat map shows abundance of OTUs in uncultured pooled vaginal specimens (1 pool = 1 cage = 5 mice) collected 1-day post inoculation with F. nucleatum . OTUs were assigned using the UPARSE-OTU algorithm. Data was clustered using hierarchical Euclidean clustering. (B, C) F. nucleatum titers in vaginal wash collected at indicated time points post inoculation (B) in mice from Envigo, and (C) in mice from Jackson. ** P < 0.01, Mann-Whitney. (D-G) Course of C57BL/6 mice (Envigo and Jackson) colonization with the F. nucleatum wild-type (WT) and ΩsiaT mutant. Number of mice colonized in percent ( y axis) was monitored on day 1 and every 2 days for 38 days (x axis). Statistically significance assessed by Log Rank test, * P < 0.05. (D) Comparison of WT vs. ΩsiaT colonization in Jackson mice. (E) Comparison of WT vs. ΩsiaT colonization in Envigo mice. (F) Comparison of WT colonization in Envigo vs. Jackson mice. (G) Comparison of ΩsiaT colonization in Envigo vs. Jackson mice. For Kaplan-Meier analysis, mice were considered cleared when no cfu were detected in undiluted wash at 2 consecutive time points. The graphs represent combined data from two experiments performed separately. OTU = operational taxonomic unit, hpi = hours post inoculation. See also .

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: MANN-WHITNEY, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A, B) F. nucleatum titers in vaginal wash collected from individual mice. (A) WT and ΩsiaT titers in mice from Envigo facility from 1 to 8 dpi. (B) WT and ΩsiaT titers in mice from Jackson facility from 1 to 8 dpi. Data is combined from 2 independent experiments, dpi = days post inoculation. Each experiment had 10 mice per group.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques:

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A-B) Sialidase activity in vaginal washes from individual animals purchased from Envigo, estrogenized, and inoculated with E. coli , or F. nucleatum from 1 to 8 dpi. (C) Sialidase activity (after estrogenization) in vaginal wash pooled specimens (1 microbiota pool = 1 cage = 5 mice) from Jackson and Envigo mice, cultured with or without F. nucleatum (WT or ΩsiaT ). No sialidase activity was observed in cultured vaginal microbial communities from Jackson mice, both with and without F. nucleatum . Data shown here is combined from 2 independent experiments.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Activity Assay, Cell Culture

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A) Sialidase activity in vaginal washes from 1 to 8 dpi from individual animals purchased from Envigo, estrogenized, and inoculated with either WT or ΩsiaT F. nucleatum . Data at later time points were compared to day 1 values using Friedman test, along with correction for multiple planned comparisons using Dunn’s test. (B) Same experiment and data as shown in A but analyzed to compare between WT or ΩsiaT- inoculated animals at each time point using the Mann-Whitney test. Data in A and B represent combined data from two independent experiments. Data points with negative values were set to 0.001 to represent them on the log scale. (C) Sialidase activity in microbiota pools from Envigo mice. Communities were cultured in the presence or absence of F. nucleatum ( F. nuc. ) WT or ΩsiaT . Each “microbiota pool” consists of a cultured vaginal community from pooled vaginal wash of 4-5 co-housed mice. Wilcoxon paired-sign rank test was used for pairwise comparison of sialidase activity in each cultured microbiome compared to the identical microbiome cultured in the presence of F. nucleatum . Data shown is combined from 4 independent biological replicates with 7-8 technical replicates for each microbiota pool. (D) Same experimental data as in C . Data shown is combined from all microbiota pools for each group and analyzed to allow for comparison of the relative sialidase boost between no added F. nucleatum ( F.n. ) versus addition of WT or ΩsiaT . A statistical comparison between the two groups was performed using Wilcoxon matched-pairs signed rank test. Data shown is combined from 3 independent biological replicates. Line in the bar indicates mean value. On all graphs *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. See also .

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Activity Assay, MANN-WHITNEY, Cell Culture, Comparison

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: Human vaginal microbial communities-collection and amplification. Step 1 - Anaerobic and aerobic vaginal swabs were collected on the same day from each participant. Aerobic swabs were eluted in sodium acetate buffer (pH 5.5) and sialidase activity was checked in the swab eluates using fluorogenic 4MU-Neu5Ac substrate. Anaerobic swabs were eluted in 2X NYCIII media (in an anaerobic chamber) and the communities were “fresh frozen”, without any amplification / overnight culture , by mixing with cryoprotectant and storing at −80°C. Step 2 - On the day of the experiment - fresh frozen anaerobic vaginal communities, from women who had detectable sialidase activity in their aerobic swab eluates, were thawed at 4°C and diluted 4-fold in NYCIII media (in an anaerobic chamber). The diluted communities were used for co-culture experiments with F. nucleatum .

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Amplification, Activity Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A) Human vaginal communities were cultivated anaerobically in Columbia media in the presence or absence of added F. nucleatum . Sialidase activity was measured following anaerobic culture. Communities from 21 individual women were used. Data are combined from 2 independent experiments. A statistical comparison between the two groups was performed using Wilcoxon matched-pairs signed rank test. Negative values were set to 0.0018 (lowest positive value) to depict them on the log scale. (B-C) G. vaginalis ( G.v. ) was co-cultivated anaerobically in Columbia media in the presence or absence of F. nucleatum ( F.n. ), followed by measurement of sialidase activity (B) and viable titers of G. vaginalis (colony forming units, C). Note that G. vaginalis was not detectable under these conditions in the absence of F. nucleatum. In this case, G. vaginalis levels were plotted at one half the limit of detection (LOD=200 CFU/mL). Heat map data is representative of two independent experiments. CFU data is combined from two independent experiments, each with 3 technical replicates each. On all graphs ***P<0.001, ****P<0.0001.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Activity Assay, Comparison