Distinct Campylobacter fetus lineages adapted as livestock pathogens and human pathobionts in the intestinal microbiota

Campylobacter fetus is a venereal pathogen of cattle and sheep, and an opportunistic human pathogen. It is often assumed that C. fetus infection occurs in humans as a zoonosis through food chain transmission. Here we show that mammalian C. fetus consists of distinct evolutionary lineages, primarily associated with either human or bovine hosts. We use whole-genome phylogenetics on 182 strains from 17 countries to provide evidence that C. fetus may have originated in humans around 10,500 years ago and may have "jumped" into cattle during the livestock domestication period. We detect C. fetus genomes in 8% of healthy human fecal metagenomes, where the human-associated lineages are the dominant type (78%). Thus, our work suggests that C. fetus is an unappreciated human intestinal pathobiont likely spread by human to human transmission. This genome-based evolutionary framework will facilitate C. fetus epidemiology research and the development of improved molecular diagnostics and prevention schemes for this neglected pathogen

Condensin II and GAIT complexes cooperate to restrict LINE-1 retrotransposition in epithelial cells

<div><p>LINE-1 (L1) retrotransposons can mobilize (retrotranspose) within the human genome, and mutagenic <i>de novo</i> L1 insertions can lead to human diseases, including cancers. As a result, cells are actively engaged in preventing L1 retrotransposition. This work reveals that the human Condensin II complex restricts L1 retrotransposition in both non-transformed and transformed cell lines through inhibition of L1 transcription and translation. Condensin II subunits, CAP-D3 and CAP-H2, interact with members of the Gamma-Interferon Activated Inhibitor of Translation (GAIT) complex including the glutamyl-prolyl-tRNA synthetase (EPRS), the ribosomal protein L13a, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and NS1 associated protein 1 (NSAP1). GAIT has been shown to inhibit translation of mRNAs encoding inflammatory proteins in myeloid cells by preventing the binding of the translation initiation complex, in response to Interferon gamma (IFN-γ). Excitingly, our data show that Condensin II promotes complexation of GAIT subunits. Furthermore, RNA-Immunoprecipitation experiments in epithelial cells demonstrate that Condensin II and GAIT subunits associate with L1 RNA in a co-dependent manner, independent of IFN-γ. These findings suggest that cooperation between the Condensin II and GAIT complexes may facilitate a novel mechanism of L1 repression, thus contributing to the maintenance of genome stability in somatic cells.</p></div

Depletion of the Condensin II subunit, CAP-D3, increases endogenous LINE-1 RNA.

<p>(A and B) qRT-PCR analysis of endogenous L1 RNA levels in HT-29 (A) or non-transformed RPE-1 (B) cells depleted of CAP-D3 or CAP-H2 by siRNA transfection compared to control siRNA transfected cells. Results were normalized to actin. (C and D) RNA FISH for L1 in HT-29 cells transfected with control siRNA (C) or CAP-D3 siRNA (D), combined with Immunostaining for CAP-D3 protein (green). Blue arrowheads indicate colocalization of foci containing both the 3’ UTR (white) and 5’ UTR (pink) of L1 RNA (full length L1 RNA) with CAP-D3. Yellow arrowheads indicate foci containing full length L1 RNA only. Open arrowheads signify nuclear localization of full length L1 RNA and filled arrowheads signify cytoplasmic localization. Nuclei were stained with DAPI (blue). (E) Quantitation of the average number of full length L1 RNA containing foci per cell (n = 10). (F) Quantitation of the average number of full length L1 RNA containing foci in the nucleus and cytoplasm (n = 10). (G) Quantitation of RNA-IP assays using CAP-D3 antibody or no antibody in fractionated HT-29 cell lysates. Binding of CAP-D3 was normalized to the signal intensity in the unbound fractions for each sample (n = 3). P-values were calculated with a student t-test.</p

Condensin II and GAIT inhibit translation initiation and eIF4G association with the LINE-1 RNA.

<p>(A, B) Immunoblotting analysis of endogenous L1 ORF1 protein in CAP-D3 or CAP-H2 siRNA transfected HT-29 (A) and CAP-D3 siRNA transfected RPE-1 cells (B) compared to control siRNA transfected cells. Actin was used as a loading control. n≥3 for each experiment. (C) Immunoblotting analysis of endogenous L1 ORF1 protein in EPRS siRNA (left panel) transfected HT-29 cells compared to control siRNA transfected cells. Actin was used as a loading control. n = 3. (D) Polysome profiles in Non-Target and CAP-D3 shRNA expressing cells (charts shown are representative of the experiments performed). Fractions containing Free RNA, Monosome-associated and Polysome-associated RNA are labelled. (E) Ratio of the area under the polysomal (P) to monosomal (M) peaks is shown (P:M). (F) qRT-PCR analysis of L1 ORF1 containing RNA levels across all polysome fractions, calculated as percentages of total RNA for the sample. Results were normalized to Actin levels for each fraction, and are presented as the average of two independent experiments. Free RNA fractions, the monosome-associated RNA fractions, and the polysome-associated RNA fractions are labeled underneath the chart. P-values were calculated with a student t-test. * p<0.05, ** p<0.005. (G) RNA-IP assays using eIF4G antibody or no antibody in lysates from Non-Target or CAP-D3 shRNA expressing cells. Binding of eIF4G to L1 RNA in the presence and absence of CAP-D3 shRNA was normalized to the signal intensity in the unbound fractions for each condition (n = 3). (H,I) Quantitation of RNA-IP assays using eIF4G antibody or no antibody in lysates expressing Non-Target, CAP-H2 (H) or EPRS (I) shRNAs. Binding of eIF4G to L1 RNA was normalized to the signal intensity in the unbound fractions for each condition (n = 2). P-values were calculated with a student t-test. (J) Quantitation of RNA-IP assays using eIF3e (left panel), eIF4E (right panel), or no antibody in lysates expressing Non-Target or CAP-D3 shRNAs. Binding of eIF3e and 4E to L1 RNA was normalized to the signal intensity in the unbound fractions for each condition (n = 2). P-values were calculated with a student t-test. A <i>p</i>-value < 0.05 was considered statistically significant.</p

CAP-D3 and EPRS cooperate to associate with LINE-1 RNA.

<p>(A,B) CAP-D3 immunoprecipitation followed by immunoblotting for CAP-D3 or EPRS in the presence and absence of RNase A (A) or Ethidium Bromide (B) in HT-29 cells transfected with a control plasmid (pCEP4) or a plasmid expressing L1 (pJM101/L1.3) to increase active retrotransposition. (C) Quantitation of RNA-IP assays using EPRS antibody or no antibody in cells fractionated into nuclear and cytoplasmic fractions. Binding of EPRS to L1 RNA was normalized to the signal intensity in the unbound fractions for each condition (n = 3). (D) RNA-IP assays using CAP-D3 antibody or no antibody in lysates from Non-Target or EPRS shRNA expressing cells. Binding of CAP-D3 to L1 RNA in the presence and absence of EPRS was normalized to the signal intensity in the unbound fractions for each condition (n = 3). Quantitation is shown in the bottom panel. (E) RNA-IP assays using EPRS antibody or no antibody in lysates from Non-Target or CAP-D3 shRNA expressing cells. Binding of EPRS to L1 RNA in the presence and absence of CAP-D3 shRNA was normalized to the signal intensity in the unbound fractions for each condition (n = 3). Quantitation is shown in the bottom panel. P-values were calculated with a student t-test.</p

Condensin II represses LINE-1 retrotransposition in human cells.

<p>(A) Immunoblotting for CAP-D3 and CAP-H2 in HT-29 colorectal adenocarcinoma cells expressing inducible Non-Target (control), CAP-D3, or CAP-H2 shRNA. Cells were mock (PBS) or IPTG treated to induce shRNA expression. Actin was used as a loading control. (B) Retrotransposition assays involving full-length, retrotransposition competent (wild type) L1s (top row) or retrotransposition-defective ORF1p mutant L1s (bottom row) in HT-29 cells expressing Non-Target, CAP-D3 or CAP-H2 shRNAs. Crystal violet stained drug-resistant foci were quantified using ImagePro. (C) Immunoblotting for CAP-D3 in Caco2 colorectal adenocarcinoma cells expressing inducible Non-Target or CAP-D3 shRNA (similar to (A)). (D) Retrotransposition assays involving wild-type (top row) or mutant/defective L1s (bottom row) in Caco2 cells deficient for CAP-D3 (similar to (B)). P-values were calculated with a student t-test.</p

Condensin II associates with another repressor of L1 retrotransposition, EPRS.

<p>(A) The top 50 CAP-D3 binding partners predicted by immunoprecipitation/mass spectrometry ranked according to total spectral count. (B) Functional annotation of all putative CAP-D3 binding partners as determined by the Database for Annotation, Visualization, and Integrated Discovery (<i>DAVID</i>). A Benjamini corrected FDR < 0.05 was considered significant. (C-E) Immunoprecipitation of CAP-D3, CAP-H2 or EPRS in HT-29 cells under both low and high salt lysis conditions, followed by immunoblotting for the indicated proteins. Immunoprecipitations with antibody only (no lysate) and with IgG antibody were performed as controls for both lysis conditions. (F) CAP-D3 immunoprecipitations in nuclear and cytoplasmic fractions of HT-29 cells, followed by immunoblotting for EPRS or RNA polymerase II and β-tubulin to confirm isolation of nuclear and cytoplasmic fractions, respectively. Percentages indicate the amount of EPRS pulled down, compared to the amount of CAP-D3 immunoprecipitated in each fraction. (G) qRT-PCR analyses of endogenous L1 ORF1 containing RNA levels in HT-29 cells transfected with siRNA directed against EPRS, as compared to control siRNA transfected cells. (H) Immunoblotting for EPRS in HT-29 colorectal adenocarcinoma cells expressing inducible Non-Target (control) or EPRS shRNA. Actin was used as a loading control. (I) Retrotransposition assays involving wild-type (top row) or mutant/defective (bottom row) L1 elements in Non-Target or EPRS shRNA expressing cells. Crystal violet stained drug-resistant foci were quantified using ImagePro for each cell type and quantifications are shown in the chart on the right.</p