Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The Shigella flexneri type 3 secretion system is required for tyrosine kinase-dependent protrusion resolution, and vacuole escape during bacterial dissemination.

Shigella flexneri is a human pathogen that triggers its own entry into intestinal cells and escapes primary vacuoles to gain access to the cytosolic compartment. As cytosolic and motile bacteria encounter the cell cortex, they spread from cell to cell through formation of membrane protrusions that resolve into secondary vacuoles in adjacent cells. Here, we examined the roles of the Type 3 Secretion System (T3SS) in S. flexneri dissemination in HT-29 intestinal cells infected with the serotype 2a strain 2457T. We generated a 2457T strain defective in the expression of MxiG, a central component of the T3SS needle apparatus. As expected, the ΔmxiG strain was severely affected in its ability to invade HT-29 cells, and expression of mxiG under the control of an arabinose inducible expression system (ΔmxiG/pmxiG) restored full infectivity. In this experimental system, removal of the inducer after the invasion steps (ΔmxiG/pmxiG (Ara withdrawal)) led to normal actin-based motility in the cytosol of HT-29 cells. However, the time spent in protrusions until vacuole formation was significantly increased. Moreover, the number of formed protrusions that failed to resolve into vacuoles was also increased. Accordingly, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to trigger tyrosine phosphorylation in membrane protrusions, a signaling event that is required for the resolution of protrusions into vacuoles. Finally, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to escape from the formed secondary vacuoles, as previously reported in non-intestinal cells. Thus, the T3SS system displays multiple roles in S. flexneri dissemination in intestinal cells, including the tyrosine kinase signaling-dependent resolution of membrane protrusions into secondary vacuoles, and the escape from the formed secondary vacuoles

Shiga toxin remodels the intestinal epithelial transcriptional response to Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that causes diarrheal disease and the potentially lethal hemolytic uremic syndrome. We used an infant rabbit model of EHEC infection that recapitulates many aspects of human intestinal disease to comprehensively assess colonic transcriptional responses to this pathogen. Cellular compartment-specific RNA-sequencing of intestinal tissue from animals infected with EHEC strains containing or lacking Shiga toxins (Stx) revealed that EHEC infection elicits a robust response that is dramatically shaped by Stx, particularly in epithelial cells. Many of the differences in the transcriptional responses elicited by these strains were in genes involved in immune signaling pathways, such as IL23A, and coagulation, including F3, the gene encoding Tissue Factor. RNA FISH confirmed that these elevated transcripts were found almost exclusively in epithelial cells. Collectively, these findings suggest that Stx potently remodels the host innate immune response to EHEC

An Oral Inoculation Infant Rabbit Model for Shigella Infection

Shigella species are the leading bacterial cause of diarrheal death globally. The pathogen causes bacillary dysentery, a bloody diarrheal disease characterized by damage to the colonic mucosa and is usually spread through the fecal-oral route. Small animal models of shigellosis that rely on the oral route of infection are lacking. Here, we found that orogastric inoculation of infant rabbits with S. flexneri led to a diarrheal disease and colonic pathology reminiscent of human shigellosis. Diarrhea, intestinal colonization, and pathology in this model were dependent on the S. flexneri type III secretion system and IcsA, canonical Shigella virulence factors. Thus, oral infection of infant rabbits offers a feasible model to study the pathogenesis of shigellosis and to develop and test new therapeutics.Shigella species cause diarrheal disease globally. Shigellosis is typically characterized by bloody stools and colitis with mucosal damage and is the leading bacterial cause of diarrheal death worldwide. After the pathogen is orally ingested, it invades and replicates within the colonic epithelium through mechanisms that rely on its type III secretion system (T3SS). Currently, oral infection-based small animal models to study the pathogenesis of shigellosis are lacking. Here, we found that orogastric inoculation of infant rabbits with Shigella flexneri resulted in diarrhea and colonic pathology resembling that found in human shigellosis. Fasting animals prior to S. flexneri inoculation increased the frequency of disease. The pathogen colonized the colon, where both luminal and intraepithelial foci were observed. The intraepithelial foci likely arise through S. flexneri spreading from cell to cell. Robust S. flexneri intestinal colonization, invasion of the colonic epithelium, and epithelial sloughing all required the T3SS as well as IcsA, a factor required for bacterial spreading and adhesion in vitro. Expression of the proinflammatory chemokine interleukin 8 (IL-8), detected with in situ mRNA labeling, was higher in animals infected with wild-type S. flexneri versus mutant strains deficient in icsA or T3SS, suggesting that epithelial invasion promotes expression of this chemokine. Collectively, our findings suggest that oral infection of infant rabbits offers a useful experimental model for studies of the pathogenesis of shigellosis and for testing of new therapeutics

Quantification of intracellular motility.

<p>(A and B) Infection of HT-29 cells with CFP-expressing wild type and Δ<i>mxiG/pmxiG</i> (Ara withdrawal) strains. (A) Graph showing the percent of bacteria with actin tail in the cytoplasm of HT-29 cells 2 h post infection. Points represent the percent of bacteria with actin tail per infected cell. Red bars indicate the mean +/- SD of three independent experiments. Statistical analysis: p = 0.73. (B) Graph showing the velocity of wild type and Δ<i>mxiG/pmxiG</i> (Ara withdrawal) bacteria in the cytoplasm of HT-29 cells 2 h post infection. Points represent the average velocity of individual bacteria. Red bars indicate the mean +/−SD of three independent experiments. Statistical analysis: p = 0.39, unpaired t test.</p

Dynamics of wild-type 2457T dissemination in HT-29 cells.

<p>(A–C) Time-lapse microscopy of plasma membrane-targeted YFP-expressing HT-29 cells infected with CFP-expressing wild-type strain 2757T. Yellow, plasma membrane; Cyan, <i>Shigella</i>. (A,B) Representative images showing the progression of a single bacterium over time. For each panel, the top image shows a low magnification image of infected cells and the bottom images show an enlargement of the tracked bacterium (merged bacterium and membrane channels, left; membrane channel only, right). (A) Successful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell), into a membrane protrusion (15′, Protrusion) that resolve into a secondary vacuole (50′, Vacuole) from which the bacterium escape and gains access to the cytoplasm of the adjacent cell (75′, Free bacteria). (B) Unsuccessful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell) into a membrane protrusion (10′, Protrusion) that retracted towards the primary infected cell (20′, Protrusion) and returned the bacterium to the cytosol of the primary infected cell (25′, Primary Cell). (C) Tracking analysis of 60 bacteria, which formed protrusions in 20 independent foci. All bacteria were tracked for at least 180 minutes and the progression of the dissemination process was depicted using the color key shown at the bottom of panel C. Primary cell, dark blue; Protrusion, light blue; Vacuole, yellow; Free bacteria in adjacent cell, red. Scale bars, 5 µm.</p

Dynamics of Δ<i>mxiG</i> dissemination in HT-29 cells.

<p>(A–C) Time-lapse microscopy of plasma membrane-targeted YFP-expressing HT-29 cells infected with the CFP-expressing Δ<i>mxiG/pmxiG</i> (Ara withdrawal) strain. Yellow, plasma membrane; Cyan, <i>Shigella</i>. (A,B) Representative images showing the progression of a single bacterium over time. For each panel, the top image shows a low magnification image of infected cells and the bottom images show an enlargement of the tracked bacterium (merged bacterium and membrane channels, left; membrane channel only, right). (A) Unsuccessful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell) into a membrane protrusion (10′, Protrusion) that resolved into a vacuole (30′, Vacuole) from which the pathogen did not escape (180′, vacuole). Note that the trapped bacterium divided into at least 5 bacteria. (B) Unsuccessful progression of a bacterium (white arrows, top panel) from the primary cell cytoplasm (0′, Primary Cell) into a membrane protrusion (15′, Protrusion) that retracted towards the primary infected cell (65′, Protrusion) and returned the pathogen to the cytosol of the primary infected cell (70′, Primary Cell). (C) Tracking analysis of 60 bacteria, which formed protrusions in 25 independent foci. All bacteria were tracked for at least 180 minutes and the progression of the dissemination process was depicted using the color key shown at the bottom of panel C. Primary cell, dark blue; Protrusion, light blue; Vacuole, yellow; Free bacteria in adjacent cell, red. Scale bars, 5 µm.</p

Quantification of <i>S. flexneri</i> dissemination in HT-29 cells.

<p>(A,B) Infection of HT-29 cells with CFP-expressing <i>S. flexneri</i>. (A) Representative images showing the size of infection foci 8 h post infection comparing wild-type 2457T to the Δ<i>mxiG</i> strain complemented with p<i>mxiG</i> with 1.0% arabinose removed after the initial 30 minutes of infection (Δ<i>mxiG/pmxiG</i> (Ara withdrawal)), and the Δ<i>mxiG</i> mutant complemented with p<i>mxiG</i> with 1.0% arabinose throughout infection (Δ<i>mxiG/pmxiG</i> 1.0% Ara). Green, <i>Shigella</i>; red, DNA. Scale bar, 200 µm. (B) Computer-assisted image analysis was used to quantify the size of the infection foci and the average focus size was determined. Individual points represent individual foci, red bars indicate the mean +/−SD. Statistical analysis; ****, p<0.0001, unpaired t-test.</p

Quantification of <i>S. flexneri</i> invasion of HT-29 cell monolayer.

<p>(A) Representative images (XY and XZ planes) of HT-29 cells expressing plasma membrane-targeted YFP at 4 h post infection with the wild type strain 2457T, the isogenic Δ<i>mxiG</i> strain and the complemented Δ<i>mxiG/pmxiG</i>. Bacteria (red) were stained with an anti-<i>Shigella</i> antibody. Arrows indicate the presence or absence of host cell membrane surrounding the bacteria. Scale bar, 2.5 µm. (B) Percent invasion of HT-29 cell monolayers with wild-type 2457T strain set to 100%. Values represent the mean +/−SD of three independent experiments. Statistical analysis; ** p = 0.0095, **** p<0.0001, unpaired t test.</p

The T3SS is required for activation of tyrosine kinase signaling in protrusions.

<p>(A) Representative images of HT-29 cells expressing a YFP-membrane marker (yellow), infected with the CFP-expressing wild-type strain 2457T (cyan) and stained for phospho-tyrosine residues (red). (B) Representative images of HT-29 cells expressing membrane-targeted YFP (yellow), infected with the CFP-expressing Δ<i>mxiG/pmxiG</i> (Ara withdrawal) strain (cyan) and stained for phospho-tyrosine residues (red). Scale bar 5 µm. (C) Graph showing the percent of phospho-tyrosine positive and phospho-tyrosine negative protrusions for the wild-type and the Δ<i>mxiG/pmxiG</i> (Ara withdrawal) strains. Values indicate the mean +/SD of three independent experiments. Statistical analysis; p-Tyr positive p<0.0001, unpaired t test.</p