Mosaic DNA imports with interspersions of recipient sequence after natural transformation of Helicobacter pylori

Helicobacter pylori colonizes the gastric mucosa of half of the human population, causing gastritis, ulcers, and cancer. H. pylori is naturally competent for transformation by exogenous DNA, and recombination during mixed infections of one stomach with multiple H. pylori strains generates extensive allelic diversity. We developed an in vitro transformation protocol to study genomic imports after natural transformation of H. pylori. The mean length of imported fragments was dependent on the combination of donor and recipient strain and varied between 1294 bp and 3853 bp. In about 10% of recombinant clones, the imported fragments of donor DNA were interrupted by short interspersed sequences of the recipient (ISR) with a mean length of 82 bp. 18 candidate genes were inactivated in order to identify genes involved in the control of import length and generation of ISR. Inactivation of the antimutator glycosylase MutY increased the length of imports, but did not have a significant effect on ISR frequency. Overexpression of mutY strongly increased the frequency of ISR, indicating that MutY, while not indispensable for ISR formation, is part of at least one ISR-generating pathway. The formation of ISR in H. pylori increases allelic diversity, and contributes to the uniquely low linkage disequilibrium characteristic of this pathogen

Epidemiologic and Molecular Analysis of Human Tularemia, United States, 1964–2004

Distinct subpopulations of F. tularensis differ in their clinical manifestations, geographic distribution, and likely modes of transmission

Non-Opsonic Phagocytosis of Legionella pneumophila by Macrophages Is Mediated by Phosphatidylinositol 3-Kinase

Background: Legionella pneumophila, is an intracellular pathogen that causes Legionnaires ’ disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis. Methodology/Principal Findings: Phagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by 32 P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3), a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K) inhibitors, wortmannin and LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and LY294002. In contrast, PI3K and protein kinase B (PKB/Akt) activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85a subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells. Conclusion/Significance: Entry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies fo

Complete Genome Sequence of Francisella tularensis Subspecies holarctica FTNF002-00

Francisella tularensis subspecies holarctica FTNF002-00 strain was originally obtained from the first known clinical case of bacteremic F. tularensis pneumonia in Southern Europe isolated from an immunocompetent individual. The FTNF002-00 complete genome contains the RD23 deletion and represents a type strain for a clonal population from the first epidemic tularemia outbreak in Spain between 1997–1998. Here, we present the complete sequence analysis of the FTNF002-00 genome. The complete genome sequence of FTNF002-00 revealed several large as well as small genomic differences with respect to two other published complete genome sequences of F. tularensis subsp. holarctica strains, LVS and OSU18. The FTNF002-00 genome shares >99.9% sequence similarity with LVS and OSU18, and is also ∼5 MB smaller by comparison. The overall organization of the FTNF002-00 genome is remarkably identical to those of LVS and OSU18, except for a single 3.9 kb inversion in FTNF002-00. Twelve regions of difference ranging from 0.1–1.5 kb and forty-two small insertions and deletions were identified in a comparative analysis of FTNF002-00, LVS, and OSU18 genomes. Two small deletions appear to inactivate two genes in FTNF002-00 causing them to become pseudogenes; the intact genes encode a protein of unknown function and a drug:H+ antiporter. In addition, we identified ninety-nine proteins in FTNF002-00 containing amino acid mutations compared to LVS and OSU18. Several non-conserved amino acid replacements were identified, one of which occurs in the virulence-associated intracellular growth locus subunit D protein. Many of these changes in FTNF002-00 are likely the consequence of direct selection that increases the fitness of this subsp. holarctica clone within its endemic population. Our complete genome sequence analyses lay the foundation for experimental testing of these possibilities

Virulent but not avirulent strains of <i>L. pneumophila</i> stimulate PI3K activity.

<p>Thin layer chromatography of lipids from <i>in vitro</i> kinase assays on infected macrophage lysates (A). Macrophages were stimulated with different ligands for 15 min. Lysates were immunoprecipitated with specific antiserum to p85^α, and immune complexes were subjected to <i>in vitro</i> kinase assays using exogenous ATP-γ³²P and PI as substrates followed by fluorography. The line (PIP) denotes the position of PI3P standard. Spots aligning at the same position as the PI3P standard were scraped and counted using scintillation counter (B). Similar results were obtained in at least two independent experiments.</p

Overexpression of PI3K mutant protein ablates <i>L. pneumophila</i> entry.

<p>Entry by <i>L. pneumophila</i> into J774A.1 macrophages expressing the p85α mutant PI3K (pSR1NeoΔp85) and containing vector alone (pSR1Neo) after 1 hour co-incubation (A). Western analysis using antibody against the PI3K p85 α subunit, demonstrating Δp85 expression in transfected macrophages (B). Macrophage lysates were immunoprecipitated with anti-p85α and run on SDS/PAGE followed by Western blot analysis. Entry into macrophages carrying the vector alone was arbitrarily set to 100%. Data are the means+/−SEM for assays done in duplicate. Similar results were obtained in at least two independent experiments.</p

L. pneumophila infection stimulates PI3K-dependent phosphorylation of Akt.

<p>Macrophages were treated or not treated with PI3K inhibitors and infected with <i>L. pneumophila</i> for 15 min. Lysates were prepared and Ser⁴⁷³ phospho Akt (activated) detected by Western blot analysis (ppAkt). The membrane was stripped and re-probed with antibody against total cellular Akt, which is normally phosphorylated at a single position (pAkt). Similar results were obtained in at least two independent experiments.</p

PI3K inhibitors block <i>L. pneumophila</i> entry into host cells.

<p>Murine J774A.1 (A, B) that were treated or not treated with different concentrations of LY294002 or (A) Wortmannin (B) for 30 min and subsequently infected with <i>L.pneumophila</i> for 1 hr. Entry in the absence of the inhibitor was arbitrarily set to 100%. Data presented are means+/−SEM for assays done in triplicate. Similar results were obtained in at least two independent experiments.</p

Virulent but not avirulent <i>L. pneumophila</i> induce association of PI3K with tyrosine-phosphorylated proteins.

<p>Macrophages were infected with the virulent (AA100) and avirulent (Lp-14) strains of <i>L. pneumophila</i> for 15 min. Lysates were then immunoprecipitated with anti-p85 antibody and probed with anti-phosphotyrosine antibody (A) or co-immunoprecipitated with anti-phosphotyrosine antibody and probed with anti-p85 (B). The levels of total protein present in each immunoprecipitate were similar as judged by Coomassie staining of identical gels run in parallel (lower panel). Similar results were obtained in at least two independent experiments.</p