J. Puls

Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8

Helicobacter pylori (Hp) carries a type IV secretion system encoded by the cag pathogenicity island (cag-PAI), which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin-8 (IL-8). The cag-PAI in Hp 26695 consists of 27 putative genes, six of which were identified as homologues to the basic type IV secretion system represented by the Agrobacterium tumefaciens virB operon. To define the role and contribution of each of the 27 genes, we applied a precise deletion/insertion mutagenesis procedure to knock out each individual gene without causing polar effects on the expression of downstream genes. Seventeen out of 27 genes were found to be absolutely essential for translocation of CagA into host cells and 14 out of 27 for the ability of Hp fully to induce transcription of IL-8. The products of hp0524 (virD4 homologue), hp0526 and hp0540 are absolutely essential for the translocation of CagA, but not for the induction of IL-8. In contrast, the products of hp0520, hp0521, hp0534, hp0535, hp0536 and hp0543 are not necessary for either translocation of CagA or for IL-8 induction. Our data argue against a translocated IL-8-inducing effector protein encoded by the cag-PAI. We isolated a variant of Hp 26695, which spontaneously switched off its capacity for IL-8 induction and translocation of CagA, but retained the complete cag-PAI. We identified a point mutation in gene hp0532, causing a premature translational stop in the corresponding polypeptide chain, providing a putative explanation for the defect in the type IV secretion system of the spontaneous mutant.

Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8: Functional dissection of the H. pylori type IV secretion system

Helicobacter pylori (Hp) carries a type IV secretion system encoded by the cag pathogenicity island (cag‐PAI), which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin‐8 (IL‐8). The cag‐PAI in Hp 26695 consists of 27 putative genes, six of which were identified as homologues to the basic type IV secretion system represented by the Agrobacterium tumefaciens virB operon. To define the role and contribution of each of the 27 genes, we applied a precise deletion/insertion mutagenesis procedure to knock out each individual gene without causing polar effects on the expression of downstream genes. Seventeen out of 27 genes were found to be absolutely essential for translocation of CagA into host cells and 14 out of 27 for the ability of Hp fully to induce transcription of IL‐8. The products of hp0524 (virD4 homologue), hp0526 and hp0540 are absolutely essential for the translocation of CagA, but not for the induction of IL‐8. In contrast, the products of hp0520, hp0521, hp0534, hp0535, hp0536 and hp0543 are not necessary for either translocation of CagA or for IL‐8 induction. Our data argue against a translocated IL‐8‐inducing effector protein encoded by the cag‐PAI. We isolated a variant of Hp 26695, which spontaneously switched off its capacity for IL‐8 induction and translocation of CagA, but retained the complete cag‐PAI. We identified a point mutation in gene hp0532, causing a premature translational stop in the corresponding polypeptide chain, providing a putative explanation for the defect in the type IV secretion system of the spontaneous mutant.

A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system

Type I strains of Helicobacter pylori (Hp) use a type IV secretion system (T4SS), encoded by the cag pathogenicity island (cag‐PAI), to deliver the bacterial protein CagA into eukaryotic cells and to induce interleukin‐8 secretion. Translocated CagA is activated by tyrosine phosphorylation involving Src‐family kinases. The mechanism and structural basis for type IV protein secretion is not well understood. We describe here, by confocal laser scanning microscopy and field emission scanning electron microscopy, a novel filamentous surface organelle which is part of the Hp T4SS. The organelle is often located at one bacterial pole but can be induced by cell contact also along the lateral side of the bacteria. It consists of a rigid needle, covered focally or completely by HP0527 (Cag7 or CagY), a VirB10‐homologous protein. HP0527 is also clustered in the outer membrane. The VirB7‐homologous protein HP0532 is found at the base of this organelle. These observations demonstrate for the first time by microscopic techniques a complex T4SS‐associated, sheathed surface organelle reminiscent to the needle structures of bacterial type III secretion systems.

Interaction of Helicobacter pylori with professional phagocytes: role of the cag pathogenicity island and translocation, phosphorylation and processing of CagA

Chronic infection of the human gastric mucosa with Helicobacter pylori is a major cause of gastroduodenal pathologies, including peptic ulcerations, mucosa‐associated lymphoid tissue (MALT) lymphoma and adenocarcinoma. Helicobacter pylori strains carrying the cag pathogenicity island, which encodes an active type IV protein secretion system (cag+ or type I strains), are preferentially associated with strong gastric inflammation and severe disease. We show here that cag+H. pylori strains use the type IV secretion system to inject the bacterial protein CagA into various types of professional phagocytes, including human polymorphonuclear leucocytes (PMNs) and the human and murine macrophage cell lines THP‐1 and J774A.1 CagA is rapidly tyrosine phosphorylated and proteolytically processed to generate a stable 35–45 kDa C‐terminally tyrosine‐phosphorylated protein fragment. H. pylori was efficiently ingested by the different types of phagocytic cells. A chromosomal deletion of the complete pathogenicity island had no significant effect on the rate of ingestion. Furthermore, the survival rate of H. pylori in the phagosome was unchanged between the wild type and a deletion mutant lacking the type IV secretion system. Thus, the type IV secretion system seems to be involved neither in active phagocytosis resistance nor in prolonged survival of the bacteria in phagocytic cells.

The VLT-FLAMES Tarantula Survey - XII. Rotational velocities of the single O-type stars

Context. The 30 Doradus (30 Dor) region of the Large Magellanic Cloud, also known as the Tarantula nebula, is the nearest starburst region. It contains the richest population of massive stars in the Local Group, and it is thus the best possible laboratory to investigate open questions on the formation and evolution of massive stars. Aims. Using ground-based multi-object optical spectroscopy obtained in the framework of the VLT-FLAMES Tarantula Survey (VFTS), we aim to establish the (projected) rotational velocity distribution for a sample of 216 presumably single O-type stars in 30 Dor. The sample is large enough to obtain statistically significant information and to search for variations among subpopulations – in terms of spectral type, luminosity class, and spatial location – in the field of view. Methods. We measured projected rotational velocities, ν_esini, by means of a Fourier transform method and a profile fitting method applied to a set of isolated spectral lines. We also used an iterative deconvolution procedure to infer the probability density, P(ν_e), of the equatorial rotational velocity, ν_e. Results. The distribution of νesini shows a two-component structure: a peak around 80 kms^(-1) and a high-velocity tail extending up to ~600 kms^(-1). This structure is also present in the inferred distribution P(ν_e) with around 80% of the sample having 0 < ν_e ≤ 300 kms^(-1) and the other 20% distributed in the high-velocity region. The presence of the low-velocity peak is consistent with what has been found in other studies for late O- and early B-type stars. Conclusions. Most of the stars in our sample rotate with a rate less than 20% of their break-up velocity. For the bulk of the sample, mass loss in a stellar wind and/or envelope expansion is not efficient enough to significantly spin down these stars within the first few Myr of evolution. If massive-star formation results in stars rotating at birth with a large portion of their break-up velocities, an alternative braking mechanism, possibly magnetic fields, is thus required to explain the present-day rotational properties of the O-type stars in 30 Dor. The presence of a sizeable population of fast rotators is compatible with recent population synthesis computations that investigate the influence of binary evolution on the rotation rate of massive stars. Even though we have excluded stars that show significant radial velocity variations, our sample may have remained contaminated by post-interaction binary products. That the high-velocity tail may be populated primarily (and perhaps exclusively) by post-binary interaction products has important implications for the evolutionary origin of systems that produce gamma-ray bursts.

A C-terminal translocation signal is necessary, but not sufficient for type IV secretion of the Helicobacter pylori CagA protein

Type IV secretion systems are increasingly recognized as important virulence determinants of Gram‐negative bacterial pathogens. While the examination of several type IV‐secreted proteins suggested that their secretion depends on C‐terminal signals, the nature of these signals and their conservation among different systems remain unclear. Here, we have characterized the secretion signal of the Helicobacter pylori CagA protein, which is translocated by the Cag type IV secretion apparatus into eucaryotic cells. The production of fusion proteins of CagA and green fluorescent protein (GFP) did not result in translocation of GFP to epithelial cells, but a fusion of GFP with the CagA C‐terminus exerted a dominant‐negative effect upon wild‐type CagA translocation. We show that CagA translocation depends on the presence of its 20 C‐terminal amino acids, containing an array of positively charged residues. Interestingly, these positive charges are neither necessary nor sufficient for CagA translocation, but replacing the C‐terminal region of CagA with that of other type IV‐secreted proteins reconstitutes CagA translocation competence. Using a novel type IV translocation assay with a phosphorylatable peptide tag, we show that removal of the N‐terminal part of the CagA protein renders the protein translocation‐incompetent as well. Thus, the Cag type IV secretion system seems to diverge from other systems not only with respect to its composition and architecture, but also in terms of substrate recognition and transport.

CagA tyrosine phosphorylation and interleukin-8 induction by Helicobacter pylori are independent from alpAB, HopZ and bab group outer membrane proteins.

In several studies Helicobacter pylori type I strains (cag-positive strains) have been described to translocate their CagA protein into epithelial cells, where it is tyrosine-phosphorylated. The intimate contact allows a Cag-dependent bacteria-to-cell signaling inducing the secretion of the chemokine interleukin-8. Although a contact between the bacterial and the eukaryotic cell is known to be necessary for these signal transduction events the bacterial adhesin and the cellular receptor are unknown, so far. In this study, we investigated the influence of several outer membrane proteins associated with adherence on CagA translocation and IL-8 induction. The quantitative assessment of a cag deletion mutant strain binding to epithelial cells revealed that the Cag secretion apparatus is not primarily necessary for attachment. In contrast, the knockout mutation of the adherence-associated alpAB locus significantly reduced the binding capacity in two independent strains. Despite this partial adherence defect, the alpAB mutation did not affect CagA translocation and IL-8 induction. The mutagenesis of the bab group genes hp317, hp896 and hp1243 in H. pylori 26695 did not influence the Cag-dependent signaling either. No causative linkage could be found between the production of the outer membrane proteins HopZ, OipA or seven additional outer membrane proteins and CagA translocation or IL-8 induction.

The VLT-FLAMES Tarantula Survey. XI. A census of the hot luminous stars and their feedback in 30 Doradus

Context. The VLT-FLAMES Tarantula Survey has an extensive view of the copious number of massive stars in the 30 Doradus (30 Dor) star forming region of the Large Magellanic Cloud. These stars play a crucial role in our understanding of the stellar feedback in more distant, unresolved star forming regions. Aims. The first comprehensive census of hot luminous stars in 30 Dor is compiled within a 10 arcmin (150 pc) radius of its central cluster, R136. We investigate the stellar content and spectroscopic completeness of the early type stars. Estimates were made for both the integrated ionising luminosity and stellar wind luminosity. These values were used to re-assess the star formation rate (SFR) of the region and determine the ionising photon escape fraction. Methods. Stars were selected photometrically and combined with the latest spectral classifications. Spectral types were estimated for stars lacking spectroscopy and corrections were made for binary systems, where possible. Stellar calibrations were applied to obtain their physical parameters and wind properties. Their integrated properties were then compared to global observations from ultraviolet (UV) to far-infrared (FIR) imaging as well as the population synthesis code, Starburst99. Results. Our census identified 1145 candidate hot luminous stars within 150 pc of R136 of which >700 were considered to be genuine early type stars and contribute to feedback. We assess the survey to be spectroscopically complete to 85% in the outer regions (>5 pc) but only 35% complete in the region of the R136 cluster, giving a total of 500 hot luminous stars in the census which had spectroscopy. Only 31 were found to be Wolf-Rayet (W-R) or Of/WN stars, but their contribution to the integrated ionising luminosity and wind luminosity was ~40% and ~50%, respectively. Similarly, stars with Minit > 100 M⊙ (mostly H-rich WN stars) also showed high contributions to the global feedback, ~25% in both cases. Such massive stars are not accounted for by the current Starburst99 code, which was found to underestimate the integrated ionising luminosity of R136 by a factor ~2 and the wind luminosity by a factor ~9. The census inferred a SFR for 30 Dor of 0.073 ± 0.04 M⊙ yr-1. This was generally higher than that obtained from some popular SFR calibrations but still showed good consistency with the far-UV luminosity tracer as well as the combined Hα and mid-infrared tracer, but only after correcting for Hα extinction. The global ionising output was also found to exceed that measured from the associated gas and dust, suggesting that ~6+55-6 % of the ionising photons escape the region. Conclusions. When studying the most luminous star forming regions, it is essential to include their most massive stars if one is to determine a reliable energy budget. Photon leakage becomes more likely after including their large contributions to the ionising output. If 30 Dor is typical of other massive star forming regions, estimates of the SFR will be underpredicted if this escape fraction is not accounted for.

Activation of Helicobacter pylori CagA by tyrosine phosphorylation is essential for dephosphorylation of host cell proteins in gastric epithelial cells

Helicobacter pylori type I strains harbour the cag pathogenicity island (cag‐PAI), a 37 kb sequence, which encodes the components of a type IV secretion system. CagA, the first identified effector protein of the cag‐PAI, is translocated into eukaryotic cells and tyrosine phosphorylated (CagAP‐tyr) by a host cell tyrosine kinase. Translocation of CagA induces the dephosphorylation of a set of phosphorylated host cell proteins of unknown identity. CagA proteins of independent H. pylori strains vary in sequence and thus in the number and composition of putative tyrosine phosphorylation motifs (TPMs). The CagA protein of H. pylori strain J99 (CagAJ99) does not carry any of three putative tyrosine phosphorylation motifs (TPM‐A, TPM‐B or TPM‐C) predicted by the MOTIF algorithm in CagA proteins. CagAJ99 is not tyrosine phosphorylated and is inactive in the dephosphorylation of host cell proteins. By site‐specific mutagenesis, we introduced a TPM‐C into CagAJ99 by replacing a single lysine with a tyrosine. This slight modification resulted in tyrosine phosphorylation of CagAJ99 and host cell protein dephosphorylation. In contrast, the removal of the indigenous TPM‐C from CagAP12 did not abolish its tyrosine phosphorylation, suggesting that further phosphorylated sites are present in CagAP12. By generation of hybrid CagA proteins, a phosphorylation of the most N‐terminal TPM‐A could be excluded. Our data suggest that tyrosine phosphorylation at TPM‐C is sufficient, but not exclusive, to activate translocated CagA. Activated CagAP‐tyr might either convert into a phosphatase itself or activate a cellular phosphatase to dephosphorylate cellular phosphoproteins and modulate cellular signalling cascades of the host.

The R136 star cluster dissected with Hubble Space Telescope/STIS. I. Far-ultraviolet spectroscopic census and the origin of He ii λ1640 in young star clusters

We introduce a Hubble Space Telescope (HST)/Space Telescope Imaging Spectrograph (STIS) stellar census of R136a, the central ionizing star cluster of 30 Doradus. We present low resolution far-ultraviolet STIS spectroscopy of R136 using 17 contiguous 52 arcsec x 0.2 arcsec slits which together provide complete coverage of the central 0.85 parsec (3.4 arcsec). We provide spectral types of 90 per cent of the 57 sources brighter than m(F555W) = 16.0 mag within a radius of 0.5 parsec of R136a1, plus 8 additional nearby sources including R136b (O4 If/WN8). We measure wind velocities for 52 early-type stars from C IV lambda lambda 1548-51, including 16 O2-3 stars. For the first time, we spectroscopically classify all Weigelt and Baier members of R136a, which comprise three WN5 stars (a1-a3), two O supergiants (a5-a6) and three early O dwarfs (a4, a7, a8). A complete Hertzsprung-Russell diagram for the most massive O stars in R136 is provided, from which we obtain a cluster age of 1.5(-0.7)(+0.3) Myr. In addition, we discuss the integrated ultraviolet spectrum of R136, and highlight the central role played by the most luminous stars in producing the prominent He II lambda 1640 emission line. This emission is totally dominated by very massive stars with initial masses above similar to 100M(circle dot). The presence of strong He II lambda 1640 emission in the integrated light of very young star clusters (e.g. A1 in NGC 3125) favours an initial mass function extending well beyond a conventional upper limit of 100M(circle dot). We include montages of ultraviolet spectroscopy for Large Magellanic Cloud O stars in the appendix. Future studies in this series will focus on optical STIS medium resolution observations.