Salmonella Pathogenicity Island 2 Mediates Protection of Intracellular Salmonella from Reactive Nitrogen Intermediates

Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid. A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is essential for virulence in mice, as well as survival and multiplication within macrophages. Reactive nitrogen intermediates (RNI) synthesized by inducible nitric oxide synthase (iNOS) are involved in the control of intracellular pathogens, including S. typhimurium. We studied the effect of Salmonella infection on iNOS activity in macrophages. Immunofluorescence microscopy demonstrated efficient colocalization of iNOS with bacteria deficient in SPI2 but not wild-type Salmonella, and suggests that the SPI2 system interferes with the localization of iNOS and Salmonella. Furthermore, localization of nitrotyrosine residues in the proximity was observed for SPI2 mutant strains but not wild-type Salmonella, indicating that peroxynitrite, a potent antimicrobial compound, is excluded from Salmonella-containing vacuoles by action of SPI2. Altered colocalization of iNOS with intracellular Salmonella required the function of the SPI2-encoded type III secretion system, but not of an individual “Salmonella translocated effector.” Inhibition of iNOS increased intracellular proliferation of SPI2 mutant bacteria and, to a lesser extent, of wild-type Salmonella. The defect in systemic infection of a SPI2 mutant strain was partially restored in iNOS−/− mice. In addition to various strategies to detoxify RNI or repair damage due to RNI, avoidance of colocalization with RNI is important in adaptation of a pathogen to an intracellular life style

Evolution mikrobieller Pathogenität: Salmonella Pathogenitätsinsel 2 als Paradigma für horizontalen Gentransfer

Diese Arbeit setzt sich mit den molekularen Mechanismen der Evolution von Virulenzfaktoren in Salmonella auseinander. Es wurde ein auf Sequenzvergleichen basierendes Verfahren eingesetzt, um neue lateral erworbene Elemente zu identifizieren, die möglicherweise Hinweise auf die Entwicklung von Virulenz und Wirtsspezifität innerhalb der Gattung Salmonella geben können. Mit Hilfe genetischer Analysen sollte darüber Aufschluss gewonnen werden, auf welche Weise diese genetischen Elemente in neue Wirtsgenome gelangen können. Des Weiteren wurde anhand der Salmonella Pathogenitätsinsel 2 (SPI2) nach möglichen Vehikeln und Transfermechanismen für den horizontalen Gentransfer gesucht. Es wurde analysiert, wie neu erworbene genetische Elemente in das regulatorische Netzwerk neuer Wirte integriert werden können und ob es Zusammenhänge zwischen der genetischen Ausstattung mit Virulenzmodulen und der Wirtsspezifität verschiedener Salmonella-Serotypen gibt. Dabei wurde festgestellt, dass einzelne Effektoren des SPI2-Virulons, die zum Teil außerhalb des SPI2-Locus im Genom kodiert sind, sehr heterogen innerhalb von Salmonella spp. verteilt sind. Diese Variabilität kann als Hinweis darauf gewertet werden, dass sich diese Faktoren nicht als ein initialer Komplex in der „Ur-Salmonella“ manifestiert haben, wie es im Invasions-Virulon von SPI1 der Fall ist. Vielmehr sind die SPI2-Effektoren vermutlich in mehreren Schritten im Laufe der Evolution in das Genom von Salmonella spp. gelangt und möglicherweise auch z.T. wieder deletiert worden. Die Bedeutung der Verteilung dieser Effektorproteine für die Virulenz und die Wirtsspezifität von Salmonella wird auch dadurch offensichtlich, dass S. bongori als Besiedler kaltblütiger Wirbeltiere keines dieser zum SPI2-Virulon gehörigen Gene besitzt, die im Zusammenspiel das intrazelluläre Replizieren innerhalb warmblütiger Wirbeltiere ermöglichen. Das Kernstück des SPI2-Virulons, das für das intrazelluläre Replizieren und die systemische Ausbreitung von S. enterica im Wirtsorganismus verantwortlich ist, konnte erfolgreich transferiert werden. Der Einbau des SPI2-TTSS im SPI2-negativen System von S. bongori ermöglichte die heterologe Expression von SPI2-abhängigen Genen und die Sekretion von SPI2-Effektorproteinen in vitro. Durch die Etablierung des SifA-Phänotyps und den Nachweis der intrazellulären Lokalisation von SseF konnte gezeigt werden, dass das transferierte SPI2-TTSS zur heterologen Translokation von SPI2-Effektorproteinen aus S. bongori in die eukaryontische Zielzelle in der Lage ist

Ultrasound screening for asymptomatic carotid stenosis in subjects with calcifications in the area of the carotid arteries on panoramic radiographs: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Directed ultrasonic screening for carotid stenosis is cost-effective in populations with > 5% prevalence of the diagnosis. Occasionally, calcifications in the area of the carotid arteries are incidentally detected on odontological panoramic radiographs. We aimed to determine if directed screening for carotid stenosis with ultrasound is indicated in individuals with such calcifications.</p> <p>Methods</p> <p>This was a cross-sectional study. Carotid ultrasound examinations were performed on consecutive persons, with findings of calcifications in the area of the carotid arteries on panoramic radiography that were otherwise eligible for asymptomatic carotid endarterectomy.</p> <p>Results</p> <p>Calcification in the area of the carotid arteries was seen in 176 of 1182 persons undergoing panoramic radiography. Of these, 117 fulfilled the inclusion criterion and were examined with carotid ultrasound. Eight persons (6.8%; 95% CI 2.2-11.5%) had a carotid stenosis - not significant over the 5% pre-specified threshold (p = 0.232, Binomial test). However, there was a significant sex difference (p = 0.008), as all stenoses were found in men. Among men, 12.5% (95%CI 4.2-20.8%) had carotid stenosis - significantly over the 5% pre-specified threshold (p = 0.014, Binomial test).</p> <p>Conclusions</p> <p>The incidental finding of calcification in the area of the carotid arteries on panoramic radiographs should be followed up with carotid screening in men that are otherwise eligible for asymptomatic carotid endarterectomy.</p> <p>Trial Registration</p> <p>The study was registered at <url>http://www.clinicaltrials.gov</url>; <a href="http://www.clinicaltrials.gov/ct2/show/NCT00514644">NCT00514644</a></p

Modelling of the regulation of the hilA promoter of type three secretion system of Salmonella enterica serovar Typhimurium

One of the most common modes of secretion of toxins in gram-negative bacteria is via the type three secretion system (TTSS), which enables the toxins to be specifically exported into the host cell. The hilA gene product is a key regulator of the expression of the TTSS located on the pathogenicity island (SPI-1) of Salmonella enterica serovar Typhimurium. It has been proposed earlier that the regulation of HilA expression is via a complex feedforward loop involving the transactivators HilD, HilC and RtsA. In this paper, we have constructed a mathematical model of regulation of hilA-promoter by all the three activators using two feedforward loops. We have modified the model to include additional complexities in regulation such as the proposed positive feedback and cross regulations of the three transactivators. Results of the various models indicate that the basic model involving two Type I coherent feedforward loops with an OR gate is sufficient to explain the published experimental observations. We also discuss two scenarios where the regulation can occur via monomers or heterodimers of the transactivators and propose experiments that can be performed to distinguish the two modes of regulator function

Differentially Evolved Genes of Salmonella Pathogenicity Islands: Insights into the Mechanism of Host Specificity in Salmonella

BACKGROUND: The species Salmonella enterica (S. enterica) includes many serovars that cause disease in avian and mammalian hosts. These serovars differ greatly in their host range and their degree of host adaptation. The host specificity of S. enterica serovars appears to be a complex phenomenon governed by multiple factors acting at different stages of the infection process, which makes identification of the cause/s of host specificity solely by experimental methods difficult. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have employed a molecular evolution and phylogenetics based approach to identify genes that might play important roles in conferring host specificity to different serovars of S. enterica. These genes are 'differentially evolved' in different S. enterica serovars. This list of 'differentially evolved' genes includes genes that encode translocon proteins (SipD, SseC and SseD) of both Salmonella pathogenicity islands 1 and 2 encoded type three secretion systems, sptP, which encodes an effector protein that inhibits the mitogen-activated protein kinase pathway of the host cell, and genes which encode effector proteins (SseF and SifA) that are important in placing the Salmonella-containing vacuole in a juxtanuclear position. CONCLUSIONS/SIGNIFICANCE: Analysis of known functions of these 'differentially evolved genes' indicates that the products of these genes directly interact with the host cell and manipulate its functions and thereby confer host specificity, at least in part, to different serovars of S. enterica that are considered in this study

Precise measurement of the W-boson mass with the CDF II detector

We have measured the W-boson mass MW using data corresponding to 2.2/fb of integrated luminosity collected in proton-antiproton collisions at 1.96 TeV with the CDF II detector at the Fermilab Tevatron collider. Samples consisting of 470126 W->enu candidates and 624708 W->munu candidates yield the measurement MW = 80387 +- 12 (stat) +- 15 (syst) = 80387 +- 19 MeV. This is the most precise measurement of the W-boson mass to date and significantly exceeds the precision of all previous measurements combined

Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane

The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K6R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K6R mutant. At later times, fewer SCV were decorated with SopB-K6R compared with SopB. Instead SopB-K6R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization

Chemistry with Controlled Ions

This chapter focuses on the application of controlled-molecule techniques for precise studies of ion-molecule reactions in the gas phase. It gives an overview of the most important techniques presently used for the control of the translational motion, internal quantum states, and structural properties of molecular ions and neutral molecules in the gas phase. Besides the control of the translational motion, preparation of the internal quantum state of molecular ions is a main prerequisite on the way to controlling chemical reactions. Since the 1980s, (multiphoton) photoionization has been a key method for preparing molecular ions in selected internal states. Various variants of this approach have been implemented. The chapter further gives a brief outline of salient concepts of ion-molecule reaction dynamics which are relevant for the present discussion. Finally, the chapter presents some illustrative examples in which cold- and controlled-molecule techniques have been used to study the mechanisms and dynamics of ionic reactions