Bacteriophages of Shiga toxin-producing Escherichia coli and their contribution to pathogenicity

Shiga toxins (Stx) of Shiga toxin-producing Escherichia coli (STEC) are generally encoded in the genome of lambdoid bacteriophages, which spend the most time of their life cycle integrated as prophages in specific sites of the bacterial chromosome. Upon spontaneous induction or induction by chemical or physical stimuli, the stx genes are co-transcribed together with the late phase genes of the prophages. After being assembled in the cytoplasm, and after host cell lysis, mature bacteriophage particles are released into the environment, together with Stx. As members of the group of lambdoid phages, Stx phages share many genetic features with the archetypical temperate phage Lambda, but are heterogeneous in their DNA sequences due to frequent recombination events. In addition to Stx phages, the genome of pathogenic STEC bacteria may contain numerous prophages, which are either cryptic or functional. These prophages may carry foreign genes, some of them related to virulence, besides those necessary for the phage life cycle. Since the production of one or more Stx is considered the major pathogenicity factor of STEC, we aim to highlight the new insights on the contribution of Stx phages and other STEC phages to pathogenicity

Coronary Spasm: Ethnic and Sex Differences

Coronary spasm (CS), which may occur at the epicardial (focal or diffuse spasm) and/or microvascular (microvascular spasm) level, is a well-established cause of myocardial ischaemia, in particular in patients with anginal chest pain despite unobstructed coronary arteries. The diagnosis of CS can be confirmed during coronary angiography by an additional provocation test with vasoactive substances such as acetylcholine. Due to partially inconsistent data from large clinical studies, especially between Asian and white CS patients, ethnic differences concerning the prevalence and angiographic patterns of CS seem to exist. Furthermore, several studies in patients with coronary vasomotor disorders pointed towards differences among male and female CS patients. This article gives an overview of ethnic- and sex-related differences in patients with CS

Prognostic association of plasma NT-proBNP levels in patients with microvascular angina -A report from the international cohort study by COVADIS-

Background The aim of this study was to assess the prognostic association of plasma levels of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) with clinical outcomes of patients with microvascular angina (MVA). Methods In this international prospective cohort study of MVA by the Coronary Vasomotor Disorders International Study (COVADIS) group, we examined the association between plasma NT-proBNP levels and the incidence of major adverse cardiovascular events (MACE), including cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, and hospitalization due to heart failure or unstable angina. Results We examined a total of 226 MVA patients (M/F 66/160, 61.9 ± 10.2 [SD] yrs.) with both plasma NT-proBNP levels and echocardiography data available at the time of enrolment. The median level of NT-proBNP level was 94 pg/ml, while mean left ventricular ejection fraction was 69.2 ± 10.9 % and E/e’ 10.7 ± 5.2. During follow-up period of a median of 365 days (IQR 365–482), 29 MACEs occurred. Receiver-operating characteristics curve analysis identified plasma NT-proBNP level of 78 pg/ml as the optimal cut-off value. Multivariable logistic regression analysis revealed that plasma NT-proBNP level ≥ 78 pg/ml significantly correlated with the incidence of MACE (odds ratio (OR) [95 % confidence interval (CI)] 3.11[1.14–8.49], P = 0.001). Accordingly, Kaplan-Meier survival analysis showed a significantly worse prognosis in the group with NT-proBNP ≥ 78 (log-rank test, P < 0.03). Finally, a significant positive correlation was observed between plasma NT-proBNP levels and E/e’ (R = 0.445, P < 0.0001). Conclusions These results indicate that plasma NT-proBNP levels may represent a novel prognostic biomarker for MVA patients

Molecular genetic examinations of the expression of type III effector NleA 4795 in Shiga toxin-producing Escherichia coli

Shiga Toxin-produzierende E. coli (STEC) gelten weltweit als wichtige Erreger von lebensmittelbedingten Infektionen und können zu schweren Erkrankungen wie der hämorrhagischen Colitis und dem lebensbedrohlichen hämolytisch-urämischen Syndrom führen. Die Bakterien kolonisieren den Dickdarm des Menschen, wo sie in der Regel zur Ausbildung von charakteristischen ?Attaching und Effacing?-Läsionen führen. Verantwortlich dafür ist eine Pathogenitätsinsel, bezeichnet als ?Locus of Enterocyte Effacement? (LEE), sowie die darauf kodierten Komponenten eines Typ III Sekretionssystems, über das die Bakterien Effektorproteine direkt in die Wirtszellen injizieren können. Zusätzlich zu den LEE-kodierten Effektoren existiert eine große Anzahl an Effektorproteinen, deren kodierende Sequenzen außerhalb der Pathogenitätsinsel lokalisiert sind. Darunter auch der ?Non-LEE encoded Effector A? (NleA), der im Genom von kryptischen oder induzierbaren Prophagen kodiert ist und unter den pathogenen E. coli Stämmen weitverbreitet vorkommt. Im Rahmen dieser Arbeit wurde die Expression und Regulation der nleA-Variante nleA4795 des E. coli O84:H4 Stammes 4795/97 untersucht, welche auf dem Shiga Toxin-konvertierenden Phagen BP-4795 lokalisiert ist. Dabei wurde mit Hilfe eines Luciferase-Reportersystems und der quantitativen Real-Time PCR der Einfluss von verschiedenen Umweltreizen getestet sowie die Abhängigkeit der nleA4795-Expression von bestimmten Regulatorproteinen untersucht. Unter den analysierten Umweltbedingungen kristallisierten sich bestimmte NaCl- und KCl-Konzentrationen als induzierend für die Expression von nleA4795 heraus und lassen daher auf eine osmotisch bedingte Aktivierung schließen. Eine zunächst vermutete Induktion der nleA4795-Expression durch Quorum Sensing in vorkonditioniertem Medium konnte nicht nachgewiesen werden, da keiner der bislang bekannten Autoinducer einen positiven Einfluss ausübte. Die erhöhte Expression von nleA4795 konnte nachfolgend mit einem reduzierten Nährstoffgehalt assoziiert und somit eine Korrelation zwischen der nleA4795-Expression und bakteriellen Stressantwort-Systemen hergestellt werden. Des Weiteren wurde untersucht, ob ein Zusammenhang zwischen der nleA4795-Expression und der Induktion des Phagen BP-4795 sowie der damit verbundenen Expression von Shiga Toxin besteht. Durch Induktionsexperimente mit Norfloxacin konnte jedoch im Gegensatz zur Expression von Shiga Toxin keine Aktivierung, sondern eine starke Repression der nleA4795-Expression nachgewiesen werden. Die Untersuchungen auf regulatorischer Ebene zeigten die Abhängigkeit der nleA4795-Expression von den drei LEE-kodierten Regulatoren Ler, GrlA und GrlR sowie von den außerhalb des LEE kodierten Pch-Regulatoren. Für den ebenfalls außerhalb der Pathogenitätsinsel kodierten Regulator EtrA konnte kein Einfluss auf die Expression von nleA4795 nachgewiesen werden. Zudem wurden die Regulatorproteine Ler, GrlA und PchA mit Hilfe von Electrophoretic Mobility Shift Assays (EMSA) auf eine direkte Bindung an die nleA4795-Promotorregion untersucht. Die beiden Regulatoren GrlA und PchA zeigten jedoch keine spezifische Bindung und wurden demzufolge als indirekte Regulatoren der nleA4795-Expression einstuft. Für den Regulator Ler konnte hingegen eine direkte Bindung an bestimmte Bereiche der nleA4795-Promotorregion nachgewiesen und somit eine Integration von nleA4795 in den Ler-vermittelten Regulationskreis des LEE bestätigt werden.Shiga toxin-producing E. coli (STEC) are the causative agents of foodborne infections in many countries and can lead to severe diseases like hemorrhagic colitis or the life-threatening hemolytic uremic syndrome. The bacteria colonize the human intestine where they normally cause the formation of characteristic ?attaching and effacing?-lesions. Essential for this effect is a pathogenicity island, termed as ?locus of enterocyte effacement? (LEE), that encodes the components of a type III secretion system and several effector proteins, which are translocated directly into the host cells by the TTSS machinery. In addition to the LEE-encoded effectors a large number of effector proteins have been identified which are encoded outside of the pathogenicity island. Among these is the ?non-LEE encoded effector A? (NleA), which is encoded on cryptic or inducible prophages and is widely distributed among pathogenic E. coli strains. In the present study, the expression and regulation of the nleA-variant nleA4795 of E. coli O84:H4 strain 4795/97 was investigated, which is located on the Shiga toxin-converting bacteriophage BP-4795. Therefore, different environmental conditions as well as certain regulatorproteins were tested on their influence on nleA4795-expression using a luciferase-reportersystem and the quantitative real-time PCR. Among the analyzed environmental factors, certain concentrations of NaCl and KCl were identified to activate nleA4795-expression, indicating an osmotic-based influence. The suggested induction of nleA4795 in preconditioned medium due to quorum sensing could not be confirmed, since none of the so far known autoinducers showed a positive influence on the expression. The increased expression of nleA4795 could be associated with a reduced amount of nutrients in subsequent investigations and therefore demonstrated a relation between nleA4795-expression and bacterial stress-response-systems. Furthermore, a possible correlation of nleA4795-expression with the induction of phage BP 4795 and Shiga toxin-expression was analyzed. Different from the expression of Shiga toxin, induction-experiments with norfloxacin showed no activation, but a strong repression of nleA4795-expression. Analysis of the regulatory level demonstrated that the expression of nleA4795 depends on the three LEE-encoded regulators Ler, GrlA und GrlR as well as on the Pch-regulators, which are encoded outside of the LEE. The non-LEE encoded regulator EtrA showed no influence on the expression of nleA4795. In addition, the regulator proteins Ler, GrlA and PchA were tested for direct binding to the nleA4795-promoterregion. Regulators GrlA and PchA showed no specific binding and were therefore classified as indirect regulators of nleA4795-expression. In contrast, regulator Ler showed a specific binding to different areas of the nleA4795-promoter region and thereby confirmed the integration of nleA4795 in the Ler-mediated circuit of LEE-regulation