Strukturelle und funktionelle Charakterisierung von Adhesinen und dem Virulenzregulator RovC aus Yersinia pseudotuberculosis

Yersinia has several multifunctional adhesins on its surface that contribute to its pathogenicity. Their expression levels are regulated by several regulatory proteins in response to the rapidly changing environment. The projects presented here involve the structural and functional characterization of adhesins and the virulence regulator RovC of Yersinia pseudotuberculosis.YPIII strain of Y. pseudotuberculosis expresses four Invasins, which are InvA, InvB, InvC and InvD. So far, details on the regulation, structure and function of InvD have been elusive. In this project, the structure of InvD was determined by X-ray crystallography. By several in vitro and ex vivo experiments, the role of InvD in binding VH3/VK1 fragment of the antibody and B cells was established.In addition to InvD, a novel Invasin, InvE has been identified in the IP31758 strain of Y. pseudotuberculosis. In this work, the crystal structure of InvE was solved and the shape was confirmed in solution by small angle X-ray scattering (SAXS). The C-terminal domain of InvE adopts a C-type lectin like fold and shares high structural similarity with the C-type lectin like domain of InvA and Intimin, even though there exists no sequence homology among these proteins.YadA of Y. enterocolitica (ent) and Y. pseudotuberculosis (pstb) exhibit differences in their specificity of extracellular matrix (ECM) substrate binding, which results from a 31 amino acid insertion in YadApstb. The aim of this work was to provide the structural basis for the functional transition by solving the structure of YadApstb and to compare it with the known YadAent structure. For that purpose, the crystal structure of the head domain of YadApstb was solved. The results show that the insertion region is located at the tip, which is likely to change the tip surface and influence ECM specificity.A novel virulence regulator, RovC has been identified in Y. pseudotuberculosis that activates transcription of the Type6-secretion system. To gain insight into the DNA binding mechanism of RovC, its crystal structure was solved. SAXS analysis revealed a hexameric ring shaped structure of RovC, which was validated by Multi Angle Laser-light Scattering and site-directed mutagenesis studies. Furthermore, site-directed mutagenesis approach was used to characterize the DNA binding activity and the model has been proposed in which DNA wraps around the surface of hexameric RovC.Yersinia weist zahlreiche multifunktionelle Adhesine auf der Zell-Oberfläche auf, die einen wichtigen Beitrag zur Pathogenizität leisten. Die Expression der Adhesine wird dabei durch verschiedene regulatorische Proteine gesteuert, um sich an die wechselnden Lebensräume und Wachstumsbedingungen anzupassen. Die hier präsentierten Projekte behandeln die strukturelle und funktionelle Charakterisierung verschiedener Adhesine und des Virulenz-Regulators RovC von Yersinia pseudotuberculosis. Der Stamm YPIII von Y. pseudotuberculosis exprimiert die vier Invasine InvA, InvB, InvC und InvD. Details zur Regulation, Struktur und Funktion von InvD sind bisher unbekannt. In diesem Projekt wurde daher die Struktur von InvD mittels Röntgenkristallographie aufgeklärt. Weiterhin konnte eine spezifische Bindung von InvD an VH3/VK1 Antikörper und B-Zellen nachgewiesen werden. Im Stamm IP31758 wurde kürzlich ein weiteres Invasin, InvE, identifiziert, dessen Struktur in dieser Arbeit aufgeklärt wurde. Die Faltung der C-terminalen Domäne entspricht der von C-Typ Lektin-ähnlichen Domänen und trotz fehlender Sequenzhomologie weist diese Domäne von InvE sehr große strukturelle Ähnlichkeiten zu den C-Typ Lektin-ähnlichen Domänen von InvA und Intimin auf. YadA-Varianten von Y. enterocolitica (ent) und Y. pseudotuberculosis (pstb) weisen Unterschiede bezüglich ihrer Bindungsspezifität gegenüber Komponenten der Extrazellulären Matrix (EZM) auf, die auf einer Insertion von 31 Aminosäuren in YadApstb basieren. Ziel dieser Arbeit war, den Einfluss dieser Insertion über die Strukturaufklärung von YadApstb und den Vergleich mit der bekannten Struktur von YadAent zu analysieren. Dazu wurde die Struktur von YadApstb in dieser Arbeit aufgeklärt. Diese zeigt, dass die Insertion an der Spitze des YadApstb-Trimers liegt, die Oberfläche dieser Region verändert und darüber wahrscheinlich auch die Bindespezifität gegenüber EZM-Komponenten verändert wird. Ein neuer Virulenz-Regulator, RovC, wurde kürzlich in Y. pseudotuberculosis identifiziert, der die Transkription von Genen des Typ6-Sekretionssystems reguliert. Um einen Einblick in den Mechanismus der DNA-Bindung zu erhalten, wurde die RovC-Struktur in dieser Arbeit aufgeklärt. Über verschiedene Experimente wurde gezeigt, dass RovC einen hexameren Ring in Lösung ausbildet. Die DNA-Bindung wurde durch Mutagenese charakterisiert und basierend auf diesen Daten wurde ein Modell postuliert, in dem sich die DNA um das RovC-Hexamer windet

A study on predictors of treatment outcome among children registered under DOTS in district Tarn Taran, Punjab

Background: Globally, tuberculosis remains an important cause of morbidity and mortality for children. Diagnosis and management of childhood TB especially Extra pulmonary tuberculosis is challenging. Method:  A cross-sectional study was conducted on 0-14 year children who were registered and being treated, in district Tarn Taran, Punjab from 1st January 2018 to 31st December 2018. The treatment outcome with their clinico-demographic determinants was ascertained. Data management and analysis was done by using Microsoft excel and SPSS. Results: Out of 62 registered patients, 62.9% of the children were in age group of 11-14 years. 67.7% were females. The various treatment outcomes observed were-cured 32.3%, treatment completed 61.3%, lost to follow up 1.6%, regimen changed 1.6%, not evaluated 3.2%. The success rate (cured+ treatment completed) was 93.6%. On statistical analysis, it was observed that age (p= 0.002), site of disease (p=0.000), contact history (p=0.012) and diagnostic method (0.000) was significantly associated with the treatment outcome whereas gender, area of residence and type of case had no association with the treatment outcome in children. Conclusions:  We also found that the overall treatment success rate was 93.6%. Increased focus be on those with household contact with TB

Indigenous uses of plants in different women ailments in Garhwal region

A survey of villages of Garhwal region was done to identify medicinally important plants used by local peoples of that region against different women ailments. Paper deals with 67 plant species of 42 families as a herbal medicines described by local peoples used for different women ailments i.e., Leucorrhoea, menstrual disorders, Menorrhagia, Poor lactation, uterus infection, abortion, Deliverythe samples

Congruence Of Record Keeping of Tuberculosis Patients by Field Interviews in A Community Block of District Amritsar

Introduction: Good recording practices are necessary for effective patient management. When high-quality data are available, success can be documented and corrective actions taken to address problems that are identified. Objective: To assess the accuracy of record keeping of TB patients by field interviews Methods: The study was conducted on a total of 326 patients residing in the area of PHC Verka and registered with District Tuberculosis Centre, Amritsar. House to house visits were done. Outcome of the patient according to the TB register and according to interview were compared. Results: According to record out of 228 patients, the outcome of 105(46.10%), patients was cured, 105(46.10%) patients had completed their treatment, 10(4.40%) had defaulted, 6(2.6%) were failure and 2(0.9%) had transferred out. On interviewing the patients the outcome of 98(43%) was cured, 95(41.70%) were treatment completed, 29(12.70%), defaulted and 6(2.6%) were failure. Discrepancies were found between outcome recorded on TB register and as obtained on interviewing the patient. The treatment outcome according to record and outcome according to interview, was found to be statistically significant (p=0.005) Conclusion: Present study reveals that there is an urgent need to strengthen performance of the programme by proper record-keeping and efficient supervision by district health officials

Prevalence of Association of Dietary Habits with Chronic Energy Deficiency in Ethnic Adult Punjabi Population

Background: Food is a basic thing without which, life is impossible. Without proper food, body will not thrive to its optimum potential and Chronic Energy Deficiency (CED) is the state, where there is a lack of food intake over a prolonged period of time. Aims & Objectives: To find the association of dietary habits with CED in ethnic Punjabi population. Methodology: The study was a cross-sectional study using simple random sampling technique. Taking the prevalence of CED as 30% in India, the sample size came out to be 336 but, 400 subjects were selected equally from rural and urban areas for convenience. Results: Subjects having lesser calorie intake than RDA, developed CED more 62(82.9%) than meeting RDA 13(17.1%). Subjects who skipped meals had higher CED rates. Pure vegetarians had higher rates of CED 71(93.4%) than non-vegetarians 5(6.6%). People consuming milk or milk products more than once daily developed lesser CED 1(1.3%) than those taking once daily 15(19.7%) and ones taking only once a week 60(79.0%). Conclusion: According to the study, persons with adequate calorie intake, who do not skip meals, take some kind of non-vegetarian food, and consume milk on daily basis have lesser tendency to develop CED

The invasin D protein fromYersinia pseudotuberculosisselectively binds the Fab region of host antibodies and affects colonization of the intestine.

Yersinia pseudotuberculosis is a Gram-negative bacterium and zoonotic pathogen responsible for a wide range of diseases, ranging from mild diarrhea, enterocolitis, lymphatic adenitis to persistent local inflammation. TheY. pseudotuberculosisinvasin D (InvD) molecule belongs to the invasin (InvA)-type autotransporter proteins, but its structure and function remain unknown. In this study, we present the first crystal structure of InvD, analyzed its expression and function in a murine infection model, and identified its target molecule in the host. We found that InvD is induced at 37°C and expressed in vivo2-4 days after infection, indicating that InvD is a virulence factor. During infection, InvD was expressed in all parts of the intestinal tract, but not in deeper lymphoid tissues. The crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold, that, apart from the canonical β-sheets, comprises various modifications of and insertions into the Ig-core structure. We identified the Fab fragment of host-derived IgG/IgA antibodies as the target of the adhesion domain. Phage display panning and flow cytometry data further revealed that InvD exhibits a preferential binding specificity toward antibodies with VH3/VK1 variable domains and that it is specifically recruited to a subset of B cells. This finding suggests that InvD modulates Ig functions in the intestine and affects direct interactions with a subset of cell surface-exposed B-cell receptors. In summary, our results provide extensive insights into the structure of InvD and its specific interaction with the target molecule in the host

Yersinia Pseudotuberculosis Modulates Regulatory T Cell Stability via Injection of Yersinia Outer Proteins in a Type III Secretion System-Dependent Manner.

Adaptive immunity is essentially required to control acute infection with enteropathogeni

Yersinia Pseudotuberculosis Modulates Regulatory T Cell Stability via Injection of Yersinia Outer Proteins in a Type III Secretion System-Dependent Manner

Adaptive immunity is essentially required to control acute infection with enteropathogenic Yersinia pseudotuberculosis (Yptb). We have recently demonstrated that Yptb can directly modulate naïve CD4+ T cell differentiation. However, whether fully differentiated forkhead box protein P3 (Foxp3+) regulatory T cells (Tregs), fundamental key players to maintain immune homeostasis, are targeted by Yptb remains elusive. Here, we demonstrate that within the CD4+ T cell compartment Yptb preferentially targets Tregs and injects Yersinia outer proteins (Yops) in a process that depends on the type III secretion system and invasins. Remarkably, Yop-translocation into ex vivo isolated Foxp3+ Tregs resulted in a substantial downregulation of Foxp3 expression and a decreased capacity to express the immunosuppressive cytokine interleukin-10 (IL-10). Together, these findings highlight that invasins are critically required to mediate Yptb attachment to Foxp3+ Tregs, which allows efficient Yop-translocation and finally enables the modulation of the Foxp3+ Tregs' suppressive phenotype

RovC - a novel type of hexameric transcriptional activator promoting type VI secretion gene expression.

Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment