Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular. / En las últimas dos décadas se ha registrado un incremento significativo en la detección de cepas hipervirulentas y multi-resistentes del patógeno bacteriano Klebsiella pneumoniae, lo cual estaría ligado con el alto dinamismo y la rápida evolución de su genoma. Dentro de los elementos genéticos móviles (EGMs) que determinan dicho dinamismo se encuentran las islas genómicas (IGs), que corresponden a fragmentos de DNA de hasta 200 kb que se integran preferentemente en genes que codifican tRNAs (tDNAs). Se ha demostrado que las IGs de K. pneumoniae y otras enterobacterias portan una gran variedad de genes relacionados con virulencia y resistencia a antibióticos, por lo que su identificación y caracterización a partir del creciente número de genomas secuenciados de esta especie resulta altamente relevante. En esta dirección, el presente trabajo describe el desarrollo de una herramienta para identificar IGs asociadas a tDNAs que se basa en el análisis del contexto genómico de dichos genes, es decir, la organización e identidad de los genes que se encuentran adyacentes en el cromosoma. En una primera etapa, el programa identifica los tDNAs y su contexto para luego clasificarlos y nombrarlos. En una segunda etapa, el programa analiza la continuidad del contexto genómico conservado de cada tDNA y determina la presencia de IGs cuando una disrupción es identificada. El programa creado, se utilizó para analizar el genoma de 50 cepas de K. pneumoniae, donde los resultados obtenidos fueron comparados con la identificación de IGs realizada manualmente y curada en un estudio previo realizado por nuestro grupo. Como resultado, se logró identificar correctamente un 99,3% de todos los tDNAs presentes en dichas cepas, además de 303 IGs de un total de 308 IGs detectadas manualmente. Adicionalmente, el programa mostró un desempeño satisfactorio, aunque mejorable en la detección de integrasas y repetidos directos, detectando ambos elementos en 148 de las IGs identificadas. Finalmente, se comparó el desempeño del programa desarrollado y otros programas para la identificación de IGs en la detección y delimitación de la isla GIE492 caracterizada experimentalmente en un trabajo previo de nuestro grupo, determinando que la detección hecha por el programa desarrollado por nuestro grupo es más sensible y precisa que cualquiera de los otros programas utilizados. / In the last two decades, there has been a significant increase in the detection of hypervirulent and multi-resistant strains of the bacterial pathogen Klebsiella pneumoniae, which would be related to the high dynamism and rapid evolution of its genome. Within the mobile genetic elements (EGMs) that determine this dynamism are Genomic Islands (IGs), which correspond to DNA fragments up to 200 kb that are preferentially integrated into genes encoding tRNAs (tDNAs). It has been shown that IGs from K. pneumoniae and other species from Enterobacteriaceae carry a wide variety of genes related to virulence and antimicrobial drug resistance. Thus, their identification and characterization among the increasing number of sequenced genomes from this species is highly relevant. In this direction, this work describes the development of a bioinformatics tool intended to identify IGs associated to tDNAs based on the analysis of the genomic context of these genes, that is, the organization and identity of the genes that are adjacent to tDNAs in the chromosome. In the first step, the program identifies all the tDNAs and their contexts to classify and name them. In a second step, the program analyzes the continuity of the conserved genomic context for each tDNA, determining the presence of a putative IG when a disruption of such continuity is detected. Our program was used to analyze the genome of a set of 50 K. pneumoniae strains, where the results obtained were compared with the identification of IGs performed and cured manually in a previous study conducted by our group. As a result, 99.3% of all the tDNAs present in the strains were correctly identified. Also, the software predicted 303 IGs from a total of 308 IGs detected manually. Additionally, the program showed a good performance in the identification of integrases and direct repeats, detecting both elements in 148 of the IGs, although there is still room for improvement. Finally, we compared the performance of our software and other programs for the identification of IGs, in the detection and precise delimitation of the xi GIE492 island, which was characterized experimentally in a previous work of our group. We determined that the detection made by our program was more sensitive and precise than any of the other programs tested.

Transcription factors (TFs) are proteins that respond to a specific chemical signal and bind to DNA. In some bacteria, TFs control transition metal ion homeostasis by specifically binding with a particular metal ion or ions and then interacting with DNA. Although most first row metal ions are required as micronutrients for life, many of them can cause cellular damage or death if their concentrations are too high; this makes these TFs and their biological interactions excellent targets for drug development. The bacteria, Streptococcus pneumoniae is a pathogenic microorganism responsible for a range of diseases that target the young and the old, including pneumonia, meningitis, and bacteremia. Herein, we describe our efforts to study the Streptococcus pneumoniae TF responsible for copper(II) ion homeostasis. This thesis describes the classical biochemical techniques used to over-express and purify CopR. It also describes a series of preliminary characterization data associated with this novel copper(II)-dependent TF.

No description available.

No description available.

As vacinas conjugadas contra Streptococcus pneumoniae mostram-se eficazes na redução da doença pneumocócica, no entanto depois de sua introdução tem se verificado a substituição de sorotipos. Para evitar estes problemas o emprego de proteínas da própria bactéria como carreadoras tem sido usado. Neste trabalho a PspA foi conjugada ao sorotipo 6B de S. pneumoniae por dois métodos de conjugação. Inicialmente a PspA clado 1 e 3 foram purificadas, recuperando as proteínas com mais de 90% de pureza. A conjugação intermediada pelo agente ativador cloreto de 4-(4,6-dimetoxi-1,3,5-triazin-2-il-)-4-metilmorfolino (DMT-MM) permitiu rendimentos de Ps entre 20 e 30%, no entanto este tipo de conjugado não foi imunogênico resultando na não indução de anticorpos anti-PspA. Por outro lado a conjugação por aminação redutiva possibilitou obter rendimentos de Ps entre 50 e 60% e os conjugados induziram elevados títulos de anticorpos anti-PspA em camundongos Balb/c, que foram capazes de promover a fagocitose da bactéria; no entanto, a resposta de anticorpos anti-Ps 6B foi baixa. / Conjugate vaccines against Streptococcus pneumonaie have had an important public health benefit; nevertheless after its introduction it has been observed a serotype replacement. To solve this problems conjugate vaccines using pneumococcal surface proteins as carriers has been studied. In this work, Pneumococal surface protein A (PspA) was employed as a carrier protein, conjugated to serotype 6B. Initially PspA clade 1 and PspA clade 3 were purified; both proteins were recovered with more than 90% purity. The conjugation mediated by the activating agent 4- (4,6-dimethoxy-1,3,5-triazin-2-yl -) - 4-methylmorpholine chloride(DMT-MM) allowed Ps yields between 20 and 30%, however this conjugation chemistry had a negative impact on the immunogenicity of the protein. On the other hand conjugation by reductive amination led to Ps yields between 50 and 60% and induced high anti-PspA antibodies titers in Balb /c mice that were able to promote phagocytosis of the bacterium; however, polysaccharide 6B induced low antibody titers.

With regard to infectious diseases, the most economical, direct, and efficient way to prevent them is timely inoculation and a comprehensive policy of vaccination. Such steps not only reduce the overall mortality rate, but also lessen a patient¡¦s susceptibility to serious complications once infected, and therefore their length of hospital stay. It is the foundation of disease prevention in all countries, and should be the primary focus of every public health department. This survey is based on a health belief model and a self-constructed questionnaire. Its sample base are parents whose children have visited one of two local hospitals, each of which is in a different administrative region. A total of 350 questionnaires were distributed. Recoveries were 270, of which 237 were useable. The effective response rate, therefore, is 67.7%. The useable recoveries were analyzed by SPSS, 17th edition, and verified and assumed by mean, standard deviation, t-test, one-way ANOVA, Pearson correlation analysis and Logistic regression analysis. The most influential factors on parents¡¦ decision whether or not to accept streptococcus pneumoniae vaccination (SPV) were as follows: 1.The greater the understanding of SPV and its policy, the greater the number of vaccinations 2.The perceived importance of good health 3. Age variability 4. The interrelationship between the perception and the policy of vaccination, the benefits of - and barriers to ¡V action, and the virulence and severity of the disease The results of this research suggest the public perception of SPV is the most important factor governing its efficacy. It is recommended, therefore, that public health departments campaign for SPV in a variety of different ways,( e.g. in newspapers and magazines, on TV, at pediatric clinics, at health centers, etc.) in order to establish a free and open flow of information to the public at large. It is in the hope of reducing the current mortality rate, length and cost of hospital stay and the serious complications arising from infection, that we offer the following data as reference for future planning.

Das hochadaptierte, zellwandlose Bakterium Mycoplasma pneumoniae gehört zu den häufigsten Erregern von ambulant erworbenen Pneumonien. Während den alle 3-7 Jahre auftretenden Epidemien könnten gefährdete Personengruppen bei Verfügbarkeit einer effektiven Vakzinierung vor einer Infektion geschützt werden. Ziel dieser Arbeit war die Erarbeitung von Grundlagen zum Aufbau einer schützenden Immunantwort durch Immunisierung mit rekombinant hergestellten Teilbereichen verschiedener Adhärenzproteine von M. pneumoniae. Da die bereits bekannten Adhäsine des Erregers einerseits unverzichtbare Virulenzfaktoren darstellen und andererseits als dominante Immunogene des Bakteriums beschrieben wurden, war die Feincharakterisierung dieser Proteine im Hinblick auf ihre Eignung als Vakzinekandidat Ausgangspunkt der Untersuchungen. Definierte Teilbereiche des Hauptadhäsins P1 und weiterer adhärenzassoziierter Proteine wurden rekombinant hergestellt und auf ihre Antigenität qualitativ und quantitativ mit Seren experimentell infizierter Tiere und Seren von Patienten mit bestätigter M. pneumoniae-Infektion geprüft. Dabei gingen der C-terminale Bereich des P1-Adhäsins (RP14; AS 1287-1518) und das Protein P30 (RP30; AS 17-274) als hoch antigene Protein(regionen) hervor. Die Oberflächenexposition beider Proteinbereiche wurde experimentell bestätigt. Parallel dazu sollte der Einfluss der monospezifischen Seren gegen die rekombinanten Proteine auf die Adhärenz von M. pneumoniae an verschiedene Zelllinien bewertet werden. Nach Entwicklung eines quantitativen und schnell durchzuführenden in vitro Adhärenzhemmtests wurde gezeigt, dass Antikörper sowohl gegen RP14 als auch gegen RP30 die Adhärenz des Erregers an humane Zellen signifikant reduzieren. Im Hinblick auf die Beeinflussung von mehr als einer an der Adhärenz beteiligten Struktur wurden beide Adhäsinregionen in einem Hybridprotein vereinigt, wodurch die adhärenzhemmende Wirkung des entsprechenden Serums in vitro mehr als additiv verstärkt werden konnte und dem Effekt des polyspezifischen Serums gegen M. pneumoniae entspricht. In Immunisierungs- und Infektionsversuchen mit dem Hybridprotein konnte über eine neu etablierte sehr sensitive real-time PCR basierend auf einer Multicopy-Targetsequenz in M. pneumoniae jedoch nur in einem Teil der Versuchstiere eine signifikante Hemmung der Kolonisierung des Respirationstraktes mit dem Erreger nachgewiesen werden. Eine Steigerung der humoralen Immunantwort durch Zugabe des mukosalen Adjuvans MALP-2 konnte nicht ausreichend belegt werden. Dagegen wurde durch Immunisierung auf intranasalem Wege eine signifikante Erhöhung des sekretorischen IgA-Titers in der BAL auf das Vierfache erreicht. Um einen stabil hohen sekretorischen Antikörperspiegel im Tiermodell zu erreichen, sind weitere Optimierungen des Immunisierungsschemas notwendig. Der Einsatz des Hybridproteins HP14/30, bestehend aus mehreren immunogenen und funktionalen Adhäsinbereichen, hat sich als sehr vielsprechend zur Entwicklung einer effektiven Vakzinierung von Risikopopulationen zur Prophylaxe von Infektionen mit M. pneumoniae erwiesen.

Thèse d'exercice : Médecine. Médecine générale : Paris 12 : 2006. / Titre provenant de l'écran-titre. Bibliogr. f. 83-88.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

A large number of studies have reported on the association between the obligate intracellular bacterium, Chlamydia pneumoniae and atherosclerosis. These studies suggest that C. pneumoniae may potentially play a role in the atherosclerotic process, as not all the current atherosclerotic risk factors account for the resulting complications, such as angina, myocardial infarction, heart failure and stroke. The research presented in this thesis analysed whether there are any reliable markers of chronic C. pneumoniae vascular infection, including chlamydial sero-prevalence as defined by two commercial serological tests, detection of C. pneumoniae DNA in the peripheral circulation, the presence or absence of risk factors and symptomatic status. The presence of the bacterium in atherosclerotic carotid specimens was diagnosed directly using a C. pneumoniae-specific polymerase chain reaction (PCR) and a genus-specific immunofluorescent (IF) assay. Eighteen of the 54 (33%) carotid artery diseased (CAD) specimens were positive for the presence of C. pneumoniae DNA by PCR detection, whereas the IF assay detected only six positive samples. PCR analysis found that only two of 43 (5%) patients had C. pneumoniae DNA present within their peripheral blood mononuclear cell (PBMC) fraction. Chlamydial antibodies were detected by Focus microimmunofluorescence and/or Medac recombinant enzyme-linked immunosorbert assay (rELISA) in 56% (24/43) of CAD patients tested. Traditional risk factors, symptomatic status, antigen detection and PCR-based detection of C. pneumoniae in PBMCs, all failed to correlate with the presence of a chlamydial vascular infection. In conclusion, the existing non-invasive diagnostic tests (serology and peripheral blood-based PCR detection) are inefficient for diagnosing a vascular Chlamydia infection, suggesting that a different chlamydial antigen should be tested targeted to identify a chronic C. pneumoniae infection in CAD patients. Given the observation that numerous previously published studies have detected C. pneumoniae in atherosclerotic arterial tissue, yet at widely different detection rates (0% to 100%), it was clear that the location and quantity of clinical specimen could directly affect the detection rate. Previous reports have not used a standard and validated procedure for sampling arterial specimens for C. pneumoniae DNA. The inconsistent detection rates of chlamydial DNA in atherosclerotic plaque are a result of low concentration and irregular distribution of the bacterium, as reported in this study. Our research concluded that a minimum of 15 (30ìm-thick) sections should be analysed by PCR to minimize these sampling variables and obtain a 95% chance of detecting all true C. pneumoniae-positive samples. All previous studies may have under estimated the prevalence of C. pneumoniae, as stringent sampling and repeat testing of the bacterium is required to minimise false-negative results. An interesting finding was that C. pneumoniae DNA was present in all 10 atherosclerotic arteries, although extensive sampling of the carotid was crucial for detection. The third area of research examined the question of possible strain differences between C. pneumoniae isolates infecting human atherosclerotic carotid arteries. Whole genome sequencing as well as specific gene typing suggests that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. We analysed the genotypes of C. pneumoniae present in human atherosclerotic carotid plaque and found several polymorphisms in the variable domain-4 (VD4) region of the outer membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD-urk) genes. Our research identified four different genotypes of C. pneumoniae in human atherosclerotic carotid arteries, including an isolate that appears genetically identical to a strain previously detected in koalas. Two genotypes of C. pneumoniae were present in both human carotid specimens and koala PBMC fractions, suggesting that these genotypes of C. pneumoniae may be capable of crossing the host barrier. The study showed that diversity exists in both the ompAVD4 gene and the ygeD-urk intergenic region enabling fine-detailed differentiation between five different genotypes found in respiratory and/or vascular C. pneumoniae isolates. The importance of the diversity of C. pneumoniae isolates in its role in atherogenesis needs to be further studied.