Colonization is prerequisite for infection and transmission of Streptococcus pneumoniae, or pneumococcus. Currently available pneumococcal conjugate and pneumococcal polysaccharide vaccines can provide protection against a limited number of capsular serotypes. Implementation of vaccines has decreased the frequency of invasive pneumococcal disease and their colonization rates, but only in a serotype-dependent manner. This has led to serotype replacement in pneumococcal ecology and increased invasive disease caused by non-vaccine serotypes. Development of conserved protein-based vaccine that can provide protection against all pneumococcal serotypes is needed. Numerous surface proteins are conserved in all serotypes, and some are known to be involved in the colonization process. Understanding how pneumococcal surface proteins interact with host cells and determining their roles in colonization will aid in vaccine development. In this dissertation, we characterized host cell receptors of pneumococcal surface proteins, and proteins involved in biofilm formation, and their effect in colonization. We utilized a novel protein expression vector, pOS1, which can express secreted proteins with no LPS, IPTG induction, or cell lysis requirement. These expressed recombinant proteins were used for further investigation. We identified that human Annexin A2 (ANXA2) interacts with pneumococcal surface adhesion A (PsaA) protein. ANXA2 transduced cells showed significant increase in binding with pneumococcus compared to non-transduced cells. We conducted proteomic profiling of planktonic and biofilm membrane proteins and identified that two lipoproteins (AmiA, SP_0148) were overexpressed during biofilm formation. Isogenic mutants lacking these individual proteins showed decreased in biofilm formation compared to their parental strain. Deletion of SP_0148 led to decreased adhesion of pneumococcus to human nasopharyngeal epithelial cells (Detroit 562). These results increased our understanding of pneumococcal surface proteins involved in biofilm-formation and colonization as well as identifying new host receptors ligands for these adhesins.

Orientadores: Jose Wilson Magalhães Bassani, Rosana Almada Bassani / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-08T13:08:09Z (GMT). No. of bitstreams: 1 ZafalonJunior_Nivaldo_D.pdf: 3812093 bytes, checksum: edd5a8af0cab279b9c0a59a80083d639 (MD5) Previous issue date: 2006 / Resumo: Taquiarritmias atriais (TA) são o tipo de arritmia crônica mais comum, e mostram associação com risco de acidentes tromboembólicos. Os mecanismos envolvidos neste tipo de arritmia têm sido alvo de numerosos estudos. Nossa equipe desenvolveu um modelo de geração de TA de origem colinérgica em átrios isolados por estimulação elétrica de alta freqüência, cuja caracterização foi o objeto do presente estudo. Neste trabalho, desenvolvemos, implementamos e validamos uma metodologia para o estudo dos padrões de propagação da atividade elétrica durante arritmias, que se baseia na determinação do vetoatriograma. Nossos principais resultados foram: a) O padrão de propagação da atividade elétrica durante TA de origem colinérgica em átrios direitos é consistente com o desenvolvimento de reentrância da propagação, enquanto que, durante arritmia de origem p-adrenérgica em átrio esquerdo (por sobrecarga celular de Ca2+), o padrão é semelhante ao observado em átrios direitos sob ritmo sinusal, portanto compatível com a formação de focos ectópicos. b) Como observado em estudos anteriores, há um antagonismo entre as vias de sinalização colinérgica muscarínica e P-adrenérgica na determinação da susceptibilidade do tecido a TA. Neste trabalho, constatamos que o efeito pró-arrítmico da ativação da primeira via em condições de estimulação P-adrenérgica é suprimido por inibição de fosfatases de proteínas. Isto nos permite concluir que a ativação de fosfatases pela via muscarínica parece ser um mecanismo adicional envolvido neste antagonismo. c) Dado que resultados prévios na literatura indicam que a redução na duração do potencial de ação atrial, por aumento da condutância de fundo da membrana ao K+, tem importante papel no favorecimento de reentrância por agonistas muscarínicos, levantamos a hipótese de que este mecanismo seja suficiente para facilitar a indução de TA. Para testá-la, investigamos o efeito do pinacidil, que promove a abertura de canais de K+ dependentes de ATP. A aplicação deste fármaco facilitou consideravelmente a indução de TA, de modo dependente de concentração e independente de estimulação de receptores muscarínicos. Além disso, o padrão de propagação da atividade elétrica atrial foi essencialmente semelhante àquele observado durante TA de origem colinérgica e compatível com o desenvolvimento de circuitos reentrantes. Estes resultados não só representam suporte para nossa hipótese, mas também são sugestivos de que condições como hipoxia e isquemia, que podem resultai em depleção do ATP citosólico e abertura dos canais de K+ dependentes de ATP, aumentem a vulnerabilidade atrial ao desenvolvimento de circuitos reentrantes e TA / Abstract: Atrial tachyarrhythmias (TA), the most common type of chronic arrhythmia, are associated with increased risk of thromboembolic accidents. Several investigators have attempted to gain insight into the mechanisms underlying TA. Our research team has developed an in vitro model of TA induction by high frequency electric stimulation of isolated atria, which is dependent on muscarinic cholinergic receptor stimulation (cholinergic TA). Aiming at further characterization of this model in the present study, we developed, implemented and validated a method to analyze the pattern of propagation of electric activity during arrhythmias, based on the determination of the vectoatriogram. Our main results were: a) The pattern of electric propagation during cholinergic TA is in agreement with that expected for reentrant propagation. On the other hand, the pattern observed during arrhythmia evoked by ß-adrenoceptor stimulation in left atria (cell Ca2+ overload) resembles that seen in right atria during spontaneous sinusal rhythm, and is thus compatible with the appearance of ectopic automatic foci. b) As previously observed, muscarinic cholinergic and p-adrenergic signaling pathways interact antagonistically to determine atrial susceptibility to arrhythmia induction. In this study, we found that protein phosphatase inhibition suppressed the cholinergic pro-arrhythmic effect in the presence of ß-adrenergic stimulation. We thus conclude that muscarinic-dependent protein phosphatase activation seems to be an additional mechanism involved in the functional antagonism of these signaling pathways. c) Based on previous results from other laboratories showing that atrial action potential abbreviation, due to increase in membrane background K+ conductance, is of paramount importance for reentry facilitation by muscarinic agonists, we hypothesized that this electrophysiological change would be sufficient for facilitation of TA induction. The hypothesis was investigated by testing the effects of pinacidil, an ATP-dependent K+ channel opener. Pinacidil caused a marked, concentration-dependent increase in TA induction, which was independent of muscarinic cholinergic receptor stimulation. In addition, the electric propagation pattern during TA was similar to that observed during cholinergic TA, which is suggestive of establishment of reentrant circuits. These results not only support our hypothesis, but also point out that conditions that may cause depletion of cytosolic ATP, such as hypoxia and ischemia, may increase atrial vulnerability to the occurrence of electric reentry and tachyarrhythmia / Doutorado / Engenharia Biomedica / Doutor em Engenharia Elétrica

Hantaviren, deren Hauptreservoir Nager bilden, sind weltweit verbreitete Pathogene. Nach Übertragung auf den Menschen prägen sie vorwiegend zwei Krankheitsbilder: Das hämorrhagische Fieber mit renalem Syndrom (HFRS) oder das Hantavirus-assoziierte cardiopulmonale Syndrom (HCPS). Je nach Virusstamm variiert die Mortalitätsrate zwischen 0,1% und 50%. Das Dobrava-Belgrad-Virus (DOBV), welches zum HFRS führt, weist eine Mortalitätsrate von bis zu 12% auf und ist somit das lebensbedrohlichste Hantavirus in Europa. Wie ihre natürlichen Wirte, die Mäuse aus dem Genus Apodemus, bilden auch die DOBV unterschiedliche phylogenetische Linien. Das aus A. flavicolis (Af) isolierte DOBV-Af ist im Balkan verbreitet und führt zu schweren Verläufen des HFRS. Das in A. agrarius (Aa) gefundene DOBV-Aa verursacht schwache bis milde HFRS-Erkrankungen in Mitteleuropa und dem zentraleuropäsichen Teil Russlands. Moderate bis schwere HFRS-Verläufe im südeuropäischen Teil Russlands wurden mit Viren der DOBV-Ap-Linie in Verbindung gebracht, welche von A. ponticus (Ap) übertragen werden. In Deutschland ist DOBV endemisch im nördlichen Teil des Landes. Epidemiologische Studien, basierend auf der serologischen Feintypisierung mittels Neutralisationstests und phylogenetischen Analysen Patienten-assoziierter Virussequenzen, zeigen, dass Viren der DOBV-Aa-Linie für die HFRS-Fälle in dieser Region verantwortlich sind. Bislang konnte das für die humane Erkrankung in Deutschland verantwortliche Virus noch nicht in Zellkultur isoliert werden. In der vorliegenden Arbeit wurde das erste Zellkulturisolat eines aus Deutschland stammenden DOBV gewonnen, dessen vollständige Genomsequenz bestimmt und als Greifswald-Virus (GRW/Aa) bezeichnet wurde. In phylogenetischen Analysen bildete GRW/Aa eine gemeinsame Gruppe mit viralen Sequenzen, die aus norddeutschen HFRS-Patienten gewonnen wurden. Folglich kann GRW/Aa als das für HFRS in Deutschland verantwortliche DOBV angesehen werden. Anhand von Wirtszellkulturen konnten wir zeigen, dass GRW/Aa ähnliche Charakteristika aufweist, wie das pathogene Hantaan-Virus; es nutzt b3-Integrine und den decay accelerating factor (DAF) als zelluläre Rezeptoren und induziert die späte Expression von Markern der angeborenen Immunantwort (z. B. IFN-b, IFN-l1, MxA). Obwohl Hantaviren auf nahezu allen Kontinenten als Humanpathogene bekannt sind, wurde das erste afrikanische Hantavirus (Sangassou-Virus, SANGV) erst kürzlich in unserer Arbeitsgruppe isoliert. Aufgrund der engen phylogenetischen Verwandtschaft zwischen SANGV und GRW/Aa haben wir untersucht, ob das potentiell humanpathogene SANGV ähnliche Eigenschaften wie GRW/Aa aufweist. Aufgrund der Tatsache, dass bislang noch kein Tiermodell für die Untersuchung der Hantaviruspathogenese existiert, haben wir die Rezeptorerkennung und Induktion der Marker der angeborenen Immunantwort zur Abschätzung des humanpathogenen Potentials von SANGV, im Vergleich zu GRW/Aa, verwendet. Wir zeigten, dass SANGV ausschließlich b1-Integrine als Rezeptor nutzt und eine starke IFN-l1-Antwort in der infizierten Zelle induziert. In der Zellkultur zeigt SANGV somit andere funktionelle Charakteristika als GRW/Aa. Ob diese in Zusammenhang mit einer durch SANGV vermittelten Pathogenese stehen, muss in zukünftigen Studien untersucht werden. / Hantaviruses are worldwide distributed pathogens which are mainly carried by rodents. When transmitted to humans, they can cause two significant diseases: hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS). Depending on the virus strain, the case fatality rates of hantavirus diseases are ranging from 0.1% up to 50%. In Europe Dobrava-Belgrade virus (DOBV) is the most life-threatening hantavirus leading to HFRS with case fatality rates of up to 12%. According to its natural hosts, mice of the genus Apodemus, DOBV forms distinct phylogenetic lineages. DOBV-Af associated with A. flavicollis (Af) causes severe HFRS cases in the Balkan region. DOBV-Aa, found in A. agrarius (Aa), is typical for Central Europe and Central European Russia where it causes mild/moderate disease. Moderate to severe HFRS cases in South European Russia have been associated with virus strains of the DOBV-Ap lineage, transmitted by A. ponticus (Ap). In Germany, DOBV is endemic in the northern part of the country. Seroepidemiological studies involving fine serotyping by neutralization assay as well as phylogenetic analyses of patient-associated virus sequences showed that strains of DOBV-Aa lineage are responsible for HFRS cases in this geographical region. However, the causative agent of human disease from Germany was not isolated in cell culture. Within the current study, we have generated the first cell culture isolate of DOBV from Germany, called Greifswald virus (GRW/Aa), and determined its complete genomic nucleotide sequence. Phylogenetic analyses revealed close clustering of GRW/Aa with sequences derived from Northern German HFRS patients. Consequently, GRW/Aa can be taken as a representative of DOBV strains causing HFRS in Germany. We have demonstrated that in cultivated host cells GRW/Aa exhibits properties similar to pathogenic Hantaan virus; it recognizes b3 integrins and Decay Accelerating Factor (DAF) as cellular entry receptors and induces late expression of innate immunity markers (IFN-b, IFN-l1, MxA). Despite hantaviruses being well recognized human pathogens in almost all continents, the first African hantavirus named Sangassou virus (SANGV) has been only very recently isolated in our group. Important to note that SANGV is most closely related to DOBV in molecular phylogenetic analyses. Therefore, it was interesting to investigate if SANGV, a potential human pathogen, displays properties similar to GRW/Aa. Given that an animal model for studying hantavirus-mediated pathogenesis is not available, we used cellular receptor recognition and induction of innate immunity markers as in vitro determinants to estimate the pathogenic potential of SANGV in comparison to GRW/Aa. We have found that SANGV exclusively recognizes b1 integrins as cellular entry receptors and elicits strong induction of IFN-l1 in infected cells. Therefore, in cultivated host cells SANGV exhibits functional characteristics distinct from GRW/Aa. Whether these properties contribute to SANGV-mediated pathogenesis in humans needs to be elucidated in future studies.