Phosphoglucosamine mutase (GlmM), an enzyme taking part in biosynthesis of cell wall, has been recently proven to be essential for Streptococcus pneumoniae. The main goal of this thesis was to prove in vivo that GlmM serine residues S99 and S101 phosphorylation is essential while the necessity of it was already proven indirectly based on transformation efficiency. For this purpose we have prepared a strain with two copies of the glmM gene - the first one with amino acid changes on monitored serine residues located at native locus; and the second ectopic copy of the wild allele of glmM gene under control of inducible zinc promoter. We have observed morphology, growth, and GlmM expression with and without the presence of an inductor. All the observed parameters show that the cells are not viable without ectopic glmM expression, thus the essential protein GlmM is functional only when phosphorylated on S99 and S101 residues. Further, we have attempted to localize the enzyme in the S. pneumoniae cell. We have fused GlmM with fluorescent marker GFP and by using the florescent microscopy we have proved that GlmM is cytoplasmic protein. Another goal of this thesis was to find an unknown third phosforylation site of the GlmM protein which is dependent on the protein kinase StkP. From in vitro kinase assay and...

Der human pathogene Erreger Streptococcus pneumoniae besiedelt symptomlos den Nasenrachenraum des Menschen, löst aber auch Infektionen wie Otitis Media und invasive Erkrankungen wie Pneumonie und Meningitis aus. Einen essentiellen Schritt für den Infektionsprozess stellt die Anheftung von S. pneumoniae an die Epithel- und Endothelzellen des Wirtes dar. Im Infektionsverlauf ist auch das nachfolgende Eindringen der pathogenen Mikroorganismen in das humane Gewebe von wichtiger Bedeutung. An dieser Interaktion zwischen Erreger und Wirtszellen sind sowohl bakterielle Virulenzfaktoren als auch Komponenten des Wirtes beteiligt. Der pneumococcal adherence and virulence factor A (PavA) von S. pneumoniae ist ein Fibronektin-bindendes Oberflächenprotein und ist essentiell für die Virulenz in einem Sepsis- und einem Pneumonie-Mausinfektionsmodell (Holmes et al., 2001; Lau et al., 2001). In der vorliegenden Arbeit konnte PavA zusätzlich in einem experimentellen Maus-Meningitis-Modell als Virulenzfaktor identifiziert werden. In in vitro Infektionsstudien zeigten pavA-defiziente Pneumokokkenmutanten eine signifikant verringerte Adhärenz an und Invasion in alveoläre Epithelzellen A549 von Typ II Pneumozyten, in Larynxkarzinomzellen HEp-2, in humane Hirnendothelzellen HBMEC und in humane Nabelschnurendothelzellen HUVEC. Diese Zelllinien repräsentieren modellhaft typische Gewebezellen, mit denen S. pneumoniae während des Infektionsprozesses in Kontakt treten kann. Die signifikante Reduktion der Adhärenz der pavA-Mutante ist auf die Mutagenese des pavA-Gens zurückzuführen, da die Komplementierung mit einem aktiven pavA-Gen die Adhärenz an die humanen Zellen wiederherstellte. In Inhibitionsstudien blockierte anti-PavA-Antiserum die bakterielle Bindung an immobilisiertes Fibronektin, die über den C-terminalen Bereich des PavA-Proteins vermittelt wird. Im Gegensatz dazu wurde die Adhärenz von S. pneumoniae an die humanen Wirtszellen in Inhibitionsstudien mit anti-PavA-Antiserum oder rekombinantem PavA-Protein nicht blockiert. Zusammenfassend ist PavA ein wichtiger Virulenzfaktor für die Infektion und das Überleben der Erreger in vivo und spielt gleichzeitig auch eine Rolle während der Adhärenz von Pneumokokken an die humanen Wirtszellen in vitro. Allerdings beeinflusst PavA den Anheftungsprozess nicht direkt als Adhäsin, sondern scheint die Funktion anderer, wichtiger, bisher unbekannter Virulenzfaktoren von S. pneumoniae zu regulieren. Im zweiten Abschnitt der Arbeit wurde die Interaktion von S. pneumoniae mit dem Glykoprotein Fibronektin und deren Bedeutung für die Kolonisierung und den Infektionsmechanismus in vitro untersucht. S. pneumoniae bindet direkt an immobilisiertes Fibronektin (van der Flier et al., 1995). In der vorliegenden Arbeit konnte gezeigt werden, dass Pneumokokken Fibronektin sowohl wirtsunabhängig aus dem Plasma rekrutieren als auch reines lösliches Fibronektin binden können. Dabei binden Pneumokokken sowohl die multimere, zelluläre Form des Fibronektins als auch das lösliche, dimere Plasma-Fibronektin. Die Zugabe von Fibronektin verstärkt die Adhärenz von S. pneumoniae an die humanen Nasopharynxepithelzellen Detroit 562, die Larynxzellen HEp-2, die Bronchialepithelzellen A549 und die Hirnendothelzellen HBMEC. Die Fibronektin-vermittelte Anheftung von Pneumokokken an die Nasopharynxzellen Detroit 562 und die Hirnendothelzellen HBMEC erfolgt wahrscheinlich über die Heparin-Bindungsdomäne, da die bakterielle Adhärenz durch die Zugabe von Heparin inhibiert wird. Weitere Inhibitionsstudien zeigten, dass weder die Präinkubation der Erreger mit anti-PavA-Antiserum noch die Zugabe von rekombinantem PavA-Protein die Fibronektin-vermittelte Adhärenz an die Wirtszellen blockierte. Die Interaktion von S. pneumoniae über das Brückenmolekül Fibronektin mit den humanen Wirtszellen findet damit nicht über das Fibronektin-bindende Oberflächenprotein PavA statt. Fibronektin vermittelt nicht nur die Adhärenz von S. pneumoniae an die Wirtszellen, sondern auch deren Internalisierung. In Inhibitionsstudien mit spezifischen Inhibitoren des Aktin-Zytoskeletts und der Mikrotubuli konnte gezeigt werden, dass die Dynamik des Zytoskeletts eine essentielle Rolle für die Fibronektin-vermittelte Internalisierung der Pneumokokken in die Wirtszellen spielt. Außerdem wurde durch die Zugabe von pharmakologischen Inhibitoren der Tyrosin Kinasen, der Familie der Src-Kinasen und der Phosphatidylinositol (PI-3)-Kinase die Fibronektin-vermittelte bakterielle Invasion in humane Zellen signifikant blockiert. Die Invasion von S. pneumoniae in Mausfibroblasten, die defizient für die fokale Adhäsionskinase (FAK) waren, war im Vergleich zu der bakteriellen Invasion in die Wildtyp-Fibroblasten reduziert. Diese Ergebnisse zeigen die Beteiligung der Src-Kinasen, der PI-3-Kinase und der FAK an der Signaltransduktion, die für die Fibronektin-vermittelte Invasion von S. pneumoniae in eukaryotische Zellen notwendig ist. / The human pathogen Streptococcus pneumoniae causes infections such as otitis media, pneumonia and meningitis. Pneumococci colonize asymptomatically the human nasopharynx. But they can migrate also to the lungs and after breaching the lung barrier spread throughout the human body and induce severe invasive diseases. Adhesion of S. pneumoniae to epithelial and endothelial host cells is an essential first step for the establishment of colonization and invasive infections. Moreover subsequent bacterial invasion into the human tissue is important for developing disease as well. Both, bacterial virulence factors and host components participate in the interaction of pneumococci and human cells. The pneumococcal adherence and virulence factor A (PavA) of S. pneumoniae is a fibronectin binding surface protein and a crucial virulence determinant in a sepsis and in a pneumonia mouse model of infection (Holmes et al., 2001; Lau et al., 2001). Within this study, PavA was identified as an important factor causing disease and promoting survival in an experimental mouse meningitis model. PavA-deficient mutants of S. pneumoniae showed significantly reduced adherence and invasion into the alveolar epithelial cell type A549 of type II pneumocytes, larynx carcinoma epithelial cells HEp-2, human brain derived endothelial cells HBMEC and human umbilical vein derived endothelial cells HUVEC. These cell lines represent typical cells of the human tissue that are involved in the progression of pneumococcal infections. The adhesion level of a pavA-knockout mutant was restored by re-introducing the entire pavA gene, indicating that the observed effect was due to pavA-deficiency. In inhibition studies the addition of anti-PavA antisera inhibited pneumococcal attachment to immobilized fibronectin. Binding of fibronectin was mediated by the C-terminal part of the PavA protein. In contrast, bacterial adherence and invasion into host cells was not influenced by addition of anti-PavA antisera or recombinant PavA protein. In conclusion, PavA plays a pivotal role in pneumococcal adherence to host cells in vitro and is an important virulence factor for the progression of pneumococcal disease and survival in vivo. PavA is not directly involved in the pneumococcal adhesion process by acting as an adhesin. The results indicated that PavA affects colonization and pneumococcal survival by modulating the function of important but yet unknown virulence determinants of S. pneumoniae. In the second part of this study the interaction of S. pneumoniae with the host glycoprotein fibronectin and its role in colonization in vitro was investigated. S. pneumoniae binds directly to immobilized fibronectin (van der Flier et al., 1995). In this study is shown that pneumococci recruite fibronectin from plasma in a species-unspecific manner and bind pure soluble fibronectin. S. pneumoniae interacts with the multimeric cellular form of fibronectin as well as with the soluble dimeric plasma fibronectin. Host-cell bound fibronectin significantly enhances pneumococcal adherence to the human nasopharyngeal epithelial cell line Detroit 562, to larynx cells HEp-2, to alveolar cells A549 and to brain endothelial cells HBMEC. Fibronectin-mediated pneumococcal adherence to the nasopharyngeal cells Detroit 562 and to the brain endothelial cells HBMEC is probably mediated by the heparin binding site due to the fact that bacterial adherence is inhibited by the addition of heparin. Further inhibition studies demonstrated that neither pneumococcal preincubation with anti-PavA antisera nor addition of recombinant PavA protein inhibited fibronectin-mediated adherence. These results indicate that fibronectin-mediated adherence was not due to binding of PavA to fibronectin. Fibronectin plays a key role in pneumococcal adherence to host cells and, in addition, promotes pneumococcal internalization. Inhibition studies with specific inhibitors of the actin cytoskeleton an the microtubulis showed an essential impact of the cytoskeleton dynamic on the fibronectin-mediated pneumococcal invasion into host cells. In addition, presence of pharmacological inhibitors of tyrosine kinases, Src-kinases and Phosphatidylinositol (PI-3)-kinase significantly blocked fibronectin-mediated bacterial invasion in human cells. Pneumococcal invasion into fibroblasts deficient for the focal adhesion kinase (FAK) was reduced compared to wildtype fibroblasts. These results indicate the involvement of Src-kinases, PI-3-kinase and FAK in signal transduction leading to fibronectin-mediated internalization of S. pneumoniae in eukaryotic cells.

Thrombospondin-1 (TSP1) ist ein matrizelluläres, Calcium-bindendes Glykoprotein, das an der Regulation verschiedener zellulärer Prozesse beteiligt ist. TSP1 wird von unterschiedlichen Zelltypen gebildet und ist vor allem in den α-Granula der Thrombozyten zu finden, aus denen es nach deren Aktivierung sekretiert wird. Streptococcus pneumoniae (Pneumokokken) sind Gram-positive humanpathogene Bakterien. Sie besiedeln asymptomatisch den menschlichen Respirationstrakt und können schwerwiegende lokale Infektionen und lebensbedrohliche Erkrankungen, wie z.B. Sepsis, bakterielle Meningitis oder invasive Pneumonien auslösen. Die Anheftung von S. pneumoniae an Wirtsstrukturen ist ein initialer Schritt für die Kolonisierung mukosaler Epitheloberflächen. In dieser Arbeit wird die Bedeutung des humanen TSP1 für die Pathogen-Wirt Interaktion analysiert und der Effekt für die Pathogenese demonstriert. Verschiedene Bindungsstudien und durchflusszytometrische Analysen zeigten eine Assoziation von S. pneumoniae an aktivierte Thrombozyten und an lösliches und immobilisiertes TSP1. In in vitro Infektionsversuchen konnte nachgewiesen werden, dass wirtszellgebundenes TSP1 die Adhärenz an und Invasion in Epithel- bzw. Endothelzellen vermittelt. TSP1 übernimmt die Funktion als Brückenmolekül zwischen S. pneumoniae und eukaryontischen Wirtszellen. Zur Charakterisierung des bakteriellen Adhäsins für TSP1 wurden die Pneumokokken mit dem proteolytischen Enzym Pronase E bzw. mit der Zucker oxidierenden Substanz Natriumperiodat inkubiert. Eine Behandlung mit Natriumperiodat reduzierte die TSP1 vermittelte Adhärenz der Pneumokokken an humane Wirtszellen. Im Gegensatz dazu hatte die Behandlung mit Pronase E keinen Einfluss auf die TSP1 vermittelte Anheftung von S. pneumoniae an eukaryontische Zellen. Diese Ergebnisse deuten an, dass es sich bei dem bakteriellen Adhäsin für TSP1 um eine oberflächenlokalisierte Glykostruktur der Pneumokokken handelt. Die TSP1 vermittelte bakterielle Adhärenz der Pneumokokken an Wirtszellen konnte durch Pneumokokken-spezifisches Phosphorylcholin bzw. durch Lipoteichonsäuren nicht reduziert werden. Im Gegensatz dazu wurde die TSP1 vermittelte Adhärenz von S. pneumoniae an Wirtszellen durch Zugabe von löslichem Peptidoglykan signifikant inhibiert. In verschiedenen Bindungsstudien wurde das Peptidoglykan als Pneumokokken-Adhäsin für TSP1 identifiziert. Weiterhin wurde herausgestellt, dass nicht nur S. pneumoniae, sondern auch andere Gram-positive pathogene Bakterien, wie Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes und verschiedene apathogene Bakterien mit TSP1 interagieren, im Gegensatz zu Gram-negativen Bakterien. Es konnte gezeigt werden, dass TSP1 das Peptidoglykan aller getesteten Gram-positiven Bakterien erkennt. Diese Beobachtung weist auf einen allgemeingültigen Mechanismus der Bakterien-Wirt Interaktion hin, der wahrscheinlich von großer Bedeutung für die Pathogenese Gram-positiver Bakterien ist. Als Rezeptoren für TSP1 auf der Wirtszellseite wurden Proteoglykane auf der Oberfläche von eukaryontischen Zellen identifiziert. Weiterhin konnte herausgestellt werden, dass eine Interaktion der Gram-positiven Bakterien mit TSP1 nicht nur eine Adhärenz an Wirtszellen vermittelt, sondern die Bakterien vor einer Phagozytose durch primäre Granulozyten schützt. Zusammenfassend beweisen diese Ergebnisse eine spezifische Interaktion von Gram-positiven Bakterien mit TSP1, die zur bakteriellen Kolonisierung des Wirtsgewebes beiträgt. Das Peptidoglykan übernimmt die Funktion eines bakteriellen Adhäsins für TSP1, so dass TSP1 als molekulare Brücke die Interaktion von Gram-positiven Bakterien und Wirtszell-Proteoglykanen vermittelt. Diese Untersuchungen tragen in bedeutender Weise zu einem besseren Verständnis der Pathogenese von Infektionen durch S. pneumoniae und anderen Gram-positiven Bakterien bei. / Thrombospondin-1 (TSP1) is a matricellular glycoprotein that has key roles in interactions between human cells and components of the extracellular matrix. TSP1 is produced by different cell types and is mainly found in the α-granules of human thrombocytes and secreted upon their stimulation. Streptococcus pneumoniae are Gram-positive human bacteria which reside asymptomatically in the human respiratory tract, but can also cause local infections and life-threatening diseases such as sepsis, bacterial meningitis and pneumonia. A prerequisite for pneumococcal colonization of mucosal epithelial cells is the bacterial interaction with host cell structures. This study reports a novel role for human TSP1 in pathogen-host interactions. Binding assays and flow cytometric analysis demonstrate that S. pneumoniae specifically interacts with human TSP1. It is shown that S. pneumoniae binds to activated thrombocytes and recruits TSP1 from human plasma. Host-cell bound TSP1 promotes adherence of S. pneumoniae to human epithelial and endothelial cells, thereby acting as a molecular bridge between pneumococci and eukaryotic cells. To identify the bacterial adhesin for TSP1, pneumococci were incubated with the proteolytic enzyme pronase E and with sodium periodate, which oxidizes surface-exposed sugars. Pretreatment of the bacteria with sodium periodate, but not pronase E substantially reduced TSP1 mediated adherence to host cells, suggesting a glycoconjugate as the pneumococcal receptor for TSP1. Pneumococcal phosphorylcholine and lipoteichoic acids did not affect TSP1 mediated adherence of S. pneumoniae to host cells. In contrast, attachment of pneumococci to host cells via TSP1 was blocked by soluble peptidoglycan, indicating the recognition of bacterial peptidoglycan by TSP1. Further studies demonstrate that in addition to S. pneumoniae other Gram-positive bacteria including Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes and several apathogenic bacteria interact with TSP1. Gram-negative bacteria did not interact with TSP1. Further it is shown that TSP1 recognizes the peptidoglycan of all tested Gram-positive bacteria, suggesting a general mechanism of bacteria-host protein interaction which probably has a significant impact on the pathogenesis of Gram-positive bacteria. Host cell proteoglycans were identified as the cellular receptors for TSP1. It is demonstrated that the bacterial binding to TSP1 does not only support adhesion to host cells, but can also protect the bacteria from phagocytosis by granulocytes. In conclusion, the results demonstrate an exploitation of TSP1 by Gram-positive bacteria which supports bacterial colonization of host tissues. In this scenario peptidoglycan functions as adhesin and TSP1 acts as a molecular bridge, linking Gram-positive bacteria with proteoglycan receptors on the host cells. These studies contribute to a better understanding of infections by S. pneumoniae and other Gram-positive bacteria.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg 2017. / Background: Nasopharyngeal colonisation is a pre-requisite for developing bacterial respiratory and invasive disease. Immunisation of children with the pneumococcal conjugate vaccine (PCV) impacts upon colonising pneumococcal serotypes, which in turn could also affect the biome of the nasopharynx in relation to colonisation by other bacteria. Due to limitations in standard culture methods, the association between PCV-immunisation and bacterial carriage density is still unclear, including among HIV-infected children. In this study we aimed to evaluate the effect of infant vaccination with the 7-valent PCV (PCV7) on vaccine-serogroup colonisation in order to determine whether the increase in non-vaccine serotype (NVT) colonisation was due to unmasking of previously low density colonising serotypes or increase in acquisition of NVT. Also, we evaluated the association between PCV7 immunisation and HIV-infection on the prevalence density of nasopharyngeal colonisation by other common potentially pathogenic bacteria. Methods: A multiplex real-time qPCR assay was set up to detect 44 common pneumococcal serotypes and 5 bacterial pathogens including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Streptococcus pyogenes. All assays were optimised according to MIQE guidelines and their ability to detect multiple pneumococcal serotype/group and bacteria in archived nasopharyngeal swabs were evaluated. The multiplex qPCR assays were then used to evaluate vaccine-serotype, non-vaccine serotype and bacterial nasopharyngeal colonisation in achieved swabs of PCV7-vaccinated (at 6, 10 and 14 weeks of age) and PCV-unvaccinated African children at 9 and 15-16 months of age, prior to routine vaccination of children with PCV through the public immunisation program. In order to address the limitations of the qPCR assays, a nanofluidic real-time PCR assay was developed to simultaneously detect 53 pneumococcal serotypes, 6 serotypes of H. influenzae and 11 bacterial pathogens. Further, all assays were optimised and evaluated according to the MIQE guidelines and findings from Fluidigm and traditional qPCR assays were compared. Lastly, Fluidigm was used to evaluate the association of HIV-infection on the prevalence and density of nasopharyngeal colonisation at 9 and 16 months of age by common, potentially pathogenic bacteria including PCV7 pneumococcal serotypes, non-PCV7 serotypes, Haemophilus influenzae, non-typable Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Streptococcus pyogenes, Neisseria meningitidis, Neisseria lactamica, Bordetella pertussis, Bordetella parapertusis, Bordetella bronchiseptica and Bordetella holmesii in achieved nasophartngeal swabs collected from PCV7-vacciniated HIV-infected and HIV-uninfected children. Results: Molecular qPCR was more sensitive than culture in detecting multiple concurrent colonising pneumococcal serotypes as well as other common nasopharyngeal colonisers, with the majority of additional isolates detected by qPCR having a low carriage density (<104 CFU/ml). Further, qPCR identified a lower prevalence of PCV7-serotype colonisation among PCV7-vaccinated compared to PCV-unvaccinated children at 9 and 16 months of age [adjusted Odds Ratio (aOR): 0.37; 95% CI; 0.19-0.7 and 0.41; 95% CI; 0.26-0.63, respectively]; and an increase in NVT-serotype [aOR: 1.88; 95% CI; 1.02-3.48 and 2.2; 95% CI; 1.18-4.1] colonisation respectively. The increase in NVT carriage among PCV7-vaccinees was driven by serotype 19A, which increased by 53.4% (p=0.021) and 70.7% (p<0.001) at 9 and 16 months of age respectively. Further, 19A had a higher density of colonisation in PCV7-vaccinated groups compared to PCV-unvaccinated groups and was more likely to be identified as a primary than non-primary isolate in PCV7-vaccinated children alone. PCV immunisation was also associated with an increased prevalence of H. influenzae at 9 months (55.8% vs. 66.3%, p<0.001) and 16 months (72% vs. 62%, p=0.017) of age, while a temporary increase in the carriage prevalence of S. aureus was found in PCV7-vaccinated (18.9%) compared to PCV-unvaccinated children (11.1%, aOR 2.1; 95% CI 1.0-1.4; p=0.049) at 9 months of age only. The density of pneumococcus (4.68 vs. 4.28 CFU/ml; p=0.007), H. influenzae (3.86 vs. 4.34 CFU/ml; p=0.008), M. catarrhalis (2.98 vs. 3.52 CFU/ml; p<0.001) and S. aureus (3.06 vs. 4.02 CFU/ml; p=0.02) were also higher among PCV7-vaccinated compared to PCV-unvaccinated children at 9 months age, although this difference diminished with increasing age. There was excellent concordance between the qPCR and Fluidigm for carriage prevalence and density of the majority of assays, with Fluidigm identifying an additional 7 pneumococcal serotypes and 11 bacterial species above those detected by qPCR. Further, discordant results between the two PCR methods were strongly associated with a low carriage density (<102 CFU/ml). Using molecular Fluidigm, a lower carriage prevalence of overall pneumococci (58.6% vs. 69.9%; p=0.02), non-vaccine serotypes (27.8% vs. 40%; p=0.047) and H. influenzae (64.2% vs. 42.3%; p=0.01) was identified in HIV-infected children compared to HIV-uninfected children who were immunised with PCV7 at 9 months of age. No difference in the carriage prevalence of overall pneumococci was however found at 16 months of age (p=0.20), although the carriage prevalence of non-vaccine serotypes (50.9% vs. 60.4%; p=0.049) and H. influenzae (56% vs. 73.4%; p=0.02) was lower in HIV-infected children at 16 months of age. In addition, the density of overall pneumococcus was found to be higher in HIV-infected children (4.81 vs. 4.44 CFU/ml; p=0.014), despite the lower carriage prevalence at 9 months of age, which was driven by a higher density of vaccine serotypes/serogroups (4.21 vs. 3.72 CFU/ml; p=0.04). By 16 months of age, there was no difference in density of pneumococcal colonisation between the HIV-infected and HIV-uninfected children (p=0.89). No difference in the density of H. influenzae was found between HIV-infected and HIV-uninfected infants at 9 months of age (p=0.08); however, by 16 months of age, HIV-uninfected children had a higher density of overall H. influenzae colonisation (4.95 vs. 4.32 CFU/ml; p<0.001), which was largely due to the higher carriage density of NThinf in HIV-uninfected children (5.0 vs. 4.23 CFU/ml; p<0.001). Conclusion: Molecular qPCR assays were shown to be a promising alternative to WHO recommended culture in that multiple pneumococcal serotypes and other bacterial pathogens could be simultaneously detected as well as the bacterial load of each colonising bacteria quantified. The mechanism behind the vaccine effect was shown to be a combination of both serotype replacement and unmasking; however, the reduction in PCV7-serotype colonisation impacted on colonisation prevalence and density of other bacterial species of the nasopharynx and the clinical relevance of this needs further exploration in relation to mucosal and invasive disease outcomes, as well as for higher valence vaccines. While the higher carriage density of overall pneumococcus in HIV-infected children, despite the lower carriage prevalence might explain the higher invasive disease burden in HIV-infected compared to HIV-uninfected children even in the era of antiretroviral therapy treatment and PCV immunisation, future studies are required to provide clarity. Nevertheless, the findings from this thesis highlight the importance of continued surveillance of the circulation of pneumococcal serotypes as well as other bacterial pathogens especially in a population with a high burden of HIV-1 infection. / MT2017

Streptococcus pneumoniae has a highly adaptable genome due at least in part to its natural transformability and its ability to recombine with other pneumococci and related species. The emergence of antibiotic resistant clones of S. pneumoniae presents an opportunity to investigate how the genome has altered, spread and diversified within a defined time frame. We postulated that the genomes of epidemic, resistant isolates of S. pneumoniae would carry evidence of the genetic mechanisms that have shaped their evolution. We investigated this using eight to fifteen isolates from each of three S. pneumoniae clones; two multiply resistant clones Spain 9V-3 and Taiwan 19F-14 and one clone that has not acquired multiple resistance, England 14-9. Genome diversity in each of the three clones was investigated using pulsed field gel electrophoresis and multilocus sequence typing. Polymorphisms identified as a result of changes in the size of restriction fragments were found to be caused mainly by genomic rearrangements rather than restriction site mutations. Several deletion/insertion events in addition to one large inversion were identified. A number of polymorphisms correlated with previously known variable regions. Database analysis of multilocus sequence data from all three clones showed that recombination leads to sequence divergence more frequently than de novo mutation, but was significantly less common in England 14-9. The lower frequency of recombination events in England14-9 was in line with a transformation deficiency observed in vitro, and may explain the rare occurrence of penicillin resistance in this clone. Analysis of competence and recombination gene sequences available from databases revealed a potential cause of transformation deficiency: a four amino acid deletion in CelA, involved in DNA uptake and transport. Recombination can act as a DNA repair mechanism, but the significantly low occurrence in England14-9 suggests other mechanisms act to repair severe damage. Although S. pneumoniae does not have a typical SOS response it does possess DNA polymerase IV, encoded by dinB, which is predicted to be involved in error-prone DNA replication and repair of double strand breaks. DinB knockout mutants were created to investigate the effect in isogenic backgrounds. DinB mutants presented a lower frequency of spontaneous rifampicin resistance mutations than wild type 3 isolates. DinB mutants were more sensitive to killing by three different DNA damaging agents as well as by hydrogen peroxide. Isolates of the natural dinB mutant clone Spain 9V-3 were also shown to be more sensitive to DNA damaging agents than clones England 14-9 and Taiwan 19F-14. It is concluded that genetic differences between the three clones investigated do influence their patterns of evolution, and may account for differences in their antibiotic resistance profiles. Furthermore DNA polymerase IV does function as an error prone repair polymerase capable of protecting pneumococci from DNA damage despite the lack of a coordinated SOS response in pneumococci, and the absence of the gene in one successful multi-resistant clone.

Orientador : Alexandra Ivo de Medeiros / Banca: Vania Luiza Deperon Bonato / Banca: Carlos Rossa Junior / Resumo: A fagocitose de células apoptóticas é um processo dinâmico importante para a homeostase dos tecidos após injúria. Os macrófagos além de atuarem na remoção de células apoptóticas, também colaboram na defesa contra microrganismos. Existem ao menos duas populações de macrófagos classificadas como macrófagos M1 (pró-inflamatórios) e macrófagos M2 (anti-inflamatórios). Estes leucócitos diferem quanto ao fenótipo e funções efetoras dependendo, primordialmente, do microambiente e dos microrganismos com que interagem no tecido. Em uma situação de injúria pulmonar estéril (inalação de agentes tóxicos), há acúmulo de células apoptóticas sem a presença de agente microbiano. Por outro lado, durante uma infecção pulmonar bacteriana, ocorre intensa migração de células para o local da infecção na tentativa de conter a proliferação bacteriana, resultando em intenso acúmulo de células apoptóticas infectadas neste tecido. Portanto, nesse trabalho foi avaliado, in vitro e in vivo, o efeito da fagocitose de células apoptóticas infectadas (AC-Sp) ou estéreis (AC) na polarização de macrófagos M1/M2, bem como suas funções efetoras, e o efeito da PGE2 nesse contexto de eferocitose. Macrófagos M0 co-cultivados com AC ou AC-Sp apresentam um fenótipo intermediário entre M1 e M2, com secreção de altos níveis de IL-6, TNF-α, PGE2, TGF-β e NO e a expressão de genes relacionados ao perfil M1, iNOS, e ao perfil M2, Arginase 1. Além disso, macrófagos M0 cultivados com AC ou AC-Sp, apresentam supressão da função microbicida quando desafiados com S. pneumoniae. A presença de PGE2 dirige a polarização de macrófagos M0 a um perfil M2 e a presença de inibidores de COX promove a reversão do fenótipo de polarização destes macrófagos. Os resultados in vivo demonstram que a instilação de AC ou AC-Sp promove distintos microambientes no pulmão destes animais, que influenciam na resolução da infecção..... / Abstract: The phagocytosis of apoptotic cells is dynamic and crucial for homeostasis after injury. Macrophages act on the clearance of apoptotic cells and collaborate with defense against microorganisms. There are at least two distinct macrophage populations classified as M1 macrophages (pro-inflammatory) and M2 macrophages (anti-inflammatory). Both cells differ on the state of polarization and effectors function depending primarily on the microenvironment and microorganisms that interact into the tissue. In a situation of sterile lung injury (inhalation of toxic agents), there are accumulation of apoptotic cells without the presence of pathogen. On the other hand, during a bacterial pulmonary infection there is intense cell migration to the infection site in an attempt to impair bacterial growth, resulting in a large accumulation of infected-apoptotic cells in the tissue. Therefore, this study has evaluated in vitro and in vivo, the effect of the phagocytosis of infected-apoptotic cells (AC-Sp) or sterile cells (AC) on the polarization of M1/M2 macrophages, as well as in their effector functions and the effect of PGE2 in the context of efferocytosis. M0 macrophages co-cultured with AC or AC-Sp show an intermediate phenotype between M1 and M2 with high secretion levels of IL-6, TNF-α, PGE2, NO and TGF-β, and gene expression profile related to M1, iNOS, and M2, arginase 1. Furthermore, M0 macrophages cultured with AC or AC-Sp show strong suppression of the macrophage microbicidal function when challenged with S. pneumonia. The presence of PGE2 drives the polarization of M0 macrophages to a M2 profile, and in the presence of COX inhibitors, it reverses the polarization of this macrophage phenotype. The in vivo results demonstrate that the instillation of AC or AC-Sp promotes distinct microenvironments in the lungs of these animals, which influence the resolution of the infection. All these results suggest that the presence of AC or AC-Sp promotes, ... / Mestre

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Streptococcus pneumoniae is a major pathogen that causes Otitis Media infections and bacterial meningitis in children as well as community acquired pneumonia in adults. Clinical isolates of S. pneumoniae exhibiting resistance to Beta-lactam antibiotics are being isolated with increased frequency in many countries. Streptococcus pneumoniae strains resistant to Beta-lactam drugs have modified forms of penicillin-binding proteins that exhibit reduced affinity for binding to chemotherapeutic Beta-lactams. Penicillin binding proteins are membrane-bound enzymes that catalyze the terminal step in cell wall synthesis, and are targets for Beta-lactam drugs. Seventeen clinical isolates and six vaccine strains of Streptococcus pneumoniae were characterized using conventional phenotypic methods, susceptibility to antimicrobial agents, capsular serotyping, and by different biochemical and genotyping methods. One strain, Sp D2, was resistant to penicillin and other Beta-lactams used in the study, to erythromycin, and to Trimethoprim/Sulfamethoxazole. Sp D2 exhibited a unique protein profile in 1D SDS-PAGE gels of whole-cell proteins. Cells of Sp D2 were fractionated, and the cytoplasmic membrane fraction was obtained by ultracentrifugation and analyzed using a 1D SDS-PAGE gel. A protein band with a mass of ~50 kDa was excised and subjected to Trypsin In-Gel Digestion, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. The resulting MALDI-TOF-MS data (peptide mass fingerprints) did not produce any significant matches with proteins in any of the published S. pneumoniae genome databases. The 50 kDa protein was further subjected to N-terminal and internal sequence analysis and database searching, and the protein could not be identified by significant matches. Sp D2 did not react with any anti-pneumococcal polysaccharide capsular antibodies, and is designated as a non-typeable strain. Sp D2 exhibited a positive reaction in the Bile Solubility Test, the Optochin Test, and also positive reactions in PCR assays for the presence of the pneumococcal surface protein gene (PsaA), the autolysin gene (LytA), and the pneumolysin gene (Ply); which confirms that Sp D2 is a strain of S. pneumoniae.

Objective: To find out the spatial autocorrelation of antibiotic resistance of S. pneumonia and test the significance of distance as a risk factor. Methods: Descriptions of penicillin and macrolide resistance in EARS-Net countries from 2006 to 2010 were given. Global moran’s I and Anselin moran’s I were used to assess the spatial autocorrelation and gravity model was used to test the significance of distance and other socio – economic factors. Results: The trend of resistance in Europe was stable. Positive spatial autocorrelation existed from 2006 to 2010 for penicillin (Z(I): 0.16-0.2) and 2009 to 2010 for macrolide (Z(I): 0.11 -0.13). Some clusters (hotspots) were identified; they were Cyprus (2006-2010 for penicillin and 2009 to 2010 for macrolide), Spain (2006 for penicillin), France (2006 for penicillin), Romania (2009 for penicillin and macrolide) and Bulgaria (2009 for penicillin and macrolide). The result of gravity model showed that only parameters of population in 2007 for penicillin (p<0.05) and parameter of distance in 2009 for penicillin (p<0.05) in Cyprus were statistically significant. Conclusion: Distance was not a risk factor of high prevalence of antibiotic resistance of S. pneumoniae although there was a positive spatial autocorrelation. Improvement in surveillance system and appropriate public action were recommended for controlling the spread of resistant strain of S. pneumoniae. / published_or_final_version / Public Health / Master / Master of Public Health

Influenza is a leading cause of respiratory disease worldwide. During pandemic and seasonal influenza, secondary Streptococcus pneumoniae infection is a severe complication that contributes to morbidity and mortality. With the clinical significance of this co-infection, it is imperative to understand the disease mechanisms and how our immune system would be modulated in dealing with the dual infection. First, in vivo co-infection model was established. Mice were sequentially infected with influenza virus and then Streptococcus pneumoniae. Co-infected mice lost their body weight significantly and had 100% mortality, whereas mice infected with either influenza virus or pneumococcus alone lost their body weight transiently and all recovered from the infection. Then, lung inflammatory response during the co-infection was examined. Although it is a common phenomenon that co-infection enhances inflammation, the kinetic of, and the relative contribution of influenza virus or pneumococcus to the lung inflammation is not well defined. Therefore, this study characterized the general lung inflammatory environment after co-infection. It was found that influenza virus and pneumococcus differentially modulated inflammatory response in terms of kinetics, leukocyte infiltration and cytokine production. At the early time point after co-infection, pneumococcal infection contributed more than the influenza virus infection to enhance inflammatory cytokine and neutrophil infiltrating the lung. At the later time point after co-infection, both influenza virus and pneumococcus contributed to synergistically increase inflammatory cytokine and macrophage infiltrating the lung. Influenza virus infection induced IFN-γ that contributed to the elevated IFN-γ level in co-infected mice. Influenza virus and pneumococcus synergistically increased Th2 associated cytokine including IL-4, IL-5, and IL-10. These up-regulated immune responses might contribute to the severe lung pathology. Next, adaptive immunity to co-infection was examined. Literature studying co-infection often reports how prior influenza virus infection impairs the immune response against subsequent bacterial infection. However, whether and how secondary pneumococcal infection would affect the immunity to the initial influenza virus is unknown. Therefore this study investigated the modulation of immunity to influenza virus by secondary pneumococcal infection. It was found that co-infection significantly enhanced virus titer in lung and depleted the number of cell in spleen. Secondary pneumococcal infection after influenza decreased influenza virus specific IgG in the lung and peripheral blood. The reduced level of virus specific IgG was associated with the decrease in the number and the percentage of follicular B cell and CD4 T follicular helper cell through both pneumococcal capsular polysaccharide dependent and independent manner. Treating co-infected mice with immune serum containing influenza virus specific IgG successfully improved survival, which suggested the important protective function of virus specific IgG to the co-infection. Taken together, these data suggested that secondary pneumococcal infection impairs the antibody response to influenza virus, which might enhance mortality after co-infection. In conclusion, this study provides new insight to understand the pathogenesis of co-infection, reveals the general lung inflammatory environment, highlights the negative role of pneumococcus to impair virus control and explores novel treatment for the co-infection. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy