Introduction Yearly, 90 million people are infected with C. trachomatis. Even though it is easily treated with antibiotics the often-asymptomatic infection often spreads prior to detection. A vaccine is therefore of great interest. A chimeric major outer membrane protein (MOMP) of C. trachomatis has in earlier studies proved to contain the epitopes necessary for immunization. In this thesis the chimeric MOMP gene was cloned and expressed in E. coli. Furthermore, the expression of the protein was analyzed in previously transformed A. thaliana. Materials and Methods The chimeric MOMP gene was cloned into E.coli. Following vector amplification, the gene was expressed and the protein purified by affinity chromatography.  Seeds from different lines of previously transformed A. thaliana were screened by PCR. Hits were then analyzed by western blot.  Results The results show successful cloning and expression of the chimeric MOMP gene in E. coli. The following protein purification did result in purified protein, however in low concentration. For the A.thaliana lines, the presence and correct orientation of the gene was verified in some of the lines screened. The B7 line was verified to express the protein. Discussion The low concentration of purified protein in E.coli was probably due to un-optimized imunnoprecipitation conditions. In expression analysis of A. thaliana, purification of plant samples by immunoprecipitation prior to running western blot gave results, whereas running un-purified samples in urea buffer did not, probably due to interfering proteins in wild type plants.

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No description available.

PagP is an integral outer membrane enzyme that transfers a palmitoyl group from a phospholipid to lipid A and the polar headgroup of phosphatidylglycerol (PG). Palmitoyl-lipid A and palmitoyl-PG (PPG) have been implicated in resistance to host immune defenses. PagP proteins are diverse, the E. coli PagP belongs to the major clade of PagP homologs and palmitoylates lipid A regiospecifically at the 2-position, whereas P. aeruginosa PagP belongs to the minor clade of PagP homologs and instead palmitoylates lipid A regiospecifically at the 3’-position. Our objective was to understand how PagP has been adapted in nature to interact with multiple lipid substrates and products. We investigated the structure-function relationships of key major clade homologs, to show that Bordetella PagP palmitoylates lipid A at the 3’-position and employs surface residue T29 in its palmitoyltransferase reaction. Legionella PagP palmitoylates lipid A at the 2-position and was confirmed to select a palmitate chain from a pool including iso-methyl branched phospholipids characteristic of this species. PagP is usually encoded as a single copy on the chromosome in most bacteria, but two copies of pagP are found in endophytic bacteria. These duplicated PagP homologs from the major clade branch into two subclades, namely chromosomal and plasmid-based PagP homologs. The chromosomal PagP homologs exhibit interacting periplasmic D61 and H67 residues, which are naturally mutated in plasmid-based PagP homologs, and are associated with a conformational change in the -barrel that determines its ability to palmitoylate PG. Chromosomal PagPs can convert PPG to bis(monoacylglycero)phosphate (BMP) and lysophosphatidylglycerol (LPG) through a periplasmic active site controlled by the invariant Y87 residue of E. coli PagP. Plasmid-based PagP homologs appear to have been adapted instead as monofunctional lipid A palmitoyltransferases. These results points to a common ancestor for PagP proteins. Knowledge gained from these studies can be applied to protein engineering. / Thesis / Doctor of Philosophy (PhD)

Anaplasma phagocytophilum and A. marginale are the etiologic agents of bovine anaplasmosis and human granulocytic anaplasmosis, respectively. As obligate intracellular pathogens, binding and entry of host cells is a prerequisite for survival. The molecular events associated with these processes are poorly understood. Identifying the adhesins mediating binding, delineating their key functional domains, and determining the molecular determinants to which they bind not only benefits better understanding of Anaplasma spp. pathobiology, but could also benefit the development of novel approaches for protecting against infection. We previously demonstrated that A. phagocytophilum outer membrane protein A (ApOmpA) is critical for bacterial binding and entry host through recognition of α2,3-sialic acid and α1,3-fucose of its receptors, including 6-sulfo-sLex. In this study, we determined that two amino acids, G61 and K64, within its binding domain (ApOmpA59-74), are essential for ApOmpA function. We also confirmed the ability of ApOmpA to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. We next extended our studies to A. marginale as it also expresses OmpA (AmOmpA) and its role in infection has not been studied. Molecular models of ApOmpA and AmOmpA were nearly identical, especially in the ApOmpA binding domain and its counterpart in AmOmpA. Antisera raised against AmOmpA or its putative binding domain inhibit A. marginale infection. AmOmpA G55 and K58 are contributory and K59 is essential for AmOmpA to bind to host cells. AmOmpA binding is dependent on α2,3-sialic acid and α1,3-fucose. Coating inert beads with AmOmpA conferred the ability to bind to and be taken up by host cells, confirming that it acts as an adhesin and invasin. 6-sulfo-sLex is dispensable for AmOmpA binding and A. marginale infection. ApOmpA works cooperatively with Asp14 (14-kDa A. phagocytophilum surface protein) to promote optimal infection of host cells. We found that Asp14 is conserved across A. phagocytophilum strains and in A. marginale and confirmed the ability of Asp14 to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. Collectively, this work advances our understanding of A. phagocytophilum and A. marginale adhesion and invasion of host cells.

A Periodontite Agressiva (PA), que atualmente pertence ao grupo das Periodontites estágios 3 e 4, distingue-se dos demais tipos de doença periodontal por seu início precoce, agregação familiar dos casos e por afetar pacientes sistemicamente saudáveis. Além disso, pode ser subclassificada em duas formas, localizada (PAL) e generalizada (PAG), em função de sua extensão. Muitas vezes, os depósitos de biofilme bacteriano são desproporcionais à quantidade de destruição óssea e perda de inserção que o paciente apresenta, independente da subclassificação. O microrganismo mais relacionado à etiopatogênese da doença é o Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), incluindo os seus principais sorotipos a, b e c, amplamente estudados. Associado a estas condições, A. actinomycetemcomitans apresenta alguns fatores de virulência como uma leucotoxina, principalmente ligada ao sorotipo b - clone JP2 (que é altamente leucotóxico) e proteínas de membrana externa (OMPs), especialmente Omp29. A resposta de imunoglobulina G (IgG) sérica contra este patógeno foi anteriormente associada à ambas as formas de PA, porém, são escassos os estudos que avaliaram longitudinalmente a resposta sérica frente a variáveis como estas. Dessa maneira, o objetivo desse estudo foi avaliar a resposta sérica, de 27 pacientes com PA e 10 pacientes periodontalmente saudáveis, contra Omp29 e sorotipos de A. actinomycetemcomitans, através de um ensaio ELISA, correlacionando com o número de cópias de JP2 (obtidos por qPCR em tempo real) e parâmetros clínicos, a partir de dados anteriormente coletados por nosso grupo. Todos os dados foram obtidos antes do início do tratamento e um ano após seu término. O tratamento consistiu de orientações de higiene bucal, tratamento mecânico e antibioticoterapia. Os dados resultantes do estudo mostraram que em ambas as formas de PA houve uma redução significativa na profundidade clínica de sondagem (PCS)(p<0,001), nível clínico de inserção (NCI)(p<0,001) e na resposta sérica contra Omp29 e sorotipo c de A. actinomycetemcomitans(p>0,005). Após 1 ano, os valores de densidade óptica (D.O.) normalizados para Omp29 e sorotipos de A. actinomycetemcomitans, bem como o número de cópias do clone JP2 tornaram-se similares aos níveis encontrados nos controles. A redução no número de cópias do clone JP2 foi correlacionada com redução da PCS em PAL(r=0.80,p=0.0042) e valores de D.O. normalizados de Omp29 em PAG(r=0.66,p=0.005). O estudo concluiu que o tratamento periodontal foi eficaz em alterar a resposta sérica contra Omp29 e sorotipos de A. actinomycetemcomitans, além de reduzir o número de cópias do clone JP2 e melhorar os parâmetros clínicos. / Aggressive Periodontitis (AP), which currently belongs to the group of Periodontites stages 3 and 4, is distinguished from other types of periodontal disease due to its early onset, familial aggregation of cases and to affect systemically healthy patients. In addition, it can be sub classified into two forms, localized (PAL) and generalized (PAG), depending on its extent. Often, bacterial biofilm deposits are disproportionate to the amount of bone destruction and loss of insertion that the patient presents, regardless of sub classification. The most important microorganism related to the etiopathogenesis of the disease is Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), including its main serotypes a, b and c, widely studied. Associated with these conditions, A. actinomycetemcomitans presents some virulence factors such as leukotoxin, mainly linked to serotype b - clone JP2 (which is highly leukotoxic) and outer membrane proteins (Omp\'s), especially Omp29. Serum immunoglobulin G (IgG) response against this pathogen was previously associated with both forms of BP; however, there are few studies that longitudinally evaluated the serum response to variables such as these. Thus, the objective of this study was to evaluate the serum response of 27 patients with AP and 10 periodontally healthy patients against Omp29 and A. actinomycetemcomitans serotypes by an ELISA, correlating with the number of copies of JP2 (obtained by qPCR in real time) and clinical parameters, from data previously collected by our group. All data were obtained prior to initiation of treatment and one year after its completion. The treatment consisted of oral hygiene guidelines, mechanical treatment and antibiotic therapy. Data from the study showed that in both forms of BP there was a significant reduction in clinical depth of sampling (PCS) (p<0,001),, clinical level of insertion (NCI)(p<0,001) and serum response against Omp29 and serotype c of A. actinomycetemcomitans(p>0,005). After 1 year, normalized optical density (O.D.) values for Omp29 and A. actinomycetemcomitans serotypes, as well as the number of copies of clone JP2 became similar to the levels found in the controls. The reduction in copy number of clone JP2 was correlated with reduction of PCS in PAL(r=0.80,p=0.0042) and O.D. normalized from Omp29 to PAG(r=0.66,p=0.005). The study concluded that periodontal treatment was effective in altering the serum response against Omp29 and A. actinomycetemcomitans serotypes, in addition to reducing the number of copies of clone JP2 and improving clinical parameters.

OMP29 é uma das principais proteínas de membrana externa de Aggregatibacter actinomycetemcomitans (Aa) e está associada à invasão de célula epitelial gengival (CEG). Os objetivos deste estudo foram: analisar filogeneticamente omp29 e omp29 parálogo (omp29par), em cepas de Aa; determinar a interação de OMP29 com proteínas de matriz extracelular e o efeito da sua interação com CEG, pela avaliação da expressão gênica e produção de mediadores inflamatórios. Variações filogenéticas foram observadas para omp29 e omp29par, bem como para os seus promotores e estas relacionam-se com os sorotipos. A proteína recombinante OMP29his interagiu com fibronectina plasmática e celular (p<0,05), mas não com os domínios F30 e 45 ou com colágenos tipo I, III, IV e V, fibrinogênio, laminina e plasminogênio. A interação das mutantes de Aa deficientes em omp29 e/ou omp29par (obtidas pelo sistema LoxP/Cre) e OMP29his com CEG OBA-09 demonstrou que OMP29 regula positivamente il-18 e negativamente il-6r e il-8 (p<0,05). Os dados sugerem que OMP29 está envolvida na evasão do sistema imune. / OMP29 is one of the major outer membrane proteins of Aggregatibacter actinomycetemcomitans (Aa) and is associated with invasion into gingival epithelial cells (GEC). This study aimed to evaluate phylogenetically omp29 and omp29 paralogue (omp29par) in Aa strains; determine the interaction of OMP29 with extracellular matrix proteins and its effect on GEC by gene expression analysis and production of inflammatory mediators. Phylogenetic variations were observed for omp29 and omp29par as well as their promoters, and they were related to the serotypes. The recombinant protein OMP29his interacted with plasma and cellular fibronectin (p <0.05), but not to F30 and F45 domains, neither to collagens I, III, IV and V, fibrinogen, laminin and plasminogen. The interaction of Aa mutants defective in omp29 and/or omp29par (obtained by LoxP system/Cre) and OMP29his with CEG OBA-09 indicated that OMP29 regulates positively il-18 and negatively il-6r and il-8 (p <0.05). Data suggested that OMP29 is involved in bacterial evasion of the immune system.

[Truncated abstract] Infectious and allergic diseases of the respiratory tract are major contributors to global mortality, morbidity and economic burden. Bacterial infections such as pneumonia and otitis media are important diseases, especially in children, while allergic diseases such as asthma and allergic rhinitis afflict up to 30% of the world's population. A confounding aspect of respiratory disease is the evidence of a complex relationship between respiratory allergy and respiratory infection, with infection suggested to both promote and prevent the pathogenesis of allergic disease. Additionally, allergy is a risk factor for bacterial infection such as otitis media, pneumonia and sinusitis, while respiratory infection can exacerbate allergic symptoms. Given the burden of bacterial respiratory disease and respiratory allergy, the development of preventative treatments for these diseases is needed and will benefit from clearer knowledge of the underlying immune mechanisms. This thesis aimed to to extend current knowledge by using Pasteurella pneumotropica, a similar bacteria to the human pathogen nontypeable Haemophilus influenzae (NTHi), to study respiratory infection and protective anti-outer membrane protein (OMP) immunity as well as the interaction of respiratory infection and allergic inflammation. Homologues of the important NTHi vaccine candidates P4, P6, P26 and D15 were found to be encoded by P. pneumotropica and a high level of amino acid sequence identity was noted between the different P. pneumotropica strains, as well as between other Pasteurellaceae members. ... In contrast, anti-P6his serum antibodies transferred to naïve mice did not confer protection. These results suggested that T-cell–mediated mechanisms were involved in P6his-mediated protection, and showed that the P. pneumotropcia model was useful for elucidating protective mechansims. The interaction of P. pneumotropica infection and papain-induced allergy was studied to investigate immune mechanisms underlying respiratory infection and allergy. Mice with ongoing allergic inflammation were intranasally challenged with bacteria and exhibited reduced pulmonary bacterial numbers, prolonged eosinophilia in the lungs and the induction of Th2 cytokines in the BALF, compared to nonallergic, infected mice. This suggested a protective role for allergic inflammation in this model. The effect of papaininduced inflammation on mice colonised by P. pneumotropica was also examined and allergic inflammation appeared to worsen infection in colonised mice. This suggested that allergic inflammation may also have a role in promoting infection in this model. In conclusion, this thesis explored mechanisms involved in vaccine-mediated immunity and the interaction of respiratory infection and allergy using a P. pneumotropica infection in its natural host. It was shown that intranasally administered recombinant P6 and P4 protected mice from lung infection, which justifies the inclusion of these OMPs as NTHi vaccine candidates. Additionally, it was demonstrated that the interaction of allergy and respiratory infection modulated immune responses. Overall, these results emphasize that a clearer understanding of the complex mechanisms underlying these interactions is required, and may be aided by the development of suitable animal models.

OMP29 é uma das principais proteínas de membrana externa de Aggregatibacter actinomycetemcomitans (Aa) e está associada à invasão de célula epitelial gengival (CEG). Os objetivos deste estudo foram: analisar filogeneticamente omp29 e omp29 parálogo (omp29par), em cepas de Aa; determinar a interação de OMP29 com proteínas de matriz extracelular e o efeito da sua interação com CEG, pela avaliação da expressão gênica e produção de mediadores inflamatórios. Variações filogenéticas foram observadas para omp29 e omp29par, bem como para os seus promotores e estas relacionam-se com os sorotipos. A proteína recombinante OMP29his interagiu com fibronectina plasmática e celular (p<0,05), mas não com os domínios F30 e 45 ou com colágenos tipo I, III, IV e V, fibrinogênio, laminina e plasminogênio. A interação das mutantes de Aa deficientes em omp29 e/ou omp29par (obtidas pelo sistema LoxP/Cre) e OMP29his com CEG OBA-09 demonstrou que OMP29 regula positivamente il-18 e negativamente il-6r e il-8 (p<0,05). Os dados sugerem que OMP29 está envolvida na evasão do sistema imune. / OMP29 is one of the major outer membrane proteins of Aggregatibacter actinomycetemcomitans (Aa) and is associated with invasion into gingival epithelial cells (GEC). This study aimed to evaluate phylogenetically omp29 and omp29 paralogue (omp29par) in Aa strains; determine the interaction of OMP29 with extracellular matrix proteins and its effect on GEC by gene expression analysis and production of inflammatory mediators. Phylogenetic variations were observed for omp29 and omp29par as well as their promoters, and they were related to the serotypes. The recombinant protein OMP29his interacted with plasma and cellular fibronectin (p <0.05), but not to F30 and F45 domains, neither to collagens I, III, IV and V, fibrinogen, laminin and plasminogen. The interaction of Aa mutants defective in omp29 and/or omp29par (obtained by LoxP system/Cre) and OMP29his with CEG OBA-09 indicated that OMP29 regulates positively il-18 and negatively il-6r and il-8 (p <0.05). Data suggested that OMP29 is involved in bacterial evasion of the immune system.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.