Electronic cigarette usage is becoming increasingly prevalent among school age children and young adults. A known bactericidal agent, propylene glycol, is often used as a carrier for nicotine, flavoring, and additional constituents of electronic cigarette juice. This study examined the relationship between propylene glycol and virulence gene expression in Streptococcus pyogenes, a respiratory tract pathogen commonly found in school-age individuals. A variety of virulence genes controlled by the three stand alone regulators mga, RofA, and Rgg/RopB were sampled in an effort to understand the pathway by which virulence is affected. The genes chosen encode C5a peptidase, fibronectin binding protein, hyaluronate lyase, NAD glycohydrolase, Streptococcal pyrogenic exotoxin A and B, streptodornase, streptokinase, Streptolysin O, and Streptolysin S. No significant change in gene expression was observed, but a novel method to test the effects of aerosols on cells was developed. This method can be used in the future to observe the effect of aerosols, including commercial electronic cigarette juice, on both bacterial and mammalian cells.

Peptide fusion tags are fundamental for the identification, detection, and capture of proteins in biological assays. Commonly used peptide fusion tags rely on temporary non-covalent interactions for binding, which can put constraints on assay sensitivity. Here, peptide fusion tags were developed that could specifically interact with protein binding partners via spontaneous and irreversible isopeptide bond formation. To develop covalently interacting peptide-protein pairs, outer-membrane proteins from Gram-positive bacteria that form autocatalyzed intramolecular isopeptide bonds were dissected to generate a short peptide fragment and a protein binding partner. Initially, the major pilin subunit Spy0128 from Streptococcus pyogenes was split to develop the 16 residue isopeptag peptide and the 31 kDa pilin-C protein partner. The isopeptag:pilin-C pair were able to react via spontaneous isopeptide bond formation between an Asn residue in isopeptag and a Lys residue in pilin-C without the requirement for any accessory factors, and with a yield of 60% after a 72 hr reaction. Reconstitution between the isopeptag:pilin-C pair was robust and occurred under all biologically relevant conditions tested, and also in the complex environment of a bacterial cytosol and on the surface of mammalian cells. A similar approach was also used to dissect the small CnaB2 domain that is part of the large FbaB fibronectin-binding protein from S. pyogenes. This led to the development of a more efficient peptide-protein pair, which was rationally modified to generate the highly optimized SpyTag:SpyCatcher pair. SpyTag is a 13 amino acid peptide with a reactive Asp that forms a spontaneous intermolecular isopeptide bond with a Lys present in the 12 kDa SpyCatcher binding partner. In a reaction with SpyTag, over 40% of SpyCatcher was depleted after 1 min and SpyCatcher could no longer be detected after 2 hr. The SpyTag and SpyCatcher reaction did not require any accessory factors and proceeded efficiently at a range of biologically relevant temperatures, pH values, concentrations, buffer compositions, and in the presence of commonly used detergents. The SpyTag:SpyCatcher technology was also used for specific cell surface labelling on mammalian cell membranes. SpyTag and SpyCatcher are both composed of the regular 20 amino acids and can therefore be genetically encoded as fusion constructs for a variety of in vitro and in vivo applications. Potential applications of the SpyTag:SpyCatcher technology include specific cell surface labelling, the development of novel protein architectures, and the covalent and irreversible capture of target proteins in biological assays.

Many pathogenic bacteria use their iron acquisition mechanisms to live inside hosts. Streptococcus pyogenes is a pathogenic bacterium that uses streptococcal iron acquisition ABC transporter to obtain heme. SiaA (HtsA, spy1795), a lipoprotein located on the cell surface, serves as a heme binding protein. To understand the iron-uptake mechanism, histidine 229, one of the two proposed axial ligands in SiaA, was mutated to alanine. SiaA H229A was expressed in E. coli, lysed by French Press, and purified by fast protein liquid chromatography (FPLC). SDS-PAGE indicated that pure protein was isolated. Nickel affinity FPLC gave purer H229A when 0.5 M imidazole was added to the binding buffer. Overall, histidine 229 is likely to be an axial ligand in wild type SiaA, as shown by the fact the mutant readily lost heme as evidenced by UV-vis spectra.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

The bacteria Streptococcus pyogenes produces a multitude of proteins which interact with and alter the functions of the host immune system. Two such proteins, Endoglycosidase S (EndoS) and Immunoglobulin G-degrading enzyme from S. pyogenes (IdeS) are able to specifically alter the effector functions of immunoglobulin G (IgG). EndoS is a glycoside hydrolase which removes the conserved <i>N</i>-linked glycan from IgG Fc whereas IdeS is a cysteine protease that cleaves the exible protein hinge of IgG. The activity of both proteins results in the reduced ability of IgG to elicit immune responses through Fc receptor binding and complement activation. Amongst other applications, both EndoS and IdeS are actively being explored as new therapeutics for IgG-mediated autoimmune diseases. Given the therapeutic potential of EndoS and IdeS, experiments were designed to investigate the structural and functional characteristics of these enzymes in an effort to understand their specficity for and activity against IgG. Here, bioinformatic and biophysical characterisation of EndoS identified subdomains outside of the catalytic domain which contribute to glycoside hydrolase activity. The substrate specificity of EndoS was also explored and showed that EndoS hydrolyses a broad range of glycans from the IgG scaffold. EndoS was also shown to have activity against alternative glycoprotein substrates, however, this non-specific activity was negligible in the context of whole serum. The effect of EndoS-mediated deglycosylation on the structure of the IgG Fc domain was explored using both X-ray crystallography and small-angle X-ray scattering. Small angle X-ray scattering was also used to characterise both EndoS and IdeS in complex with IgG Fc. Solution-state models of each complex were produced providing preliminary data towards how these enzymes interact with IgG. Overall, the results presented here contribute to our understanding of these enzymes which is of importance as they go forward into clinical applications.

Introduction – La place des tests de diagnostic rapide (TDR) et des règles de décision cliniques (RDC) pour le diagnostic des pharyngites à streptocoque du groupe A (SGA) chez l’enfant varie selon les recommandations internationales en raison de doutes sur la stabilité des performances diagnostiques du TDR et d’une validation insuffisante des RDC. Méthodes – Dans une étude prospective multicentrique (n=17) ambulatoire réalisée au sein du réseau clinique pédiatrique ACTIV de 2009 à 2011, 1776 enfants avec pharyngite ou sains ont été soumis à des prélèvements de gorge pour réaliser un TDR et une mise en culture (test de référence). Nous avons étudié l’effet indépendant de variables liées aux patients et aux médecins sur les performances diagnostiques du TDR, exploré systématiquement les faux-Positifs (FP) du TDR et réalisé une validation externe et une comparaison des RDC existantes. Résultats – La sensibilité du TDR (en moyenne 87%) variait selon la présentation clinique (âge, signes cliniques), l’inoculum bactérien et le phénomène de portage (paramètres aussi liés entre eux), et selon des variables liées aux médecins (dont le type d’activité clinique). La valeur prédictive négative du TDR était élevée (autour de 90%) et stable. Les FP du TDR étaient positifs pour le SGA en PCR. Aucune RDC n’était satisfaisante en termes de calibration et de discrimination. Conclusion – Le TDR est suffisant pour le diagnostic de pharyngite à SGA si les cliniciens évaluent leurs propres performances et les améliorent si besoin. Aucune RDC ne peut être recommandée en pratique clinique en pédiatrie. / Background – The roles of rapid antigen detection tests (RADT) and clinical prediction rules (CPR) for the diagnosis of group A streptococcus (GAS) in children with pharyngitis vary across international clinical guidelines. This might be related to unstable diagnostic accuracy of RADTs and insufficient validation of CPRs. Methods – In a prospective multicenter (n=17) office-Based study that took place in France within the ACTIV network between 2009 and 2011, 1776 children with pharyngitis or healthy controls underwent throat swabs to perform a RADT and a throat culture (reference standard). We assessed the independent effect of patient- and physician-Level characteristics on the accuracy of a RADT, systematically re-Analyzed RADT false-Positive results, and externally validated and compared existing CPRs. Results – RADT sensitivity (overall 87%) varied according to clinical signs and symptoms, bacterial inoculum size and GAS throat carriage (factors also related to each other), and according to physician-Level characteristics (including type of clinical practice). RADT negative predictive value was high (about 90%) and stable. RADT false-Positives were positive for GAS when using a new PCR technique. No CPR had sufficient performances regarding calibration and discrimination. Conclusions – RADTs are sufficient for diagnosing GAS pharyngitis if clinicians accept diagnostic accuracy monitoring and adequate training when needed. No CPR can be recommended for use in pediatrics.

Há um amplo espectro de doenças causadas por estreptococos do grupo A (GAS), e são consideradas um problema de saúde pública em vários países, principalmente os em desenvolvimento, com aproximadamente 600 milhões de casos/ano. As infecções causadas por GAS podem ocasionar doenças invasivas como faringite e pioderma com seqüelas auto-imunes graves como a febre reumática (FR) e glomerulonefrite. A FR acomete principalmente crianças e jovens adultos. A FR apresenta diversas manifestações, sendo a doença reumática cardíaca (DRC) a seqüela mais grave, caracterizada por lesões cardíacas valvares progressivas e permanentes. O tratamento, frequentemente envolve cirurgia cardíaca para a correção de lesões valvulares, o que acarreta alto custo para o Sistema Único de Saúde no Brasil e em vários países. Em trabalhos anteriores sobre os mecanismos desencadeadores das lesões reumáticas no coração, foi possível identificar o papel do linfócito T como mediador principal da autoimunidade, através da análise do receptor de células T infiltrantes de lesão cardíaca de indivíduos com DRC. Várias expansões oligoclonais com diferentes tamanhos da região que reconhece o antígeno, CDR3 foram encontradas. No presente trabalho, analisou-se a atividade tímica através da quantificação de fragmentos circulares excisados pelo rearranjo do gene do receptor do linfócito T (TREC) em linfócitos T de sangue periférico de indivíduos com FR e DRC. Também foi avaliada a presença de células T naïve e de memória através de citometria de fluxo. Os resultados do presente trabalho mostraram que a quantidade de TREC em amostras de sangue periférico do grupo de pacientes com FR/DRC foi significantemente menor quando comparada a observada em indivíduos saudáveis. Interessantemente, em ambos os grupos a quantidade de TREC apresentou correlação negativa com a idade dos indivíduos estudados. Os resultados indicaram diferenças na atividade tímica em pacientes com FR/DRC, provavelmente decorrente do processo autoimune que envolve linfócitos T / There is a wide spectrum of diseases caused by group A streptococci (GAS), that still being considered a public health problem in developing countries, with about 600 million cases per year. Infections by GAS can cause invasive diseases such as pharyngitis and pyoderma leading to serious autoimmune complications such as rheumatic fever (RF) and glomerulonephritis. RF mainly affects children and young adults, and presents different manifestations. Rheumatic heart disease (RHD) is considered the most serious complication leading to valvular lesions that are characterized by progressive and permanent heart damage, which entails high cost to the Public Health System in Brazil and worldwide. In previous works that focused on the mechanisms leading to rheumatic heart lesions, we identified the role of T lymphocytes as principal mediator of autoimmune reactions. Through the in deep analysis of infiltrating T-cell receptor repertoire of patients with RHD, we identified oligoclonal expansions with different sizes of CDR3 that is the region of antigen recognition. In the present study we analyzed the thymic activity through T cell receptor excision circles (TREC) quantification in T cells from peripheral blood of RF/RHD patients. We also evaluated naïve and memory T cells from peripheral blood by flow cytometry. Our results showed that the amount of TREC in the peripheral blood of patients was significantly lower when compared to the healthy individuals. In addition, both groups showed that the amount of TREC is negatively correlated with age. These results indicated that the thymic activity in RF/RHD patients is altered probably due to the autoimmune process that involves T lymphocytes

Há um amplo espectro de doenças causadas por estreptococos do grupo A (GAS), e são consideradas um problema de saúde pública em vários países, principalmente os em desenvolvimento, com aproximadamente 600 milhões de casos/ano. As infecções causadas por GAS podem ocasionar doenças invasivas como faringite e pioderma com seqüelas auto-imunes graves como a febre reumática (FR) e glomerulonefrite. A FR acomete principalmente crianças e jovens adultos. A FR apresenta diversas manifestações, sendo a doença reumática cardíaca (DRC) a seqüela mais grave, caracterizada por lesões cardíacas valvares progressivas e permanentes. O tratamento, frequentemente envolve cirurgia cardíaca para a correção de lesões valvulares, o que acarreta alto custo para o Sistema Único de Saúde no Brasil e em vários países. Em trabalhos anteriores sobre os mecanismos desencadeadores das lesões reumáticas no coração, foi possível identificar o papel do linfócito T como mediador principal da autoimunidade, através da análise do receptor de células T infiltrantes de lesão cardíaca de indivíduos com DRC. Várias expansões oligoclonais com diferentes tamanhos da região que reconhece o antígeno, CDR3 foram encontradas. No presente trabalho, analisou-se a atividade tímica através da quantificação de fragmentos circulares excisados pelo rearranjo do gene do receptor do linfócito T (TREC) em linfócitos T de sangue periférico de indivíduos com FR e DRC. Também foi avaliada a presença de células T naïve e de memória através de citometria de fluxo. Os resultados do presente trabalho mostraram que a quantidade de TREC em amostras de sangue periférico do grupo de pacientes com FR/DRC foi significantemente menor quando comparada a observada em indivíduos saudáveis. Interessantemente, em ambos os grupos a quantidade de TREC apresentou correlação negativa com a idade dos indivíduos estudados. Os resultados indicaram diferenças na atividade tímica em pacientes com FR/DRC, provavelmente decorrente do processo autoimune que envolve linfócitos T / There is a wide spectrum of diseases caused by group A streptococci (GAS), that still being considered a public health problem in developing countries, with about 600 million cases per year. Infections by GAS can cause invasive diseases such as pharyngitis and pyoderma leading to serious autoimmune complications such as rheumatic fever (RF) and glomerulonephritis. RF mainly affects children and young adults, and presents different manifestations. Rheumatic heart disease (RHD) is considered the most serious complication leading to valvular lesions that are characterized by progressive and permanent heart damage, which entails high cost to the Public Health System in Brazil and worldwide. In previous works that focused on the mechanisms leading to rheumatic heart lesions, we identified the role of T lymphocytes as principal mediator of autoimmune reactions. Through the in deep analysis of infiltrating T-cell receptor repertoire of patients with RHD, we identified oligoclonal expansions with different sizes of CDR3 that is the region of antigen recognition. In the present study we analyzed the thymic activity through T cell receptor excision circles (TREC) quantification in T cells from peripheral blood of RF/RHD patients. We also evaluated naïve and memory T cells from peripheral blood by flow cytometry. Our results showed that the amount of TREC in the peripheral blood of patients was significantly lower when compared to the healthy individuals. In addition, both groups showed that the amount of TREC is negatively correlated with age. These results indicated that the thymic activity in RF/RHD patients is altered probably due to the autoimmune process that involves T lymphocytes

Sulfonated phthalocyanines were characterized using capillary electrophoresis and mass spectrometry. Derivatives investigated included the copper, cobalt, zinc and metal-free sulfonated phthalocyanines. The electropherograms of commercially available copper phthalocyanine-3,4',4'',4'''-tetrasulfonic acid and 4,4',4'',4'''-tetrasulfonic acid were very different, consistent with the latter compound having a structure that is not fully sulfonated. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) were used to characterize the sulfonated phthalocyanines. Mass spectral evidence was obtained for a pentasulfonated species of both the metal-free phthalocyanine and zinc phthalocyanine when these species were made by sulfonation of the metal-free phthalocyanine (followed by zinc insertion in the latter case). Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The heme binding protein (HBP) of this transporter is SiaA (HtsA). Several biophysical techniques were used to determine the coordination state, and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggested that the heme is six-coordinate and low spin in both oxidation states of the protein, with methionine and histidine as axial ligands. The pKa of SiaA was determined, as were the reductive and oxidative midpoint potentials. Guanidinium titration studies of wild-type SiaA showed that the ferric state is less stable than the ferrous state. Free energy of unfolding values [ÄG(H2O)] for the oxidized and reduced proteins were 7.3 ± 0.8 and 16.0 ± 3.6 kcal mol−1, respectively. Denaturation of the histidine mutant H229A was not able to be followed via absorbance spectrometry, possibly due to the large amount of apoprotein present or to non-specific binding of the heme in the binding pocket. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

No description available.