Repetitive tetramers of the DNA sequence 5'-CAAT-3' are present in several loci associated with lipooligosaccharide (LOS) phase variation in Haemophilus influenzae type b (Hib). In an attempt to identify H. somnus phase-variable LOS genes, the presence of CAAT repeats within the H. somnus 738 genome was confirmed using a (CAAT)7 probe. A 3.9 kb EcoRI fragment that reacted with the probe was cloned and sequenced. Sequence analysis confirmed the presence of 31 CAAT repeats downstream of two potential start codons, and indicated that small or large proteins would be encoded depending on the number of CAAT repeats. The larger gene products showed 46% amino acid homology to Lex2b from Hib, which influences LOS phase variation in that species. In H. somnus, this gene was named lob1 (lipooligosaccharide biosynthesis gene). Sequence analysis showed that randomly selected colonies most frequently contained 33 CAAT repeats in lob1, corresponding to a 294 amino acid product. Colonies selected for negative reactivity to mAb 5F5 were significantly more likely to have different numbers of CAAT repeats in lob1 than randomly selected colonies. The presence of lob1 in trans altered the LOS profile of a non-phase variable strain of H. somnus, and caused increased levels of reactivity to polyclonal antisera made to purified LOS from strain 738. Based on the ability of this gene to alter the LOS profile of a non-phase varying strain and the correlation of changes in CAAT repeats with mAb 5F5 reactivity, lob1 appears to be involved in LOS biosynthesis and phase variation. / Master of Science

Lipooligosaccharide (LOS) is the major outer membrane component of many Gram-negative bacteria inhabiting the mucosal membranes, including pathogenic species of <I>Haemophilus</I> and <I>Neisseria</I>. LOS phase variation is one mechanism by which some of these bacteria avoid the host immune response. To better understand LOS phase variation as a virulence mechanism of <I>H. somnus</I>, knowledge of the antigenic diversity of LOS epitopes must be increased. Monoclonal antibodies (MAbs) to <I>H. somnus</I> LOS were produced and used with cross-reacting MAbs to <I>H. aegyptius</I> LOS (MAb 5F5) and <I>Neisseria</I> <I>gonorrhoeae</I> LOS (MAb 3F11) in an ELISA to investigate LOS heterogeneity among forty-five strains of <I>H. somnus</I>. Using three MAbs, thirty-nine of these <I>H. somnus</I> strains were grouped into six antigenic types. Three groups, associated solely with the cross-reacting MAbs 5F5 and 3F11, included the majority (76%) of <I>H. somnus</I> strains. The anti-<I>H. somnus</I> LOS MAb 5D7 recognized a low frequency epitope associated with each of the remaining three groups, which included 11% of the <I>H. somnus</I> strains. Six strains (13%) were not recognized by any of these MAbs. Inhibition ELISA experiments showed that the MAb 5F5 epitope contained phosphocholine (PCho) and this epitope was present in 56% of the strains tested. The MAb 5F5 epitope is phase variable in <I>H. somnus</I> LOS. How PCho negative variants could allow for systemic infection after initial colonization of the mucosa by PCho positive variants is discussed. / Master of Science

More than half of all people worldwide are infected with H. pylori. The infection always cause a gastric inflammation that may develop into peptic ulcer disease or gastric cancer. Attachment proteins, adhesins, mediate specific adherence of H. pylori to receptor structures on the human gastric mucosa. The best-characterized H. pylori adhesin-receptor interactions are the BabA adhesin and the binding to the fucosylated blood group antigens ABO/Lewis b (Leb) and the SabA adhesin and its binding to the inflammation associated sialyl-Lewis x antigen. During H. pylori infection the availability of receptor structures on the human gastric mucosa changes as a consequence of the host inflammatory and immune responses. Consequently the bacterial population need to adjust its adherence properties to stay colonized. This thesis describes mechanisms that generate H. pylori populations with variable adherence properties and mechanisms for adjustment of adhesin expression levels.In H. pylori strains devoid of Leb-binding, we found bacterial cells with Leb-binding. Isolation of such H. pylori clones demonstrated that the change in receptor binding phenotype was obtained via the mechanisms of homologous recombination and slipped strand mispairing (SSM). Disease presentation in relation to BabA expression was studied in H. pylori infected Mongolian gerbils. We showed that BabA was not essential for colonization but caused severe injury to the gastric mucosa and was turned off during long-term infection by nucleotide changes within the babA gene. Gerbils infected with BabA-weak-expressing strains maintained BabA expressing clones for a longer period than gerbils that were infected with BabA-high-expressing strains. Studies of the gerbil gastric mucosal glycosylation showed that gerbils respond in a similar way as humans and Rhesus monkeys which support gerbils to be a model suitable for studying H. pylori infection and disease outcome in relation to adherence.We studied the SSM mechanism of SabA phase variation and the cognate shift in sLex-binding phenotype and we show sLex-binding activity to be growth phase dependent. H. pylori vesicles were characterized for the major phosholipid and protein components. Virulence factors e.g., VacA, and CagA were identified and both the BabA and the SabA adhesins was shown to be located on the vesicle surface and to mediate specific binding to their cognate receptors present on the human gastric mucosa. H. pylori generate bacterial cells with different receptor binding phenotypes via the mechanisms of homologous recombination, SSM and nucleotide changes. These mechanisms will probably contribute to bacterial fitness by the generation of quasi species populations where some of the clones will be better adapted to the environmental chances during persistent infection.

Previous work carried out in our laboratory has identified that phase variation of type III R-M systems found in Haemophilus influenzae, Neisseria meningitidis and N. gonorrhoeae is reversible, and occurs at high frequency, as seen both through mod::lacZ fusions, and by measuring changes in repeat tract length. In addition, phase variation of the methyltransferases results in coordinated switching of expression of a distinct group of genes in each of the strains studied so far. WE have termed this phenomenon the PHASEVARION, for phase variable regulon, to identify the set of genes whose expression is affected by moe phase variation. Many of the genes found to be regulated by mod phase variation are known virulence factors and even include some genes investigated as candidates for vaccine development (Srikhanta et al., 2005 and 2009. The aims of this project was to further the investigation of how these R-M systems regulated the expression of genes which hitherto had not been predicted to phase vary. The first step in the process of investigating how phase variable R-M systems influence expression of unrelated genes is to identify the DNA sequences methylated by the methyltransferases of interest. As discussed in Chapter 3, elucidation of the ModA1 methylation target site was in part facilitated by predictions that the phase variable methyltransferase found in H. influenzae strain Rd methylated the same sequence as did HinfIII, isolated from H. influenzae strain Rf. This hypothesis was confirmed by methylation dependent inhibition of digestion, revealing that ModA1 methylates the second A in its recognition sequence, 5’-CGAAT-3’. Once confirmed, the genes found to be regulated by modA1 phase variation in the initial phasevarion study could be investigated for the presence of ModA1 methylation sites within their promoters or upstream of their transcriptional regulators. Two such methylation target sites were located just upstream of the dnaK ORF. Transcriptional start site analysis of the dnaK gene revealed three transcripiotnal start sites, one of which is unduced by heat shock. Exactly 10 nucleotides upstream of this heat shock induced transcriptional start site lies one of these ModA1 methylation target sequences. Ongoing invetigations are looking into the importance of this ModA1 site located within the dnaK promoter, and whether this is the site responsible for ModA1 dependent variations in dnaK expression. Although numerous methods were investigated for their potential to identify all sites methylated by the different modA alleles, the only method which resulted in identification of any methylation target sites was methylation dependent inhibition of restriction. This method allowed us to confirm the ModA1 recongition sequence, and to discover the methylation sequence, and adenine targeted by the modA13 allele, which is found in many clinically relevant N. gonorrhoeae strains. As will be discussed in Chapter 5, ModA13 dependent inhibition of restriction was first observed when the Neisserial plasmid pCmGFP was extracted from modA13 ON and modA13::kan cells, and further investigated and confirmed using a Southern blot approach to determine whether ModA13 dependent inhibtion could be detected as differential methylation of the chromosome. It was found that ModA13 recognised the sequence 5’-AGAAA-3’, with methylation occurring on the second last A. This sequence was mapped not only to the genes found to be regulated by modA13 phase variation, but also to the entire FA1090 chromosome, and this information will be used in future studies to investigate the direct molecular mechanisms by which modA13 phase variation results in subpopulations with different phenotypes in relation to antimicrobial resistance and biofilm/cell invasion.

Being among the most prevalent of persistent infectious agents in humans worldwide, Helicobacter pylori induces chronic inflammation (gastritis), which may progress to peptic ulceration and stomach cancer. The ability to adhere to the gastric mucosa is considered to be both a colonization and virulence property of H. pylori. For adherence, H. pylori expresses surface-located attachment proteins (adhesins) that bind to specific receptors in the gastric mucosa. The best characterized H. pylori adhesin-receptor interaction is that between the blood group antigen binding adhesin (BabA) and the fucosylated blood group antigens, which are glycans highly expressed in the gastric mucosa. Our recent results have changed the view of the blood group antigen-specific binding mode of H. pylori. We have tested clinical isolates of H. pylori from human populations worldwide for their ability to bind to ABO blood group antigens. The results revealed that more than 95% of isolates from Sweden, Germany, Spain, Japan and Alaska that bind fucosylated blood group antigens, bind both the Lewis b antigen (Leb) (of blood group O) and the blood group A-related antigen A-Lewis b, i.e. they exhibit a generalist type of binding mode. In contrast, the majority of strains (62%) from South American Amerindians bound best to Leb, i.e. they exhibit a specialist blood group “O antigen” binding mode. This specialization in binding coincides with the unique predominance of blood group O in the South American Amerindian populations. Furthermore, we also showed that H. pylori could switch from specialist to generalist binding modes by chromosomal integration of foreign babA gene fragments. A mutant strain lacking the babA gene turned out to adhere to inflamed gastric epithelium, despite the fact that it did not bind Leb. We identified the receptor to which the mutant binds to as the sialyl-dimeric-Lewis x antigen (sdiLex) and found its expression to be associated with persistent H. pylori infection and chronic inflammation, both in humans and Rhesus monkeys. The cognate sialic acid binding adhesin (SabA) was identified by our ReTagging technique. Deletion of sabA caused loss of H. pylori binding to sialylated glycans, and screening of single colony isolates revealed a high frequency of spontaneous on⇒off phase variation in sLex binding. Using erythrocytes as a model for sialyl dependent cell adhesion, we could show that SabA is the sought-after H. pylori sialyl-dependent hemagglutinin. Swedish clinical H. pylori isolates were analyzed for sialyl-dependent hemagglutination (sia-HA), and the sia-HA titers were found to be highly correlated to the levels of sLex binding. Clinical isolates were shown to exhibit several distinct binding modes for sialylated glycans, which suggest that SabA exhibit polymorphism in binding. We also found that SabA binds to sialylated glycans on neutrophil surfaces by mechanisms involving “selectin mimicry”, and that SabA plays an important role in nonopsonic activation of neutrophils. In the human stomach, H. pylori is exposed to selective pressures such as immune and inflammatory responses, and this is reflected by changes in mucosal glycosylation patterns. The high mutation and recombination rates of H. pylori in combination with bio selection will continuously generate clones that are adapted to changes in individual gastric mucosa. Such adaptive selection contributes to the remarkable diversity in binding modes and to the extraordinary chronicity of H. pylori infections worldwide.

F1651, les pili Pap et l’antigène CS31A associé aux antigènes de surface K88 sont tout trois des membres de la famille de type P des facteurs d’adhérence jouant un rôle prépondérant lors de l’établissement d’une maladie causée par des souches Escherichia coli pathogènes, en particulier des souches d’E. coli pathogènes extra-intestinales (ExPEC, Extra-intestinal pathogenic E. coli). Leur expression est sous le contrôle d’un mécanisme de régulation transcriptionnel dépendant de l’état de méthylation de l’ADN, résultant dans l’existence de deux populations définies, l’une exprimant l’adhésine (population ON) et l’autre ne l’exprimant pas (population OFF). Malgré de fortes identités de séquences, ces trois systèmes diffèrent l’un de l’autre, principalement par le pourcentage de cellules ON rencontrées. Ainsi, quand CS31A est systématiquement orienté vers un état considéré comme OFF, F1651 présente une phase ON particulièrement élevée et Pap montre deux états OFF et ON bien distincts, selon le phénotype de départ. La protéine régulatrice sensible à la leucine (Lrp, Leucine-responsive regulatory protein) joue un rôle essentiel dans la réversibilité de ce phénomène épigénétique et il est supposé que les différences de séquences au niveau de la région régulatrice modifient la localisation à ces sites de fixation de Lrp; ce qui résulte, en final, aux différences de phase existant entre CS31A, F1651 et Pap.À l’aide de divers techniques parmi lesquelles l’utilisation de gènes rapporteurs, mutagénèses dirigées et d’analyse des interactions ADN-protéines in vitro, nous montrons dans ce présent projet que la phase OFF prédominante chez CS31A est principalement due à une faible interaction de Lrp avec la région distale de l’opéron clp, et que la présence d’un homologue du régulateur local PapI joue un rôle également clef dans la production de CS31A. Dans le cas de F1651, nous montrons dans cette étude que le taux élevé de cellules en phase ON est dû à une altération dans le maintien de Lrp sur les sites répresseurs 1-3. Ceci est dû à la présence de deux nucléotides spécifiques, situé de part et d’autre du site répresseur 1, qui défavorisent la fixation de Lrp sur ce site précis. Tout comme dans le cas de CS31A, la formation d’un complexe, activateur ou répresseur de la phase ON, dépend également de l’action de du régulatuer local FooI, qui favorise alors le déplacement de Lrp des sites répresseurs 1-3 vers les sites activateurs 4-6. / F1651, the pyelonephritis-associated pili (Pap) and the K88-related surface antigen CS31A are three members of the type P family of adhesive factors that play a key role in the establishment of disease caused by Extra-intestinal Escherichia coli (ExPEC) strains. They are all under the control of methylation-dependent transcriptional regulation that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Despite a high similarity in DNA sequence, these three adhesive systems nonetheless differ in the ratio of ON cells. While CS31A is always turned toward the OFF state, F1651 presents a particularly high level of ON cells and Pap shows two distinct OFF and ON states, depending on the starting phenotype. The leucine-responsive regulatory protein (Lrp) plays an essential role in the reversibility of this epigenetic switch and it is believed that the difference in nucleotides within the regulatory region of each operons could modify the binding of Lrp and, in turn, CS31A, Pap and F1651 phase variation. Using a variety of techniques including gene expression, site-directed mutagenesis, and in vitro protein–DNA interaction analysis, we demonstrate that the preferential OFF state observed in CS31A-positive cells is mainly due to a weak interaction of Lrp with the clp distal region and that the presence of a PapI homologue within the cell plays a key role in CS31A production. For F1651, we show in this study that the high level of ON cells found during F1651 phase variation is due to an altered stability of the DNA complex formed by Lrp at its repressor binding sites 1-3. Again, after each cell cycle, complex formation is modulated by the local regulator FooI (homologue to PapI) which promotes the transit of Lrp toward its activator binding sites 4-6. Furthermore, we identify two nucleotides (T490, G508) surrounding the Lrp-binding site 1 that are critical to maintaining a high OFF to ON switch rate during F1651 phase variation, as well switching Pap fimbriae toward the OFF state.

Medical Laboratories Accreditation is covered by ISO 15189:2012 - Medical Laboratories — Requirements for Quality and Competence. In Portugal, accreditation processes are held under the auspices of the Portuguese Accreditation Institute (IPAC), which applies the Portuguese edition (NP EN ISO 15189:2014). Accordingly, Medical Laboratories accreditation processes now require the estimate of measurement uncertainty (MU) associated to the results. The Guide to the Expression of Uncertainty in Measurement (GUM) describes the calculation of MU, not contemplating the specific aspects of medical laboratory testing. Several models have been advocated, yet without a final consensus. Given the lack of studies on MU in Portugal, especially on its application in the medical laboratory, it is the objective of this thesis to reach to a model that fulfils the IPAC’s accreditation regulations, in regards to this specific requirement. The study was based on the implementation of two formulae (MU-A and MU-B), using the Quality Management System (QMS) data of an ISO 15189 Accredited Laboratory. Including the laboratory’s two Cobas® 6000–c501 (Roche®) analysers (C1 and C2) the work focused three analytes: creatinine, glucose and total cholesterol. The MU-B model formula, combining the standard uncertainties of the method’s imprecision, of the calibrator’s assigned value and from the pre-analytical variation, was considered the one best fitting to the laboratory's objectives and to the study's purposes, representing well the dispersion of values reasonably attributable to the measurand final result. Expanded Uncertainties were: Creatinine - C1 = 9,60%; C2 = 5,80%; Glucose - C1 = 8,32%; C2 = 8,34%; Cholesterol - C1 = 4,00%; C2 = 3,54 %. ...[cont.].

No description available.

Les Escherichia coli pathogènes extra-intestinaux (ExPEC) sont responsables d’une grande variété de maladies. Plus particulièrement, certaines souches ExPEC, du sous-groupe d’E. coli uropathogènes, sont porteuses de fimbriae de type P. Cette famille d’adhésines est soumise à une régulation transcriptionnelle appelée variation de phase; un mécanisme du tout ou rien. Il s’agit d’une compétition entre deux protéines régulatrices : la Dam méthylase et la nucléoprotéine Lrp. Ce mécanisme est aussi soumis à l’influence des régulateurs locaux PapB et PapI, deux régulateurs essentiels. Afin d’étudier PapI et ses homologues ainsi que leur impact sur la variation de phase des fimbriae F1651, Pap et CS31A. Grâce à une fusion chromosomique entre la région régulatrice de clp et les gènes lacZYA, nous avons étudié l’effet, en trans, de PapI et FooI qui ont pu restaurer la variation de phase avec une forte tendance pour la phase OFF. Pour étudier l’action de ces protéines sur foo et pap, nous avons utilisé un système utilisant gfp comme gène rapporteur de l’activité des promoteurs des opérons pap et foo. Cela a permis d’observer la variation de phase au niveau cellulaire par cytométrie en flux et en temps réel par microscopie à fluorescence. Ces expériences ont confirmé que la population de cellules F1651 positives a un phénotype d’expression de F1651 partielle alors que les cellules Pap sont en majorité en phase OFF. PapI et FooI n’ont pas la même influence sur la variation de phase, puisque FooI favorise une plus grande fréquence de variation de phase. / Escherichia coli extra-intestinal pathogenic (ExPEC) are responsible for a wide variety of diseases. Particularly ExPEC strains from the subset called uropathogenic E.coli (UPEC) are carrying fimbriae type P. This adhesin family is subject to transcriptional regulation called phase variation, an all or nothing mechanism. It is a competition between two regulatory proteins: the Dam methylase and the nucleoprotein Lrp. This mechanism is also under the influence of the local regulators PapB and PapI. These two regulators are essential to the phase variation. We therefore sought to investigate PapI and its homologs and their impact on the phase variation of fimbriae F1651, Pap, and CS31A. By means of a chromosomal fusion between the regulatory region of clp gene and lacZYA, we studied the effect in trans of PapI and FooI which could restore the phase variation with a strong tendency to phase OFF. To study the action of PapI and FooI, we used a system with gfp as a reporter gene in operons pap and foo. This allowed the observation of the phase variation at the cellular level by flow cytometry and real-time fluorescence microscopy. These experiments confirmed that the population of F165 positive cells have a partial expression state whereas Pap cells mostly have an OFF expression state. We also confirmed that FooI and PapI do not have the same influence on phase variation and that FooI promotes greater frequency of phase variation.

Neisseria meningitidis causes 500,000 cases of meningitis and septicaemia annually worldwide, with a mortality rate of approximately 10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opa proteins are major meningococcal outer membrane proteins, and a limited number of Opa variants have been associated with hyperinvasive serogroup B meningococci, suggesting their use as a potential novel vaccine. Immunisation of mice with recombinant Opa elicited high levels of meningococcal-specific serum bactericidal antibody (SBA), demonstrating proof in principle of this approach. Opa proteins mediate bacterial adherence to host cells and modulate human cellular immunity, and there are conflicting data regarding their effects on CD4⁺ T cells. opa genes from N. meningitidis strain H44/76 were cloned into the plasmid vector pBluescript, disrupted using antibiotic resistance cassettes and transformed into H44/76 to sequentially disrupt the four opa genes. This produced a unique panel of 15 isogenic Opa-deficient strains, including an Opa-negative strain, which enabled investigation of the immunomodulatory role of surface-expressed Opa proteins. There was no consistent effect of Opa expressed on the surface of OMVs and inactivated bacteria on CD4⁺ T cells, with significant heterogeneity of responses between individuals. The rate of Opa phase variation was between 10^-3 and 10^-4, and increased 180-fold following transformation of bacteria with unrelated DNA. These data support further investigation of Opa as a potential meningococcal vaccine component, and highlight the importance of host and bacterial factors in the development of OMV vaccines.