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Characterization of genes conferring V factor independence in haemophilus parainfluenzae and haemophilus ducreyi.Windsor, Helen Marie January 1994 (has links)
A thesis submitted to the Faculty of Medicine, University of the
Witwatersrand, Johannesburg. in fulfilment of the requirements
of Doctor of Philosophy / Haemophilus influenzae and Haemophilus parainfluenzae are obligate
human parasites that form part of the flora of the mucous membranes.
They are normally present in the mouth and upper respiratory tract of
healthy individuals. H. influenzae is known as a major pathogen in
children while H. parainfluenzae is an opportunistic pathogen which is
also found in the urogenital tract. H. ducreyi, however; has only been
isolated from genital ulcers in patients suffering from chancroid. The
various species of the genus Haemophilus are characterised by their
requirements for two growth factors, X factor or haemin and V factor or
nicotinamide adenine dinucleotide (NAD). According to present
taxonomic criteria and laboratory practice, isolates belonging to the genus
Haemophilus that grow in the absence of NAD cannot be identified as H.
influenzae or H. poraintluenzae. However, in 1989 four unusual clinical
strains of H. parainfluenzae were isolated which were found to be V
factor-independent. (Abbreviation abstract) / Andrew Chakane 2019
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Antibiotic resistance in Haemophilus influenzaeGroot, Ronald de, January 1991 (has links)
Thesis Erasmus University Rotterdam. / ook verschenen in gedrukte versie. With bibliogr., with a summary in Dutch.
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Nasopharynx and mucosa associated lymphatic tissue : studies on mucosal immunity, nasopharyngeal colonization with non-encapsulated non-typable Haemophilus influenzae and local administration of immunoglobulin in the upper respiratory tract /Lindberg, Karin, January 1900 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.
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Charakterisierung der initialen Aufnahme des Kofaktors V (NAD) bei Haemophilus influenzaeKemmer, Gabriele. January 1900 (has links) (PDF)
Würzburg, Univ., Diss., 2001. / Erscheinungsjahr an der Haupttitelstelle: 2001
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Antimicrobial resistance in Haemophilus speciesMak, Chun-kit, Gannon., 麥俊傑. January 2006 (has links)
published_or_final_version / abstract / Microbiology / Master / Master of Philosophy
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Receptor-mediated iron and haem transport in HaemophilusParsons, Tina January 1995 (has links)
No description available.
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NAD+-Abhängigkeit bei Pasteurellaceae : Charakterisierung der Nikotinamid-Ribosid-Aufnahme bei Haemophilus influenzae / "NAD+-dependence in Pasteurellaceae:characterisation of the nicotinamide riboside uptake in Haemophilus influenzae"Sauer, Elizabeta January 2006 (has links) (PDF)
Haemophilus influenzae ist ein fakultativ anaerobes, Gram-negatives Bakterium aus der Familie der Pasteurellacaea. Das physiologische Merkmal von H. influenzae ist die essentielle, aber defiziente Hämin- und Nikotinamid-Adenin-Dinukleotid (NAD+) Biosynthese. Während Hämin für aerobes Wachstum benötigt wird, ist NAD+ sowohl für aerobes, als auch für anaerobes Wachstum essentiell. Als NAD+-abhängiger Organismus fehlen H. influenzae die meisten Enzyme für die NAD+-Biosynthese. Daher kann dieses Bakterium nur eine begrenzte Anzahl an Vorläufermolekülen, wie NAD+(P), Nikotinamid-Mono-Nukleotid (NMN) und Nikotinamid-Ribosid (NR) aus der Umwelt zur NAD+-Synthese nutzen. Andere NAD+-unabhängige Pasteurellacaea-Spezies, wie Haemophilus ducreyi, Pasteurella multocida und Actinobacillus actinomycetemcomitans, können auch kein NAD+ aus der de novo Biosynthese bereitstellen, diese Arten können aber zusätzlich auf Nikotinamid (NAm) wachsen. Die Erforschung des NAD+-Aufnahmesystems kann für die Entwicklung antimikrobieller Therapeutika von großem Interesse sein. Von unserer Arbeitsgruppe wurde die NAD+-Aufnahmeroute aufgeklärt; so werden NAD+(P) und NMN von e(P4) und NadN zu NR degradiert, nachdem NAD+ und NMN durch OmpP2 in das Periplasma gelangt sind. NR wird schließlich, als einziges Substrat, durch einen putativen Transporter in das Zytoplasma aufgenommen. In dieser Arbeit wurde der putative NR-Transporter als das hypothetische Gen HI1077.1 im Genom von H. influenzae identifiziert, welches nur 13,8% Identität zu Escherichia coli pnuC und 43,6% Identität zu P. multocida pnuC aufwies. Es konnten zwei Sequenzierungsfehler im original-annotierten Genom von H. influenzae gefunden werden, die zu einer Leserasterverschiebung geführt haben. HI1077.1 wurde zu pnuCHi reannotiert und weist nun eine 19,7% Identität zu pnuCEc und 71,4% Identität zu pnuCPm auf. Es wurde eine HI1077.1 Mutante konstruiert, die ein Wachstumsdefizit auf BHI-Platten bis zu einer NAD+-Konzentration von 15 mM bzw. unterhalb einer NR-Konzentration von 0,1 mM zeigte. Im Transport-Assay transportierte die HI1077.1 Mutante nur noch etwa 1% des eingesetzten NAD+- bzw. NR-Labels. Im Rattenversuch konnte gezeigt werden, dass das in vitro nicht essentielle pnuC in vivo sehr wohl essentiell ist. Die HI1077.1 Mutante verursachte, im Gegensatz zum Wildtyp und zur pnuC-Komplementante keine Bakteriämie mehr. Bei einer Komplementation mit NadV, kodierend für eine Nikotinamid-Phosphoribosyltransferase von H. ducreyi, wurde ebenfalls eine mit dem Wildtyp vergleichende Bakteriämie verursacht. Da bisher noch keine H. influenzae Isolate bekannt sind, die nadV besitzen, würde sich der hier identifizierte NR-Transporter von H. influenzae gut als antimikrobielles Ziel eignen. Die Protein-Topologie von PnuC wurde hinsichtlich der Membranlokalisation analysiert und PnuC konnte als ein Transmembranprotein bestätigt werden, das in der cytosolischen Membran lokalisiert ist. Durch PhoA und LacZ Analysen konnte die Topologie von PnuC aufgeklärt werden. Demnach besitzt PnuC acht Transmembrandomänen (TMD), wobei die N- und C-Termini im Cytosol lokalisiert sind. Die Analyse der strukturellen Funktion ergab, dass PnuC nur eine Unterbrechung der sechs letzten C-terminalen Aminosäuren (AS) toleriert, während die Proteinfunktion durch einen fusionierten C- und N-terminaler His-Tag nur mäsig beeinflusst wird. Des Weiteren wurde die Substratspezifität von PnuC untersucht. Dabei zeigte sich, dass der lange geglaubte NMN-Transporter von E. coli ebenfalls als NR-Transporter fungiert. Die Substratspezifität wurde auch bei A. actinomycetemcomitans und P. multocida untersucht. Es konnte festgestellt werden, dass A. actinomycetemcomitans und P. multocida ebenfalls als einziges Substrat NR transportieren können, aber im Gegensatz zu H. influenzae NAD+ und NMN nicht als NR-Quellen verwerten können, was wahrscheinlich auf das Fehlen von e(P4) und NadN zurückzuführen ist. Zusätzlich wurde in dieser Arbeit das Gen HI0308 untersucht. Um dem Aspekt der Energetisierung des PnuC-Transporters näher zu kommen, sind die Gencluster HI1078-HI1080 und HI0164-HI0171 in die Untersuchung mit einbezogen worden. Die Na+-NQR-Oxidoreduktase wurde im Hinblick ihrer Dehydrogenase-Aktivität genauer charakterisiert. Die Suche nach möglichen Inhibitoren von PnuC führte zu 3-Aminopyridin-Analoga. Durch eine Mutantenanalyse konnte gezeigt werden, dass 3-AADP, 3-AAD und 3-AmPR der selben Aufnahmeroute folgen wie NADP+, NAD+ und NR, und via PnuC in die Zelle aufgenommen werden. Darüber hinaus konnten wir nachweisen, dass NadR aus 3-AmPR und ATP das 3-AAD synthetisiert, welches als kompetitiver Inhibitor mit NAD+ in den zellulären Redoxreaktionen konkurriert und dadurch den Stoffwechsel hemmt. Des Weiteren wurden mehrere spontan 3-Aminopyridin-resistente H. influenzae Mutanten isoliert. / Haemophilus influenzae is a facultativ anaerobic, Gram-negative bacterium of the family of Pasteurellaceae. A physiological characteristic of H. influenzae is its essential but deficient hemin and NAD+ biosynthesis. While hemin is required for aerobic growth, NAD+ is essential for anaerobic and aerobic growth. As a NAD+ dependent organism H. influenzae lacks the enzymes for NAD+ biosynthesis and that is why it can only incorporate a limited number of precursor molecules, such as NAD+(P), NMN, and NR. Other NAD+-independent Pasteurellaceae such as Haemophilus ducreyi, Pasteurella multocida und Actinobacillus actinomycetemcomitans can grow on NAm, additionally to NR. Hence, the investigation of the NAD+ uptake system harbors some potential to be utilized for the development of antimicrobial therapeutics. Our laboratory has clarified the NAD+ uptake route; NAD+(P) and NMN are degraded by e(P4) and NadN to NR, after NAD+ and NMN reached the periplasm through OmpP2. Finally, NR is taken up in the cytoplasm through a putative transporter. In this study, the putative NR transporter was identified as the hypothetical gene HI1077.1 in the genome of H. influenzae, which showed only 13,8% identity to the pnuC of Escherichia coli and 43,6% identity to the pnuC of Pasteurella multocida. Two sequencing errors were found within the pnuC encoding region in the original annotated genome of H. influenzae, which produced frame shifts. After sequence correction HI1077.1 was re-annotated to pnuCHi now showing 19,7% identity to the pnuC of E. coli, and 71,4% identity to the pnuC of P. multocida. A knock-out mutation was constructed for HI1077.1, which showed a growth deficiency on BHI-plates, if supplemented with NAD+ on concentrations below 15 mM, and NR concentrations below 0,1 mM. In transport assay the HI1077.1 mutant only transported 1% of the provided NAD+ and NR label, respectively. In the rat model it could be shown that the pnuC knock-out mutation was essential. There in respect to bacterial growth in the animal model, the HI1077.1 mutant was not able to cause a bacteremia in contrast to the wild type and the pnuC-complemented mutant. When the mutant was complemented with nadV, encoding a nicotinamide phosphoribosyltransferase of H. ducreyi, bacteremia was maintained that was comparable with the one produced by the wild type. Since, so far there are no H. influenzae isolates known, which carry the nadV gene, hence the NR transporter serves for essential gene function and could be further investigated as a potential antimicrobial target. The topology of PnuC was also analysed. The localisation of PnuC was confirmed at the cytosolic membrane. The membrane topology of PnuC was resolved with the investigation of PhoA and LacZ insertion analysis. According to this, PnuC possesses eight transmembrane domains (TMD) with the N-and C-termini localized within the cytosole. The research of the structural function indicates that PnuC tolerates a truncation of the last six C-terminal amino acids, while the function of the protein was only moderately affected by fusions of a C- or N-terminal His-Tags. In addition, the substrate specificity of PnuC was investigated. Thereby, it was demonstrated that the NMN transporter in E. coli in fact acts as an NR transporter. The substrate specificity of A. pleuropneumoniae and P. multocida was also under examination. It was established that A. pleuropneumoniae and P. multocida can transport NR as the unique substrate. However, NAD+ and NMN could not serve as Substrats to deliver NR, raising the question, whether e(P4) or/and NadN function is present in these bacteria. Additionally, in this study the gene HI0308 was investigated. To approach the aspect of energizing the PnuC transporter and the residual low affinity NR uptake the gene cluster HI1078-HI1080 and HI0164-HI0171 were characterised. The Na+-NQR oxidoreductase was more precisely characterised with regard to the activity of the dehydrogenase. The search for putative inhibitors of PnuC yielded in the analysis of 3-aminopyridine analogs. In this work it was demonstrated, that 3-AADP, 3-AAD and 3-AmPR follow the same uptake route as NADP+, NAD+ and NR, and are taken up via PnuC into the cell. Furthermore, we were able to show that 3-AAD can be synthesized from 3-AmPR and ATP. 3-AAD most likely competes versus NAD+ in metabolic relevant redox reactions and thereby inhibits the biosynthesis rate of the cells. In addition, several 3-aminopyridin resistant H. influenzae mutants were isolated.
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Charakterisierung von NadR : das essentielle Enzym der NAD-Synthese bei Haemophilus influenzae / Characterization of NadR: an essential enzyme of NAD synthesis in Haemophilus influenzaeMerdanovic, Melisa January 2005 (has links) (PDF)
I Zusammenfassung Haemophilus influenzae, ein Gram-negatives, Bakterium der Familie Pasteurellaceae, kann beim Menschen eine Vielzahl an Erkrankungen auslösen: Die bekapselte Stämme, v. a. mit Typ b Kapsel können Cellulitis, septische Arthritis, Epiglottitis und Meningitis verursachen. Die nicht-bekapselte Stämme können Otitis media, Sinusitis, Pneumonie und in selteneren Fällen Bakterämie verursachen. Ein besonderes Merkmal des Metabolismus von H. influenzae ist dessen Unfähigkeit Nikotinamid-Adenin-Dinukleotid (NAD+) de novo zu synthetisieren. Daher sind die Enzyme bzw. Transporter, die an NAD+ Aufnahme und Resynthese beteiligt sind, als putative antimikrobielle Ziele von Interesse. In unserer Arbeitsgruppe konnte gezeigt werden, dass NAD+ zu Nikotinamidribosyl degradiert werden muss, bevor es in die Zelle aufgenommen werden kann. Auch Proteine, die an der Degradation des exogenen NAD+ zu Nikotinamidribosyl und dessen anschließender Aufnahme in die Zelle verantwortlich sind, konnten identifiziert und charakterisiert werden. Wie Nikotinamidribosyl im Cytoplasma wiederum zu NAD+ synthetisiert wird, ist auch erst kürzlich geklärt worden: für NadR konnte sowohl eine Ribosyl-Nukleotid-Kinase (RNK) Aktivität als auch eine Nikotinamid-Mononukleotid-Adenylyltransferase (NMNAT) Aktivität in vitro gezeigt werden. Die Kristallstruktur von hiNadR im Komplex mit NAD+ wurde auch aufgeklärt. In dieser Arbeit sollte NadR, insbesondere dessen RNK Domäne, in vivo und in vitro näher charakterisiert werden. Um zu untersuchen, ob beide Domänen in vivo essentiell sind, wurden Deletionsmutanten erzeugt, bei welchen die komplette bzw. der C-terminale Teil der RNK Domäne fehlten. Diese Deletionen konnten im nadV+ Hintergrund erzeugt werden. Die Deletionen konnten in H. influenzae nur zusammen mit dem nadV-Gen transferiert werden oder alternativ nur in die Zellen, die mit pNadRKan Plasmid transformiert wurden. Dies verdeutlicht, dass nicht nur die NMNAT Domäne sondern auch die RNK Domäne bzw. sogar nur wenige C-terminal fehlende Aminosäuren des NadR Proteins essentiell für die Lebensfähigkeit von H. influenzae sind. Gleichzeitig zeigen diese Experimente, dass die RNK-Domäne in Anwesenheit von NadV redundant ist. Ein weiterer Phänotyp der RNK-Deletionsmutante zeigte sich beim Nikotinamidribosyl-Transport. Im Gegensatz zum Wt, welcher ca. 60-80% des 14C-Nikotinamidribosyls aufnahm, konnte für die RNK-Deletionsmutante nur 2-5% Aufnahme gemessen werden. Dies konnte durch das pNadRKan Plasmid komplementiert werden. Weiterhin wurde festgestellt, dass spontan Aminopyridin-resistente H. influenzae Zellen Mutationen im nadR Gen haben, insbesondere im Walker A-Motif (P-Loop) der RNK Domäne. Zusätzlich konnte in dieser Arbeit gezeigt werden, dass NadR aus Aminopyridin und ATP Aminopyridin-Adenin-Dinukleotid synthetisieren kann. Somit konnte gezeigt werden, dass die wachstumshemmende Wirkung eigentlich durch das aus Aminopyridin synthetisierte Aminopyridin-Adenin-Dinukleotid entsteht, welches NAD+ in Redox-Reaktionen verdrängt, wodurch es letztendlich zum Stillstand des Metabolimus kommt. Durch Einführen von gezielten AS-Substitutionen im Walker A und B Motif und in der LID-Domäne von NadR, konnten einige Aminosäuren identifiziert werden, welche essentiell für die Aktivität der RNK Domäne sind. Alle Aminosäuren-Substitutionen führten zum Verlust der RNK Aktivität, die NMNAT Aktivität jedoch war nicht beeinträchtigt. Desweiteren wurden diese NadR Punktmutanten in vivo untersucht. Für alle konnte eine signifikante Defizienz in der Nikotinamidribosyl-Aufnahme beobachtet werden, die gemessene Aufnahme lag im Bereich der RNK-Deletionsmutante. Dadurch konnte eine direkte Korrelation zwischen der RNK Aktivität und der Nikotinamidribosyl-Aufnahme gezeigt werden. In weiteren in vitro Experimenten konnte für NadR eine Feedback-Inhibition durch das NAD+ gezeigt werden, wobei NAD+ in erster Linie die RNK Domäne von NadR inhibiert. Eine graduelle Erhöhung der NAD+ Konzentration führte in den in vitro Assays zu einer graduellen Abnahme der RNK. Bei der NMNAT Aktivität jedoch zeigte sich keine signifikante Inhibition in Anwesenheit von NAD+. Begleitende in vivo Experimente, zeigten eine 2/3 Reduktion der Nikotinamidribosyl-Aufnahme bei den Zellen, die mit NAD+ inkubiert wurden, d. h. höhere intrazelluläre NAD+ Konzentration hatten. Für die genauere Analyse der Feedback-Inhibition durch NAD+ wurden weitere Punktmutanen hergestellt. Bei zwei der Punktmutanten wurde eine Beeinträchtigung der NadR-Aktivität beobachtet, daher wurden diese Punktmutanten von weiteren Analysen im Bezug auf NAD+-Feedback Inhibition ausgeschlossen. Eine Mutante (NadRW256F) jedoch, zeigte ähnliche Aktivität wie das Wt-NadR. In Anwesenheit von NAD+ wurde die RNK Aktivität dieser Punktmutante, im Gegensatz zum Wt-Protein, kaum gehemmt. Dadurch konnte W256 als eine der Aminosäuren identifiziert werden, die an der Vermittlung der NAD+-bedingten Inhibition der RNK-Domäne beteiligt ist. / I Summary Haemophilus influenzae, a gram-negative human pathogen belonging to a family of Pasteurellaceae is a causative agent of several distinct diseases. Whereas capsulated strains, particulary those with tybe b capsule can cause severe invasive infections such as cellulitis, septic arthrithis, epiglottitis and meningitis, non-capsulated strains generally tend to cause localized disease including otitis media, sinusitis, pneumonia and in rare cases bacteremia. The inability to synthesize NAD+ de novo is one of the hallmarks of H. influenzae metabolism, therefore proteins involved in NAD+ uptake and utilization respresent interesting putative targets for development of novel antimicrobial treatment. In our lab we were able to show, that prior to uptake, NAD+ has to be degraded to NR. Several proteins involved in NAD+ degradation and NR uptake were identified and characterized: OmpP2 (an outer-membrane porin), e(P4) (a membrane-bound acid phosphoesterase), NadN (a periplasmatic nucleotidase) and PnuC (a nicotinamidribosyl transporter localized in inner membrane). Enzyme responsible for resynthesis of nicotinamidribosyl to NAD+ was recently found to be NadR: A bifunctional protein containing a nicotinamidribosyl kinase (RNK) and a nicotinamid mononucleotide adenylyltransferase (NMNAT) activity, both of which were confirmed in vitro. Also, the crystal structure of NadR complexed with NAD+ was recently resolved. The aim of this work was to characterize the in vivo function of NadR, particular interest was laid on the characterization of the nicotinamidribosyl kinase domain. To test if both domains of NadR are essential for survival, deletion mutants lacking the entire RNK domain and the C-terminal 58 amino acids were constructed. Initially, these mutants were made in a H. influenzae strain which contains a chromosomal copy of H. ducreyi nadV gene. In following transformation experiments, transfer of the RNK deletion mutants to H. influenzae strain was always accompanied with an nadV transfer as well. Only in strain containing pNadRKan plasmid, no nadV transfer along with RNK-deletions took place. Indirectly, this shows that not only the entire RNK domain is essential for H. influenzae, but also the last 58 amino acids as well. It also shows that in presence of NadV the RNK domain is redundant. RNK deletion mutant displayed a significant deficiency in nicotinamidribosyl transport as well: whereas the Wt strain can accumulate up to 80% of 14C labeled nicotinamidribosyl, RNK mutant is able to accumulate only 2-5%. Introduction of pNadRKan plasmid to RNK mutant restored transport efficiency to Wt level. Studies using spontanous 3-aminopyridine (3-AmPR) resistant H. influenzae isolates, revealed that almost all 3-AmPR resistant isolates have mutations in the nadR gene. A clustering of mutations in Walker A motif of the RNK domain could be observed. Further studies represented in this work, show that 3-AmPR can act as a subtrate for NadR, therefore in ATP consuming reactions aminopyridine-adenindinucleotide can be synthesized. Intracellular aminopyridine-adenindinucleotide replaces NAD+ in redox reactions, which ultimately leads to inhibition of cell metabolism, thereby explaining the mechanism of 3-AmPR based growth inhibition. Using site-directed mutagenesis to introduce amino acid substitutions in distinct parts of the NadR-RNK domain, active sites of the RNK domain were revealed and amino acids essential for the RNK activity were identified. These defined amino acid exchanges resulted in loss of the RNK activity in vitro, but had no effect on the NMNAT activity, which remained intact in these mutant variants of NadR. Following in vivo studies revealed that all mutant NadR proteins caused a severe nicotinamidribosyl uptake deficiency, similar to the one observed in the RNK deletion mutant. Therefore, a direct correlation between the RNK activity and nicotinamidribosyl uptake was shown. Further in vitro studies revealed a feedback inhibition of NadR by NAD+, especially for the RNK domain. In case of RNK domain a gradual increase of NAD+ concentration led to gradual decrease in RNK activity. In contrast, for NMNAT domain no significant inhibition in the presence of NAD+ was observed. Also, in in vivo experiments a 3 fold reduction of nicotinamidribosyl uptake rate was observed when intracellular NAD+ concentrations were higher. To adress the mechanism of NAD+ feedback inhibition, once again, distinct amino acid exchanges were introduced. In vitro, two of the mutant proteins were impaired in their activity, especially if lower protein contrations were used. Therefore, further test concerning inhibtion were not preformed with these mutants. However, a W256F protein displayed activity similar to that of the native protein and furthermore was not inhibited in presence of NAD+. This indicates an involvement of the amino acid W256 in mediating the NAD+ dependent feedback inhibition on NadR activity.
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A Tale of Two Proteins: Insights into the Haemophilus influenzae Hap and Hia AutotransportersSpahich, Nicole Ann January 2011 (has links)
<p>Nontypeable Haemophilus influenzae (NTHi) is a common commensal in the human nasopharynx that can cause localized respiratory tract diseases such as otitis media, bronchitis, and pneumonia. NTHi adheres to respiratory epithelial cells, a critical step in the process of colonization enabled by bacterial surface adhesive structures called adhesins. One group of NTHi adhesins are autotransporters, proteins that have an N-terminal signal sequence, a C-terminal β-barrel domain, and an internal passenger domain with effector function. The goal of this work was to increase our understanding of two NTHi autotransporters, Hap and Hia.</p><p>Hap is a monomeric autotransporter that mediates adherence to epithelial cells and extracellular matrix (ECM) proteins. Hap also self-associates with protein on neighboring bacteria, resulting in bacterial aggregation and microcolony formation. The Hap passenger domain contains the regions responsible for adhesive activity. To define the molecular mechanism of Hap adhesive activity, we crystallized the Hap passenger domain. Characterization of the crystal structure revealed an N-terminal globular domain and a more ordered, prism-like C-terminal domain. Interestingly, Hap crystallized as a multimer, suggesting that Hap-Hap interactions occurred in the passenger domain. Progressive deletions of the β-loops that comprise the C-terminal region disrupted Hap-Hap interactions and led to a defect in bacterial settling. To further support that the C-terminal domain was responsible for Hap-Hap interactions,</p><p>7</p><p>we purified the wild type and truncated passenger domains and conjugated the proteins to latex beads. By light microscopy we visualized bead aggregation when the wild type passenger domain was conjugated to the beads, but not when the truncated passenger domain was conjugated. These results show that the C-terminal portion of the Hap passenger domain is responsible for Hap-Hap interactions leading to multimerization. Hap multimerization could be important in microcolony formation that leads to biofilm formation in vivo.</p><p>The ECM binding domain in located in the final 511 amino acids of the Hap passenger domain. To pin-point the region of the ECM protein fibronectin that is recognized by Hap, we spotted small fragments of fibronectin onto nitrocellulose membranes and incubated the membrane with purified Hap passenger domain. Far Western analysis using Hap antibody revealed that the smallest fibronectin region necessary for binding was comprised of the first two type III repeats, FNIII(1-2). To define the regions of Hap responsible for interaction with fibronectin, we mutated motifs in the Hap passenger domain that are important for fibronectin binding in other bacterial proteins. Based on assessment by ELISA, many of the mutations located between amino acids 525-725 caused reduced bacterial binding to fibronectin. However, no mutation totally ablated binding, suggesting that a larger Hap region is involved in fibronectin binding.</p><p>8</p><p>In an additional study, we identified a relationship between Hap levels in the outer membrane and the expression of lipopolysaccharide (LPS) biosynthesis enzymes. Through Western and qPCR analysis, we found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB and lsgD genes involved in the synthesis of LPS oligosaccharide core in H. influenzae strain Rd/HapS243A resulted in loss of Hap in the bacterial outer membrane and a decrease in hap transcript. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 and IgA1 protease or levels of the p5 or iga1 transcripts, suggesting a Hap-specific effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to the outer membrane and restoration of wild type levels of hap transcript. We speculate that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease in hap transcript. lgtC is one of several phase variable LPS biosynthesis genes. Using an antibody against the epitope formed in part by the lgtC gene product, we identified lgtC phase-off bacteria by Western analysis of colony blots. Consistent with our previous observations, in lgtC phase off bacteria Hap was absent from the outer membrane and hap transcript was reduced. By analyzing a lgtC/lic2A double mutant, we found that Hap localization in the outer membrane and hap transcript levels were not related to LPS size but instead to the functions of the LPS synthesis enzymes themselves. This relationship could be beneficial to bacteria in vivo as a way to regulate Hap expression.</p><p>9</p><p>Early models suggested that autotransporters do not require accessory factors for folding and OM insertion. However, mounting recent evidence has suggested that the Bam complex is required for OM localization of most β-barrel proteins, including autotransporters. We studied the role of the Bam complex in OM localization of the trimeric autotransporter Hia. We expressed Hia in E. coli strains with mutations in the Bam complex and found that BamA and BamD were needed for Hia localization, while BamB, BamC, and BamE were not necessary. In further studies, we mutated the C-terminus of Hia and found that the final and third-to-last amino acids were the most important for outer membrane localization.</p><p>In summary, this work provides insights into the regulation and adhesive activity of Hap and the outer membrane localization of Hia. We have learned important details about these factors that shed light on aspects of H. influenzae disease and could lead to new antimicrobial therapies.</p> / Dissertation
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Molecular Characterization of IgA1 protease from Non-typable Haemophilus influenzaeChang, Hui-hsuan 01 August 2006 (has links)
IgA1 (immunoglobulin A1), a predominant immunoglobulin, is at the first defense line against microbial pathogens infection and invasion, to neutralize pathogenic antigens. Some bacterial pathogens, such as Neisseria meningitidis and Haemophilus influenzae, however, secrete site-specific IgA1 proteases to counteract with the human defense system. The protease is capable of cleaving at the hinge region of immunoglobulin A1 to destroy the structure and function of human IgA1, impairing the role of the immunoglobulin from the host defense. The protease has therefore been implicated as a putative virulence factor that contributes to bacterial colonization, but bacterial isolates from patients with invasive diseases contain both positive and negative IgA1 proteases. To clarify the role of IgA1 protease in bacterial infection, this project is designed to reveal the molecular mechanism of the protease in bacterial infection and colonization. To do this, iga genes encoding non-typable H. influenzae type 1, type 3 and Neisseria meningitidis type 3 IgA1 proteases were isolated, sequenced and then expressed in IgA1 protease-negative E. coli BL21 (DE3). The recombinant proteases have been purified to homogeneity using ion exchange chromatography. Comparison of the deduced amino acid sequences from non-typable H. influenzae IgA1 proteases with other published H. influenzae IgA1 protease revealed a high degree of homology. Sequence analysis indicates that both type 1 and type 3 non-typable H. influenzae IgA1 proteases lack £\-protein in comparison with the iga from N. meningitidis. The role of IgA1 protease in relation to deposition and invasion has also been evaluated in human lung carcinoma cell (A549) model. The results suggest that the IgA1 protease plays a role in the adherence of H. influenzae on epithelial cell surface though the best effectiveness varies upon different pathogenic bacterial strains at different concentrations.
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