Thesis (Ph.D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on March 10, 2009) Includes bibliographical references.

University, Diss., 2005--Würzburg.

Neutrophils are the innate immune system's first line of defense in response to an infection. During an infection in the tissue, chemical cues called chemoattractants are released, which signal neutrophils to exit circulation and enter the tissue. Once in the tissue, neutrophils directionally migrate in response to the chemoattractant and toward the site of infection in a process called chemotaxis. At the site of infection, they initiate antimicrobial responses to clear the infection and resolve inflammation, restoring homeostasis. However, neutrophils are exposed to multiple chemoattractants and must prioritize these signals in order to correctly migrate to the appropriate site. The ability of neutrophils to properly undergo chemotaxis in the presence of infection and inflammation is crucial for resolution of inflammation and pathogen clearance. It has been recently shown that when pre-conditioned with bacterial endotoxin (LPS), innate immune function can become dysregulated. Neutrophils start to display altered antimicrobial response as well as dysfunctional migration patterns. This behavior has been seen in patients with sepsis, where a person's immune system overreacts to an infection, leading to systemic inflammation throughout the body, causing tissue damage, multiple organ failure, and in many cases, death. We explore the effects of inflammation on neutrophil migratory patterns and decision-making within chemotaxis. Additionally, to understand how inflammation within disease impacts chemotaxis, we measure the difference between neutrophils from healthy individuals and those from septic patients. We approached this using a combination of experimental and computational techniques. We developed a microfluidic assay to measure neutrophil decision-making in a competitive chemoattractant environment between an end-target (fMLP) and intermediary (LTB4) chemoattractant. Additionally, we probed for the expression level of molecules related to neutrophil chemotaxis. We also built a system of ordinary differential equations to model the dynamics of the molecular interactions underlying neutrophil chemotaxis. Our results showed that when neutrophils were induced into a highly inflammatory state, they prioritized pro-inflammatory signals over pro-resolution signals and displayed dysfunctional migration patterns. Similarly, neutrophils from patients with sepsis also displayed dysregulated migration patterns. This aberrant neutrophil chemotaxis may be implicated in the pathogenesis of sepsis, where accumulation of neutrophils in off-target organs is often seen. These results shed light onto the directional migratory decision-making of neutrophils exposed to inflammatory signals. Understanding these mechanisms may lead to the development of pro-resolution therapies that correct the neutrophil compass and reduce off-target organ damage. / Doctor of Philosophy / Neutrophils are innate immune cells that act as the first line of defense toward an infection. During an infection, chemical signals are released, stimulating neutrophils to migrate toward that specific site of infection. Once the cells are in the tissue, they can clear the pathogen and resolve inflammation. However, when neutrophils are migrating in the tissue, they are overwhelmed with multiple signals, directing them toward different sites. These signals must be prioritized by the cell so they can properly migrate toward the correct location. It has been recently shown that neutrophils that have been preconditioned into inflammatory states will display dysfunctional migration patterns. They are unable to migrate to the site of infection and instead migrate to healthy tissue, where they can cause damage. This has been shown in patients with sepsis, which is a condition where a person's immune system overreacts to an infection, causing inflammation throughout the body, leading to tissue damage and multiple organ failure. Our work explores the impact of inflammation on neutrophil migration patterns and the ability of the cell to properly prioritize when stimulated by multiple chemical signals. Additionally, we look at how neutrophils from healthy individuals differ from neutrophils from patients with sepsis, to understand how inflammation within disease impacts cellular migration. We approach this both experimentally and computationally. We designed a microfluidic assay to measure neutrophil migration in the presence of two competing chemical signals. We also measured the expression levels of molecules relevant to cell migration. We also built a mathematical model to investigate the molecular interactions underlying these processes. These results shed light on how inflammation impacts neutrophil migration and its role in inflammatory diseases.

Perceiving and responding to the environment is key to survival. Using the prokaryotic equivalent of a nervous system – the chemotaxis system – bacteria sense chemical stimuli and respond by adjusting their movement accordingly. In chemotactic bacteria, such as the well-studied E. coli, environmental nutrient sensing is achieved through a membrane embedded protein array that specifically clusters at the cell poles. Signalling to the motor is performed by activation of the CheA kinase, which phosphorylates CheY and CheB. CheY-P tunes the activity of the flagellar motor while CheB-P, together with CheR is involved in adaptation to the stimulus. In E. coli, a dedicated phosphatase terminates the signal. Most bacterial species however, have a much more complex chemotaxis network. Rhodobacter sphaeroides, a model organism for complex chemotaxis systems, has one membrane-embedded chemosensory array and one cytoplasmic chemosensory array, plus several homologs of the E. coli chemotaxis proteins. Signals from both arrays are integrated to control the rotation of a single start-stop flagellar motor. The phosphorelay network has been studied extensively through in vitro phosphotransfer while in vivo studies have established the components of each array and the requirements for formation. Mathematical modelling has also contributed towards inferring connectivities within the signalling network. Starting by constructing a two-hybrid-based interaction network focused on the components of the cytoplasmic chemosensory array, this thesis further addresses its associated adaptation network through a series of in vivo techniques. The swimming behaviour of series of deletion mutants involving the adaptation network of R. sphaeroides is characterised under steady state conditions as well as upon chemotactic stimulation. New connectivities within the R. sphaeroides chemotaxis network are inferred from analysing these data together with results from in vivo photoactivation localisation microscopy of CheB₂. The experimental results are used to propose a new model for chemotaxis in R. sphaeroides.

Apoptosis is a physiological form of cell death. It is a highly evolutionarily conserved process that is non-inflammatory or anti-inflammatory in nature. This anti-inflammatory nature of apoptosis is evident by the fact that neutrophils are histologically absent from sites where homeostatic apoptosis rates are high. The rapid phagocytosis of apoptotic cells as a means to prevent the release of noxious inflammatory compounds also accounts for the anti-inflammatory environment of such sites. However, the mechanisms that enable mononuclear phagocytes to migrate to sites where homeostatic apoptosis rates are high, and not granulocytes, the professional phagocytes that accumulate at sites of inflammation, have not been determined yet. Using Burkitt’s lymphoma (BL) as a model of apoptosis, the aim of this thesis was to identify the regulatory mechanisms or factors underlying the non-phlogistic features of sites where homeostatic apoptosis rates are high and in particular, those preventing the recruitment of neutrophils - a major granulocyte subclass to these sites. BL is a highly aggressive B cell lymphoma that is mainly characterised by a high rate of apoptosis. By carrying out a series of in vitro chemotaxis assays and biochemical approaches, it was found in this thesis that BL cells actively inhibit neutrophil migration by releasing factors that were identified to be lactoferrin, a 80 kDa iron-binding glycoprotein with anti-bacterial and anti-inflammatory properties. It was further demonstrated that lactoferrin selectively inhibited migration of granulocytes (both neutrophils and eosinophils) but not mononuclear phagocytes and this effect was irrespective of its iron saturation status and the chemoattractant used. Also, lactoferrin potently inhibited neutrophil migration, as assessed by thioglycollate-induced in vivo model of mouse peritonitis. This anti-inflammatory function of lactoferrin was attributed to its effect on granulocyte signalling pathways that regulate cell adhesion and motility. Finally, it was demonstrated that in cell types of diverse lineages, induction of apoptosis results in de novo synthesis and secretion of lactoferrin. In subsequent proliferation assays determining the in vitro growth of a number of BL cell types, it was demonstrated that lactoferrin is an essential component of BL cells and promotes their proliferation, as its antibody-mediated neutralisation or shRNA-mediated expression knockdown, reduced BL cell growth. Together, the results of this thesis identified lactoferrin as one of the few characterised antiinflammatory components of the apoptosis milieu that negatively regulate granulocyte migration. This effect may provide opportunities for broad therapeutic interventions concerning the use of lactoferrin in chronic inflammatory conditions characterised by aberrant neutrophil influx as well as atopic allergic disorders, such as asthma. Moreover, based on the tumour-supporting role of lactoferrin described in this study, targeting its expression in tumours could lead to tumour regression and thus, be a promising therapeutic molecule in tumour immunotherapy.

Root cap cells of two cotton species elicited a specific chemotactic response in zoospores of Pythium dissotocum. When roots of cotton seedlings were placed into a suspension of P. dissotocum zoospores, there was immediate attraction, accumulation and encystment exclusively in the root cap cell region. Furthermore, root cap cells remained attractive when isolated from the root: attraction, accumulation, and encystment on individual root cap cells occurred within seconds after contact. Zoospores penetrated and killed isolated root cap cells within 15-30 minutes, and seedlings died within 24 hours. In contrast, zoospores of P. catenulatum, which exhibited a chemotactic response to roots of Bentgrass, were not attracted to and did not infect seedlings or isolated root cap cells of cotton. Preliminary studies indicate that both Pythium species are capable of infecting cotton seedlings in sand culture, though it is not known if either are pathogens on cotton grown in the field.

The internal organisation of bacteria is far more complex than originally thought. Many components of the cell have specific localisation patterns. Proteins are localised to many different regions of the cell by numerous mechanisms, and often their function depends on correct localisation. Bacterial and plasmid DNA are also highly organised and actively positioned. These tightly regulated positioning patterns ensure stable maintenance of genetic material. Members of the ParA/MinD family of ATPases are responsible for the segregation of a large number of bacterial chromosomes and plasmids. Recently members of this family have been shown to position and segregate protein complexes. One such complex is the cytoplasmic chemosensory cluster of Rhodobacter sphaeroides. This large complexes are segregated from a single cluster positioned at the mid-cell to two clusters at 1/4, 3/4 positions by the ParA homologue PpfA using the nucleoid as a scaffold. This ensures that each daughter cell inherits a cluster. This study sought to investigate this cytoplasmic chemosensory cluster, and its positioning and segregation by PpfA through the cell cycle. The use of fluorescence recovery after photobleaching revealed that like membrane bound chemoreceptor arrays the cytoplasmic cluster of R. sphaeroides is a highly stable complex. The difference seen between the cytoplasmic cluster and the data reported for the membrane bound cluster of Escherichia coli is probably due to the lack of membrane helping hold the array together. Investigation of the role of PpfA in segregation of the cytoplasmic cluster, using fluorescence imaging and single molecule tracking with a range of mutants through the cell cycle, suggest that it uses a mechanism unlike any reported for ParA homologues. Single molecule tracking of PpfA molecules shows that the chemoreceptor TlpT stabilises PpfA molecules resulting in slower diffusion of PpfA molecules at the cluster. The use of a ΔppfA mutant shows that PpfA restrains the movement of the cluster, together these results suggest a model in which TlpT stabilises PpfA’s interaction with the nucleoid and PpfA positions the cluster.

Immune cell migration and chemotaxis plays a key role in immune response. Further research to study the mechanisms of immune cell migration and to develop clinical applications requires advanced experimental tools. Microfluidic devices can precisely apply chemical gradient signals to cells, which is advantageous in quantifying cell migratory response. However, most existing microfluidic systems are impractical to use without specialized facilities and research skills, which hinders their broad use in biological and medical research communities. In this thesis, we integrated several new developments in microfluidic gradient generating devices, compact imaging systems, on-chip cell isolation, cell patterning, and rapid data analysis, to provide an easy-to-use and practical solution for immune cell migration and chemotaxis experiments. Using these systems, we quantitatively studied neutrophil migration for both research and clinical applications. First, we developed a compact USB microscope-based Microfluidic Chemotaxis Analysis System (UMCAS), which integrates microfluidic devices, live cell imaging, environmental control, and data analysis to provide an inexpensive and compact solution for rapid microfluidic cell migration and chemotaxis experiments with real-time result reporting. To eliminate the lengthy cell preparation from large amounts of blood, we developed a simple all-on-chip method for magnetic isolation of untouched neutrophils directly from small volumes of blood, followed by chemotaxis testing on the same microfluidic device. Using these systems, we studied neutrophil migration in gradients of different chemoattractants, such as interleukin-8 (IL-8), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and clinical sputum samples from Chronic Obstructive Pulmonary Disease (COPD) patients. Previous studies have shown that COPD is correlated with neutrophil infiltration into the airways through chemotactic migration. The thesis work is the first application of the microfluidic platform to quantitatively characterizing neutrophil chemotaxis to sputum samples from COPD patients. Our results show increased neutrophil chemotaxis to COPD sputum compared to control sputum from healthy individuals. The level of COPD sputum induced neutrophil chemotaxis was correlated with the patient’s spirometry data. Collectively, the research in this thesis provides novel microfluidic systems for neutrophil migration and chemotaxis analysis in both basic research and clinical applications. The developed microfluidic systems will find broad use in cell migration related applications. / May 2016

In der vorliegenden Arbeit wurde zunächst die Konservierung bekannter RpoS-assoziierter Funktionen für das V. cholerae Homolog untersucht. Dabei ergab die phänotypische Analyse der rpoS-Deletionsmutante, dass analog zu der Bedeutung als Regulator des Stationärphasen-Wachstums in E. coli, definierte Zelldichte-abhängige Eigenschaften in V. cholerae gleichermaßen der Kontrolle von RpoS unterliegen. In weiterführenden Experimenten konnte daraufhin die Konservierung der entsprechenden Promotorstrukturen über die funktionelle Komplementierung rpoS-abhängiger Gene durch das jeweils speziesfremde Protein aufgedeckt werden. Dahingegen konnte die Bedeutung von RpoS bei der Ausprägung der generellen Stress-Resistenz u. a. in E. coli für das V. cholerae Homolog über den gewählten experimentellen Ansatz nicht belegt werden. So wurden in Survival-Assays für keine der getesteten Stress-Bedingungen signifikante Unterschiede zwischen rpoS-Mutante und Wildtyp ermittelt. Die in E. coli gezeigte intrazelluläre Anreicherung des Sigmafaktors unter diversen Stress-Situationen konnte ebenfalls nicht nachgewiesen werden. Hinsichtlich der potentiellen Stellung von RpoS als globaler Regulator für Virulenz-assoziierte Gene, unterstützen und ergänzen die Ergebnisse der vorliegenden Arbeit die gegenwärtige Theorie, wonach RpoS das Ablösen der V. cholerae Zellen vom Darm-Epithel fördert. Die postulierte Bedeutung des alternativen Sigmafaktors in der letzten Phase der Pathogenese wurde über die RpoS-abhängige Sekretion der Mukin-degradierenden Protease HapA und die hier unabhängig nachgewiesene Transkriptionskontrolle von Chemotaxis-Genen bestätigt. In E. coli gilt als entscheidender Parameter für die dargelegten RpoS-Funktionen die intrazelluläre Konzentration des Masterregulators. Deshalb war ein weiteres zentrales Thema dieser Arbeit die Regulation des RpoS-Levels in V. cholerae. Neben der Identifizierung von Bedingungen, welche die RpoS-Expression beeinflussen, wurde vorrangig der Mechanismus der Proteolyse analysiert. Dabei wurden als RpoS-degradierende Komponenten in V. cholerae die Homologe des Proteolyse-Targetingfaktors RssB und des Protease-Komplexes ClpXP identifiziert. Die weitere Untersuchung der RpoS-Proteolyse ergab außerdem, dass bestimmte Stress-Signale den Abbau stark verzögern. Interessanterweise resultierten die gleichen Signale jedoch nicht in der Akkumulation von RpoS. Als weiterer Unterschied zu der bekannten Proteolysekaskade in E. coli zeigte sich, dass das V. cholerae Homolog der RssB-aktivierenden Kinase ArcB (FexB) an der RpoS-Proteolyse nicht beteiligt ist. Indessen deuten die Ergebnisse weiterführender Experimente auf den Einfluss der Kinasen CheA-1 und CheA-3 des V. cholerae Chemotaxis-Systems auf die RpoS-Degradation. Aus diesem Grund wurde in der vorliegenden Arbeit ein zu E. coli abweichendes Modell der RpoS-Proteolyse postuliert, in welchem die aktiven CheA-Kinasen den Targetingfaktor RssB phosphorylieren und somit den Abbau einleiten. Die Beteiligung von MCP-Rezeptoren an der Kontrolle der intrazellulären RpoS-Konzentration und damit an der Transkription der Chemotaxisgene selbst, beschreibt erstmalig ein Regulationssystem, wonach innerhalb der Chemotaxis-Kaskade die Rezeptoraktivität wahrscheinlich über einen positiven „Feedback-Loop“ mit der eigenen Gen-Expression gekoppelt ist. Darüber hinaus deutete sich die Beteiligung der ATP-abhängigen Protease Lon an der RpoS-Proteolyse-Kaskade in V. cholerae an. Die Inaktivierung der in E. coli unter Hitzeschock-Bedingungen induzierten Protease resultierte in einem extrem beschleunigten RpoS-Abbau. Ein letztes Teilprojekt dieser Arbeit adressierte die Regulationsmechanismen der V. cholerae Osmostress-Adaptation. Während in E. coli der alternative Sigmafaktor dabei eine zentrale Rolle spielt, konnte die Beteiligung des V. cholerae RpoS an der Osmostress-Regulation jedoch nicht aufgedeckt werden. Dafür ergab die Funktionsanalyse eines neu definierten Osmostress-Sensors (OsmRK) die Kontrolle von ompU durch dieses Zwei-Komponentensystems unter hypertonen Bedingungen. Dieses Ergebnis überraschte, da bislang nur der Virulenzfaktor ToxR als Regulator für das Außenmembranporin beschrieben wurde. Die nachgewiesene ompU-Transkriptionskontrolle durch zwei Regulatoren führte zu der Hypothese eines unbekannten regulativen Netzwerkes, welchem mindestens 52 weitere Gene zugeordnet werden konnten. Insgesamt ist festzuhalten, dass die in dieser Arbeit durchgeführte molekulare Charakterisierung der RpoS-Proteolyse in V. cholerae Beweise für eine mögliche Verbindung zwischen der Transkriptionskontrolle für Motilitäts- und Chemotaxisgene mit der Chemotaxis-Reizwahrnehmung erbrachte. Eine derartige intermolekulare Verknüpfung wurde bislang für keinen anderen Organismus beschrieben und stellt somit eine neue Variante der Signaltransduktion innerhalb der Virulenz-assoziierten Genregulation dar. / In the present work conserved function of RpoS in E. coli was approached for its homolog in V. cholerae. Comprehensive phenotypical analysis of rpoS-mutant and wildtype revealed the involvement of RpoS in growth-phase-dependent processes, according to RpoS-function as stationary phase regulator in E. coli. In further experiments the conservation of RpoS-promoters in both species could be shown. To the contrary, the well-known function of E. coli RpoS as general stress-regulator could not be demonstrate for V. cholerae: By testing several stress conditions in survival assays, no significant differences were determined between rpoS mutant and wildtype. Additionally, the intracellular mode of RpoS accumulation in E. coli due to different stress conditions was also not observed in V. cholerae. Regarding the putative role of RpoS as a regulator for virulence-associated genes, the inhere described data support and complement the current theory of RpoS being involved in mucosal detachment of V. cholerae cells. In E. coli the intracellular concentration of RpoS is a decisive parameter for its described function. So far the homologs of the proteolysis targeting factor RssB and the ATP-depending terminal protease complex ClpXP were identified to be involved in V. cholerae RpoS-proteolysis. Further characterization also unravelled, that various stress signals slow down that degradation. But such conditions did not yield in the RpoS accumulation. Based on these differences to the E. coli dynamics of RpoS-degradation additional investigations were performed to gain more insights into the regulatory path of RpoS degradation in V. cholerae. In E. coli the ArcB kinase ist the sensor kinase for regulating the activity of RssB. In this study fexB was identified as arcB homolog in V. cholerae. But by monitoring the RpoS stability in the corresponding knock-out mutant no effect could be observed. Therefore the ArcB-system is not influencing RpoS stability in V. cholerae. Knowing, that RpoS is a major regulator for motility and chemotaxis in V. cholerae, it was investigated next whether other signal-kinases are involved in RpoS proteolysis. Thereby, the known chemotaxis kinases were tested. Knockout mutants of cheAs and subsequent analysis of RpoS half-life revealed, that cheA-1 and cheA-3 did alter RpoS proteolysis to slow down the degradation, whereas cheA-2 mutant did not. Therefore, it can be postulated, that a different mode of RpoS-proteolysis is operating in V. cholerae in which active CheA-1 and CheA-3 may be responsible for RssB phosphorylation, hence leading to RpoS degradation. That kind of interaction may also include the output signalling of the MCP-receptors regulating CheA kinase activity. Since the cheA genes are also under transcriptional control by RpoS a new regulation system can be postulated, where MCP signal output links transcriptional regulation of motility and chemotaxis via RpoS stability in a “positive feedback loop”. Additionally, data are presented, where the ATP depending protease Lon is also involved in RpoS proteolysis in an inverted manner. Lon, which in E. coli is a heat shock induced protease, seems to recognize and degrade substrates in V. cholerae operating in RpoS degradation in the RssB-depending branch. That phenotype was observed as an accelerated RpoS degradation in a lon background. Finally, the complex regulatory pathway of osmo-regulation was characterized. In E. coli RpoS plays a central role. However, in V. cholerae RpoS could not be identified to participate in osmo-regulation, instead a new defined osmostress-sensor (OsmRK) was characterized. In first analysis, it was found that osmRK knockout mutants showed a deregulated ompU expression under hyperosmotic conditions. Considering, that so far only the well known virulence regulator ToxR was identified to act on the ompU promoter, a novel regulatory network was suggested, which regulates at least further 52 genes. In summary, the components of RpoS proteolysis in V. cholerae were unravelled and characterized. Additionally, evidence could be gathered, which indicates a linkage between transcriptional control of motility and chemotaxis genes and the chemotaxis-signalling pathway. So far, such an regulatory pathway has not been described before and would represent a novel branch of signal transduction in bacteria.

Flagellen-basierte Motilität und Chemotaxis stellen essentielle Pathogenitätsfaktoren dar, die für die erfolgreiche Kolonisierung der Magenschleimhaut durch H. pylori notwendig sind. Die Mechanismen der Regulation der Flagellensynthese und das Chemotaxis-System von H. pylori weisen trotz einiger Ähnlichkeiten fundamentale Unterschiede zu den Systemen anderer Bakterien auf. In H. pylori ist die Flagellensynthese durch eine komplex regulierte Kaskade kontrolliert, die Regulatorkomponenten wie das Zweikomponentensystem HP244/FlgR, die Sigma Faktoren 54 und 28 und den Sigma Faktor28-Antagonisten FlgM enthält. Das Signal, welches über die Histidinkinase des Zweikomponentensystems HP244/FlgR die Expression der Sigma Faktor54-abhängigen Klasse 2 Flagellengene reguliert, ist bisher noch nicht bekannt. Allerdings konnte mit HP137 ein Protein identifiziert werden, das im „yeast two-hybrid“ System sowohl mit der korrespondierenden Kinase HP244 des Flagellenregulators FlgR, als auch mit der Flagellenkomponente FlgE´ interagiert (Rain et al., 2001). In dieser Arbeit wurde eine mögliche Rolle von HP137 in einem Rückkopplungsmechanismus untersucht, welcher die Aktivität der Histidinkinase in der Flagellenregulation kontrollieren könnte. Obwohl die Deletion des ORF hp137 zu einer unbeweglichen Mutante führte, legen die erfolglosen Komplementations Experimente, sowie die Beobachtung, dass HP137 in vitro keinen bedeutenden Effekt auf die Aktivität der Histidinkinase HP244 hat nahe, dass HP137 weder in H. pylori noch im nahe verwandten C. jejuni direkt an der Flagellenregulation beteiligt ist. Das Chemotaxis-System von H. pylori unterscheidet sich vom gutuntersuchten Chemotaxis-System der Enterobakterien in einigen Aspekten. Zusätzlich zu dem CheY Response Regulator Protein (CheY1) besitzt H. pylori eine weitere CheY-artige Receiver-Domäne (CheY2) welche C-terminal an die Histidinkinase CheA fusioniert ist. Zusätzlich finden sich im Genom von H. pylori Gene, die für drei CheV Proteine kodieren die aus einer N-terminalen Domäne ähnlich CheW und einer C-terminalen Receiver Domäne bestehen, während man keine Orthologen zu den Genen cheB, cheR, and cheZ findet. Um einen Einblick in den Mechanismus zu erhalten, welcher die chemotaktische Reaktion von H. pylori kontrolliert, wurden Phosphotransferreaktionen zwischen den gereinigten Signalmodulen des Zweikomponentensystems in vitro untersucht. Durch in vitro-Phosphorylierungsexperimente wurde eine ATP-abhängige Autophosphorylierung der bifunktionellen Histidinkinase CheAY2 und von CheA´, welches ein verkürztes Derivat von ChAY2 ohne Receiver-Domäne darstellt, nachgewiesen. CheA´ zeigt eine für an der Chemotaxis beteiligte Histidinkinasen typische Phosphorylierungskinetik mit einer ausgeprägten exponentiellen Phase, während die Phosphorylierungskinetik von CheAY2 nur eine kurze exponentielle Phase aufweist, gefolgt von einer Phase in der die Hydrolyse von CheAY2~P überwiegt. Es wurde gezeigt, dass die Anwesenheit einer der CheY2 Domäne die Stabilität der phosphorylierten P1 Domäne im CheA Teil des bifunktionellen Proteins beeinflusst. Außerdem wurde gezeigt, dass sowohl CheY1 als auch CheY2 durch CheAY2 phosphoryliert werden und dass die drei CheV Proteine die Histidinkinase CheA´~P dephosphorylieren, wenn auch mit einer im Vergleich zu CheY1 und CheY2 geringeren Affinität. Außerdem ist CheA´ in der Lage seine Phosphatgruppen auf CheY1 aus C. jejuni und CheY aus E. coli zu übertragen. Retrophosphorylierungsexperimente weisen darauf hin, dass CheY1~P die Phosphatgruppe zurück auf die Histidinkinase CheAY2 übertragen kann und dass die CheY2-Domäne in dem bifunktionellen Protein CheAY2 als „Phosphat Sink“ agiert der den Phosphorylierungszustand und damit die Aktivität des frei diffundierbaren Proteins CheY1 reguliert, das vermutlich es mit dem Flagellenmotor interagiert. Es konnte weiterhin gezeigt werden, dass die unabhängige Funktion der beiden Domänen CheA´ und CheY2 für eine normale chemotaktische Signalgebung in vivo nicht ausreicht. In dieser Arbeit wurden also Hinweise auf eine komplexe Kaskade Phosphatübertragungsreaktionen im chemotaktischen System von H. pylori gefunden, welches Ähnlichkeiten zu dem Syteme-Chemotaxis von S. meliloti aufweist an denen multiple CheY Proteine beteiligt sind. Die Rolle der CheV Proteine bleibt im Moment unklar, jedoch könnte es sein, dass sie an einer weiteren Feinregulierung der Phosphatgruppenübertragungsreaktionen in diesem komplexen chemotaktischen System beteiligt sind / Flagellar motility and chemotaxis are essential virulence traits required for the ability of Helicobacter pylori to colonize the gastric mucosa. The flagellar regulatory network and the complex chemotaxis system of H. pylori are fundamentally different from other bacteria, despite many similarities. In H. pylori expression of the flagella is controlled by a complex regulatory cascade involving the two-component system FlgR-HP244, the sigma factors 54 and 28 and the anti-sigma 28 factor FlgM. Thus far, the input signal for histidine kinase HP244, which activates the transcriptional regulator FlgR, which triggers sigma factor 54-dependent transcription of the flagellar class 2 genes, is not known. Based on a yeast two-hybrid screen a highly significant protein-protein interaction between the H. pylori protein HP137 and both the histidine kinase HP244 and the flagellar hook protein HP908 (FlgE´) has been reported recently (Rain et al., 2001). So far, no function could be assigned to HP137. Interestingly, the interaction between HP137 and histidine kinase HP244 was observed in the characteristic block N sequence motif of the C-terminal ATP-binding kinase domain. In this work a potential role of HP137 in a feedback regulatory mechanism controlling the activity of histidine kinase HP244 in the flagellar regulation of H. pylori was investigated. Although the substitution of the gene encoding HP137 by a kanamycin cassette resulted in non-motile bacteria, the failure to restore motility by the reintroduction of hp137 in cis into the mutant strain, and the observation that HP137 has no significant effect on the activity of histidine kinase HP244 in vitro indicated that HP137 is not directly involved in flagellar regulation. Therefore, it was demonstrated that HP137 does not participate in the regulation of flagellar gene expression, neither in H. pylori nor in the closely related bacterium C. jejuni. Chemotactic signal transduction in H. pylori differs from the enterobacterial paradigm in several respects. In addition to a CheY response regulator protein (CheY1) H. pylori contains a CheY-like receiver domain (CheY2) which is C-terminally fused to the histidine kinase CheA. Furthermore, the genome of H. pylori encodes three CheV proteins consisting of an N-terminal CheW-like domain and a C-terminal receiver domain, while there are no orthologues of the chemotaxis genes cheB, cheR, and cheZ. To obtain insight into the mechanism controlling the chemotactic response of H. pylori the phosphotransfer reactions between the purified two-component signalling modules were investigated in vitro. Using in vitro phosphorylation assays it was shown that both H. pylori histidine kinases CheAY2 and CheA´ lacking the CheY-like domain (CheY2) act as ATP-dependent autokinases. Similar to other CheA proteins CheA´ shows a kinetic of phosphorylation represented by an exponential time course, while the kinetics of phosphorylation of CheAY2 is characterized by a short exponential time course followed by the hydrolysis of CheAY2~P. Therefore, it was demonstrated that the presence of the CheY2-like receiver domain influences the stability of the phosphorylated P1 domain of the CheA part of the bifunctional protein. Furthermore, it was proven that both CheY1 and CheY2 are phosphorylated by CheAY2 and CheA´~P and that the three CheV proteins mediate the dephosphorylation of CheA´~P, although with a clearly reduced efficiency as compared to CheY1 and CheY2. Moreover, CheA´ is capable of donating its phospho group to the CheY1 protein from C. jejuni and to CheY protein from E. coli. Retrophosphorylation experiments indicated that CheY1~P is able to transfer the phosphate group back to the HK CheAY2 and the receiver domain present in the bifunctional CheAY2 protein acts as a phosphate sink fine tuning the activity of the freely diffusible CheY1 protein, which is thought to interact with the flagellar motor. Hence, in this work evidence of a complex phosphorelay in the chemotaxis system was obtained which has similarities to other systems with multiple CheY proteins. The role of the CheV proteins remain unclear at the moment, but they might be engaged in a further fine regulation of the phosphate flow in this complex chemotaxis system and the independent function of the two domains CheA´ and CheY2 is not sufficient for normal chemotactic signalling in vivo.