Thesis (Ph. D.)--Auburn University. / Abstract. Includes bibliographical references (p. 129-151).

Würzburg, Univ., Diss., 2000. / Dateien im PDF-Format. Computerdatei im Fernzugriff.

Würzburg, Univ., Diss., 2000. / Dateien im PDF-Format. Computerdatei im Fernzugriff.

Würzburg, Universiẗat, Diss., 2000. / Dateien im PDF-Format.

Ineffective bacterial monitoring in water systems represents a danger to public health and can result in costly disease outbreaks. Of interest is Legionella pneumophila, a deadly water-borne bacterial pathogen that causes Legionnaires’ disease - a severe form of pneumonia. The Center for Disease Control stated that reported cases of Legionnaires’ disease have quadrupled since 2000 and ranks L. pneumophila as the number one cause of waterborne disease outbreaks in the United States. This threat is expected to increase given an aging population who are more susceptible to L. pneumophila infection and rising global temperatures that can promote L. pneumophila growth. Presently, Public Health agencies recommend bacterial culturing for the detection of L. pneumophila in environmental samples, however, this process can take up to ten days to complete. Consequently, there is a delay between sample collection and subsequent L. pneumophila detection, creating an opportunity for a Legionnaires’ disease outbreak to occur. There is a great need to develop a field-appropriate device that can provide early-stage detection of L. pneumophila in water as a means of mitigating Legionnaires’ disease outbreaks. We propose the use of DNAzymes for the development of such a device. DNAzymes are small, catalytically-active single-stranded DNA molecules that demonstrate target-specific enzymatic activity. We have successfully isolated an RNA-cleaving fluorescent DNAzyme (RFD) specific for the detection of L. pneumophila using in vitro selection. Thorough characterization of the DNAzyme has revealed key structural features influencing kinetics, specificity and sensitivity. In addition, the ability of the DNAzyme to function in cooling tower water, and conservation of the DNAzyme target across Legionella bacteria, has been investigated. In the future we plan to incorporate this RFD into a field-appropriate paper-based device which would play a key role in managing infectious diseases and preventing large-scale outbreaks. / Thesis / Master of Health Sciences (MSc) / Legionella pneumophila is a deadly water-borne bacterial pathogen that causes Legionnaires’ disease - a severe form of pneumonia. Numerous Legionnaires’ disease outbreaks have occurred, with the most common source of exposure to L. pneumophila coming from contaminated cooling towers. Presently, bacterial culturing is used to determine if a cooling tower is contaminated with L. pneumophila, however this process can take up to 10 days to complete. To address this delay, we plan to develop a rapid paper-based test for L. pneumophila detection in cooling tower water using DNAzymes. DNAzymes are small, catalytically-active single-stranded DNA molecules that demonstrate target-specific enzymatic activity. We have isolated a DNAzyme that can specifically detect L. pneumophila and characterized its properties. In the future we plan to incorporate this DNAzyme into a field-appropriate paper-based test which would play a key role in managing Legionnaires’ disease outbreaks.

Outer membrane proteins (OMPs) were recovered from eleven strains (eight serogroups) of Legionella pneumophila by sequential treatment with Tris buffer (pH 8), citrate buffer(pH 2.75) and Tris buffer (pH 8). Transmission electron microscopy revealed clearly the separation of the outer membrane from the bacteria. The development of delayed hypersensitivity was also noted by measuring the area of arythema and induration produced by intradermal injections of the MPSs from Chicago 8 strain. The adjuvants enhanced greatly both active and cell-meditated immunity (CMI). Transient lymphocytopenia with a slight rise in neutrophils was noted in each of the immunized groups. Intraperitoneal challenge, seven days after the OMP booster, of one LD (1.5 x10^6) of legionellae resulted in lymphocytopenia with elevated neutrophils. All immunized rats survived the challenge, although those in the saline-OMP group were clearly the sickest. Post-challenge, legionella antibody titers rose greatly and CMI was heightened. Passive immunization (homologous and heterologous) was found to protect the rats from a challenge of on LD. Actively-immunized rats retained their immunity for at least six months as determined by their resistance to a second challenge.

The bacterial pathogen Legionella pneumophila replicates intracellularly in protozoa, but can also cause severe pneumonia, called Legionnaires' disease. The bacteria invade and proliferate in the alveolar macrophages of the human lung. L. pneumophila bacteria exhibit a biphasic life cycle: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Primarily aim of this thesis was to evaluate the impact of the regulatory proteins FleQ, FleR, and RpoN in flagellar gene regulation. Phenotypic analysis, Western blot and electron microscopy of regulatory mutants in the genes coding for FleQ, RpoN and FleR demonstrated that flagellin expression is strongly repressed and that these mutants are non-flagellated in transmissive phase. Transcriptomic studies of these putative flagellar gene expression regulators demonstrated that fleQ controls the expression of numerous flagellar biosynthetic genes. Together with RpoN, FleQ controls transcription of 14 out of 31 flagellar class II genes, coding for the basal body, hook, and regulatory proteins. Unexpectedly, 7 out of 15 late flagellar genes class III and IV) are expressed dependent on FleQ but independent of RpoN. Thus, in contrast to the commonly accepted view that enhancer binding proteins as FleQ always interact with RpoN to initiate transcription, our results strongly indicate that FleQ of L. pneumophila regulates gene expression RpoN-dependent as well as RpoN-independent. Moreover, transcriptome analysis of a fleR mutant strain elucidated that FleR does not regulate the flagellar class III genes as previously suggested. Instead FleR regulates together with RpoN numerous protein biosynthesis and metabolic genes. Based on these experimental results our modified model for the transcriptional regulation of flagellar genes in L. pneumophila is that flagellar class II genes are controlled by FleQ and RpoN, while flagellar class III and IV genes are controlled in a fleQ-dependent but rpoN-independent manner. Although all L. pneumophila strains share the same complex life style, various pathotypes have evolved. This is reflected by the genomes, which contain e.g. genomic islands. The genomic island Trb-1 of L. pneumophila Corby, carries all genes necessary for a type-IV conjugation system, an integrase gene and a putative oriT site. The second aim of this thesis was to investigate the implication of this genomic island in conjugative DNA transfer. Using conjugation assays we showed that the oriT site located on Trb-1 is functional and contributes to conjugation between different L. pneumophila strains. As this is the first oriT site of L. pneumophila known to be functional our results provide evidence that conjugation is a major mechanism for the evolution of new pathotypes in L. pneumophila. / Das pathogene Bakterium Legionella pneumophila repliziert sich in der Natur intrazellulär in Protozoen. Beim Menschen kann das Bakterium eine schwere Pneumonie, die sogenannte Legionärskrankheit auslösen. Hierbei vermehren sich die Bakterien in Alveolarmakrophagen der Lunge. Der Lebenszyklus von L. pneumophila Bakterien ist gekennzeichnet durch zwei Phase: replikative Bakterien sind avirulent; im Gegensatz dazu sind transmissive Bakterien virulent und flagelliert. Hauptziel dieser Arbeit war es die Beteiligung der regulatorischen Proteins FleQ, FleR, and RpoN an der Flagellengenregulation zu ermitteln. Mutanten für die Gene welche für FleQ, FleR oder RpoN codieren exprimieren in der transmissiven Phase im Genesatz zum Wildtyp nur wenig Flagellin und sind nicht flagelliert. Nachgewiesen wurde dies durch eine phänotypische Analyse, Western blot und Ektronenmikroskopie. Studien des Transkripoms dieser Mutanten zeigten, daß FleQ die Expression zahlreicher Flagellenbiosynthesegenen kontrolliert. Gemeinsam mit RpoN kontrolliert FleQ die Transkription von 14 der 31 Klasse II Flagellengene, welche für Basalkörper, Haken und regulatorische Proteine codieren. Überraschenderweise sind 7 der 15 späten Flagellengenen (Klasse III und IV) abhängig von FleQ, aber unabhängig von RpoN exprimiert. Daher und entgegen der allgemeinen Auffassung dass sogenannte ‚enhancer binding' Proteine wie FleQ zur Transkriptionsinitiation immer mit RpoN interagieren, deuten unsere Ergebnisse darauf hin, dass FleQ von L. pneumophila Genexpression sowohl RpoN-abhängig, als auch RpoN-unabhängig reguliert. Ebenso anders als zuvor vorgeschlagen, verdeutlichen Studien des Transkriptoms einer fleR Mutante, dass FleR nicht die Expression der Klasse III Flagellengene induziert. Statt dessen reguliert FleR gemeinsam mit RpoN zahlreiche Gene der Proteinbiosynthese und des Metabolismus. Basierend auf diesen experimentellen Ergebnissen sind in unserem modifizierten Modell für die transkriptionelle Regulation der L. pneumophila Flagellengene die Flagellengene der Klasse II von FleQ und RpoN kontrolliert, während die Flagellengene der Klasse III und IV in einer fleQ-abhängigen aber rpoN-unabhängigen Weise kontrolliert sind. Obwohl alle L. pneumophila Stämme den zweiphasigen Lebenszyklus aufweisen haben sich unterschiedliche Pathotypen evolviert. Das ist auch in den Genomen sichtbar, die z. B. genomische Inseln enthalten. Die genomische Insel Trb-1 von L. pneumophila Corby trägt alle Gene eines Typ-IV Konjugationssystem, ein ntegrase-Gen und einen putative oriT-Bereich. Das zweite Ziel dieser Arbeit war es also zu untersuchen, inwieweit Trb-1 an konjugativem DNA-Transfer beteiligt ist. Mit Hilfe von Konjugationsexperimenten, zeigten wir, dass der oriT-Bereich von Trb-1 funktional ist und zur Konjugation zwischen verschiedenen L. pneumophila Stämmen beiträgt. Dies ist der erste oriT Bereich von L. pneumophila, dessen Funktionalität nachgewiesen wurde. Damit bekräftigen unsere Ergebnisse, dass Konjugation eine treibende Kraft für die Evolution neuer Pathotypen in L. pneumophila ist.

The intracellular bacterial pathogen Legionella pneumophila is the etiological agent of Legionnaires’ disease, a severe pneumonia; it has also served as a valuable tool in studying host-pathogen interactions. The study of L. pneumophila pathogenesis has led to the discovery of novel biochemical and enzymatic mechanisms and a better understanding of host cell immune responses and signaling. L. pneumophila replicates within eukaryotic cells through the use of a type IV secretion system and over 330 effector proteins injected into the host cell. Only approximately 10% of these effectors have been characterized, but regardless of the small fraction, the complexity of L. pneumophila infection is clear. A good demonstration of this complexity is the large number of effector activities the bacteria uses to manipulate the small GTPase involved in ER to Golgi trafficking, Rab1. Six different effectors with eight separate activities modulate the activity of Rab1 to aid in the replication of the bacteria. We recently discovered that the protein SetA is yet another effector targeting Rab1. SetA glucosylates Rab1 using a canonical DxD motif and the glucose moiety interferes with both GTP hydrolysis and guanosine nucleotide dissociation inhibitor (GDI) binding. Based on our findings, the role of SetA is likely to aid in maintaining a pool of free Rab1, increasing availability for use by other L. pneumophila effectors. Another example of the complexity of L. pneumophila pathogenesis is the use of metaeffectors. Metaeffectors are effectors that regulate other effectors, both being produced by L. pneumophila. Three mechanisms of metaeffector regulation have been identified: 1) removal of a modification on host proteins placed by the cognate effector, 2) direct modification of the cognate effector or 3) direct binding to the cognate effector. Through the use of Size Exclusion Chromatography (SEC), binding assays with purified proteins and bacterial two-hybrid analysis, we found the mechanism of regulation for the SidI metaeffector Lpg2505 to be inactivation through direct binding. Atypical of previously identified effector characteristics, the binding of SidI by Lpg2505 occurs within the bacterial cell prior to translocation. The expression pattern of both effectors in L. pneumophila in addition to the other findings suggest a temporal role for Lpg2505 activity in which inactivation of SidI occurs after sufficient bacterial replication has occurred.<br>

The intracellular pathogen Legionella pneumophila thrives in both natural and man-made water habitats where it replicates inside freshwater amoebae. L. pneumophila follows a developmental cycle as it grows in amoebae. The actively-multiplying intracellular replicative forms (RFs) differentiate into highly virulent mature infectious forms (MIFs) late in the amoeba infection, and are then released extracellularly. L. pneumophila accidentally infects susceptible humans causing the non-communicable Legionnaires’ disease (LD). MIFs play a central role in the life cycle of L. pneumophila and are thought to be responsible for the transmission of LD. Early reports demonstrated that MIFs were poorly produced inside human macrophages, suggesting that the L. pneumophila progeny from human macrophages has fitness and infectivity disadvantages. Direct comparisons of the L. pneumophila progenies from amoebae and human macrophages have demonstrated that the progeny from amoebae is more morphologically differentiated, resistant to antibiotic challenges, and able to adhere to and initiate infections in host cells than the progeny from macrophages. Analysis of the transcriptomic and proteomic profiles of L. pneumophila inside different hosts has revealed a specific set of genes that are upregulated during differentiation of L. pneumophila into MIFs inside freshwater protozoa but not inside human macrophages, suggesting that these genes may be required for the full differentiation of L. pneumophila and, therefore, for the transmission of LD to susceptible humans. Since the expression of the gene lpg1669, which encodes a putative α-amylase, was upregulated in amoebae (highest level of upregulation among the tested genes) and inside Tetrahymena ciliates, but not inside human macrophages, the role of lpg1669 in the differentiation of L. pneumophila into MIFs was investigated. An isogenic lpg1669 deletion mutant did not display defects in morphological differentiation, in vitro (BYE broth) or in vivo (A. castellanii or U937 human macrophages) growth when compared to its parent strain, suggesting that the gene lpg1669 is not essential for the intracellular differentiation of L. pneumophila. Collectively, these findings demonstrate that L. pneumophila can reach different developmental end points in different hosts and could also provide a clue for the lack of transmission of LD among humans.

Würzburg, Univ., Diss., 2009. / Zsfassung in dt. Sprache.