Legionella Research Topics
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ir. Leen Vranckx Merel Ruttens
Contact person: ir. Leen Vranckx |
Legionella pneumophila, a Gram-negative, facultative intracellular, parasite of several protozoa and human macrophages, is the causative agent of a very severe form of pneumonia, also called Legionaires' disease, or a milder, flu-like self-limiting disease named Pontiac fever (www.legionella.org). L. pneumophila is found ubiquitously in freshwater environments, free-living or associated in biofilms. In the environment, growth of Legionella within protozoa may be one of the primary means of proliferation. Infection occurs through inhalation of contaminated aerosols. The Legionella bacteria are then transmitted to the lungs where they are engulfed by alveolar macrophages and cause disease. In general, the understanding of L. pneumophila pathogenesis is still rather limited.
To identifiy novel virulence factors and their regulatory pathwaysOur goal
PROTEIN SECRETION PATHWAYS IN LEGIONELLA PNEUMOPHILA
Protein secretion pathways have been characterized as important determinants of virulence in L. pneumophila since several identified virulence factors are likely to be secreted into the extracellular environment.
Study of the L. pneumophila Tat pathway
The aim of this research project is the characterization of the Tat secretion pathway in L. pneumophila and the search for a possible role of this secretion system in the virulence of this pathogen. The Tat pathway is a secretory pathway for protein translocation across the cytoplasmic membrane. Proteins translocated through this pathway are folded proteins that typically carry two arginine residues or a lysine/arginine pair in their signal peptide. It is known that this secretion pathway plays a role in the virulence of different human and plant pathogens. In this research project we identified this secretion pathway in L. pneumophila and it was shown that the Tat pathway is a functional secretion system. Furthermore it was shown that the Tat pathway plays a role in intracellular replication in amoebae and macrophages, in stress respons and in biofilm formation. For the moment Tat substrates are being identified.
Study of L. pneumophila signal peptidases
Signal peptidases (SPases) are membrane embedded proteins responsible for removal of the signal peptide from secretory proteins during or after translocation across the cytoplasmic membrane. While signal peptides from most secretory proteins are enzymatically removed by a type I SPase, type II SPase is especially dedicated to the processing of signal peptides from lipoproteins. Based on our results, we believe that both L. pneumophila proteins might be promising new targets for therapeutic intervention.
Study of outer membrane proteins
Proteins in the outer membrane (Omps) of Gram-negative bacteria play a crucial role in the interaction of bacteria with the environment or with the host cell. In this project a detailed study of L. pneumophila Omps is envisaged. We aim at analyzing
· the biogenesis of L. pneumophila Omps and identification of proteins involved in their membrane insertion
· Omp profiles of different strains from different sources using 1D or 2D protein gel electrophoresis
· whether L. pneumophila Omps can be suitable candidates to be used as targets in diagnostics
A better understanding of the function of L. pneumophila Omps, their biogenesis and their role in infection and immunity, is very important in order to get a better insight into the host-parasite interaction.
ROLE OF TANDEM REPEATS IN VIRULENCE OF L. PNEUMOPHILA
Tandemly repeated DNA sequences, especially within coding regions, have been relatively well-studied in some eukaryotes, but are just beginning to be examined in prokaryotes. Yet, in bacteria, they may provide a critical source of variation for an adaptable response to a variety of environmental stresses. The aim of this project is to investigate the presence and physiological relevance of intragenic tandem repeats in the genome of Legionella pneumophila. The goals for this project are:
· Characterize intragenic tandem repeats from the published Legionella pneumophila genome.
· Screen for genes which exhibit natural variation in the number of repeats between different isolates of L. pneumophila and are involved in antigenic variation, biofilm formation or virulence.
· Study the behaviour of these genes and their variants in a number of phenotypic assays, especially those related to virulence, biofilm formation, and host-pathogen interactions.
Conducting a thorough and extensive analysis of these repeats and their phenotypic consequences in L. pneumophila will shed new light on the importance and mechanisms underlying this phenomenon in prokaryotes. Additionally, the knowledge gained relating to virulence factors, immune avoidance, and biofilm formation may have important implications for treatment and prevention of Legionnaires’ disease.
LPNR-DEPENDENT TRANSCRIPTIONAL REGULATION IN L. PNEUMOPHILA
The life cycle of the pathogen Legionella pneumophila is composed of two reciprocal phases: replication and transmission. When conditions are favourable for replication, traits that promote transmission are repressed and the intracellular bacteria multiply. As nutrients become limiting, the progeny differentiate into the transmissive phase, repressing multiplication while expressing a number of traits that are believed to equip L. pneumophila to escape from its host cell, survive as a planktonic cell and re-establish a replicative niche within a new phagocyte. Although many aspects of regulation of virulence of L. pneumophila have been elucidated the past years, further research is necessary to fully link all components of the proposed regulatory cascades. Recently 3 L. pneumophila Philadelphia-1 transcriptional regulators with homology to proteins of the LuxR family, quorum sensing related regulators, were identified. They were designated LpnR1 (lpg2557), LpnR2 (lpg1946) and LpnR3 (lpg1448) (Legionella pneumophila regulators). The similarity with proteins of the LuxR family was mostly based on the presence of a C-terminal DNA binding helix-turn-helix motif. This implies that the LpnR proteins are able to bind DNA and might therefore act as transcriptional regulators. Identification of the specific function of the LpnR proteins was achieved by the characterization of mutants wherein the lpnR gene was replaced by an antibiotic resistance gene. Based on these results, a possible role of the LpnR proteins in the regulation of the virulence of this pathogen is assumed. Further research will help to position these proteins in the regulatory cascade of expression of virulence traits of L. pneumophila.
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