Exploitation of Phosphoinositides by the Intracellular Pathogen, <em>Legionella pneumophila</em>

Manipulation of host phosphoinositide lipids has emerged as a key survival strategy utilized by pathogenic bacteria to establish and maintain a replication-permissive compartment within eukaryotic host cells. The human pathogen, Legionella pneumophila, infects and proliferates within the lung’s innate immune cells causing severe pneumonia termed Legionnaires’ disease. This pathogen has evolved strategies to manipulate specific host components to construct its intracellular niche termed the Legionella-containing vacuole (LCV). Paramount to LCV biogenesis and maintenance is the spatiotemporal regulation of phosphoinositides, important eukaryotic lipids involved in cell signaling and membrane trafficking. Through a specialized secretion system, L. pneumophila translocates multiple proteins that target phosphoinositides in order to escape endolysosomal degradation. By specifically binding phosphoinositides, these proteins can anchor to the cytosolic surface of the LCV or onto specific host membrane compartments, to ultimately stimulate or inhibit encounters with host organelles. Here, we describe the bacterial proteins involved in binding and/or altering host phosphoinositide dynamics to support intracellular survival of L. pneumophila

Primal Eukaryogenesis:On the Communal Nature of Precellular States, Ancestral to Modern Life

This problem-oriented, exploratory and hypothesis-driven discourse toward the unknown combines several basic tenets: (i) a photo-active metal sulfide scenario of primal biogenesis in the porespace of shallow sedimentary flats, in contrast to hot deep-sea hydrothermal vent conditions; (ii) an inherently complex communal system at the common root of present life forms; (iii) a high degree of internal compartmentalization at this communal root, progressively resembling coenocytic (syncytial) super-cells; (iv) a direct connection from such communal super-cells to proto-eukaryotic macro-cell organization; and (v) multiple rounds of micro-cellular escape with streamlined reductive evolution—leading to the major prokaryotic cell lines, as well as to megaviruses and other viral lineages. Hopefully, such nontraditional concepts and approaches will contribute to coherent and plausible views about the origins and early life on Earth. In particular, the coevolutionary emergence from a communal system at the common root can most naturally explain the vast discrepancy in subcellular organization between modern eukaryotes on the one hand and both archaea and bacteria on the other

Molecular Interactions Of Tectonin Proteins In Host and Pathogen Recognition

Ph.DDOCTOR OF PHILOSOPH

Genomic conflict and disparity within basidiomycete mycelia

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN013205 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Bioinformatics for comparative cell biology

For hundreds of years biologists have studied the naturally occurring diversity in plant and animal species. The invention of the electron microscope in the rst half of the 1900's reveled that cells also can be incredible complex (and often stunningly beautiful). However, despite the fact that the eld of cell biology has existed for over 100 years we still lack a formal understanding of how cells evolve: It is unclear what the extents are in cell and organelle morphology, if and how diversity might be constrained, and how organelles change morphologically over time.(...

Analysis of the distribution and molecular interactions of the novel protein muskelin

Muskelin is a novel intracellular protein involved in cell responses to the adhesion modulating matrix component, Thrombospondin-1 (TSP-1). The muskelin polypeptide sequence contains six motifs with homology to the tandem kelch motifs, initially identified in the Drosophila Kelch protein, a component of ring canals. The focus of the thesis project has been to identify the molecular interactions of muskelin by use of a number of approaches including the yeast dihybrid system, copprecipitation, pharmacological agents and gel overlays. The two hybrid approach did not prove to be appropriate, because expression of muskelin had toxic effects on yeast cells. Treatment of cell extracts with a panel of pharmacological agents, to target components of cell signalling pathways, had no effect on the distribution of muskelin between detergent soluble (cytosol and membranes) and insoluble (cytoskeleton) fractions. Using a panel of cell lines including skeletal myoblasts, a set of proteins migrating with apparent molecular weights between 45-57 kDa were specifically detected upon muskelin overlays of SDS-polyacrylamide gels. These proteins did not correspond to the most abundant proteins within cell extract. Interaction of muskelin with these proteins was detectable throughout myoblast fusion into myotubes. Fascin, tubulin and actin were investigated as potential candidates for the proteins detected by muskelin gel overlay. A combination of muskelin and fascin overlays of cell extracts demonstrated that muskelin did not directly bind fascin. Muskelin did not interact with purified tubulin and actin in gel overlay assays and cosedimentation studies revealed that muskelin does not interact direct with microtubules or microfilaments. The most prominant interaction of muskelin in a gel overlay was with a 45 kDa protein of pI 6.0. This interaction was enhanced during myoblast fusion. Apparent postranslational modification of the 45 kDa protein was detected by muskelin overlay of extracts resolved on 2-Dimensional IEF/SDS-PAGE gels. A two step approach was developed to isolate the 45 kDa protein by using a strong anion exchange column to enrich for the 45 kDa protein, followed by 2D gel anaKsis. Identifying muskelin's binding partners will enhance understanding of its functional role

Functional studies of selected actin binding proteins by point mutations and GFP fusions

Profilin is an ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cellular physiology. Site-directed mutagenesis of profilin II from Dictyostelium discoideum by PCR resulted in the point mutations W3N and K114E, whereby the W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin-binding, whereas the K114E profilin shows profound decrease in its ability to interact with actin but its affinity for poly-(L)-proline remained unaltered. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in the profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., 1994). Expression resulted in normal cell physiology, a reduction in the F-actin content, and a complete development. Interestingly, only cells which overexpressed W3N could restore the aberrant phenotype, while the K114E profilin with its fully functional poly-(L)-proline binding and its strongly reduced actinbinding activities rescued the phenotype at low concentrations. Both the wild-type and pointmutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities. To have a better understanding of the in vivo role of profilin, D. discoideum profilin II has been tagged at its C-terminus with the green fluorescent protein (GFP) with a 100-aa linker separating profilin and GFP. This fusion construct was introduced in D. discoideum profilinminus cells and expression of the fusion protein could restore the aberrant phenotype partially. The partial rescue might be due to the uneven expression of the fusion protein leading to mixed populations even after repeated recloning. The profilin-GFP transformants showed normal cell morphology, could be cultivated in shaking suspensions, and could develop fruiting bodies which closely resembled those of the wild-type. In vivo studies revealed the distribution of the fusion protein in highly active regions of the cells such as phagocytic cups, macropinocytotic crowns, cell cortex and at the leading edges of locomoting cells. Thus profilin appears to play a significant role in the regulation of the dynamic actinbased cellular processes. A second actin-regulatory protein from D. discoideum namely, severin, a Ca2+-dependent Factin fragmenting and capping protein, was also investigated via fusion to GFP at its C-terminus. Although severin is a very active F-actin fragmenting protein in in vitro assays, the severin null D. discoideum mutant exhibits normal growth, cell motility, chemotaxis and development. Examination of the live dynamics of severin-GFP should clarify the in vivo role of severin and other functionally redundant cytoskeletal proteins. The 70 kDa severin-GFP fusion protein has been sufficiently expressed and partially purified from the severin null cells whereby in vitro assays confirmed the ability of this fusion protein to sever F-actin only in the presence of Ca2+. Data from confocal microscopy showed that the fusion protein was transiently detected in macropinocytotic crowns, phagocytic cups, membrane ruffles, at the leading edges of motile cells and cell-cell contacts of aggregating cells in directed motion. These data suggest an in vivo role for severin in the remodulation of existing F-actin structures as supported by the in vitro data. The highly dynamic cytoskeleton also plays a significant part in the defence of the cells against pathogens. The behaviour of the actin cytoskeleton of cultured mammalian cells in response to Yersinia enterocolitica infection was examined by confocal microscopy with the aid of GFP-tagged actin, cofilin and profilin II. The translocated Yersinia outer proteins (Yops) encoded by a virulence plasmid in the wild-type bacteria have been observed to disrupt the actin microfilaments, resulting in diffuse actin staining which subsequently disappeared completely upon prolonged bacterial infection. In addition, F-actin structures resembling phagocytic cups were found at the sites of bacterial adherence, suggesting the likelihood of the involvement of the Rho family of small GTPases in the regulation of the actin cytoskeleton. The secreted Yops appeared to have no major effect on the distribution of GFP-profilin whereas the staining pattern of GFP-cofilin seemed to be modified by the Yops, resulting in a decrease in length of the actin-cofilin rods and a diffuse localization of cofilin. The exact mechanisms of interaction between the Yops and their host targets remain to be determined. However, a clearer insight into the interaction between pathogens and the host cytoskeleton will certainly aid in the cellular defence and the prevention of pathogenesis.Profilin ist ein ubiquitäres Zytoskelettprotein, dessen Funktion für die Zellphysiologie von fundamentaler Bedeutung ist. Durch gerichtete Mutagenese von Profilin II aus Dictyostelium discoideum wurden die Punktmutationen W3N und K114E erzeugt. Das W3N-Profilin interagiert nicht mehr mit poly-(L)-Prolin und zeigt geringfügig verminderte Aktinbindung. Dagegen ist die Aktinbindung des K114E-Profilin stark reduziert, während seine Affinität zu poly-(L)-Prolin unverändert bleibt. Die in vivo Eigenschaften der punktmutierten Profiline wurden durch Expression von W3N- bzw. K114E-Profilin in D. discoideum Profilin- Nullmutanten (Haugwitz et al., 1994) untersucht, die Defekte im Aktingehalt, der Cytokinese und der Entwicklung aufweisen. Die Expression führte zur Wiederherstellung eines normalen Phänotyps. Interessanterweise ist dafür eine W3N-Profilin-Überexpression erforderlich, während K114E-Profilin mit seiner stark reduzierten Aktin-, aber voll funktionellen poly-(L)- Prolin-Bindung den mutanten Phänotyp schon in geringen Mengen aufhebt. Sowohl das Wildtyp-Profilin als auch die beiden punktmutierten Profiline sind während der Phagozytose von Hefezellen in „phagocytic cups“ angereichert. Diese Befunde sprechen dafür, dass a) eine funktionelle poly-(L)-Prolin-Bindungsaktivität für die Suppression des mutanten Phänotyps wichtiger ist als die G-Aktin-Bindungsaktivität, und b) die Anreicherung von Profilin bei der Phagozytose weder von seiner poly-(L)-Prolin-Bindung, noch von einer intakten Aktinbindung abhängt. Für ein besseres Verständnis der Funktion von Profilin in vivo wurde D. discoideum Profilin II am C-Terminus über ein 100 Aminosäuren langes Verbindungsstück mit dem grünfluoreszierenden Protein (GFP) verknüpft. Die Expression dieses Fusionskonstrukts in der D. discoideum Profilin-Nullmutante führte zu weitgehender Kompensation des mutanten Phänotyps. In vivo Untersuchungen zeigten die Lokalisation des Fusionsproteins in hochaktiven Regionen der Zelle, wie z. B. in Phagozytose Strukturen, dem Zellkortex und Pseudopodien. Profilin scheint also eine signifikante Rolle bei der Regulation dynamischer, Aktin-abhängiger, zellulärer Prozesse zu spielen. Severin, ein weiteres Aktin-regulatorisches Protein aus D. discoideum mit Ca2+-abhängiger Aktin-Fragmentierungs- und „Capping“-Aktivität, wurde ebenfalls mit Hilfe C-terminaler Verknüpfung an GFP untersucht. Das 70 kDa große Severin-GFP Fusionsprotein wurde in den Severin-Nullmutanten ausreichend stark exprimiert und aus diesen Zellen angereichert. Dabei zeigten in vitro Versuche, dass die Fragmentierung von F-Aktin durch das Fusionsprotein nur in Gegenwart von Ca2+ erfolgt. Die Ergebnisse der Konfokalmikroskopie belegten die transiente Lokalisation des Fusionsproteins in aktiven Regionen bei der Zellbewegung. Diese Befunde deuten auf eine Rolle von Severin beim Umbau bereits existierender Aktinstrukturen in vivo hin. Das hochdynamische Zytoskelett spielt auch eine bedeutende Rolle bei der Verteidigung von Zellen gegen pathogene Organismen. Durch Konfokalmikroskopie an kultivierten Säugerzellen wurde das Verhalten des Aktin-Zytoskeletts als Antwort auf eine Yersinia enterocolitica Infektion mit Hilfe von GFP-verknüpftem Aktin, Cofilin und Profilin II untersucht. Es wurde beobachtet, dass die von einem Virulenzplasmid der Wildtyp-Bakterien kodierten Yops (Yersinia outer proteins) die Aktin-Mikrofilamente abbauen, was sich zunächst in einer diffusen Aktinfärbung äußerte. Zusätzlich wurden an den Stellen bakterieller Anheftung F-Aktin-Strukturen gefunden, die Phagozytose Strukturen ähnlich sahen. Dies macht eine Beteiligung der Rho-Familie kleiner GTPasen bei der Regulation des Aktin- Zytoskeletts wahrscheinlich. Die sezernierten Yops hatten keinen deutlichen Effekt auf die Verteilung von GFP-Profilin, dagegen schien das Färbungsmuster von GFP-Cofilin durch die Yops modifiziert zu werden, was zu einer Verkürzung der Aktin-Cofilin-Stäbchen und diffuser Lokalisation von Cofilin führte. Der genaue Mechanismus der Interaktion der Yops mit den Zieldomänen der Wirtszelle ist noch unbekannt, aber ein genauerer Einblick in die Interaktionen zwischen pathogenen Organismen und dem Zytoskelett der Wirtszelle kann sicherlich zum Schutz der Zellen und der Prävention der Pathogenese beitragen