Cells defend their cytosol against pathogen invasion using cell-autonomous immunity. When pathogens enter the cytosol they can damage host endomembranes, causing the mislocalisation of host molecules not normally found in the cytosol that are sensed as Danger Associated Molecular Patterns (DAMPs). Glycans exposed on damaged endomembranes are detected by danger receptors such as Galectin8. Galectin8 is recognised by the autophagy cargo receptor NDP52, specifically targeting the bacteria to autophagy. I hypothesised that other proteins would also be recruited to damaged endomembranes, which may initiate downstream mechanisms involved in cell-autonomous immunity or endomembrane repair. Identifying novel damage recruited proteins (DRPs) is difficult due to the short-lived and dynamic nature of damaged endomembranes. Therefore, I developed an unbiased approach for the identification of novel DRPs by proximity-dependent biotinylation using the ascorbate peroxidise enzyme APEX. This approach preferentially labels proteins located at damaged endomembranes for subsequent identification by TMT mass spectrometry. Four enriched proteins CCDC50, FBXO21, STAMBP and PDCD6 were identified as novel damage recruited proteins, recognising damaged SCVs. An alternative form of cell-autonomous immunity is the induction of cell death, for example by pyroptosis. Cell death destroys the bacteria's replicative niche and exposes them to the extracellular space where they may be phagocytosed. I hypothesised that host cells might tag cytoplasmic bacteria with intracellular opsonins to assist in their phagocytosis following their release from host cells. However, my work revealed that intracellular Salmonella Typhimurium acquire phagocytosis protection, thus becoming internalised by phagocytes less efficiently than control bacteria. Phagocytosis protection was acquired rapidly after S.Typhimurium infection and was not observed with dead bacteria. Phagocytosis protection is only partially reversed by opsonisation in human serum. My results indicate that intracellular S.Typhimurium-induces an evasion mechanism to prevent its subsequent recognition by extracellular phagocytes.

The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) is the causative agent of visceral leishmaniasis in South America. The flagellated promastigote life stage of the parasite undergoes receptor-mediated phagocytosis by macrophages. This process is followed by a transient delay in phagolysosome maturation that allows for conversion into the amastigotes, a stage that is resistant to degradation inside host cells. We hypothesized that events occurring early during parasite-host interaction influence whether the pathogen ultimately survives or is eliminated in the intracellular environment, and that these processes are facilitated by determinants from both the macrophage and the incoming Leishmania. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). The macrophage surface receptors that ligated the two forms of promastigotes differed, guiding metacyclic promastigotes into a compartment that supported their replication and logarithmic promastigotes into a vacuole that rapidly assembled its microbicidal machinery. Survival of metacyclic promastigotes following their phagocytosis also varied greatly on characteristics of the host macrophage. U937 cells, a model monocytic cell line lacking the third complement receptor (CR3) on their surface, took up parasites via a unique "coiling" mode of pseudopod extension, leading to a formation of a phagosome that did not fully mature. Since the parasites never demonstrated escape into the macrophage cytosol, it is logical to predict that they synthesize and release virulence factors that localize within the parasitophorous vacuole (PV) in order to establish communication with the host cell. Using a previously assembled bioinformatic catalogue of putatively secreted or excreted (E/S) proteins encoded in the Leishmania infantum genome, we chose four candidate proteins for further analysis. Two of these, serine carboxypeptidase (CBP) and a flavodoxin domain-containing protein (HP) coding sequences, were overexpressed or removed in Lic. Parasites lacking one allele of either CBP or HP were defective in survival within MDMs. Furthermore, recombinant overexpressed HP was detected from parasite lysate in a stage-specific manner, paralleling expression in wild type Lic. This implies that the regulatory elements within the protein coding sequence remain functional outside of their native locus. Taken together, our study shows that quiescent entry of virulent Leishmania spp. into macrophages is accounted for by i) the ability of metacyclic promastigotes to selectively bypass macrophage components leading to deleterious pathways, as well as ii) tightly regulated parasite virulence factors for deliberately enhancing intracellular survival.

Bei der Teilung einer Zelle werden das Genom und die Zellbestandteile zwischen zwei Tochterzellen aufgeteilt. Dies erfordert verschiedene fein aufeinander abgestimmte Vorgänge. Unter anderem ist eine proteinreiche Struktur beteiligt, die 1891 entdeckt wurde: der Mittelkörper. In vorliegender Arbeit wurden gezielt gekennzeichnete Mittelkörperproteine analysiert und verschiedene Phasen des Transports unterschieden. Es erfolgten erstmals Messungen unter Nutzung der ZF1-Methode. Zudem wird anhand der ZF1-Technik nachgewiesen, dass im Rahmen der Zellteilung die Trennung der interzellulären Brücke zu beiden Seiten des Mittelkörpers stattfindet, woraufhin dieser nach extrazellulär abgegeben wird und über einen der Phagozytose ähnlichen und von Aktin abhängigen Mechanismus von einer Tochterzelle oder unverwandten Nachbarzelle aufgenommen wird. / In animals, the midbody coordinates the end of cytokinesis. Using the ZF1-mediated degradation technique it is shown that midbodies are released outside the cell in C. elegans embryos. Furthermore it is shown that midbodies are released after abscission cuts on both sides of the midbody and that released midbodies are internalized via actin-driven phagocytosis.

Phagocytosis is an innate immune response that is paramount in the clearance of pathogenic particles. Recognition of target particles by phagocytic receptors expressed on phagocytes induces modifications in the underlying actin cytoskeleton to form pseudopods that encircle and internalize the target particle into a membrane bound organelle called the phagosome. The nascent phagosome undergoes a maturation sequence that is characterized by substantial remodeling of the membrane and its luminal contents through interactions with components of the endocytic pathway, culminating in an acidic and hydrolytic organelle capable of digesting and elminating pathogens. Phagosome maturation is a complicated pathway that involves many protein and lipid signaling molecules. Several factors that influence phagosome maturation particularly the participation of pH, lysosome-associated membrane proteins-1 and –2, cholesterol, in addition to the survival and escape mechanisms used by, Burkholderica cenocepacia were explored. All three tenets are essential for phagosome maturation, although each factor has different mechanistic consequences. Acidification alters Rab5 activation, while ablation of LAMPs and accumulation of cholesterol interferes with various aspects of Rab 7 turnover in phagosomes and/or endosome membranes. Moreover, Burkholderia cenocepacia, an intracellular pathogen, inactivates Rab7 on phagosome membranes from within the vacuole lumen. Herein, mechanisms that govern phagosome maturation are explored and several molecules are added to the long list of essential players in this complicated pathway.

Phagocytosis is an innate immune response that is paramount in the clearance of pathogenic particles. Recognition of target particles by phagocytic receptors expressed on phagocytes induces modifications in the underlying actin cytoskeleton to form pseudopods that encircle and internalize the target particle into a membrane bound organelle called the phagosome. The nascent phagosome undergoes a maturation sequence that is characterized by substantial remodeling of the membrane and its luminal contents through interactions with components of the endocytic pathway, culminating in an acidic and hydrolytic organelle capable of digesting and elminating pathogens. Phagosome maturation is a complicated pathway that involves many protein and lipid signaling molecules. Several factors that influence phagosome maturation particularly the participation of pH, lysosome-associated membrane proteins-1 and –2, cholesterol, in addition to the survival and escape mechanisms used by, Burkholderica cenocepacia were explored. All three tenets are essential for phagosome maturation, although each factor has different mechanistic consequences. Acidification alters Rab5 activation, while ablation of LAMPs and accumulation of cholesterol interferes with various aspects of Rab 7 turnover in phagosomes and/or endosome membranes. Moreover, Burkholderia cenocepacia, an intracellular pathogen, inactivates Rab7 on phagosome membranes from within the vacuole lumen. Herein, mechanisms that govern phagosome maturation are explored and several molecules are added to the long list of essential players in this complicated pathway.

No description available.

No description available.

The aim of this thesis is to incorporate the smallest organisms, in particular the heterotrophic bacteria and their predators, more fully into current descriptions of aquatic community structure and dynamics. A strong, positive empirical relationship was found between bacterial abundance and chlorophyll concentration in freshwater and marine systems. Common members of the photosynthetic phytoplankton (all chrysophyceans) were shown to be major, even dominant, grazers of these bacteria. This phagotrophic capability is quantitatively important to the bacterioplankton, to the mixotrophs themselves, and in some cases, to the structure of the limnological community as a whole. Grazing by mixo- and heterotrophic protozoans is concentrated on the largest bacterial cells that also have the greatest growth rates. Relatively lower removal rates of the tiniest cells, with low growth rates, is proposed to explain their numerical dominance in lakes.

Typescript (Photocopy) / Addendum inserted. / Bibliography: leaves 202-216. / vii, 216 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1989

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 151-166).