Date on Master's Thesis/Doctoral Dissertation

5-2006

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Committee Chair

Kwaik, Yousef Abu

Author's Keywords

Molecular pathogenesis; Legionella longbeachae; Endocytic markers; Legionnaires' disease; Phagosomes; Legionella pneumophila

Subject

Legionella pneumophila

Abstract

The ability of L. pneumophila to cause disease depends on its replication in alveolar macrophages. Infectivity and the expression of various virulence traits is triggered at post exponential growth phase. We show that unlike quiescent macrophages, L. longbeachae does not replicate in IFN-ã activated macrophages and its intracellular replication is independent of the growth phase of the inocula. L. pneumophila replicates in a phagosome that excludes early and late endocytic markers, and is surrounded by the rough endoplasmic reticulum (RER). In contrast, the L. longbeachae phagosome colocalizes with early endosomal marker EEA1, late endosomal markers LAMP-2 and M6PR, and ER maker KDEL, but excludes vacuolar ATPase (vATPase), and lysosomal markers cathepsin D, and Texas red ovalbumin. During late stages of infection, L. longbeachae, like L. pneumophila escapes into the cytoplasm, prior to lysis of the macrophage. Despite the different trafficking of L. longbeachae and L. pneumophila, both can replicate in communal phagosomes harboring both species. The L. pneumophila dotA mutant is rescued for intracellular replication if it co-inhibits the same phagosome with L. longbeachae. Most inbred mouse strains are resistant to infection by L. pneumophila , which is associated with polymorphism in the naip5 allele. Most human homologues of Naip proteins inhibit caspase-3 and caspase-7 activity. L. pneumophila activates caspase-3 which is necessary for arrest of phagosome biogenesis. We show that genetic susceptibility of several inbred mice strains to L. longbeachae is independent of allelic polymorphism of naip5 , and infection of cultured murine macrophages by L. longbeachae is independent of polymorphism in the naip5 allele. Real time PCR on permissive and resistant mouse macrophages shows that both L. longbeachae and L. pneumophila trigger similar level of naip5 expression but the level of naip5 is higher in macrophages resistant to L. pneumophila. Unlike L. pneumophila, L. longbeachae induces low level of caspase-3 activation and apoptosis during late stages of infection of macrophages in vitro or in the lungs of infected mice. Our data indicate a unique trafficking of L. longbeachae compared to other intracellular pathogens, and divergence in the intracellular life style of L. longbeachae from that of L. pneumophila.

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