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The Impact of Type II Secretion on the Intracellular Lifecycles of Legionella pneumophila
Başlık:
The Impact of Type II Secretion on the Intracellular Lifecycles of Legionella pneumophila
Yazar:
White, Richard Christopher, author.
ISBN:
9780438116610
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (299 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-11(E), Section: B.
Advisors: Nicholas P. Cianciotto Committee members: Vladimir I. Gelfand; Alan R. Hauser; Hank S. Seifert.
Özet:
Previous work demonstrated that the type II secretion (T2S) system of Legionella pneumophila secreted at least 25 different proteins, several of which promote infection of protozoan hosts. In order to uncover new roles for T2S effector proteins during protozoan infection, I assessed gene expression during the intracellular replication phase of L. pneumophila. Genes encoding LapA, LapB, and PlaC were the most highly upregulated T2S genes during infection of Acanthamoeba castellanii, although these genes were thought dispensable on the basis of the behavior of mutants lacking either lapA and lapB or plaC. I determined that the role of the LapA/B aminopeptidase is compensatory to that of the PlaC acyltransferase. Hence, I made double mutants and found that lapA plaC mutants have a ~50-fold defect during infection of A. castellanii. These data revealed, for the first time, the importance of LapA in any sort of infection. I documented LapA's activation by another T2S-dependent protease (ProA), and its broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of the lapA plaC mutant was reversed, implying that LapA generates amino acids for nutrition. Based on past proteomic analysis, I began characterization of a novel secreted protein (NttD) that promotes infection of A. castellanii. A lapA plaC nttD mutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data largely explain the importance of T2S in L. pneumophila infection of amoebae. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside the Legionella genus. LapA was notable for having as its closest homologue an A. castellanii protein. To determine the extent to which these effectors promote intracellular replication, I utilized fluorescent L. pneumophila to monitor the intracellular compartment, and implicated ProA in late stages of replication, as opposed to the entire T2S system, which I documented is required for the onset of replication.
Past work also documented that T2S promotes intracellular infection of macrophages. I sought to identify infection events that are modulated by T2S by comparing the behaviors of wild-type and T2S mutant bacteria in murine bone marrow-derived macrophages and human U937 cells, to refine the role of T2S within macrophages. While T2S was dispensable for entry into the host cells and evasion of lysosomal fusion, T2S promoted the initiation of replication between 4 and 8 h postentry and was required for replication to large numbers within the Legionella-containing vacuole (LCV). At 4 h postinoculation, mutant LCVs had a significantly reduced association with Rab1B, a host GTPase that facilitates the tethering of endoplasmic reticulum-derived vesicles to LCVs. Interestingly, the mutant's growth defect was exacerbated in macrophages that had been depleted of Rab1B by short hairpin RNA treatment, indicating that T2S potentiates events beyond Rab1B association with the LCV. In support of a Rab1-independent role for T2S, a sidM lspF double mutant had an intracellular growth defect that was more dramatic than that of the lspF mutant (and a sidM mutant) and showed a growth difference up to 400-fold compared to the wild type. Together, these findings reveal a new role for T2S in intracellular infection that involves both Rab1B-dependent and Rab1B-independent processes.
Notlar:
School code: 0163
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(693017.1) | 693017-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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