Macroautophagy is dispensable for intracellular replication of <i>Legionella pneumophila</i> in <i>Dictyostelium discoideum</i>

Otto, Grant; Wu, Mary; Clarke, Margaret; Lu, Hao; Anderson, O. Roger; Hilbi, Hubert; Shuman, Howard A.; Kessin, Richard H.

doi:10.1046/j.1365-2958.2003.03826.x

Cited by 115 publications

(100 citation statements)

References 56 publications

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“…Unlike many bacteria that replicate intracellularly in mammalian macrophages, Salmonella is rapidly degraded after internalization and is nonpathogenic in Dictyostelium (12). We found that GFP-labeled Salmonella invaded wild-type Dictyostelium and previously described Dictyostelium mutants (19) lacking the atg genes, ATG1 (a serine/threonine kinase involved in autophagy induction), ATG6 (the ortholog of bec-1), and ATG7 with similar kinetics and to a similar degree ( Fig. 2A).…”

Section: Atg Genes Restrict Intracellular Bacterial Replication and Hmentioning

confidence: 49%

Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance

Jia¹,

Thomas²,

Akbar³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

199

195

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A conserved insulin-like pathway modulates both aging and pathogen resistance in Caenorhabditis elegans. However, the specific innate effector functions that mediate this pathogen resistance are largely unknown. Autophagy, a lysosomal degradation pathway, plays a role in controlling intracellular bacterial pathogen infections in cultured cells, but less is known about its role at the organismal level. We examined the effects of autophagy gene inactivation on Salmonella enterica Serovar Typhimurium (Salmonella typhimurium) infection in 2 model organisms, Caenorhabditis elegans and Dictyostelium discoideum. In both organisms, genetic inactivation of the autophagy pathway increases bacterial intracellular replication, decreases animal lifespan, and results in apoptotic-independent death. In C. elegans, genetic knockdown of autophagy genes abrogates pathogen resistance conferred by a loss-of-function mutation, daf-2(e1370), in the insulin-like tyrosine kinase receptor or by overexpression of the DAF-16 FOXO transcription factor. Thus, autophagy genes play an essential role in host defense in vivo against an intracellular bacterial pathogen and mediate pathogen resistance in long-lived mutant nematodes.

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Section: Atg Genes Restrict Intracellular Bacterial Replication and Hmentioning

confidence: 49%

Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance

Jia¹,

Thomas²,

Akbar³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

199

195

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“…E. coli K-12 strains HfrH and AE2386/JCFL0 were from the authors' strain collection and have been described (23). The strains expressed the cytoplasmic, IPTG-inducible fluorescent marker DsRed Express (31). Cultures were grown with aeration at 37°C in Luria-Bertani (LB) medium containing chloramphenicol (5 g/ml) and IPTG (0.5 mM).…”

Section: Methodsmentioning

confidence: 99%

F-pili dynamics by live-cell imaging

Clarke

Maddera

Harris

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

130

139

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Bacteria have evolved numerous mechanisms for cell-cell communication, many of which have important consequences for human health. Among these is conjugation, the direct transfer of DNA from one cell to another. For Gram-negative bacteria, conjugation requires thin, flexible filaments (conjugative pili) that are elaborated by DNA donor cells. The structure, function, and especially the dynamics of conjugative pili are poorly understood. Here, we have applied live-cell imaging to characterize the dynamics of F-pili (conjugative pili encoded by the F plasmid of Escherichia coli). We establish that F-pili normally undergo cycles of extension and retraction in the absence of any obvious triggering event, such as contact with a recipient cell. When made, such contacts are able to survive the shear forces felt by bacteria in liquid media. Our data emphasize the role of F-pilus flexibility both in efficiently sampling a large volume surrounding donor cells in liquid culture and in establishing and maintaining cell-cell contact. Additionally and unexpectedly, we infer that extension and retraction are accompanied by rotation about the long axis of the filament.conjugation ͉ conjugative pilus ͉ extension ͉ retraction A mong Gram-negative bacteria, conjugative DNA transfer is mediated by multicomponent type IV secretion systems commonly encoded by large plasmids (1, 2). By virtue of such activity, these plasmids rapidly spread within bacterial populations, contributing to the dissemination of antibiotic resistance (3) among other traits pertinent to human health (2, 4-6).A hallmark of Gram-negative bacterial cells capable of acting as DNA donors is the presence of surface filaments collectively designated conjugative pili (7). F-pili, characteristic of Escherichia coli carrying the conjugative plasmid F, are helical polymers of one subunit, F-pilin (8). Conjugation commences when the tips of F-pili make contact with recipient cells (9, 10). F-pili tips also bind filamentous DNA bacteriophages, such as M13, whereas icosohedral RNA bacteriophages such as R17 bind along the filament sides (11).Indirect evidence has suggested that F-pili are dynamic structures, capable of both extension and retraction. F-pilus retraction as an essential stage of DNA transfer was first proposed by Marvin and Hohn (12) and by Curtiss (13). Various lines of indirect evidence since then have supported the retraction hypothesis (14-18), although fundamental uncertainties remain. For example, retraction was initially regarded as a triggered event, occurring only when a recipient cell or bacteriophage bound to the filament tip (12). Later it was proposed that F-pili normally undergo cycles of extension and retraction (14,17,19). The idea that DNA can be conducted from donor to recipient through extended F-pili (10, 20) has also received experimental support (21,22).Here, we have applied laser-scanning confocal microscopy to study F-pilus dynamics with living cells. Our results clarify some of the uncertainties surrounding F-pilus function and sugge...

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“…Moreover, the icmG mutant is only partially defective for intracellular growth within A. castellanii at 30 u C (Fig. 4), the icmG and icmF mutants persist in D. discoideum (Otto et al, 2004), and the icmS and icmW mutants are not impaired in Icm/Dotdependent immediate cytotoxicity (Coers et al, 2000;Zuckman et al, 1999). However, LcsC was cytotoxic specifically in an icmG mutant but neither in other mutant strains only partially defective for intracellular growth, nor in wild-type L. pneumophila (data not shown).…”

Section: Mechanism Of Lcsc Cytotoxicitymentioning

confidence: 99%

“…Intracellular growth in macrophages and amoebae, including Acanthamoeba castellanii and Dictyostelium discoideum, is mechanistically similar and requires the L. pneumophila Icm/Dot transporter, a type IV secretion apparatus related to conjugation systems (Hagele et al, 2000;Otto et al, 2004;Segal & Shuman, 1999b;Solomon et al, 2000;Vogel et al, 1998). The Icm/Dot secretion system determines the initial contact of L. pneumophila with host cells and phagosome biogenesis (Hilbi et al, 2001;Watarai et al, 2001), is required to evade immediate endocytic maturation (Roy et al, 1998;Wiater et al, 1998) and governs subsequent formation of the ER-derived, replicative vacuole (reviewed by Nagai & Roy, 2003).…”

Section: Introductionmentioning

confidence: 99%

The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

et al. 2005

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Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.

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Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum

Cited by 115 publications

References 56 publications

Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance

Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance

F-pili dynamics by live-cell imaging

The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

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