Structure, Dynamics and Cellular Insight Into Novel Substrates of the Legionella pneumophila Type II Secretion System

AbstractThe type II secretion system (T2SS) is a multi-protein envelope-spanning assembly that translocates a wide range of virulence factors, enzymes and effectors through the outer membrane (OM) of many Gram-negative bacteria. Here, using electron cryotomography and subtomogram averaging methods, we present the first in situ structure of an intact T2SS, imaged within the human pathogen Legionella pneumophila. Although the T2SS has only limited sequence and component homology with the evolutionarily-related Type IV pilus (T4P) system, we show that their overall architectures are remarkably similar. Despite similarities, there are also differences, including for instance that the T2SS-ATPase complex is usually present but disengaged from the inner membrane, the T2SS has a much longer periplasmic vestibule, and it has a short-lived flexible pseudopilus. Placing atomic models of the components into our ECT map produced a complete architectural model of the intact T2SS that provides new insights into the structure and function of its components, its position within the cell envelope, and the interactions between its different subcomplexes. Overall, these structural results strongly support the piston model for substrate extrusion.

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icmT Is Essential for Pore Formation-Mediated Egress of Legionella pneumophila from Mammalian and Protozoan Cells

Infection and Immunity ◽

10.1128/iai.70.1.69-78.2002 ◽

2002 ◽

Vol 70 (1) ◽

pp. 69-78 ◽

Cited By ~ 64

Author(s):

Maelle Molmeret ◽

O. A. Terry Alli ◽

Steven Zink ◽

Antje Flieger ◽

Nicholas P. Cianciotto ◽

...

Keyword(s):

Host Cell ◽

Legionella Pneumophila ◽

Type Strain ◽

Pore Formation ◽

Wild Type Strain ◽

Secretion System ◽

Type Ii Secretion ◽

Type Ii ◽

Wild Type ◽

Type Ii Secretion System

ABSTRACT The final step of the intracellular life cycle of Legionella pneumophila and other intracellular pathogens is their egress from the host cell after termination of intracellular replication. We have previously isolated five spontaneous mutants of L. pneumophila that replicate intracellularly similar to the wild-type strain but are defective in pore formation-mediated cytolysis and egress from mammalian and protozoan cells, and the mutants have been designated rib (release of intracellular bacteria). Here, we show that the rib mutants are not defective in the activity of enzymes secreted through the type II secretion system, including phospholipase A, lysophospholipase A, and monoacylglycerol lipase, although they are potential candidates for factors that lyse host cell membranes. In addition, the pilD and lspG mutants, which are defective in the type II secretion system, are not defective in the pore-forming toxin. We show that all five rib mutants have an identical point mutation (deletion) following a stretch of poly(T) in the icmT gene. Spontaneous revertants of the rib mutants, due to an insertion of a nucleotide following the poly(T) stretch in icmT, have been isolated and shown to have regained the wild-type phenotype. We constructed an icmT insertion mutant (AA100kmT) in the chromosome of the wild-type strain by allelic exchange. The AA100kmT mutant was as defective as the rib mutant in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. Both the rib mutant and the AA100kmT mutant were complemented by the icmT gene for their phenotypic defect. rtxA, a gene that is thought to have a minor role in pore formation, was not involved in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. We conclude that the icmT gene is essential for pore formation-mediated lysis of mammalian and protozoan cells and the subsequent bacterial egress.

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The Type II Secretion System of Legionella pneumophila Elaborates Two Aminopeptidases, as Well as a Metalloprotease That Contributes to Differential Infection among Protozoan Hosts

Applied and Environmental Microbiology ◽

10.1128/aem.01944-07 ◽

2007 ◽

Vol 74 (3) ◽

pp. 753-761 ◽

Cited By ~ 49

Author(s):

Ombeline Rossier ◽

Jenny Dao ◽

Nicholas P. Cianciotto

Keyword(s):

Legionella Pneumophila ◽

Acanthamoeba Castellanii ◽

Secretion System ◽

Type Ii Secretion ◽

Type Ii ◽

Intracellular Infection ◽

Type Ii Secretion System ◽

Protozoan Infection ◽

Key Factor ◽

Protein Secretion System

ABSTRACT Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor for L. pneumophila in intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found that L. pneumophila secretes two distinct aminopeptidase activities encoded by the genes lapA and lapB. Whereas lapA conferred activity against leucine, phenylalanine, and tyrosine aminopeptides, lapB was linked to the cleavage of lysine- and arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities of lapA and lapB mutants to infect human U937 cell macrophages as well as Hartmannella vermiformis and Acanthamoeba castellanii amoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. Whereas proA mutants were not defective for macrophage or A. castellanii infection, they (but not their complemented derivatives) were impaired for growth upon coculture with H. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore, proA represents an L. pneumophila gene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts.

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Disruption of the Phagosomal Membrane and Egress of Legionella pneumophila into the Cytoplasm during the Last Stages of Intracellular Infection of Macrophages and Acanthamoeba polyphaga

Infection and Immunity ◽

10.1128/iai.72.7.4040-4051.2004 ◽

2004 ◽

Vol 72 (7) ◽

pp. 4040-4051 ◽

Cited By ~ 57

Author(s):

Maëlle Molmeret ◽

Dina M. Bitar ◽

Lihui Han ◽

Yousef Abu Kwaik

Keyword(s):

Legionella Pneumophila ◽

Amorphous Material ◽

Hydrolytic Enzymes ◽

Secretion System ◽

Type Ii Secretion ◽

Type Ii ◽

Acanthamoeba Polyphaga ◽

Intracellular Infection ◽

Type Ii Secretion System ◽

Phagosomal Membrane

ABSTRACT Although the early stages of intracellular infection by Legionella pneumophila are well established at the ultrastructural level, a detailed ultrastructural analysis of late stages of intracellular replication has never been done. Here we show that the membrane of the L. pneumophila-containing phagosome (LCP) is intact for up to 8 h postinfection of macrophages and Acanthamoeba polyphaga. At 12 h, 71 and 74% of the LCPs are disrupted within macrophages and A. polyphaga, respectively, while the plasma membrane remains intact. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic. At 18 h, 77% of infected macrophages and 32% of infected A. polyphaga amoebae harbor cytoplasmic bacteria. At 24 h, 99 and 78% of infected macrophages and amoebae, respectively, contain cytoplasmic bacteria. On the basis of lysosomal acid phosphatase staining of infected macrophages and A. polyphaga, the lysosomal enzyme is present among the bacteria when host vesicles are dispersed among bacteria. Our data indicate that bacterial replication proceeds despite physical disruption of the phagosomal membrane. We also show that an lspG mutant that is defective in the type II secretion system and therefore does not secrete the hydrolytic enzymes metalloprotease, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A is as efficient as the wild-type strain in disruption of the LCP. Therefore, L. pneumophila disrupts the phagosomal membrane and becomes cytoplasmic at the last stages of infection in both macrophages and A. polyphaga. Lysosomal elements, mitochondria, cytoplasmic vesicles, and amorphous material are all dispersed among the bacteria, after phagosomal disruption, within both human macrophages and A. polyphaga. The disruption of the LCP is independent of the hydrolytic enzymes exported by the type II secretion system.

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