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

2007 ◽  
Vol 74 (3) ◽  
pp. 753-761 ◽  
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.

scholarly journals 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

2004 ◽  
Vol 72 (7) ◽  
pp. 4040-4051 ◽  
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.

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

2020 ◽  
Vol 7 ◽  
Author(s):  
Theo J. Portlock ◽  
Jessica Y. Tyson ◽  
Sarath C. Dantu ◽  
Saima Rehman ◽  
Richard C. White ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Structure Dynamics ◽  
Type Ii Secretion System ◽  
Insight Into

scholarly journals The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2732-2744 ◽  
Author(s):  
Jessica Y. Tyson ◽  
Paloma Vargas ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Type Ii Secretion ◽  
The Novel ◽  
Type Ii ◽  
Intracellular Infection ◽  
Novel Proteins ◽  
Hartmannella Vermiformis ◽  
The Individual ◽  
Naegleria Lovaniensis

The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires’ disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts.

scholarly journals In vivo structure of the Legionella type II secretion system by electron cryotomography

2019 ◽  
Author(s):  
Debnath Ghosal ◽  
Ki Woo Kim ◽  
Huaixin Zheng ◽  
Mohammed Kaplan ◽  
Joseph P. Vogel ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Cell Envelope ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Iv ◽  
Type Ii Secretion System ◽  
Wide Range ◽  
Electron Cryotomography

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.

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

2002 ◽  
Vol 70 (1) ◽  
pp. 69-78 ◽  
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.

scholarly journals The Type II Protein Secretion System of Legionella pneumophila Promotes Growth at Low Temperatures

2004 ◽  
Vol 186 (12) ◽  
pp. 3712-3720 ◽  
Author(s):  
Maria A. Söderberg ◽  
Ombeline Rossier ◽  
Nicholas P. Cianciotto
Keyword(s):  
Low Temperature ◽  
Protein Secretion ◽  
Legionella Pneumophila ◽  
Low Temperatures ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Temperature Growth ◽  
Type Iv ◽  
Protein Secretion System

ABSTRACT The gram-negative bacterium Legionella pneumophila grows in both natural and man-made water systems and in the mammalian lung as a facultative intracellular parasite. The PilD prepilin peptidase of L. pneumophila promotes type IV pilus biogenesis and type II protein secretion. Whereas pili enhance adherence, Legionella type II secretion is critical for intracellular growth and virulence. Previously, we observed that pilD transcript levels are greater in legionellae grown at 30 versus 37°C. Using a new pilD::lacZ fusion strain, we now show that pilD transcriptional initiation increases progressively as L. pneumophila is grown at 30, 25, and 17°C. Legionella pilD mutants also had a dramatically reduced ability to grow in broth and to form colonies on agar at the lower temperatures. Whereas strains specifically lacking type IV pili were not defective for low-temperature growth, mutations in type II secretion (lsp) genes greatly impaired the capacity of L. pneumophila to form colonies at 25, 17, and 12°C. Indeed, the lsp mutants were completely unable to grow at 12°C. The growth defect of the pilD and lsp mutants was complemented by reintroduction of the corresponding intact gene. Interestingly, the lsp mutants displayed improved growth at 25°C when plated next to a streak of wild-type but not mutant bacteria, implying that a secreted, diffusible factor promotes low-temperature growth. Mutants lacking either the known secreted acid phosphatases, lipases, phospholipase C, lysophospholipase A, or protease grew normally at 25°C, suggesting the existence of a critical, yet-to-be-defined exoprotein(s). In summary, these data document, for the first time, that L. pneumophila replicates at temperatures below 20°C and that a bacterial type II protein secretion system facilitates growth at low temperatures.

scholarly journals A type II secreted RNase of Legionella pneumophila facilitates optimal intracellular infection of Hartmannella vermiformis

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 882-890 ◽  
Author(s):  
Ombeline Rossier ◽  
Jenny Dao ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Mutational Analysis ◽  
Critical Role ◽  
Rnase Activity ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Intracellular Infection ◽  
Zinc Metalloprotease ◽  
Type Ii Secretion System ◽  
Hartmannella Vermiformis

Type II protein secretion plays a role in a wide variety of functions that are important for the ecology and pathogenesis of Legionella pneumophila. Perhaps most dramatic is the critical role that this secretion pathway has in L. pneumophila intracellular infection of aquatic protozoa. Recently, we showed that virulent L. pneumophila strain 130b secretes RNase activity through its type II secretion system. We now report the cloning and mutational analysis of the gene (srnA) encoding that novel type of secreted activity. The SrnA protein was defined as being a member of the T2 family of secreted RNases. Supernatants from mutants inactivated for srnA completely lacked RNase activity, indicating that SrnA is the major secreted RNase of L. pneumophila. Although srnA mutants grew normally in bacteriological media and human U937 cell macrophages, they were impaired in their ability to grow within Hartmannella vermiformis amoebae. This finding represents the second identification of a L. pneumophila type II effector being necessary for optimal intracellular infection of amoebae, with the first being the ProA zinc metalloprotease. Newly constructed srnA proA double mutants displayed an even larger infection defect that appeared to be the additive result of losing both SrnA and ProA. Overall, these data represent the first demonstration of a secreted RNase promoting an intracellular infection event, and support our long-standing hypothesis that the infection defects of L. pneumophila type II secretion mutants are due to the loss of multiple secreted effectors.

scholarly journals Ancestral mitochondrial apparatus derived from the bacterial type II secretion system

2019 ◽  
Author(s):  
Lenka Horváthová ◽  
Vojtěch Žárský ◽  
Tomáš Pánek ◽  
Romain Derelle ◽  
Jan Pyrih ◽  
...  
Keyword(s):  
Functional Data ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Ii Secretion System ◽  
Biochemical Assays ◽  
Protein Secretion System ◽  
Functional Pathway ◽  
Bacterial Type

AbstractThe type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the formation of a mitochondrial T2SS-derived protein secretion system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.

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