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 Evidence for HrpXo-Dependent Expression of Type II Secretory Proteins in Xanthomonas oryzae pv. oryzae

2004 ◽  
Vol 186 (5) ◽  
pp. 1374-1380 ◽  
Author(s):  
Ayako Furutani ◽  
Seiji Tsuge ◽  
Kouhei Ohnishi ◽  
Yasufumi Hikichi ◽  
Takashi Oku ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Gene ◽  
Secretion System ◽  
Xanthomonas Oryzae ◽  
Nucleotide Sequence Analysis ◽  
Secretory Proteins ◽  
Type Ii ◽  
Wild Type ◽  
Type Iii

ABSTRACT Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by hrp genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that cysP2 has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a cysP2::gus fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the hrp regulatory gene hrpXo is also involved in the expression of not only hrp genes and type III secretory proteins but also some type II secretory proteins.

scholarly journals Type IV pili and type II secretion play a limited role in Legionella pneumophila biofilm colonization and retention

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3569-3573 ◽  
Author(s):  
Claressa E. Lucas ◽  
Ellen Brown ◽  
Barry S. Fields
Keyword(s):  
Legionella Pneumophila ◽  
Wild Type Strain ◽  
Water Systems ◽  
Type Iv Pili ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Intracellular Replication ◽  
Type Iv

Legionellae colonize biofilms in building water systems, yet little is known about their interaction with the organisms in these microbial communities. The role of Legionella pneumophila type IV pili and the type II secretion pre-pilin peptidase was evaluated in a model biofilm system. L. pneumophila strains 130b (wild-type), BS100 (a type IV pili mutant) and NU243 (a pre-pilin peptidase mutant) were assessed for attachment and retention in an established biofilm. Strains 130b and NU243 colonized the biofilm at a similar level while BS100 attached at a tenfold lower level. Over time, NU243 dropped below the level of detection while BS100 remained in the biofilm throughout the course of the experiment. The wild-type strain decreased but remained at a considerably higher level than either of the mutants. Inclusion of amoebae with BS100 allowed for attachment and retention at a level similar to 130b. NU243, which displays reduced intracellular replication, was able to establish itself and persist in the presence of amoebae. Thus, type IV pili and the pre-pilin peptidase facilitate L. pneumophila colonization of biofilms but are not required in the presence of a host for intracellular replication.

scholarly journals Requirement of the Type II Secretion System for Utilization of Cellulosic Substrates by Cellvibrio japonicus

2010 ◽  
Vol 76 (15) ◽  
pp. 5079-5087 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
David H. Keating
Keyword(s):  
Secretion System ◽  
Biofuel Production ◽  
Growth Defect ◽  
Cellulolytic Bacterium ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Gram Negative ◽  
Type Ii Secretion System ◽  
Cellvibrio Japonicus

ABSTRACT Cellulosic biofuels represent a powerful alternative to petroleum but are currently limited by the inefficiencies of the conversion process. While Gram-positive and fungal organisms have been widely explored as sources of cellulases and hemicellulases for biomass degradation, Gram-negative organisms have received less experimental attention. We investigated the ability of Cellvibrio japonicus, a recently sequenced Gram-negative cellulolytic bacterium, to degrade bioenergy-related feedstocks. Using a newly developed biomass medium, we showed that C. japonicus is able to utilize corn stover and switchgrass as sole sources of carbon and energy for growth. We also developed tools for directed gene disruptions in C. japonicus and used this system to construct a mutant in the gspD gene, which is predicted to encode a component of the type II secretion system. The gspD::pJGG1 mutant displayed a greater-than-2-fold decrease in endoglucanase secretion compared to wild- type C. japonicus. In addition, the mutant strain showed a pronounced growth defect in medium with biomass as a carbon source, yielding 100-fold fewer viable cells than the wild type. To test the potential of C. japonicus to undergo metabolic engineering, we constructed a strain able to produce small amounts of ethanol from biomass. Collectively, these data suggest that C. japonicus is a useful platform for biomass conversion and biofuel production.

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 Legionella pneumophila Catalase-Peroxidases Are Required for Proper Trafficking and Growth in Primary Macrophages

2003 ◽  
Vol 71 (8) ◽  
pp. 4526-4535 ◽  
Author(s):  
Purnima Bandyopadhyay ◽  
Brenda Byrne ◽  
Yolande Chan ◽  
Michele S. Swanson ◽  
Howard M. Steinman
Keyword(s):  
Legionella Pneumophila ◽  
Type Strain ◽  
Wild Type Strain ◽  
Acanthamoeba Castellanii ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Wild Type ◽  
Type Iv ◽  
Virulence Traits

ABSTRACT Legionella pneumophila, a parasite of aquatic amoebae and pathogen of pulmonary macrophages, replicates intracellularly, utilizing a type IV secretion system to subvert the trafficking of Legionella-containing phagosomes. Defense against host-derived reactive oxygen species has been proposed as critical for intracellular replication. Virulence traits of null mutants in katA and katB, encoding the two Legionella catalase-peroxidases, were analyzed to evaluate the hypothesis that L. pneumophila must decompose hydrogen peroxide to establish a replication niche in macrophages. Phagosomes containing katA or katB mutant Legionella colocalize with LAMP-1, a late endosomal-lysosomal marker, at twice the frequency of those of wild-type strain JR32 and show a decreased frequency of bacterial replication, in similarity to phenotypes of mutants with mutations in dotA and dotB, encoding components of the Type IV secretion system. Quantitative similarity of the katA/B phenotypes indicates that each contributes to virulence traits largely independently of intracellular compartmentalization (KatA in the periplasm and KatB in the cytosol). These data support a model in which KatA and KatB maintain a critically low level of H2O2 compatible with proper phagosome trafficking mediated by the type IV secretion apparatus. During these studies, we observed that dotA and dotB mutations in wild-type strain Lp02 had no effect on intracellular multiplication in the amoeba Acanthamoeba castellanii, indicating that certain dotA/B functions in Lp02 are dispensable in that experimental model. We also observed that wild-type JR32, unlike Lp02, shows minimal contact-dependent cytotoxicity, suggesting that cytotoxicity of JR32 is not a prerequisite for formation of replication-competent Legionella phagosomes in macrophages.

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 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.

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