scholarly journals More than 18,000 effectors in theLegionellagenus genome provide multiple, independent combinations for replication in human cells

2019 ◽  
Vol 116 (6) ◽  
pp. 2265-2273 ◽  
Author(s):  
Laura Gomez-Valero ◽  
Christophe Rusniok ◽  
Danielle Carson ◽  
Sonia Mondino ◽  
Ana Elena Pérez-Cobas ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Chromatin Modification ◽  
Severe Pneumonia ◽  
Rab Gtpase ◽  
Functional Screening ◽  
Evolutionary Analysis ◽  
Human Pathogen ◽  
Human Pathogens ◽  
Intracellular Replication ◽  
Type Iv

The genusLegionellacomprises 65 species, among whichLegionella pneumophilais a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80Legionellagenomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes andLegionella. Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted toL. pneumophilaand seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.

scholarly journals More than 16,000 effectors in theLegionellagenus genome provide multiple, independent combinations for replication in human cells

2018 ◽  
Author(s):  
Laura Gomez-Valero ◽  
Christophe Rusniok ◽  
Danielle Carson ◽  
Sonia Mondino ◽  
Ana Elena Pérez-Cobas ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Secreted Proteins ◽  
Rab Gtpase ◽  
Functional Screening ◽  
Evolutionary Analysis ◽  
Human Pathogen ◽  
Human Pathogens ◽  
Intracellular Replication ◽  
Bacterial Genus ◽  
Gene Acquisition

SignificanceLegionella pneumophilais a bacterial pathogen causing outbreaks of a lethal pneumonia. The genusLegionellacomprises 65 species for which aquatic amoebae are the natural reservoirs. Using functional and comparative genomics to deconstruct the entire bacterial genus we reveal the surprising parallel evolutionary trajectories that have led to the emergence of human pathogenicLegionella.An unexpectedly large and unique repository of secreted proteins (>16,000) containing eukaryotic-like proteins acquired from all domains of life (plant, animal, fungal, archaea) is contrasting with a highly conserved type 4 secretion system. This study reveals an unprecedented environmental reservoir of bacterial virulence factors, and provides a new understanding of how reshuffling and gene-acquisition from environmental eukaryotic hosts, may allow for the emergence of human pathogens.AbstractThe bacterial genusLegionellacomprises 65 species among, whichLegionella pneumophilais a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80Legionellagenomes spanning 58 species. Uniquely, an immense repository of 16,000 secreted proteins encoding 137 different eukaryotic-like domains and more than 200 eukaryotic-like proteins is paired with a highly conserved T4SS. Specifically, we show that eukaryotic Rho and Rab GTPase domains are found nearly exclusively in eukaryotes andLegionellaspecies. Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F/U-box and SET domains were present in >70% of all species suggesting that manipulation of host signal transduction, protein turnover and chromatin modification pathways, respectively are fundamental intracellular replication strategies forLegionellae. In contrast, the Sec-7 domain was restricted toL. pneumophilaand seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by inter-domain gene transfer. Furthermore we revealed the surprising extent to which legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts and provides a new understanding of how dynamic reshuffling and gene-acquisition has led to the emergence of major human pathogens.

scholarly journals Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires

2015 ◽  
Vol 83 (10) ◽  
pp. 4081-4092 ◽  
Author(s):  
Rebecca E. Wood ◽  
Patrice Newton ◽  
Eleanor A. Latomanski ◽  
Hayley J. Newton
Keyword(s):  
Legionella Pneumophila ◽  
A549 Cells ◽  
Severe Pneumonia ◽  
Rab Gtpase ◽  
Human Pathogens ◽  
Immunofluorescence Analysis ◽  
Content Type ◽  
Extensive Analysis ◽  
Legionnaires Disease ◽  
Environmental Bacteria

Legionellaorganisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed theLegionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis ofLegionella pneumophila. Here, we have characterized theLegionella longbeachaereplicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV ofL. pneumophila(pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deletingdotBand demonstrated the inability of this mutant to replicate inside THP-1 cells.L. longbeachaedoes not enter THP-1 cells as efficiently asL. pneumophila, and this is reflected in the observation that translocation of BlaM-RalFLLO(where RalFLLOis theL. longbeachaehomologue of RalF) into THP-1 cells by theL. longbeachaeDot/Icm system is less efficient than that byL. pneumophila. This difference is negated in A549 cells whereL. longbeachaeandL. pneumophilainfect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, theRab-likeeffector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on thelongbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to thelongbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of theL. longbeachaereplicative vacuole, highlighting phenotypic similarities to the vacuole ofL. pneumophilaas well as unique aspects of LCV biology.

The Legionella pneumophila effector RavY contributes to a replication-permissive vacuolar environment during infection

2021 ◽  
Author(s):  
Luying Liu ◽  
Craig R. Roy
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Effector Protein ◽  
Intracellular Replication ◽  
Phagocytic Cells ◽  
Host Molecules ◽  
Type Iv ◽  
Legionnaires Disease

Legionella pneumophila is the causative agent of Legionnaires’ Disease and is capable replicating inside phagocytic cells such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila , but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, nor does RavY contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild type L. pneumophila . Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.

scholarly journals Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Yoon-Suk Kang ◽  
James E. Kirby
Keyword(s):  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Life Cycles ◽  
Wild Type ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Reporter Systems ◽  
Brucella Neotomae

ABSTRACT We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila. Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.

scholarly journals How bacterial effectors hijack small GTPases to divert membrane traffic

2014 ◽  
Vol 70 (a1) ◽  
pp. C802-C802
Author(s):  
Jacqueline Cherfils ◽  
Marcia Folly-Klan ◽  
Valérie Campanacci
Keyword(s):  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Membrane Traffic ◽  
Severe Pneumonia ◽  
Small Gtpases ◽  
Covalent Attachment ◽  
Rab Gtpase ◽  
Postal Service ◽  
Nucleotide Exchange ◽  
Vesicular Traffic

Membrane traffic, which is the "cellular postal service" that shuttles biomolecules around the cell and organizes the structure of organelles, is among the primary targets of effectors injected by intracellular pathogenic bacteria to invade their host and avoid from being destroyed. I will present our recent structural and biochemical studies of effectors from Legionella pneumophila (the bacteria that causes the legionnaire's disease, a severe pneumonia) that divert membrane traffic to generate a membrane-bound vacuole where the pathogen hides and replicates. One of these effectors, AnkX, is a FIC domain-containing toxin that alters the functions of a Rab GTPase involved in vesicular traffic at the endoplasmic reticulum, by covalent attachment of a phosphocholine molecule. The other one, RalF, functions as an illegitimate guanine nucleotide exchange factor to activate an Arf GTPase on the vacuole. Our studies showed how AnkX binds and processes CDP-choline to transfer phosphocholine onto Rab1 [1], and uncover a novel membrane sensor in RalF that controls its localization and activity [2]

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 Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion

2014 ◽  
Vol 82 (10) ◽  
pp. 4325-4336 ◽  
Author(s):  
Alan M. Copenhaver ◽  
Cierra N. Casson ◽  
Hieu T. Nguyen ◽  
Thomas C. Fung ◽  
Matthew M. Duda ◽  
...  
Keyword(s):  
Immune Response ◽  
Alveolar Macrophages ◽  
Legionella Pneumophila ◽  
Pulmonary Infection ◽  
Severe Pneumonia ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Content Type ◽  
Type Iv

ABSTRACTLegionella pneumophila, an intracellular pathogen responsible for the severe pneumonia Legionnaires' disease, uses itsdot/icm-encoded type IV secretion system (T4SS) to translocate effector proteins that promote its survival and replication into the host cell cytosol. However, by introducing bacterial products into the host cytosol,L. pneumophilaalso activates cytosolic immunosurveillance pathways, thereby triggering robust proinflammatory responses that mediate the control of infection. Thus, the pulmonary cell types thatL. pneumophilainfects not only may act as an intracellular niche that facilitates its pathogenesis but also may contribute to the immune response againstL. pneumophila. The identity of these host cells remains poorly understood. Here, we developed a strain ofL. pneumophilaproducing a fusion protein consisting of β-lactamase fused to the T4SS-translocated effector RalF, which allowed us to track cells injected by the T4SS. Our data reveal that alveolar macrophages and neutrophils both are the primary recipients of T4SS-translocated effectors and harbor viableL. pneumophiladuring pulmonary infection of mice. Moreover, both alveolar macrophages and neutrophils from infected mice produced tumor necrosis factor and interleukin-1α in response to T4SS-sufficient, but not T4SS-deficient,L. pneumophila. Collectively, our data suggest that alveolar macrophages and neutrophils are both an intracellular reservoir forL. pneumophilaand a source of proinflammatory cytokines that contribute to the host immune response againstL. pneumophiladuring pulmonary infection.

scholarly journals Induction of Rapid Cell Death by an Environmental Isolate of Legionella pneumophila in Mouse Macrophages

2013 ◽  
Vol 81 (9) ◽  
pp. 3077-3088 ◽  
Author(s):  
Lili Tao ◽  
Wenhan Zhu ◽  
Bi-Jie Hu ◽  
Jie-Ming Qu ◽  
Zhao-Qing Luo
Keyword(s):  
Cell Death ◽  
Legionella Pneumophila ◽  
Immune Surveillance ◽  
Human Macrophage ◽  
Aqueous Environment ◽  
Intracellular Replication ◽  
Content Type ◽  
Type Iv ◽  
Extensive Cell Death ◽  
Extensive Cell

ABSTRACTLegionella pneumophila, the etiological agent for Legionnaires' disease, is ubiquitous in the aqueous environment, where it replicates as an intracellular parasite of free-living protozoa. Our understanding ofL. pneumophilapathogenicity is obtained mostly from study of derivatives of several clinical isolates, which employ almost identical virulent determinants to exploit host functions. To determine whether environmentalL. pneumophilaisolates interact similarly with the model host systems, we analyzed intracellular replication of several recently isolated such strains and found that these strains cannot productively grow in bone marrow-derived macrophages of A/J mice, which are permissive for all examined laboratory strains. By focusing on one strain called LPE509, we found that its deficiency in intracellular replication in primary A/J macrophages is not caused by the lack of important pathogenic determinants because this strain replicates proficiently in two protozoan hosts and the human macrophage U937 cell. We also found that in the early phase of infection, the trafficking of this strain in A/J macrophages is similar to that of JR32, a derivative of strain Philadelphia 1. Furthermore, infection of these cells by LPE509 caused extensive cell death in a process that requires the Dot/Icm type IV secretion system. Finally, we showed that the cell death is caused neither by the activation of the NAIP5/NLRC4 inflammasome nor by the recently described caspase 11-dependent pathway. Our results revealed that some environmentalL. pneumophilastrains are unable to overcome the defense conferred by primary macrophages from mice known to be permissive for laboratoryL. pneumophilastrains. These results also suggest the existence of a host immune surveillance mechanism differing from those currently known in responding toL. pneumophilainfection.

scholarly journals Legionella pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins

2010 ◽  
Vol 192 (22) ◽  
pp. 6001-6016 ◽  
Author(s):  
Gunnar N. Schroeder ◽  
Nicola K. Petty ◽  
Aurélie Mousnier ◽  
Clare R. Harding ◽  
Adam J. Vogrin ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Draft Genome ◽  
Severe Pneumonia ◽  
Gene Clusters ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Secretion Systems ◽  
Type Iv ◽  
Core Set

ABSTRACT Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.

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