scholarly journals Evolution of the Arsenal ofLegionella pneumophilaEffectors To Modulate Protist Hosts

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Ashley Best ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Host Cells ◽  
Human Pathogen ◽  
Intracellular Growth ◽  
Content Type ◽  
Human Macrophages ◽  
Cellular Processes ◽  
Host Interaction

ABSTRACTWithin the human host,Legionella pneumophilareplicates within alveolar macrophages, leading to pneumonia. However,L. pneumophilais an aquatic generalist pathogen that replicates within a wide variety of protist hosts, including amoebozoa, percolozoa, and ciliophora. The intracellular lifestyles ofL. pneumophilawithin the two evolutionarily distant hosts macrophages and protists are remarkably similar. Coevolution with numerous protist hosts has shaped plasticity of the genome ofL. pneumophila, which harbors numerous proteins encoded by genes acquired from primitive eukaryotic hosts through interkingdom horizontal gene transfer. The Dot/Icm type IVb translocation system translocates ∼6,000 effectors amongLegionellaspecies and >320 effector proteins inL. pneumophilainto host cells to modulate a plethora of cellular processes to create proliferative niches. Since many of the effectors have likely evolved to modulate cellular processes of primitive eukaryotic hosts, it is not surprising that most of the effectors do not contribute to intracellular growth within human macrophages. Some of the effectors may modulate highly conserved eukaryotic processes, while others may target protist-specific processes that are absent in mammals. The lack of studies to determine the role of the effectors in adaptation ofL. pneumophilato various protists has hampered the progress to determine the function of most of these effectors, which are routinely studied in mouse or human macrophages. Since many protists restrictL. pneumophila, utilization of such hosts can also be instrumental in deciphering the mechanisms of failure ofL. pneumophilato overcome restriction of certain protist hosts. Here, we review the interaction ofL. pneumophilawith its permissive and restrictive protist environmental hosts and outline the accomplishments as well as gaps in our knowledge ofL. pneumophila-protist host interaction andL. pneumophila’s evolution to become a human pathogen.

scholarly journals Interaction of the Ankyrin H Core Effector of Legionella with the Host LARP7 Component of the 7SK snRNP Complex

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Juanita Von Dwingelo ◽  
Ivy Yeuk Wah Chung ◽  
Christopher T. Price ◽  
Lei Li ◽  
Snake Jones ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Host Protein ◽  
Host Cells ◽  
Transcriptional Elongation ◽  
Intracellular Growth ◽  
Pol Ii ◽  
Content Type ◽  
Small Nuclear Ribonucleoprotein

ABSTRACT Species of the Legionella genus encode at least 18,000 effector proteins that are translocated through the Dot/Icm type IVB translocation system into macrophages and protist hosts to enable intracellular growth. Eight effectors, including ankyrin H (AnkH), are common to all Legionella species. The AnkH effector is also present in Coxiella and Rickettsiella. To date, no pathogenic effectors have ever been described that directly interfere with host cell transcription. We determined that the host nuclear protein La-related protein 7 (LARP7), which is a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, interacts with AnkH in the host cell nucleus. The AnkH-LARP7 interaction partially impedes interactions of the 7SK snRNP components with LARP7, interfering with transcriptional elongation by polymerase (Pol) II. Consistent with that, our data show AnkH-dependent global reprogramming of transcription of macrophages infected by Legionella pneumophila. The crystal structure of AnkH shows that it contains four N-terminal ankyrin repeats, followed by a cysteine protease-like domain and an α-helical C-terminal domain. A substitution within the β-hairpin loop of the third ankyrin repeat results in diminishment of LARP7-AnkH interactions and phenocopies the ankH null mutant defect in intracellular growth. LARP7 knockdown partially suppresses intracellular proliferation of wild-type (WT) bacteria and increases the severity of the defect of the ΔankH mutant, indicating a role for LARP7 in permissiveness of host cells to intracellular bacterial infection. We conclude that the AnkH-LARP7 interaction impedes interaction of LARP7 with 7SK snRNP, which would block transcriptional elongation by Pol II, leading to host global transcriptional reprogramming and permissiveness to L. pneumophila. IMPORTANCE For intracellular pathogens to thrive in host cells, an environment that supports survival and replication needs to be established. L. pneumophila accomplishes this through the activity of the ∼330 effector proteins that are injected into host cells during infection. Effector functions range from hijacking host trafficking pathways to altering host cell machinery, resulting in altered cell biology and innate immunity. One such pathway is the host protein synthesis pathway. Five L. pneumophila effectors have been identified that alter host cell translation, and 2 effectors have been identified that indirectly affect host cell transcription. No pathogenic effectors have been described that directly interfere with host cell transcription. Here we show a direct interaction of the AnkH effector with a host cell transcription complex involved in transcriptional elongation. We identify a novel process by which AnkH interferes with host transcriptional elongation through interference with formation of a functional complex and show that this interference is required for pathogen proliferation.

scholarly journals Cyclic Diguanylate Signaling Proteins Control Intracellular Growth of Legionella pneumophila

mBio ◽  
2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Assaf Levi ◽  
Marc Folcher ◽  
Urs Jenal ◽  
Howard A. Shuman
Keyword(s):  
Causative Agent ◽  
Legionella Pneumophila ◽  
Second Messenger ◽  
Host Cells ◽  
Diguanylate Cyclase ◽  
Intracellular Growth ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Legionnaires Disease

ABSTRACTProteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle ofLegionella pneumophila, the causative agent of Legionnaires’ disease, has not previously been examined. TheL. pneumophilagenome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes ascdgS(cyclicdiguanylatesignaling) genes. Strains ofL. pneumophilacontaining deletions of all individualcdgSgenes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, severalcdgSgenes strongly decreased the ability ofL. pneumophilato grow inside host cells. Expression of thesecdgSgenes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth inL. pneumophila.L. pneumophilastrains overexpressing thesecdgSgenes were less cytotoxic to THP-1 macrophages than wild-typeL. pneumophilabut retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused bycdgSgene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of thecdgSgenes in aSalmonella entericaserovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that severalcdgSgenes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability ofL. pneumophilato grow in host cells.IMPORTANCEAll bacteria must sense and respond to environmental cues. Intracellular bacterial pathogens must detect and respond to host functions that limit their ability to carry out a successful infection. Small-molecule second messengers play key roles in transmitting signals from environmental receptors to the proteins and other components that respond to signals. Cyclic diguanylate is a ubiquitous bacterial second messenger known to play an important role in many sensing and signaling systems in bacteria. The causative agent of Legionnaires’ disease,Legionella pneumophila, is an intracellular pathogen that grows inside environmental protists and human macrophages by subverting the normal processes that these cells use to capture and destroy bacteria. We show that the several cyclic diguanylate signaling components inLegionellaplay a role in the ability to grow inside both kinds of host cells. This work highlights the role of cyclic diguanylate signaling during intracellular growth.

scholarly journals Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
A. Leoni Swart ◽  
Bernhard Steiner ◽  
Laura Gomez-Valero ◽  
Sabina Schütz ◽  
Mandy Hannemann ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Host Cells ◽  
Divergent Evolution ◽  
Ran Gtpase ◽  
Content Type ◽  
Gtpase Cycle ◽  
Gtpase Ran

ABSTRACT Legionella pneumophila governs its interactions with host cells by secreting >300 different “effector” proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG. The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975. legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection. IMPORTANCE Legionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires’ disease. The opportunistic pathogen grows in amoebae and macrophages by employing a “type IV” secretion system, which secretes more than 300 different “effector” proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.

scholarly journals Live-Cell Imaging of Phosphoinositide Dynamics and Membrane Architecture during Legionella Infection

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Stephen Weber ◽  
Maria Wagner ◽  
Hubert Hilbi
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Effector Proteins ◽  
Host Cells ◽  
Wild Type ◽  
Host Cell Membrane ◽  
Content Type ◽  
Temporal And Spatial

ABSTRACTThe causative agent of Legionnaires’ disease,Legionella pneumophila, replicates in amoebae and macrophages in a distinct membrane-bound compartment, theLegionella-containing vacuole (LCV). LCV formation is governed by the bacterial Icm/Dot type IV secretion system that translocates ~300 different “effector” proteins into host cells. Some of the translocated effectors anchor to the LCV membrane via phosphoinositide (PI) lipids. Here, we use the soil amoebaDictyostelium discoideum, producing fluorescent PI probes, to analyze the LCV PI dynamics by live-cell imaging. Upon uptake of wild-type or Icm/Dot-deficientL. pneumophila, PtdIns(3,4,5)P3transiently accumulated for an average of 40 s on early phagosomes, which acquired PtdIns(3)Pwithin 1 min after uptake. Whereas phagosomes containing ΔicmTmutant bacteria remained decorated with PtdIns(3)P, more than 80% of wild-type LCVs gradually lost this PI within 2 h. The process was accompanied by a major rearrangement of PtdIns(3)P-positive membranes condensing to the cell center. PtdIns(4)Ptransiently localized to early phagosomes harboring wild-type or ΔicmT L. pneumophilaand was cleared within minutes after uptake. During the following 2 h, PtdIns(4)Psteadily accumulated only on wild-type LCVs, which maintained a discrete PtdIns(4)Pidentity spatially separated from calnexin-positive endoplasmic reticulum (ER) for at least 8 h. The separation of PtdIns(4)P-positive and ER membranes was even more pronounced for LCVs harboring ΔsidC-sdcAmutant bacteria defective for ER recruitment, without affecting initial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI lipids implicated in LCV formation and provide insight into host cell membrane and effector protein interactions.IMPORTANCEThe environmental bacteriumLegionella pneumophilais the causative agent of Legionnaires’ pneumonia. The bacteria form in free-living amoebae and mammalian immune cells a replication-permissive compartment, theLegionella-containing vacuole (LCV). To subvert host cell processes, the bacteria secrete the amazing number of ~300 different proteins into host cells. Some of these proteins bind phosphoinositide (PI) lipids to decorate the LCV. PI lipids are crucial factors involved in host cell membrane dynamics and LCV formation. UsingDictyosteliumamoebae producing one or two distinct fluorescent probes, we elucidated the dynamic LCV PI pattern in high temporal and spatial resolution. Notably, the endocytic PI lipid PtdIns(3)Pwas slowly cleared from LCVs, thus incapacitating the host cell’s digestive machinery, while PtdIns(4)Pgradually accumulated on the LCV, enabling critical interactions with host organelles. The LCV PI pattern underlies the spatiotemporal configuration of bacterial effector proteins and therefore represents a crucial aspect of LCV formation.

scholarly journals The Legionella longbeachae Icm/Dot Substrate SidC Selectively Binds Phosphatidylinositol 4-Phosphate with Nanomolar Affinity and Promotes Pathogen Vacuole-Endoplasmic Reticulum Interactions

2014 ◽  
Vol 82 (10) ◽  
pp. 4021-4033 ◽  
Author(s):  
Stephanie Dolinsky ◽  
Ina Haneburger ◽  
Adam Cichy ◽  
Mandy Hannemann ◽  
Aymelt Itzen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Legionella Pneumophila ◽  
Severe Pneumonia ◽  
Biological Data ◽  
Effector Proteins ◽  
Host Cells ◽  
Titration Calorimetry ◽  
Dependent Manner ◽  
Content Type ◽  
Phosphatidylinositol 4

ABSTRACTLegionellaspp. cause the severe pneumonia Legionnaires' disease. The environmental bacteria replicate intracellularly in free-living amoebae and human alveolar macrophages within a distinct, endoplasmic reticulum (ER)-derived compartment termed theLegionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS) that translocates into host cells a plethora of different “effector” proteins, some of which anchor to the pathogen vacuole by binding to phosphoinositide (PI) lipids. Here, we identified by unbiased pulldown assays inLegionella longbeachaelysates a 111-kDa SidC homologue as the major phosphatidylinositol 4-phosphate [PtdIns(4)P]-binding protein. The PI-binding domain was mapped to a 20-kDa P4C [PtdIns(4)Pbinding of SidC] fragment. Isothermal titration calorimetry revealed that SidC ofL. longbeachae(SidCLlo) binds PtdIns(4)Pwith aKd(dissociation constant) of 71 nM, which is 3 to 4 times lower than that of the SidC orthologue ofLegionella pneumophila(SidCLpn). Upon infection of RAW 264.7 macrophages withL. longbeachae, endogenous SidCLloor ectopically produced SidCLpnlocalized in an Icm/Dot-dependent manner to the PtdIns(4)P-positive LCVs. AnL. longbeachaeΔsidCdeletion mutant was impaired for calnexin recruitment to LCVs inDictyostelium discoideumamoebae and outcompeted by wild-type bacteria inAcanthamoeba castellanii. Calnexin recruitment was restored by SidCLloor its orthologues SidCLpnand SdcALpn. Conversely, calnexin recruitment was restored by SidCLloinL. pneumophilalackingsidCandsdcA. Together, biochemical, genetic, and cell biological data indicate that SidCLlois anL. longbeachaeeffector that binds through a P4C domain with high affinity to PtdIns(4)Pon LCVs, promotes ER recruitment to the LCV, and thus plays a role in pathogen-host interactions.

scholarly journals A Novel Legionella Genomic Island Encodes a Copper-Responsive Regulatory System and a Single Icm/Dot Effector Protein Transcriptionally Activated by Copper

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Marika Linsky ◽  
Yevgeniya Vitkin ◽  
Gil Segal
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Legionella Pneumophila ◽  
Genomic Island ◽  
Regulatory System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Effector Protein ◽  
Content Type

ABSTRACT The intracellular pathogen Legionella pneumophila utilizes the Icm/Dot type IV secretion system to translocate >300 effector proteins into host cells during infection. The regulation of some of these effector-encoding genes was previously shown to be coordinated by several global regulators, including three two-component systems (TCSs) found in all the Legionella species examined. Here, we describe the first Legionella genomic island encoding a single Icm/Dot effector and a dedicated TCS, which regulates its expression. This genomic island, which we named Lci, undergoes horizontal gene transfer in the Legionella genus, and the TCS encoded from this island (LciRS) is homologous to TCSs that control the expression of various metal resistance systems found in other bacteria. We found that the L. pneumophila sensor histidine kinase LciS is specifically activated by copper via a unique, small periplasmic sensing domain. Upon activation by LciS, the response regulator LciR directly binds to a conserved regulatory element and activates the expression of the adjacently located lciE effector-encoding gene. Thus, LciR represents the first local regulator of effectors identified in L. pneumophila. Moreover, we found that the expression of the lciRS operon is repressed by the Fis1 and Fis3 regulators, leading to Fis-mediated effects on copper induction of LciE and silencing of the expression of this genomic island in the absence of copper. This island represents a novel type of effector regulation in Legionella, shedding new light on the ways by which the Legionella pathogenesis system evolves its effector repertoire and expands its activating signals. IMPORTANCE Legionella pneumophila is an intracellular human pathogen that utilizes amoebae as its environmental host. The adaptation of L. pneumophila to the intracellular environment requires coordination of expression of its multicomponent pathogenesis system, which is composed of a secretion system and effector proteins. However, the regulatory factors controlling the expression of this pathogenesis system are only partially uncovered. Here, we discovered a novel regulatory system that is activated by copper and controls the expression of a single effector protein. The genes encoding both the regulatory system and the effector protein are located on a genomic island that undergoes horizontal gene transfer within the Legionella genus. This regulator-effector genomic island represents the first reported case of local regulation of effectors in Legionella. The discovery of this regulatory mechanism is an important step forward in the understanding of how the regulatory network of effectors functions and evolves in the Legionella genus.

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 Legionella pneumophila targets autophagosomes and promotes host autophagy during late infection

2021 ◽  
Author(s):  
Rebecca R. Noll ◽  
Colleen M. Pike ◽  
Stephanie S. Lehman ◽  
Chad Williamson ◽  
Ramona Neunuebel
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Pathogen ◽  
Nutrient Release ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Effector Protein ◽  
Wild Type ◽  
Cellular Processes ◽  
Type Iv

Autophagy is a fundamental eukaryotic process that mediates clearance of unwanted molecules and facilitates nutrient release. The bacterial pathogen Legionella pneumophila establishes an intracellular niche within phagocytes by manipulating host cellular processes, such as autophagy. Effector proteins translocated by L. pneumophila's Dot/Icm type IV secretion system have been shown to suppress autophagy. However evidence suggests that overall inhibition of autophagy may be detrimental to the bacterium. As autophagy contributes to cellular homeostasis and nutrient acquisition, L. pneumophila may translocate effectors that promote autophagy for these benefits. Here, we show that effector protein Lpg2411 binds phosphatidylinositol-3-phosphate lipids and preferentially binds autophagosomes. Translocated Lpg2411 accumulates late during infection and co-localizes with the autophagy receptor p62 and ubiquitin. Furthermore, autophagy is inhibited to a greater extent in host cells infected with a mutant strain lacking Lpg2411 compared to those infected with wild-type L. pneumophila, indicating that Lpg2411 stimulates autophagy to support the bacterium's intracellular lifestyle.

scholarly journals Potentiation of Cytokine-Mediated Restriction ofLegionellaIntracellular Replication by a Dot/Icm-Translocated Effector

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Tshegofatso Ngwaga ◽  
Alex J. Hydock ◽  
Sandhya Ganesan ◽  
Stephanie R. Shames
Keyword(s):  
Legionella Pneumophila ◽  
Defense Mechanisms ◽  
Effector Proteins ◽  
Host Cells ◽  
Content Type ◽  
Host Restriction ◽  
Type Iv ◽  
Legionnaires Disease ◽  
Ifn Γ ◽  
Necrosis Factor

ABSTRACTLegionella pneumophilais ubiquitous in freshwater environments, where it replicates within unicellular protozoa. However,L. pneumophilais also an accidental human pathogen that can cause Legionnaires’ disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes,L. pneumophilautilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction ofL. pneumophila. We found previously that the effector LegC4 is important forL. pneumophilareplication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires’ disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuatesL. pneumophilareplication. We found that LegC4 enhanced restriction ofL. pneumophilareplication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-γ). In addition, expression oflegC4was sufficient to restrictLegionella longbeachaereplication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes toL. pneumophilaclearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.IMPORTANCELegionellaspp. are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controlsLegionellainfection due in part to rapid and robust production of proinflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances proinflammatory host restriction ofLegionellaby macrophages. These data suggest that LegC4 may augment proinflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.

scholarly journals Toxicity and SidJ-Mediated Suppression of Toxicity Require Distinct Regions in the SidE Family of Legionella pneumophila Effectors

2015 ◽  
Vol 83 (9) ◽  
pp. 3506-3514 ◽  
Author(s):  
James C. Havey ◽  
Craig R. Roy
Keyword(s):  
Central Region ◽  
Legionella Pneumophila ◽  
Genetic Interaction ◽  
Terminal Region ◽  
Effector Proteins ◽  
Host Cells ◽  
Toxic Activity ◽  
Content Type ◽  
Amino Terminal ◽  
Low Levels

Intracellular bacteria use a variety of strategies to evade degradation and create a replicative niche.Legionella pneumophilais an intravacuolar pathogen that establishes a replicative niche through the secretion of more than 300 effector proteins. The function of most effectors remains to be determined. Toxicity in yeast has been used to identify functional domains and elucidate the biochemical function of effectors. A library ofL. pneumophilaeffectors was screened using an expression plasmid that produces low levels of each protein. This screen identified the effector SdeA as a protein that confers a strong toxic phenotype that inhibits yeast replication. The toxicity of SdeA was suppressed in cells producing the effector SidJ. The effector SdeA is a member of the SidE family ofL. pneumophilaeffector proteins. All SidE orthologs encoded by the Philadelphia isolate ofLegionella pneumophilawere toxic to yeast, and SidJ suppressed the toxicity of each. We identified a conserved central region in the SidE proteins that was sufficient to mediate yeast toxicity. Surprisingly, SidJ did not suppress toxicity when this central region was produced in yeast. We determined that the amino-terminal region of SidE was essential for SidJ-mediated suppression of toxicity. Thus, there is a genetic interaction that links the activity of SidJ and the amino-terminal region of SidE, which is required to modulate the toxic activity displayed by the central region of the SidE protein. This suggests a complex mechanism by which theL. pneumophilaeffector SidJ modulates the function of the SidE proteins after translocation into host cells.

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