scholarly journals Effector-mediated subversion of proteasome activator (PA)28αβ enhances lysosomal targeting of Legionella pneumophila within cytokine-activated macrophages

2021 ◽  
Author(s):  
Tshegofatso Ngwaga ◽  
Deepika Chauhan ◽  
Abigail G Salberg ◽  
Stephanie R Shames
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Activated Macrophages ◽  
Proteasome Activator ◽  
Proteotoxic Stress ◽  
Effector Triggered Immunity ◽  
A Subunit ◽  
Legionnaires Disease ◽  
Proteasome Regulators ◽  
Pathogen Clearance

Legionella pneumophila causes Legionnaires' Disease via replication within host macrophages using an arsenal of hundreds of translocated virulence factors termed effector proteins. Effectors are critical for intracellular replication but can also enhance pathogen clearance in mammalian hosts via effector-triggered immunity. The effector LegC4 confers a fitness disadvantage on L. pneumophila within mouse models of Legionnaires' Disease and uniquely potentiates the antimicrobial activity of macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-γ. Here, we investigated the mechanism of LegC4 function. We found that LegC4 binds proteasome activator (PA)28α, a subunit of the PA28αβ (11S) proteasome regulator, and that the LegC4 restriction phenotype is abolished within PA28αβ-deficient macrophages. PA28αβ facilitates ubiquitin-independent proteasomal degradation of oxidant-damaged proteins. Impaired proteasome activity results in compensatory upregulation of lysosomal degradation pathways to relieve oxidative proteotoxic stress. We found that LegC4 impairs the resolution of oxidative proteotoxic stress and enhances phagolysosomal fusion with the Legionella-containing vacuole. PA28αβ has been traditionally associated with antigen presentation and adaptive immunity; however, our data support a model whereby suppression of PA28αβ by LegC4 impairs resolution of oxidative proteotoxic stress, which culminates in the lysosomal killing of L. pneumophila within activated macrophages. This work provides a solid foundation on which to evaluate induced proteasome regulators as mediators of cell-autonomous immunity.

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 Quorum sensing modulates the formation of virulent Legionella persisters within infected cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Nicolas Personnic ◽  
Bianca Striednig ◽  
Emmanuelle Lezan ◽  
Christian Manske ◽  
Amanda Welin ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Legionella Pneumophila ◽  
Interferon Γ ◽  
Activated Macrophages ◽  
Sensing System ◽  
High Virulence ◽  
Quorum Sensing System ◽  
Infected Cells ◽  
Legionnaires Disease ◽  
Active Display

AbstractThe facultative intracellular bacterium Legionella pneumophila replicates in environmental amoebae and in lung macrophages, and causes Legionnaires’ disease. Here we show that L. pneumophila reversibly forms replicating and nonreplicating subpopulations of similar size within amoebae. The nonreplicating bacteria are viable and metabolically active, display increased antibiotic tolerance and a distinct proteome, and show high virulence as well as the capacity to form a degradation-resistant compartment. Upon infection of naïve or interferon-γ-activated macrophages, the nonreplicating subpopulation comprises ca. 10% or 50%, respectively, of the total intracellular bacteria; hence, the nonreplicating subpopulation is of similar size in amoebae and activated macrophages. The numbers of nonreplicating bacteria within amoebae are reduced in the absence of the autoinducer synthase LqsA or other components of the Lqs quorum-sensing system. Our results indicate that virulent, antibiotic-tolerant subpopulations of L. pneumophila are formed during infection of evolutionarily distant phagocytes, in a process controlled by the Lqs system.

scholarly journals The Legionella pneumophila Metaeffector Lpg2505 (MesI) Regulates SidI-Mediated Translation Inhibition and Novel Glycosyl Hydrolase Activity

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Ashley M. Joseph ◽  
Adrienne E. Pohl ◽  
Theodore J. Ball ◽  
Troy G. Abram ◽  
David K. Johnson ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Glycosyl Hydrolase ◽  
Protein Translation ◽  
Effector Proteins ◽  
Hydrolase Activity ◽  
Intracellular Replication ◽  
Content Type ◽  
Translation Inhibition ◽  
Legionnaires Disease ◽  
The Impact

ABSTRACT Legionella pneumophila, the etiological agent of Legionnaires’ disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

scholarly journals Cryo-EM reveals new species-specific proteins and symmetry elements in the Legionella pneumophila Dot/Icm T4SS

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Michael J Sheedlo ◽  
Clarissa L Durie ◽  
Jeong Min Chung ◽  
Louise Chang ◽  
Jacquelyn Roberts ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Genetic Locus ◽  
Opportunistic Pathogen ◽  
Effector Proteins ◽  
Type Iv ◽  
Specific Proteins ◽  
Legionnaires Disease ◽  
Multiple Conformations ◽  
Membrane Spanning ◽  
Species Specific

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' Disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.

scholarly journals Structural studies of Legionella effectors

2014 ◽  
Vol 70 (a1) ◽  
pp. C583-C583
Author(s):  
Kathy Wong ◽  
Yinglu Zhang ◽  
Guennadi Kozlov ◽  
Kalle Gehring
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Homology Search ◽  
Structural Homology ◽  
Nucleotide Synthesis ◽  
Type Iv ◽  
Conserved Domain ◽  
Middle Domain ◽  
Legionnaires Disease

Legionella pneumophila is a gram-negative bacterium that causes Legionnaires' disease. It uses a Dot/Icm type IV secretion system to inject effector proteins into the host cell to manipulate host processes. Currently, about 300 Icm/Dot dependent effectors of L.pneumophila have been identified. Lpg1496 is an effector protein, which contains a conserved domain from the SidE family. To date, the middle domain and the conserved SidE domain have been crystallized and the structure solved at a resolution of 1.15Å and 2.3Å, respectively. A structural homology search using the middle domain suggested a similarity to phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase, an ATPase involved in purine nucleotide synthesis. We performed 1H–15N HSQC NMR titrations to show that this domain binds ATP, ADP and AMP, with the highest binding affinity for ADP. A structural homology search using the SidE domain showed a similarity to cyclic nucleotide phosphodiesterases. To further elucidate the function of lpg1496, other fragments have been cloned, expressed, and subjected to crystallization trials. Currently, we have successfully crystallized the N-terminal domain, with crystals diffracting to <2.0Å. Obtaining the crystal structure of lpg1496 and revealing its function will not only lead to a better understanding of the virulence of L. pneumophila, but also contribute to the development of novel therapeutic treatments of Legionnaires' disease.

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 Structural and Functional Characterization of Legionella pneumophila Effector MavL

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1802
Author(s):  
Kevin Voth ◽  
Shivani Pasricha ◽  
Ivy Yeuk Wah Chung ◽  
Rachelia R. Wibawa ◽  
Engku Nuraishah Huda E. Zainudin ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Functional Characterization ◽  
Effector Proteins ◽  
Type Iv ◽  
Ubiquitin Conjugating Enzyme ◽  
Legionnaires Disease ◽  
Organelle Trafficking ◽  
Binding Cleft ◽  
Adp Ribosyltransferase

Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires' disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose “reader” proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitin-conjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila.

scholarly journals The Legionella pneumophila metaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity

2019 ◽  
Author(s):  
Ashley M. Joseph ◽  
Adrienne E. Pohl ◽  
Theodore J. Ball ◽  
Troy G. Abram ◽  
David K. Johnson ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Protein Translation ◽  
Effector Proteins ◽  
Intracellular Replication ◽  
Unknown Mechanism ◽  
Translation Inhibition ◽  
Legionnaires Disease ◽  
The Impact ◽  
Insight Into ◽  
Cytotoxic Effector

AbstractLegionella pneumophila, the etiological agent of Legionnaires Disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and SusF is not mutually exclusive and these proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyltransferase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505, SusF (suppressor of SidI function). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

scholarly journals The Dot/Icm-translocated effector LegC4 potentiates cytokine-mediated restriction of Legionella within accidental hosts.

2018 ◽  
Author(s):  
Tshegofatso Ngwaga ◽  
Alex J Hydock ◽  
Sandhya Ganesan ◽  
Stephanie Rochelle Shames
Keyword(s):  
Legionella Pneumophila ◽  
Defense Mechanisms ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Innate Immune ◽  
Host Cells ◽  
Type Iv ◽  
Legionnaires Disease ◽  
Ifn Γ ◽  
Necrosis Factor

Legionella pneumophila is ubiquitous in freshwater environments where it replicates within unicellular protozoa. However, L. pneumophila is 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. pneumophila utilizes 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 of L. pneumophila. We found previously that the effector LegC4 is important for L. pneumophila replication 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 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon (IFN)-γ. Specifically, TNF-mediated signaling was required for LegC4-mediated attenuation of L. pneumophila replication within macrophages. In addition, expression of legC4 was sufficient to restrict L. longbeachae replication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.

scholarly journals Direct targeting of membrane fusion by SNARE mimicry: Convergent evolution of Legionella effectors

2016 ◽  
Vol 113 (31) ◽  
pp. 8807-8812 ◽  
Author(s):  
Xingqi Shi ◽  
Partho Halder ◽  
Halenur Yavuz ◽  
Reinhard Jahn ◽  
Howard A. Shuman
Keyword(s):  
Legionella Pneumophila ◽  
Lethal Factor ◽  
Effector Proteins ◽  
Host Cells ◽  
Snare Proteins ◽  
Stable Complex ◽  
Hybrid Complex ◽  
Infected Cells ◽  
Legionnaires Disease ◽  
Direct Targeting

Legionella pneumophila, the Gram-negative pathogen causing Legionnaires’ disease, infects host cells by hijacking endocytic pathways and forming a Legionella-containing vacuole (LCV) in which the bacteria replicate. To promote LCV expansion and prevent lysosomal targeting, effector proteins are translocated into the host cell where they alter membrane traffic. Here we show that three of these effectors [LegC2 (Legionella eukaryotic-like gene C2)/YlfB (yeast lethal factor B), LegC3, and LegC7/YlfA] functionally mimic glutamine (Q)-SNARE proteins. In infected cells, the three proteins selectively form complexes with the endosomal arginine (R)-SNARE vesicle-associated membrane protein 4 (VAMP4). When reconstituted in proteoliposomes, these proteins avidly fuse with liposomes containing VAMP4, resulting in a stable complex with properties resembling canonical SNARE complexes. Intriguingly, however, the LegC/SNARE hybrid complex cannot be disassembled by N-ethylmaleimide-sensitive factor. We conclude that LegCs use SNARE mimicry to divert VAMP4-containing vesicles for fusion with the LCV, thus promoting its expansion. In addition, the LegC/VAMP4 complex avoids the host’s disassembly machinery, thus effectively trapping VAMP4 in an inactive state.

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