A Legionella pneumophila Effector Protein Encoded in a Region of Genomic Plasticity Binds to Dot/Icm-Modified Vacuoles

Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy.

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Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ

eLife ◽

10.7554/elife.51162 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 12

Author(s):

Alan Sulpizio ◽

Marena E Minelli ◽

Min Wan ◽

Paul D Burrowes ◽

Xiaochun Wu ◽

...

Keyword(s):

Crystal Structure ◽

Mass Spectrometry ◽

Ubiquitin Ligase ◽

Legionella Pneumophila ◽

Kinase Activity ◽

Effector Protein ◽

Dependent Manner ◽

Biochemical Analyses ◽

Human And Mouse

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here, we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analyses demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

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

Infection and Immunity ◽

10.1128/iai.00261-21 ◽

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.

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MultipleLegionella pneumophilaeffector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1708553114 ◽

2017 ◽

Vol 114 (48) ◽

pp. E10446-E10454 ◽

Cited By ~ 31

Author(s):

Stephanie R. Shames ◽

Luying Liu ◽

James C. Havey ◽

Whitman B. Schofield ◽

Andrew L. Goodman ◽

...

Keyword(s):

Legionella Pneumophila ◽

Severe Pneumonia ◽

Effector Proteins ◽

Host Cells ◽

Limiting Factor ◽

Effector Protein ◽

Host Immune System ◽

Loss Of Function ◽

High Throughput Analysis ◽

Single Strain

Legionella pneumophilais the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain ofL. pneumophilaencodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number ofL. pneumophilaeffectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhancedL. pneumophilain the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute toL. pneumophilavirulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.

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Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

10.1101/738567 ◽

2019 ◽

Cited By ~ 1

Author(s):

Alan Sulpizio ◽

Marena E. Minelli ◽

Min Wan ◽

Paul D. Burrowes ◽

Xiaochun Wu ◽

...

Keyword(s):

Crystal Structure ◽

Mass Spectrometry ◽

Ubiquitin Ligase ◽

Legionella Pneumophila ◽

Biochemical Analysis ◽

Kinase Activity ◽

Effector Protein ◽

Dependent Manner ◽

Human And Mouse

AbstractPseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here we report the crystal structure of theLegionella pneumophilaeffector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, Calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analysis demonstrate that SidJ modifies anotherLegionellaeffector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

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LegC3, an Effector Protein from Legionella pneumophila, Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

PLoS ONE ◽

10.1371/journal.pone.0056798 ◽

2013 ◽

Vol 8 (2) ◽

pp. e56798 ◽

Cited By ~ 20

Author(s):

Terry L. Bennett ◽

Shannon M. Kraft ◽

Barbara J. Reaves ◽

Joji Mima ◽

Kevin M. O’Brien ◽

...

Keyword(s):

Legionella Pneumophila ◽

Effector Protein ◽

Yeast Vacuole ◽

Vacuole Fusion

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

mBio ◽

10.1128/mbio.03232-19 ◽

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.

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Legionella pneumophila targets autophagosomes and promotes host autophagy during late infection

10.1101/2021.07.08.451723 ◽

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.

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Detection and quantification of the vacuolar H ATPase using the Legionella effector protein SidK

10.1101/2021.07.29.454369 ◽

2021 ◽

Author(s):

Michelle Maxson ◽

Yazan M. Abbas ◽

Jing Ze Wu ◽

Sergio Grinstein ◽

John L Rubinstein

Keyword(s):

Legionella Pneumophila ◽

High Specificity ◽

Proton Pumping ◽

Eukaryotic Cells ◽

Effector Protein ◽

Receptor Recycling ◽

Phagosome Maturation ◽

A Subunit ◽

Endocytic Vesicles ◽

Vacuolar Atpases

Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar ATPases (V ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive nor specific to test this hypothesis. We introduce a new probe to localize and quantify V ATPases in eukaryotic cells. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1 278, and labeled recombinant SidK1 278 with AlexaFluor-568 to visualize and quantify V ATPases with high specificity in live and fixed cells, respectively. We show that V ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on the subcellular localization of the lysosome.

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Structure of the N-terminal domain of the effector protein LegC3 fromLegionella pneumophila

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s139900471302991x ◽

2014 ◽

Vol 70 (2) ◽

pp. 436-441 ◽

Cited By ~ 7

Author(s):

Deqiang Yao ◽

Maia Cherney ◽

Miroslaw Cygler

Keyword(s):

Legionella Pneumophila ◽

Coiled Coil ◽

Human Cells ◽

Effector Protein ◽

Protein Fold ◽

Sequence Motifs ◽

Transmembrane Helices ◽

Helical Bundle ◽

Terminal Domain ◽

Antiparallel Coiled Coil

Legionella pneumophilasecretes over 300 effectors during the invasion of human cells. The functions of only a small number of them have been identified. LegC3 is one of the identified effectors, which is believed to act by inhibiting vacuolar fusion. It contains two predicted transmembrane helices that divide the protein into a larger N-terminal domain and a smaller C-terminal domain. The function of LegC3 has been shown to be associated primarily with the N-terminal domain, which contains coiled-coil sequence motifs. The structure of the N-terminal domain has been determined and it is shown that it is highly α-helical and contains a helical bundle followed by a long antiparallel coiled-coil. No similar protein fold has been observed in the PDB. A long loop at the tip of the coiled-coil distal from the membrane is disordered and may be important for interaction with an as yet unidentified protein.

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