Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole

SummaryLegionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonize vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilize the replication vacuole, EEA1 and Rab11FIP1 showed enhanced colocalization with the vacuole surrounding the sdhA mutant compared with the WT vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant colocalization. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilize vacuole integrity during infection.

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Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

Infection and Immunity ◽

10.1128/iai.72.10.5983-5992.2004 ◽

2004 ◽

Vol 72 (10) ◽

pp. 5983-5992 ◽

Cited By ~ 56

Author(s):

Jessica A. Sexton ◽

Jennifer L. Miller ◽

Aki Yoneda ◽

Thomas E. Kehl-Fie ◽

Joseph P. Vogel

Keyword(s):

Cell Surface ◽

Legionella Pneumophila ◽

Bacterial Species ◽

Wide Distribution ◽

Host Cells ◽

Growth Defect ◽

Intracellular Growth ◽

Homologous Proteins ◽

Type Iv ◽

Cell Surface Structure

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

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Exploitation of Phosphoinositides by the Intracellular Pathogen, Legionella pneumophila

Pathogenic Bacteria ◽

10.5772/intechopen.89158 ◽

2020 ◽

Author(s):

Colleen M. Pike ◽

Rebecca R. Noll ◽

M. Ramona Neunuebel

Keyword(s):

Membrane Trafficking ◽

Legionella Pneumophila ◽

Pathogenic Bacteria ◽

Severe Pneumonia ◽

Bacterial Proteins ◽

Specific Host ◽

Spatiotemporal Regulation ◽

Host Membrane ◽

Membrane Compartments ◽

Legionnaires Disease

Manipulation of host phosphoinositide lipids has emerged as a key survival strategy utilized by pathogenic bacteria to establish and maintain a replication-permissive compartment within eukaryotic host cells. The human pathogen, Legionella pneumophila, infects and proliferates within the lung’s innate immune cells causing severe pneumonia termed Legionnaires’ disease. This pathogen has evolved strategies to manipulate specific host components to construct its intracellular niche termed the Legionella-containing vacuole (LCV). Paramount to LCV biogenesis and maintenance is the spatiotemporal regulation of phosphoinositides, important eukaryotic lipids involved in cell signaling and membrane trafficking. Through a specialized secretion system, L. pneumophila translocates multiple proteins that target phosphoinositides in order to escape endolysosomal degradation. By specifically binding phosphoinositides, these proteins can anchor to the cytosolic surface of the LCV or onto specific host membrane compartments, to ultimately stimulate or inhibit encounters with host organelles. Here, we describe the bacterial proteins involved in binding and/or altering host phosphoinositide dynamics to support intracellular survival of L. pneumophila.

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Legionella Subvert the Functions of Rab1 and Sec22b to Create a Replicative Organelle

Journal of Experimental Medicine ◽

10.1084/jem.20031706 ◽

2004 ◽

Vol 199 (9) ◽

pp. 1201-1211 ◽

Cited By ~ 212

Author(s):

Jonathan C. Kagan ◽

Mary-Pat Stein ◽

Marc Pypaert ◽

Craig R. Roy

Keyword(s):

Endoplasmic Reticulum ◽

Legionella Pneumophila ◽

Molecular Mechanisms ◽

Bacterial Pathogen ◽

Host Cells ◽

Intracellular Growth ◽

Host Proteins ◽

Morphological Studies ◽

Bacterial Replication ◽

Inhibition Studies

Legionella pneumophila is a bacterial pathogen that infects eukaryotic host cells and replicates inside a specialized organelle that is morphologically similar to the endoplasmic reticulum (ER). To better understand the molecular mechanisms governing transport of the Legionella-containing vacuole (LCV), we have identified host proteins that participate in the conversion of the LCV into a replicative organelle. Our data show that Rab1 is recruited to the LCV within minutes of uptake. Rab1 recruitment to the LCV precedes remodeling of this compartment by ER-derived vesicles. Genetic inhibition studies demonstrate that Rab1 is important for the recruitment of ER-derived vesicles to the LCV and that inhibiting Rab1 function abrogates intracellular growth of Legionella. Morphological studies indicate that the Sec22b protein is located on ER-derived vesicles recruited to the LCV and that Sec22b is delivered to the LCV membrane. Sec22b function was found to be important for biogenesis of the specialized organelle that supports Legionella replication. These studies demonstrate that Legionella has the ability to subvert Rab1 and Sec22b function to facilitate the transport and fusion of ER-derived vesicles with the LCV, resulting in the formation of a specialized organelle that can support bacterial replication.

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The Legionella pneumophila PilT Homologue DotB Exhibits ATPase Activity That Is Critical for Intracellular Growth

Journal of Bacteriology ◽

10.1128/jb.186.6.1658-1666.2004 ◽

2004 ◽

Vol 186 (6) ◽

pp. 1658-1666 ◽

Cited By ~ 80

Author(s):

Jessica A. Sexton ◽

Jerome S. Pinkner ◽

Robyn Roth ◽

John E. Heuser ◽

Scott J. Hultgren ◽

...

Keyword(s):

Atpase Activity ◽

Legionella Pneumophila ◽

Specific Activity ◽

Host Cells ◽

Mutant Protein ◽

Wild Type ◽

Intracellular Growth ◽

Type Iv ◽

Molecular Behavior ◽

Membrane Spanning

ABSTRACT The ability of Legionella pneumophila to grow and cause disease in the host is completely dependent on a type IV secretion system known as the Dot/Icm complex. This membrane-spanning apparatus translocates effector molecules into host cells in a process that is poorly understood but that is known to require the putative ATPase DotB. One possible role for DotB is suggested by its similarity to the PilT family of proteins, which mediate pilus retraction. To better understand the molecular behavior of DotB, we have purified the protein and shown that it forms stable homohexameric rings and hydrolyzes ATP with a specific activity of 6.4 nmol of ATP/min/mg of protein. ATPase activity is critical to the function of DotB, as alteration of the conserved Walker box lysine residue resulted in a mutant protein, DotB K162Q, which failed to bind or hydrolyze ATP and which could not complement a ΔdotB strain for intracellular growth in macrophages. Consistent with the ability of DotB to interact with itself, the dotBK162Q allele exhibited transdominance over wild-type dotB, providing the first example of such a mutation in L. pneumophila. Finally, the DotB K162Q mutant protein had a significantly enhanced membrane localization in L. pneumophila compared to wild-type DotB, suggesting a relationship between nucleotide binding and membrane association. These results are consistent with a model in which DotB cycles between the cytoplasm and the Dot/Icm complex at the membrane, where it hydrolyzes nucleotides to provide energy to the complex.

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Macrophage-Induced Genes of Legionella pneumophila: Protection from Reactive Intermediates and Solute Imbalance during Intracellular Growth

Infection and Immunity ◽

10.1128/iai.70.7.3637-3648.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3637-3648 ◽

Cited By ~ 47

Author(s):

Susannah Rankin ◽

Zhiru Li ◽

Ralph R. Isberg

Keyword(s):

Legionella Pneumophila ◽

Response Regulator ◽

Protein A ◽

Host Cells ◽

Intracellular Growth ◽

Selection Strategies ◽

Promoter Probe ◽

Genes Encoding ◽

Chromosomal Loci ◽

Reducing Potential

ABSTRACT A promoter-probe strategy was devised to identify genes specifically expressed by Legionella pneumophila during growth within the macrophage. Random fragments from the L. pneumophila chromosome were inserted upstream of a promoterless phage T4 td gene, and fragments that led to complementation of thymine auxotrophy during intracellular growth of the bacterium were identified. Two different selection strategies were employed to eliminate promoters that were also active during extracellular growth of the bacterium. Some of these genes were identified independently by using both of the selection strategies. The factors identified include orthologs of efflux-mediated resistance determinants and transporters, a transporter involved in protection from osmotic stress, a stress response GTP-binding protein, a response regulator, a sensor kinase, and two systems that increase the reducing potential of the bacterium, one of which encodes the L. pneumophila ortholog of ahpC. Five of the clones analyzed here were fusions to promoters that were closely linked to genes encoding three-component chemiosmotic efflux pumps that export heavy metals or toxic organic compounds. Analysis of ahpC gene expression indicates that levels increased at least sevenfold during intracellular growth of the bacterium. Inactivation of several of the genes at their chromosomal loci had no effect on the intracellular growth rate of L. pneumophila in cultured macrophages. This suggests that a number of genes with increased expression during intracellular growth may be part of redundant systems that allow survival and growth under the conditions encountered within host cells.

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ArgR-Regulated Genes Are Derepressed in the Legionella-Containing Vacuole

Journal of Bacteriology ◽

10.1128/jb.00465-10 ◽

2010 ◽

Vol 192 (17) ◽

pp. 4504-4516 ◽

Cited By ~ 29

Author(s):

Galadriel Hovel-Miner ◽

Sebastien P. Faucher ◽

Xavier Charpentier ◽

Howard A. Shuman

Keyword(s):

Legionella Pneumophila ◽

Acanthamoeba Castellanii ◽

Transcriptional Response ◽

Global Gene Expression ◽

Host Cells ◽

Intracellular Growth ◽

Arginine Repressor ◽

Legionnaires Disease ◽

Global Gene Expression Analysis ◽

Eukaryotic Organisms

ABSTRACT Legionella pneumophila is an intracellular pathogen that infects protozoa in aquatic environments and when inhaled by susceptible human hosts replicates in alveolar macrophages and can result in the often fatal pneumonia called Legionnaires' disease. The ability of L. pneumophila to replicate within host cells requires the establishment of a specialized compartment that evades normal phagolysosome fusion called the Legionella-containing vacuole (LCV). Elucidation of the biochemical composition of the LCV and the identification of the regulatory signals sensed during intracellular replication are inherently challenging. l-Arginine is a critical nutrient in the metabolism of both prokaryotic and eukaryotic organisms. We showed that the L. pneumophila arginine repressor homolog, ArgR, is required for maximal intracellular growth in the unicellular host Acanthamoeba castellanii. In this study, we present evidence that the concentration of l-arginine in the LCV is sensed by ArgR to produce an intracellular transcriptional response. We characterized the L. pneumophila ArgR regulon by global gene expression analysis, identified genes highly affected by ArgR, showed that ArgR repression is dependent upon the presence of l-arginine, and demonstrated that ArgR-regulated genes are derepressed during intracellular growth. Additional targets of ArgR that may account for the argR mutant's intracellular multiplication defect are discussed. These results suggest that l-arginine availability functions as a regulatory signal during Legionella intracellular growth.

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Evolution of the Arsenal ofLegionella pneumophilaEffectors To Modulate Protist Hosts

mBio ◽

10.1128/mbio.01313-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 13

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.

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

mBio ◽

10.1128/mbio.01942-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 5

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.

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Legionella pneumophila Invasion of MRC-5 Cells Induces Tyrosine Protein Phosphorylation

Infection and Immunity ◽

10.1128/iai.67.9.4490-4498.1999 ◽

1999 ◽

Vol 67 (9) ◽

pp. 4490-4498 ◽

Cited By ~ 5

Author(s):

Milorad Susa ◽

Reinhard Marre

Keyword(s):

Protein Phosphorylation ◽

Legionella Pneumophila ◽

Structural Changes ◽

Kinase Inhibitor ◽

Sodium Dodecyl ◽

Host Cells ◽

Lung Fibroblasts ◽

Intracellular Growth ◽

Tyrosine Residues ◽

Cytoskeletal Filament

ABSTRACT After uptake and intracellular multiplication of Legionella pneumophila in MRC-5 lung fibroblasts, important cytoskeletal filament structures, like actin, tubulin, or vimentin, and a cell membrane-associated fibronectin were rearranged during early infection, resulting in a loss of cell adhesion and collapse of the cytoskeleton. Dysregulation of the cellular phosphorylation and dephosphorylation cascade may contribute to the observed changes and may support intracellular survival and multiplication of L. pneumophila. We therefore studied expression of phosphoproteins during intracellular growth of L. pneumophila. By using an anti-tyrosine phosphoprotein antibody we showed that proteins phosphorylated on tyrosine residues accumulated progressively during late infection exclusively around or in phagosomes filled with bacteria. In contrast, expression of serine/threonine phosphoproteins did not change. To discern the origin of phosphorylated proteins, the host cells were treated with cycloheximide, an inhibitor of eukaryotic protein synthesis. The newly synthesized proteins were labeled metabolically with [35S]methionine-cysteine and immunoprecipitated with a phosphotyrosine-specific antibody. Sodium dodecyl sulfate gel electrophoresis gave evidence for synthesis of at least three protein clusters (160 to 200, 35 to 60, and 19 to 28 kDa) of Legionella origin that were phosphorylated on tyrosine residues 24 h after infection. Treatment of infected host cells with genistein, a tyrosine kinase inhibitor, revealed that tyrosine protein phosphorylation was not important for bacterial uptake but contributed to intracellular growth of L. pneumophila. Bacterial tyrosine phosphoproteins and the observed intracellular structural changes may be important to understanding the process involved in intracellular growth of L. pneumophila.

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Legionella pneumophila LegC7 effector protein drives aberrant ER:endosome fusion in yeast

10.1101/2020.11.20.391441 ◽

2020 ◽

Author(s):

Nathan K. Glueck ◽

Kevin M. O’Brien ◽

Vincent J. Starai

Keyword(s):

Membrane Trafficking ◽

Legionella Pneumophila ◽

Molecular Mechanisms ◽

Bacterial Pathogen ◽

Severe Form ◽

Effector Proteins ◽

Effector Protein ◽

Host Membrane ◽

Membrane Tethering

AbstractLegionella pneumophila is a facultative intracellular bacterial pathogen, causing the severe form of pneumonia known as Legionnaires’ disease. Legionella actively alters host organelle trafficking through the activities of ‘effector’ proteins secreted via a TypeIVB secretion system, in order to construct the bacteria-laden Legionella-containing vacuole (LCV) and prevent lysosomal degradation. The LCV is derived from membrane derived from host ER, secretory vesicles, and phagosomes, although the precise molecular mechanisms that drive its synthesis remain poorly understood. In an effort to characterize the in vivo activity of the LegC7/YlfA SNARE-like effector protein from Legionella in the context of eukaryotic membrane trafficking in yeast, we find that LegC7 interacts with the Emp46p/Emp47p ER-to-Golgi glycoprotein cargo adapter complex, alters ER morphology, and induces aberrant ER:endosome fusion, as measured by visualization of ER cargo degradation, reconstitution of split-GFP proteins, and enhanced oxidation of the ER lumen. LegC7-dependent toxicity, disruption of ER morphology, and ER:endosome fusion events were dependent upon endosomal VPS class C tethering complexes and the endosomal t-SNARE, Pep12p. This work establishes a model in which LegC7 functions to recruit host ER material to the bacterial phagosome during infection by inducing membrane fusion, potentially through interaction with host membrane tethering complexes and/or cargo adapters.

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