Characterization of the icmH and icmF Genes Required for Legionella pneumophila Intracellular Growth, Genes That Are Present in Many Bacteria Associated with Eukaryotic Cells

ABSTRACT Legionella pneumophila, the causative agent of Legionnaires' disease, replicates intracellularly within a specialized phagosome of mammalian and protozoan host cells, and the Icm/Dot type IV secretion system has been shown to be essential for this process. Unlike all the other known Icm/Dot proteins, the IcmF protein, which was described before, and the IcmH protein, which is characterized here, have homologous proteins in many bacteria (such as Yersinia pestis, Salmonella enterica, Rhizobium leguminosarum, and Vibrio cholerae), all of which associate with eukaryotic cells. Here, we have characterized the L. pneumophila icmH and icmF genes and found that both genes are present in 16 different Legionella species examined. The icmH and icmF genes were found to be absolutely required for intracellular multiplication in Acanthamoeba castellanii and partially required for intracellular growth in HL-60-derived human macrophages, for immediate cytotoxicity, and for salt sensitivity. Mutagenesis of the predicted ATP/GTP binding site of IcmF revealed that the site is partially required for intracellular growth in A. castellanii. Analysis of the regulatory region of the icmH and icmF genes, which were found to be cotranscribed, revealed that it contains at least two regulatory elements. In addition, an icmH::lacZ fusion was shown to be activated during stationary phase in a LetA- and RelA-dependent manner. Our results indicate that although the icmH and icmF genes probably have a different evolutionary origin than the rest of the icm/dot genes, they are part of the icm/dot system and are required for L. pneumophila pathogenesis.

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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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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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Intracellular Growth in Acanthamoeba castellanii Affects Monocyte Entry Mechanisms and Enhances Virulence of Legionella pneumophila

Infection and Immunity ◽

10.1128/iai.67.9.4427-4434.1999 ◽

1999 ◽

Vol 67 (9) ◽

pp. 4427-4434 ◽

Cited By ~ 162

Author(s):

Jeffrey D. Cirillo ◽

Suat L. G. Cirillo ◽

Ling Yan ◽

Luiz E. Bermudez ◽

Stanley Falkow ◽

...

Keyword(s):

Legionella Pneumophila ◽

Acanthamoeba Castellanii ◽

Host Cells ◽

Intracellular Pathogen ◽

Activated Macrophages ◽

Standard Laboratory ◽

Intracellular Growth ◽

Growth Environment ◽

Pathogen Entry ◽

First Time

ABSTRACT Since Legionella pneumophila is an intracellular pathogen, entry into and replication within host cells are thought to be critical to its ability to cause disease. L. pneumophilagrown in one of its environmental hosts, Acanthamoeba castellanii, is phenotypically different from L. pneumophila grown on standard laboratory medium (BCYE agar). Although amoeba-grown L. pneumophila displays enhanced entry into monocytes compared to BCYE-grown bacteria, the mechanisms of entry used and the effects on virulence have not been examined. To explore whether amoeba-grown L. pneumophila differs from BCYE-grown L. pneumophila in these characteristics, we examined entry into monocytes, replication in activated macrophages, and virulence in mice. Entry of amoeba-grown L. pneumophilainto monocytes occurred more frequently by coiling phagocytosis, was less affected by complement opsonization, and was less sensitive to microtubule and microfilament inhibitors than was entry of BCYE-grown bacteria. In addition, amoeba-grown L. pneumophila displays increased replication in monocytes and is more virulent in A/J, C57BL/6 Beige, and C57BL/6 mice. These data demonstrate for the first time that the intra-amoebal growth environment affects the entry mechanisms and virulence of L. pneumophila.

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Additive Effect on Intracellular Growth by Legionella pneumophila Icm/Dot Proteins Containing a Lipobox Motif

Infection and Immunity ◽

10.1128/iai.73.11.7578-7587.2005 ◽

2005 ◽

Vol 73 (11) ◽

pp. 7578-7587 ◽

Cited By ~ 19

Author(s):

Gal Yerushalmi ◽

Tal Zusman ◽

Gil Segal

Keyword(s):

Legionella Pneumophila ◽

Acanthamoeba Castellanii ◽

Cysteine Residue ◽

Secretion System ◽

Host Cells ◽

Intracellular Growth ◽

Type Iv ◽

Essential Components ◽

Legionnaires Disease ◽

Type Ivb Secretion

ABSTRACT Legionella pneumophila, the causative agent of Legionnaires' disease, utilizes a type IVB secretion system to subvert its host cells and grow intracellularly. This type IV secretion system is composed of 25 icm (or dot) genes that probably constitute parts of a secretion complex as well as more than 30 proteins that are translocated via this system into the host cells. Three of the Icm/Dot proteins (DotD, DotC, and IcmN) contain a lipobox motif at their N terminals and are predicted to be lipoproteins. Two of these lipoproteins (DotD and DotC) were found to be essential for intracellular growth in both HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii, while the third lipoprotein (IcmN) was found to be partially required for intracellular growth only in A. castellanii. Mutation analysis of the lipobox cysteine residue, which was shown previously to be indispensable for the lipobox function, indicated that both DotC and DotD are partially functional without this conserved residue. Cysteine mutations in both DotC and DotD or in DotC together with an icmN deletion or in DotD together with an icmN deletion were found to be additive, indicating that each of these lipoproteins performs its function independently from the others. Analysis of the transcriptional regulation of both the dotDC operon and the icmN gene revealed that both had higher levels of expression at stationary phase which were partially dependent on the LetA regulator. Our results indicate that the lipoproteins of the L. pneumophila icm (or dot) system are essential components of the secretion system and that they perform their functions independently.

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Subinhibitory Concentrations of Antimicrobial Agents Reduce the Uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 Cells by Altering the Expression of Virulence-Associated Antigens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.11.2870 ◽

1998 ◽

Vol 42 (11) ◽

pp. 2870-2876 ◽

Cited By ~ 8

Author(s):

P. Christian Lück ◽

Jürgen W. Schmitt ◽

Arne Hengerer ◽

Jürgen H. Helbig

Keyword(s):

Monoclonal Antibodies ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Legionella Pneumophila ◽

Antimicrobial Agents ◽

Acanthamoeba Castellanii ◽

Host Cells ◽

Uncharacterized Protein ◽

Subinhibitory Concentrations

ABSTRACT We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reducedLegionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionellaantigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected.

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Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector of Legionella pneumophila

Journal of Experimental Medicine ◽

10.1084/jem.20100771 ◽

2010 ◽

Vol 207 (8) ◽

pp. 1713-1726 ◽

Cited By ~ 97

Author(s):

Christopher T.D. Price ◽

Tasneem Al-Quadan ◽

Marina Santic ◽

Snake C. Jones ◽

Yousef Abu Kwaik

Keyword(s):

Legionella Pneumophila ◽

Biological Function ◽

Host Cells ◽

Dependent Manner ◽

Protein Farnesyltransferase ◽

Type Iv ◽

Eukaryotic Proteins ◽

Bacterial Effectors

Farnesylation involves covalent linkage of eukaryotic proteins to a lipid moiety to anchor them into membranes, which is essential for the biological function of Ras and other proteins. A large cadre of bacterial effectors is injected into host cells by intravacuolar pathogens through elaborate type III–VII translocation machineries, and many of these effectors are incorporated into the pathogen-containing vacuolar membrane by unknown mechanisms. The Dot/Icm type IV secretion system of Legionella pneumophila injects into host cells the F-box effector Ankyrin B (AnkB), which functions as platforms for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) to enable intravacuolar proliferation in macrophages and amoeba. We show that farnesylation of AnkB is indispensable for its anchoring to the cytosolic face of the LCV membrane, for its biological function within macrophages and Dictyostelium discoideum, and for intrapulmonary proliferation in mice. Remarkably, the protein farnesyltransferase, RCE-1 (Ras-converting enzyme-1), and isoprenyl cysteine carboxyl methyltransferase host farnesylation enzymes are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB. In conclusion, this study shows novel localized recruitment of the host farnesylation machinery and its anchoring of an F-box effector to the LCV membrane, and this is essential for biological function in vitro and in vivo.

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Inherent hepatocytic heterogeneity determines expression and retention of edited F9 alleles post-AAV/CRISPR infusion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2110887118 ◽

2021 ◽

Vol 118 (42) ◽

pp. e2110887118

Author(s):

Qiang Wang ◽

Lin Zhang ◽

Guo-Wei Zhang ◽

Jian-Hua Mao ◽

Xiao-Dong Xi ◽

...

Keyword(s):

Viral Vectors ◽

Gene Editing ◽

Regulatory Region ◽

Factor Ix ◽

Host Cells ◽

Clotting Factor ◽

Dependent Manner ◽

Coding Region ◽

Therapeutic Benefits

Infusing CRISPR/donor-loaded adeno-associated viral vectors (AAV/CRISPR) could enable in vivo hepatic gene editing to remedy hemophilia B (HB) with inherited deficiency of clotting factor IX (FIX). Yet, current regimens focus on correcting HB with simple mutations in the coding region of the F9, overlooking those carrying complicated mutations involving the regulatory region. Moreover, a possible adverse effect of treatment-related inflammation remains unaddressed. Here we report that a single DNA cutting-mediated long-range replacement restored the FIX-encoding function of a mutant F9 (mF9) carrying both regulatory and coding defects in a severe mouse HB model, wherein incorporation of a synthetic Alb enhancer/promoter-mimic (P2) ensured FIX elevation to clinically meaningful levels. Through single-cell RNA sequencing (scRNA-seq) of liver tissues, we revealed that a subclinical hepatic inflammation post-AAV/CRISPR administration regulated the vulnerability of the edited mF9-harboring host cells to cytotoxic T lymphocytes (CTLs) and the P2 activity in a hepatocytic subset–dependent manner via modulating specific sets of liver-enriched transcription factors (LETFs). Collectively, our study establishes an AAV/CRISPR-mediated gene-editing protocol applicable to complicated monogenetic disorders, underscoring the potentiality of improving therapeutic benefits through managing inflammation.

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

10.1101/781849 ◽

2019 ◽

Author(s):

Ila S. Anand ◽

Won Young Choi ◽

Ralph R. Isberg

Keyword(s):

Membrane Trafficking ◽

Legionella Pneumophila ◽

Host Cells ◽

Rab Gtpases ◽

Intracellular Growth ◽

Mutant Proteins ◽

Host Cytoplasm ◽

Downstream Effectors ◽

Cell Death Response ◽

Host Membrane

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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The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

Microbiology ◽

10.1099/mic.0.27563-0 ◽

2005 ◽

Vol 151 (1) ◽

pp. 167-182 ◽

Cited By ~ 36

Author(s):

Urs Albers ◽

Katrin Reus ◽

Howard A. Shuman ◽

Hubert Hilbi

Keyword(s):

Legionella Pneumophila ◽

Lipid A ◽

Sequence Similarity ◽

Acanthamoeba Castellanii ◽

Growth Defect ◽

Wild Type ◽

Intracellular Growth ◽

Plate Test ◽

Raw264.7 Macrophages ◽

Mutant Strains

Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.

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Pseudomonas aeruginosa injects NDK into host cells through a type III secretion system

Microbiology ◽

10.1099/mic.0.078139-0 ◽

2014 ◽

Vol 160 (7) ◽

pp. 1417-1426 ◽

Cited By ~ 25

Author(s):

Dennis Neeld ◽

Yongxin Jin ◽

Candace Bichsel ◽

Jinghua Jia ◽

Jianhui Guo ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Eukaryotic Cells ◽

Mammalian Host ◽

Type I ◽

Dependent Manner ◽

Type Iii

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen possessing a type III secretion system (T3SS) which injects toxic effector proteins into mammalian host cells. In previous studies, P. aeruginosa strains lacking all of the known type III effectors were shown to cause cytotoxicity upon prolonged infection time. In this study, we report the identification of a new cytotoxin, nucleoside diphosphate kinase (NDK), which is injected into eukaryotic cells in a T3SS-dependent manner. Injection of NDK is inhibited by the presence of previously known effectors of the T3SS, with an effectorless strain injecting the highest amount, suggesting active competition with the known T3SS effectors. NDK is shown to cause a cytotoxic response when expressed in eukaryotic cells, and P. aeruginosa strains harbouring NDK also show a greater toxicity than strains lacking it. Interestingly, the cytotoxic effect of intracellular NDK is independent of its kinase activity. In previous studies, NDK was shown to be secreted into culture supernatants via a type I secretion system and cause cytotoxicity in a kinase-dependent manner. Therefore, the current study highlights an alternative route of NDK secretion as well as two different cytotoxic mechanisms of NDK, depending on the extra- or intra-cellular location of the protein.

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