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

2004 ◽  
Vol 72 (10) ◽  
pp. 5983-5992 ◽  
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.

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

2004 ◽  
Vol 186 (6) ◽  
pp. 1658-1666 ◽  
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.

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

2005 ◽  
Vol 73 (11) ◽  
pp. 7578-7587 ◽  
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.

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

2004 ◽  
Vol 72 (6) ◽  
pp. 3398-3409 ◽  
Author(s):  
Tal Zusman ◽  
Michal Feldman ◽  
Einat Halperin ◽  
Gil Segal
Keyword(s):  
Legionella Pneumophila ◽  
Rhizobium Leguminosarum ◽  
Regulatory Region ◽  
Acanthamoeba Castellanii ◽  
Regulatory Elements ◽  
Host Cells ◽  
Eukaryotic Cells ◽  
Dependent Manner ◽  
Intracellular Growth ◽  
Homologous Proteins

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.

scholarly journals A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Nicole A. Ellis ◽  
Byoungkwan Kim ◽  
Jessica Tung ◽  
Matthias P. Machner
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Polymerase Activity ◽  
Growth Defect ◽  
Great Promise ◽  
Macrophage Infection ◽  
Crispr Interference ◽  
Crispr Array ◽  
Virulence Protein

AbstractCatalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

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

2010 ◽  
Vol 207 (8) ◽  
pp. 1713-1726 ◽  
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.

scholarly journals MavN is aLegionella pneumophilavacuole-associated protein required for efficient iron acquisition during intracellular growth

2015 ◽  
Vol 112 (37) ◽  
pp. E5208-E5217 ◽  
Author(s):  
Dervla T. Isaac ◽  
Rita K. Laguna ◽  
Nicole Valtz ◽  
Ralph R. Isberg
Keyword(s):  
Legionella Pneumophila ◽  
Transmembrane Protein ◽  
Iron Acquisition ◽  
Broth Culture ◽  
Growth Defect ◽  
Secretion Systems ◽  
Intracellular Growth ◽  
Iron Starvation ◽  
Macrophage Infection ◽  
Intracellular Iron

Iron is essential for the growth and virulence of most intravacuolar pathogens. The mechanisms by which microbes bypass host iron restriction to gain access to this metal across the host vacuolar membrane are poorly characterized. In this work, we identify a unique intracellular iron acquisition strategy used byLegionella pneumophila.The bacterial Icm/Dot (intracellular multiplication/defect in organelle trafficking) type IV secretion system targets the bacterial-derived MavN (more regions allowing vacuolar colocalization N) protein to the surface of theLegionella-containing vacuole where this putative transmembrane protein facilitates intravacuolar iron acquisition. TheΔmavNmutant exhibits a transcriptional iron-starvation signature before its growth is arrested during the very early stages of macrophage infection. This intracellular growth defect is rescued only by the addition of excess exogenous iron to the culture medium and not a variety of other metals. Consistent with MavN being a translocated substrate that plays an exclusive role during intracellular growth, the mutant shows no defect for growth in broth culture, even under severe iron-limiting conditions. Putative iron-binding residues within the MavN protein were identified, and point mutations in these residues resulted in defects specific for intracellular growth that are indistinguishable from the ΔmavNmutant. This model of a bacterial protein inserting into host membranes to mediate iron transport provides a paradigm for how intravacuolar pathogens can use virulence-associated secretion systems to manipulate and acquire host iron.

scholarly journals Molecular architecture of theLegionellaDot/Icm type IV secretion system

2018 ◽  
Author(s):  
Debnath Ghosal ◽  
Yi-Wei Chang ◽  
Kwang Cheol Jeong ◽  
Joseph P. Vogel ◽  
Grant J. Jensen
Keyword(s):  
Legionella Pneumophila ◽  
Cell Envelope ◽  
Secretion System ◽  
Host Cells ◽  
Molecular Architecture ◽  
Intact Cells ◽  
Type Iv ◽  
Type Ivb Secretion ◽  
Electron Cryotomography ◽  
First Time

AbstractLegionella pneumophilasurvives and replicates inside host cells by secreting ~300 effectors through the Dot/Icm type IVB secretion system (T4BSS). Understanding this machine’s structure is challenging because of its large number of components (27) and integration into all layers of the cell envelope. Previously we overcame this obstacle by imaging the Dot/Icm T4BSS in its native state within intact cells through electron cryotomography. Here we extend our observations by imaging a stabilized mutant that yielded a higher resolution map. We describe for the first time the presence of a well-ordered central channel that opens up into a windowed large (~32 nm wide) secretion chamber with an unusual 13-fold symmetry. We then dissect the complex by matching proteins to densities for many components, including all those with periplasmic domains. The placement of known and predicted structures of individual proteins into the map reveals the architecture of the T4BSS and provides a roadmap for further investigation of this amazing specialized secretion system.

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

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.

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

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 167-182 ◽  
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.

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.

Sign in / Sign up

Export Citation Format

Share Document