scholarly journals A Unique cis -Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Giulia Oliva ◽  
Tobias Sahr ◽  
Monica Rolando ◽  
Maike Knoth ◽  
Carmen Buchrieser
Keyword(s):  
Life Cycle ◽  
Virulence Factors ◽  
Legionella Pneumophila ◽  
Noncoding Rna ◽  
Acanthamoeba Castellanii ◽  
Severe Pneumonia ◽  
Dependent Manner ◽  
Rnase Iii ◽  
Small Noncoding Rna ◽  
Cycle Dependent

ABSTRACT Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires’ disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis -encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5′ untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti- hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria. IMPORTANCE The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra- and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and adaptation traits is crucial. Yet, the regulatory circuits governing the life cycle of L. pneumophila from replicating to virulent bacteria are only partly uncovered. Here we show that the life cycle-dependent regulation of the RNA chaperone Hfq relies on a small regulatory RNA encoded antisense to the hfq -encoding gene through a base pairing mechanism. Furthermore, Hfq regulates its own expression in an autoregulatory loop. The discovery of this RNA regulatory mechanism in L. pneumophila is an important step forward in the understanding of how the switch from inoffensive, replicating to highly virulent, transmissive L. pneumophila is regulated.

scholarly journals The Epigenetic Repertoire of Daphnia magna Includes Modified Histones

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Nicole F. Robichaud ◽  
Jeanette Sassine ◽  
Margaret J. Beaton ◽  
Vett K. Lloyd
Keyword(s):  
Life Cycle ◽  
Histone H3 ◽  
Dependent Manner ◽  
Nurse Cells ◽  
Important Species ◽  
Clonal Population ◽  
A Cell ◽  
Cycle Dependent

Daphnids are fresh water microcrustaceans, many of which follow a cyclically parthenogenetic life cycle. Daphnia species have been well studied in the context of ecology, toxicology, and evolution, but their epigenetics remain largely unexamined even though sex determination, the production of sexual females and males, and distinct adult morphological phenotypes, are determined epigenetically. Here, we report on the characterization of histone modifications in Daphnia. We show that a number of histone H3 and H4 modifications are present in Daphnia embryos and histone H3 dimethylated at lysine 4 (H3K4me2) is present nonuniformly in the nucleus in a cell cycle-dependent manner. In addition, this histone modification, while present in blastula and gastrula cells as well as the somatic cells of adults, is absent or reduced in oocytes and nurse cells. Thus, the epigenetic repertoire of Daphnia includes modified histones and as these epigenetic forces act on a genetically homogeneous clonal population Daphnia offers an exceptional tool to investigate the mechanism and role of epigenetics in the life cycle and development of an ecologically important species.

scholarly journals The Legionella longbeachae Icm/Dot Substrate SidC Selectively Binds Phosphatidylinositol 4-Phosphate with Nanomolar Affinity and Promotes Pathogen Vacuole-Endoplasmic Reticulum Interactions

2014 ◽  
Vol 82 (10) ◽  
pp. 4021-4033 ◽  
Author(s):  
Stephanie Dolinsky ◽  
Ina Haneburger ◽  
Adam Cichy ◽  
Mandy Hannemann ◽  
Aymelt Itzen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Legionella Pneumophila ◽  
Severe Pneumonia ◽  
Biological Data ◽  
Effector Proteins ◽  
Host Cells ◽  
Titration Calorimetry ◽  
Dependent Manner ◽  
Content Type ◽  
Phosphatidylinositol 4

ABSTRACTLegionellaspp. cause the severe pneumonia Legionnaires' disease. The environmental bacteria replicate intracellularly in free-living amoebae and human alveolar macrophages within a distinct, endoplasmic reticulum (ER)-derived compartment termed theLegionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS) that translocates into host cells a plethora of different “effector” proteins, some of which anchor to the pathogen vacuole by binding to phosphoinositide (PI) lipids. Here, we identified by unbiased pulldown assays inLegionella longbeachaelysates a 111-kDa SidC homologue as the major phosphatidylinositol 4-phosphate [PtdIns(4)P]-binding protein. The PI-binding domain was mapped to a 20-kDa P4C [PtdIns(4)Pbinding of SidC] fragment. Isothermal titration calorimetry revealed that SidC ofL. longbeachae(SidCLlo) binds PtdIns(4)Pwith aKd(dissociation constant) of 71 nM, which is 3 to 4 times lower than that of the SidC orthologue ofLegionella pneumophila(SidCLpn). Upon infection of RAW 264.7 macrophages withL. longbeachae, endogenous SidCLloor ectopically produced SidCLpnlocalized in an Icm/Dot-dependent manner to the PtdIns(4)P-positive LCVs. AnL. longbeachaeΔsidCdeletion mutant was impaired for calnexin recruitment to LCVs inDictyostelium discoideumamoebae and outcompeted by wild-type bacteria inAcanthamoeba castellanii. Calnexin recruitment was restored by SidCLloor its orthologues SidCLpnand SdcALpn. Conversely, calnexin recruitment was restored by SidCLloinL. pneumophilalackingsidCandsdcA. Together, biochemical, genetic, and cell biological data indicate that SidCLlois anL. longbeachaeeffector that binds through a P4C domain with high affinity to PtdIns(4)Pon LCVs, promotes ER recruitment to the LCV, and thus plays a role in pathogen-host interactions.

scholarly journals Human GBP1 promotes pathogen vacuole rupture and inflammasome activation during Legionella pneumophila infection

2020 ◽  
Author(s):  
Antonia R. Bass ◽  
Sunny Shin
Keyword(s):  
Legionella Pneumophila ◽  
Severe Pneumonia ◽  
Inflammasome Activation ◽  
Dependent Manner ◽  
Cytokine Release ◽  
Legionnaires Disease ◽  
Cell Cytosol ◽  
Ifn Γ ◽  
Host Cell Cytosol ◽  
Family Cytokine

AbstractThe inflammasome is an essential component of host defense against intracellular bacterial pathogens, such as Legionella pneumophila, the causative agent of the severe pneumonia Legionnaires’ disease. Inflammasome activation leads to recruitment and activation of caspases, which promote IL-1 family cytokine release and pyroptosis. In mice, interferon (IFN) signaling promotes inflammasome responses against L. pneumophila, in part through the functions of a family of IFN-inducible GTPases known as guanylate binding proteins (GBPs) (1). Within murine macrophages, IFN signaling promotes rupture of the L. pneumophila-containing vacuole (LCV), whereas GBPs are dispensable for vacuole rupture. Instead, GBPs facilitate the lysis of cytosol-exposed L. pneumophila. In contrast to mouse GBPs, the functions of human GBPs in inflammasome responses to L. pneumophila are poorly understood. Here, we show that IFN-γ promotes caspase-1, caspase-4, and caspase-5 inflammasome activation during L. pneumophila infection and upregulates GBP expression in primary human macrophages. We find that human GBP1 is important for maximal IFN-γ-driven inflammasome responses to L. pneumophila. Furthermore, IFN-γ signaling promotes the rupture of LCVs. Intriguingly, in contrast to murine GBPs, human GBP1 targets the LCV in a T4SS-dependent manner and promotes vacuolar lysis, resulting in increased bacterial access to the host cell cytosol. Our findings show a key role for human GBP1 in targeting and disrupting pathogen-containing vacuoles and reveal mechanistic differences in how mouse and human GBPs promote inflammasome responses to L. pneumophila.

scholarly journals Characterization of the Major Secreted Zinc Metalloprotease- Dependent Glycerophospholipid:Cholesterol Acyltransferase, PlaC, of Legionella pneumophila

2005 ◽  
Vol 73 (5) ◽  
pp. 2899-2909 ◽  
Author(s):  
Sangeeta Banerji ◽  
Mayte Bewersdorff ◽  
Björn Hermes ◽  
Nicholas P. Cianciotto ◽  
Antje Flieger
Keyword(s):  
Legionella Pneumophila ◽  
Hydrolytic Enzymes ◽  
Acanthamoeba Castellanii ◽  
Severe Pneumonia ◽  
Phospholipase A ◽  
Type Ii Secretion ◽  
Lipolytic Enzyme ◽  
Acyltransferase Activity ◽  
Zinc Metalloprotease ◽  
Bacterial Culture Supernatant

ABSTRACT Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.

scholarly journals Legionella norrlandica sp. nov., isolated from the biopurification systems of wood processing plants

2015 ◽  
Vol 65 (Pt_2) ◽  
pp. 598-603 ◽  
Author(s):  
Kristina Rizzardi ◽  
Jadwiga Winiecka-Krusnell ◽  
Miriam Ramliden ◽  
Erik Alm ◽  
Sabina Andersson ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Legionella Pneumophila ◽  
Type Species ◽  
Sequence Similarity ◽  
Acanthamoeba Castellanii ◽  
Content Type ◽  
Wood Processing ◽  
Link Type ◽  
Processing Plants ◽  
Biopurification Systems

Fourteen isolates of an unknown species identified as belonging to the genus Legionella by selective growth on BCYE agar were isolated from the biopurification systems of three different wood processing plants. The mip gene sequence of all 14 isolates was identical and a close match alignment revealed 86 % sequence similarity with Legionella pneumophila serogroup 8. The whole genome of isolate LEGNT was sequenced, and a phylogenetic tree based on the alignment of 16S rRNA, mip, rpoB, rnpB and the 23S–5S intergenic region clustered LEGNT with L. pneumophila ATCC 33152T. Analysis of virulence factors showed that strain LEGNT carries the majority of known L. pneumophila virulence factors. An amoeba infection assay performed to assess the pathogenicity of strain LEGNT towards Acanthamoeba castellanii showed that it can establish a replication vacuole in A. castellanii but does not significantly affect replication of amoebae. Taken together, the results confirm that strain LEGNT represents a novel species of the genus Legionella , for which the name Legionella norrlandica sp. nov. is proposed. The type strain is LEGNT ( = ATCC BAA-2678T = CCUG 65936T).

scholarly journals Reverting the mode of action of the mitochondrial FOF1-ATPase by Legionella pneumophila preserves its replication niche

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Pedro Escoll ◽  
Lucien Platon ◽  
Mariatou Dramé ◽  
Tobias Sahr ◽  
Silke Schmidt ◽  
...  
Keyword(s):  
Cell Death ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
Severe Pneumonia ◽  
Human Monocyte ◽  
Dependent Manner ◽  
Delayed Cell Death ◽  
Mitochondrial Functions ◽  
Infected Cells ◽  
Bacterial Replication

Legionella pneumophila, the causative agent of Legionnaires'; disease, a severe pneumonia, injects via a type-IV-secretion-system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here we show that despite reduced OXPHOS, the mitochondrial membrane potential (Δψm) is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the Δψm, preserves mitochondrial polarization and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the Δψm, but not LncP and MitF. The inhibition of the L. pneumophila-induced 'reverse mode' of the FOF1-ATPase collapsed the Δψm and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the Δψm and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.

scholarly journals Downregulation of miR-214-3p May Contribute to Pathogenesis of Ulcerative Colitis via Targeting STAT6

2017 ◽  
Vol 2017 ◽  
pp. 1-7
Author(s):  
Jin-an Li ◽  
Yong-duo Wang ◽  
Kui Wang ◽  
Zhen-lan Wang ◽  
Dao-yong Jia ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Noncoding Rna ◽  
Intestinal Inflammation ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Altered Expression ◽  
Rna Molecules ◽  
Small Noncoding Rna ◽  
Ht29 Cells ◽  
Tight Association

MicroRNAs (miRs) are small noncoding RNA molecules and recently have demonstrated that altered expression and functions are their tight association with ulcerative colitis (UC). Previous microarray study reported that miR-214 was downregulated in the sigmoid colon of patients with active UC, but the roles of miR-214 in the pathogenesis of UC remain to be elucidated. In this study, significant lower level of miR-214-3p and higher level of STAT6 in the intestinal mucosa of active UC patients compared with the health controls were confirmed by quantitative real-time PCR. Results of luciferase reporter assays and western blot demonstrated that miR-214-3p directly targets STAT6 and negatively regulates the expression of STAT6 at the posttranscriptional level. Furthermore, the expression of miR-214-3p was decreased in TNF-αtreated HT29 cells and STAT6 protein level was increased in a time-dependent manner. Silenced STAT6 and upregulation of miR-214-3p could decrease the level of INF-γin TNF-αtreated HT29 cells. Additionally, the results of the present study indicate that miR-214-3p and STAT6 axis may be a novel therapeutic target for intestinal inflammation of patients with active UC.

scholarly journals Virulence Conversion of Legionella pneumophila by Conjugal Transfer of Chromosomal DNA

2003 ◽  
Vol 185 (22) ◽  
pp. 6712-6718 ◽  
Author(s):  
Hiroshi Miyamoto ◽  
Shin-ichi Yoshida ◽  
Hatsumi Taniguchi ◽  
Howard A. Shuman
Keyword(s):  
Legionella Pneumophila ◽  
Virulent Strain ◽  
Acanthamoeba Castellanii ◽  
Severe Pneumonia ◽  
Conjugal Transfer ◽  
Avirulent Strain ◽  
Donor Strain ◽  
Chromosomal Dna ◽  
Conjugation System ◽  
Sublethal Doses

ABSTRACT In this study, we examined whether virulence conversion occurs in Legionella pneumophila by conjugal transfer of chromosomal DNA. A virulent strain, K6, which has the genes for Kmr and LacZ+ transposed in the chromosome of strain Philadelphia-1, which belongs to serogroup 1, was used as one parent, and an avirulent strain, Chicago-2S, which is a spontaneous streptomycin-resistant derivative of strain Chicago-2 belonging to serogroup 6, was used as the other parent. Experiments in which K6 (approximately 2.6 × 109 CFU) and Chicago-2S (approximately 8.9 × 109 CFU) were mated typically yielded 103 Kmr Smr LacZ+ transconjugants. Thirty-two (about 2.8%) of 1,152 transconjugants belonging to serogroup 6 acquired the ability to grow intracellularly in Acanthamoeba castellanii and guinea pig macrophages. When guinea pigs were infected with sublethal doses of Legionella aerosols generated from one of these transconjugants (HM1011), they developed a severe pneumonia similar to that caused by donor strain K6. These results show that avirulent strain Chicago-2S changed into virulent strain HM1011 through conjugation with virulent strain K6. Furthermore, we showed that Legionella chromosomal virulence genes (icm-dot locus) were horizontally transferred by the conjugation system. The chromosomal conjugation system may play a role(s) in the evolution of L. pneumophila.

scholarly journals Virulence Phenotypes of Legionella pneumophila Associated with Noncoding RNA lpr0035

2012 ◽  
Vol 80 (12) ◽  
pp. 4143-4153 ◽  
Author(s):  
Deepak Jayakumar ◽  
Julie V. Early ◽  
Howard M. Steinman
Keyword(s):  
Legionella Pneumophila ◽  
Noncoding Rna ◽  
Deletion Mutant ◽  
Acanthamoeba Castellanii ◽  
Direct Repeat ◽  
Repeat Sequence ◽  
Open Reading Frames ◽  
Causative Organism ◽  
Content Type ◽  
Intracellular Multiplication

ABSTRACTThe Philadelphia-1 strain ofLegionella pneumophila, the causative organism of Legionnaires' disease, contains a recently discovered noncoding RNA, lpr0035. lpr0035 straddles the 5′ chromosomal junction of a 45-kbp mobile genetic element, pLP45, which can exist as an episome or integrated in the bacterial chromosome. A 121-bp deletion was introduced in strain JR32, a Philadelphia-1 derivative. The deletion inactivated lpr0035, removed the 49-bp direct repeat at the 5′ junction of pLP45, and locked pLP45 in the chromosome. Intracellular multiplication of the deletion mutant was decreased by nearly 3 orders of magnitude inAcanthamoeba castellaniiamoebae and nearly 2 orders of magnitude in J774 mouse macrophages. Entry of the deletion mutant into amoebae and macrophages was decreased by >70%. The level of entry in both hosts was restored to that in strain JR32 by plasmid copies of two open reading frames immediately downstream of the 5′ junction and plasmid lpr0035 driven by its endogenous promoter. When induced from atacpromoter, plasmid lpr0035 completely reversed the intracellular multiplication defect in macrophages but was without effect in amoebae. These data are the first evidence of a role for noncoding RNA lpr0035, which has homologs in six otherLegionellagenomes, in entry ofL. pneumophilainto amoebae and macrophages and in host-specific intracellular multiplication. The data also demonstrate that deletion of a direct-repeat sequence restricts the mobility of pLP45 and is a means of studying the role of pLP45 mobility inLegionellavirulence phenotypes.

scholarly journals Identification of a σB-Dependent Small Noncoding RNA in Listeria monocytogenes

2008 ◽  
Vol 190 (18) ◽  
pp. 6264-6270 ◽  
Author(s):  
Jesper Sejrup Nielsen ◽  
Anders Steno Olsen ◽  
Mette Bonde ◽  
Poul Valentin-Hansen ◽  
Birgitte H. Kallipolitis
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Tolerance ◽  
Sigma Factor ◽  
Noncoding Rna ◽  
Noncoding Rnas ◽  
Alternative Sigma Factor ◽  
Dependent Manner ◽  
Small Noncoding Rna ◽  
Small Noncoding Rnas ◽  
Regulatory Circuits

ABSTRACT In Listeria monocytogenes, the alternative sigma factor σB plays important roles in stress tolerance and virulence. Here, we present the identification of SbrA, a novel small noncoding RNA that is produced in a σB-dependent manner. This finding adds the σB regulon to the growing list of stress-induced regulatory circuits that include small noncoding RNAs.

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