Journal articles on the topic 'Legionella pneumophila Dot'
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Berk, Sharon G., Gary Faulkner, Elizabeth Garduño, Mark C. Joy, Marco A. Ortiz-Jimenez, and Rafael A. Garduño. "Packaging of Live Legionella pneumophila into Pellets Expelled by Tetrahymena spp. Does Not Require Bacterial Replication and Depends on a Dot/Icm-Mediated Survival Mechanism." Applied and Environmental Microbiology 74, no. 7 (February 1, 2008): 2187–99. http://dx.doi.org/10.1128/aem.01214-07.
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ABSTRACT The freshwater ciliate Tetrahymena sp. efficiently ingested, but poorly digested, virulent strains of the gram-negative intracellular pathogen Legionella pneumophila. Ciliates expelled live legionellae packaged in free spherical pellets. The ingested legionellae showed no ultrastructural indicators of cell division either within intracellular food vacuoles or in the expelled pellets, while the number of CFU consistently decreased as a function of time postinoculation, suggesting a lack of L. pneumophila replication inside Tetrahymena. Pulse-chase feeding experiments with fluorescent L. pneumophila and Escherichia coli indicated that actively feeding ciliates maintain a rapid and steady turnover of food vacuoles, so that the intravacuolar residence of the ingested bacteria was as short as 1 to 2 h. L. pneumophila mutants with a defective Dot/Icm virulence system were efficiently digested by Tetrahymena sp. In contrast to pellets of virulent L. pneumophila, the pellets produced by ciliates feeding on dot mutants contained very few bacterial cells but abundant membrane whorls. The whorls became labeled with a specific antibody against L. pneumophila OmpS, indicating that they were outer membrane remnants of digested legionellae. Ciliates that fed on genetically complemented dot mutants produced numerous pellets containing live legionellae, establishing the importance of the Dot/Icm system to resist digestion. We thus concluded that production of pellets containing live virulent L. pneumophila depends on bacterial survival (mediated by the Dot/Icm system) and occurs in the absence of bacterial replication. Pellets of virulent L. pneumophila may contribute to the transmission of Legionnaires’ disease, an issue currently under investigation.
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Takamatsu, Reika, Eriko Takeshima, Chie Ishikawa, Kei Yamamoto, Hiromitsu Teruya, Klaus Heuner, Futoshi Higa, Jiro Fujita, and Naoki Mori. "Inhibition of Akt/GSK3β signalling pathway by Legionella pneumophila is involved in induction of T-cell apoptosis." Biochemical Journal 427, no. 1 (March 15, 2010): 57–67. http://dx.doi.org/10.1042/bj20091768.
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Legionella pneumophila is the causative agent of human Legionnaires' disease. L. pneumophila has been shown to induce apoptosis of T-cells and this may be important pathologically and clinically. The present study has determined the molecular mechanisms underlying L. pneumophila-induced apoptosis, which were unclear. Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T-cells. However, apoptosis was efficiently induced in T-cells only by wild-type L. pneumophila, and not flagellin-deficient or Dot/Icm-deficient Legionella. Induction of apoptosis involved activation of the initiator caspase 9 and effector caspase 3. Infection with L. pneumophila inhibited phosphorylation of Akt (also known as protein kinase B) and the Akt substrate GSK3β (glycogen synthase kinase 3β), and reduced the levels of β-catenin, a transcriptional activator regulated by GSK3β. It also caused the activation of the pro-apoptotic protein Bax and inhibited the expression of the anti-apoptotic protein XIAP (X-linked inhibitor of apoptosis) via inhibition of the Akt pathway. In conclusion, L. pneumophila induces mitochondria-mediated T-cell apoptosis through inhibition of the Akt/GSK3β signalling pathway.
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Kozak, Natalia A., Meghan Buss, Claressa E. Lucas, Michael Frace, Dhwani Govil, Tatiana Travis, Melissa Olsen-Rasmussen, Robert F. Benson, and Barry S. Fields. "Virulence Factors Encoded by Legionella longbeachae Identified on the Basis of the Genome Sequence Analysis of Clinical Isolate D-4968." Journal of Bacteriology 192, no. 4 (December 11, 2009): 1030–44. http://dx.doi.org/10.1128/jb.01272-09.
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ABSTRACT Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.
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Santic, Marina, Rexford Asare, Miljenko Doric, and Yousef Abu Kwaik. "Host-Dependent Trigger of Caspases and Apoptosis by Legionella pneumophila." Infection and Immunity 75, no. 6 (April 9, 2007): 2903–13. http://dx.doi.org/10.1128/iai.00147-07.
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ABSTRACT The Dot/Icm system of Legionella pneumophila triggers activation of caspase-3 during early stages of infection of human macrophages, but apoptosis is delayed until late stages of infection. During early stages of infection of mouse macrophages, the organism triggers rapid caspase-1-mediated cytotoxicity, which is mediated by bacterial flagellin. However, it is not known whether caspase-1 is triggered by L. pneumophila in human macrophages or whether caspase-3 is activated in permissive or nonpermissive mouse macrophages. Using single-cell analyses, we show that the wild-type strain of L. pneumophila does not trigger caspase-1 activation throughout the intracellular infection of human monocyte-derived macrophages (hMDMs), even when the flagellated bacteria escape into the cytoplasm during late stages. Using single-cell analyses, we show that the Dot/Icm system of L. pneumophila triggers caspase-3 but not caspase-1 within permissive A/J mouse bone marrow-derived primary macrophages by 2 to 8 h, but apoptosis is delayed until late stages of infection. While L. pneumophila triggers a Dot/Icm-dependent activation of caspase-1 in nonpermissive BALB/c mouse-derived macrophages, caspase-3 is not activated at any stage of infection. We show that robust intrapulmonary replication of the wild-type strain of L. pneumophila in susceptible A/J mice is associated with late-stage Dot/Icm-dependent pulmonary apoptosis and alveolar inflammation. In the lungs of nonpermissive BALB/c mice, L. pneumophila does not replicate and does not trigger pulmonary apoptosis or alveolar inflammation. Thus, similar to hMDMs, L. pneumophila does not trigger caspase-1 but triggers caspase-3 activation during early and exponential replication in permissive A/J mouse-derived macrophages, and apoptosis is delayed until late stages of infection. The Dot/Icm type IV secretion system is essential for pulmonary apoptosis in the genetically susceptible A/J mice.
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Hovel-Miner, Galadriel, Sergey Pampou, Sebastien P. Faucher, Margaret Clarke, Irina Morozova, Pavel Morozov, James J. Russo, Howard A. Shuman, and Sergey Kalachikov. "σS Controls Multiple Pathways Associated with Intracellular Multiplication of Legionella pneumophila." Journal of Bacteriology 191, no. 8 (February 13, 2009): 2461–73. http://dx.doi.org/10.1128/jb.01578-08.
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ABSTRACT Legionella pneumophila is the causative agent of the severe and potentially fatal pneumonia Legionnaires' disease. L. pneumophila is able to replicate within macrophages and protozoa by establishing a replicative compartment in a process that requires the Icm/Dot type IVB secretion system. The signals and regulatory pathways required for Legionella infection and intracellular replication are poorly understood. Mutation of the rpoS gene, which encodes σS, does not affect growth in rich medium but severely decreases L. pneumophila intracellular multiplication within protozoan hosts. To gain insight into the intracellular multiplication defect of an rpoS mutant, we examined its pattern of gene expression during exponential and postexponential growth. We found that σS affects distinct groups of genes that contribute to Legionella intracellular multiplication. We demonstrate that rpoS mutants have a functional Icm/Dot system yet are defective for the expression of many genes encoding Icm/Dot-translocated substrates. We also show that σS affects the transcription of the cpxR and pmrA genes, which encode two-component response regulators that directly affect the transcription of Icm/Dot substrates. Our characterization of the L. pneumophila small RNA csrB homologs, rsmY and rsmZ, introduces a link between σS and the posttranscriptional regulator CsrA. We analyzed the network of σS-controlled genes by mutational analysis of transcriptional regulators affected by σS. One of these, encoding the L. pneumophila arginine repressor homolog gene, argR, is required for maximal intracellular growth in amoebae. These data show that σS is a key regulator of multiple pathways required for L. pneumophila intracellular multiplication.
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Aurass, P., B. Pless, K. Rydzewski, G. Holland, N. Bannert, and A. Flieger. "bdhA-patD Operon as a Virulence Determinant, Revealed by a Novel Large-Scale Approach for Identification of Legionella pneumophila Mutants Defective for Amoeba Infection." Applied and Environmental Microbiology 75, no. 13 (May 1, 2009): 4506–15. http://dx.doi.org/10.1128/aem.00187-09.
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ABSTRACT Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular parasite of eukaryotic cells. In the environment, it colonizes amoebae. After being inhaled into the human lung, the bacteria infect and damage alveolar cells in a way that is mechanistically similar to the amoeba infection. Several L. pneumophila traits, among those the Dot/Icm type IVB protein secretion machinery, are essential for exploiting host cells. In our search for novel Legionella virulence factors, we developed an agar plate assay, designated the scatter screen, which allowed screening for mutants deficient in infecting Acanthamoeba castellanii amoebae. Likewise, an L. pneumophila clone bank consisting of 23,000 transposon mutants was investigated here, and 19 different established Legionella virulence genes, for example, dot/icm genes, were identified. Importantly, 70 novel virulence-associated genes were found. One of those is L. pneumophila bdhA, coding for a protein with homology to established 3-hydroxybutyrate dehydrogenases involved in poly-3-hydroxybutyrate metabolism. Our study revealed that bdhA is cotranscribed with patD, encoding a patatin-like protein of L. pneumophila showing phospholipase A and lysophospholipase A activities. In addition to strongly reduced lipolytic activities and increased poly-3-hydroxybutyrate levels, the L. pneumophila bdhA-patD mutant showed a severe replication defect in amoebae and U937 macrophages. Our data suggest that the operon is involved in poly-3-hydroxybutyrate utilization and phospholipolysis and show that the bdhA-patD operon is a virulence determinant of L. pneumophila. In summary, the screen for amoeba-sensitive Legionella clones efficiently isolated mutants that do not grow in amoebae and, in the case of the bdhA-patD mutant, also human cells.
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Zusman, Tal, Gal Yerushalmi, and Gil Segal. "Functional Similarities between the icm/dot Pathogenesis Systems of Coxiella burnetii and Legionella pneumophila." Infection and Immunity 71, no. 7 (July 2003): 3714–23. http://dx.doi.org/10.1128/iai.71.7.3714-3723.2003.
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ABSTRACT Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular pathogen, whereas Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen. During infection of humans both of these pathogens multiply in alveolar macrophages inside a closed phagosome. L. pneumophila intracellular multiplication was shown to be dependent on the icm/dot system, which probably encodes a type IV-related translocation apparatus. Recently, genes homologous to all of the L. pneumophila icm/dot genes (besides icmR) were found in C. burnetii. To explore the similarities and differences between the icm/dot pathogenesis systems of these two pathogens, interspecies complementation analysis was performed. Nine C. burnetii icm homologous genes (icmT, icmS, icmQ, icmP, icmO, icmJ, icmB, icmW, and icmX) were cloned under regulation of the corresponding L. pneumophila icm genes and examined for the ability to complement L. pneumophila mutants with mutations in these genes. The C. burnetii icmS and icmW homologous genes were found to complement the corresponding L. pneumophila icm mutants to wild-type levels of intracellular growth in both HL-60-derived human macrophages and Acanthamoeba castellanii. In addition, the C. burnetii icmT homologous gene was found to completely complement an L. pneumophila insertion mutant for intracellular growth in HL-60-derived human macrophages, but it only partially complemented the same mutant for intracellular growth in A. castellanii. Moreover, as previously shown for L. pneumophila, the proteins encoded by the C. burnetii icmS and icmW homologous genes were found to interact with one another, and interspecies protein interaction was observed as well. Our results strongly indicate that the Icm/Dot pathogenesis systems of C. burnetii and L. pneumophila have common features.
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Vincent, Carr D., Benjamin A. Buscher, Jonathan R. Friedman, Lee Anne Williams, Patrick Bardill, and Joseph P. Vogel. "Identification of Non-dot/icm Suppressors of the Legionella pneumophila ΔdotL Lethality Phenotype." Journal of Bacteriology 188, no. 23 (September 22, 2006): 8231–43. http://dx.doi.org/10.1128/jb.00937-06.
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ABSTRACT Legionella pneumophila, a causative agent of bacterial pneumonia, survives inside phagocytic cells by avoiding rapid targeting to the lysosome. This bacterium utilizes a type IVB secretion system, encoded by the dot/icm genes, to replicate inside host cells. DotL, a critical component of the Dot/Icm secretion apparatus, functions as the type IV coupling protein. In contrast to most dot/icm genes, which are dispensable for growth on bacteriological media, dotL is required for the viability of wild-type L. pneumophila. Previously we reported that ΔdotL lethality could be suppressed by inactivation of the Dot/Icm complex via mutations in other dot/icm genes. Here we report the isolation of non-dot/icm suppressors of this phenotype. These ΔdotL suppressors include insertions that disrupt the function of the L. pneumophila homologs of cpxR, djlA, lysS, and two novel open reading frames, lpg0742 and lpg1594, that we have named ldsA and ldsB for lethality of ΔdotL suppressor. In addition to suppressing ΔdotL lethality, inactivation of these genes in a wild-type strain background causes a range of defects in L. pneumophila virulence traits, including intracellular growth, implicating these factors in the proper function of the Dot/Icm complex. Consistent with previous data showing a role for the cpx system in regulating expression of several dot/icm genes, the cpxR insertion mutant produced decreased levels of three Dot/Icm proteins, DotA, IcmV, and IcmW. The remaining four suppressors did not affect the steady-state levels of any Dot/Icm protein and are likely to represent the first identified factors necessary for assembly and/or activation of the Dot/Icm secretion complex.
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Newton, Hayley J., Fiona M. Sansom, Vicki Bennett-Wood, and Elizabeth L. Hartland. "Identification of Legionella pneumophila-Specific Genes by Genomic Subtractive Hybridization with Legionella micdadei and Identification of lpnE, a Gene Required for Efficient Host Cell Entry." Infection and Immunity 74, no. 3 (March 2006): 1683–91. http://dx.doi.org/10.1128/iai.74.3.1683-1691.2006.
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ABSTRACT Legionella pneumophila is a ubiquitous environmental organism and a facultative intracellular pathogen of humans. To identify genes that may contribute to the virulence of L. pneumophila, we performed genomic subtractive hybridization between L. pneumophila serogroup 1 strain 02/41 and L. micdadei strain 02/42. A total of 144 L. pneumophila-specific clones were sequenced, revealing 151 genes that were absent in L. micdadei strain 02/42. Low-stringency Southern hybridization was used to determine the distribution of 41 sequences, representing 40 open reading frames (ORFs) with a range of putative functions among L. pneumophila isolates of various serogroups as well as strains of Legionella longbeachae, L. micdadei, Legionella gormanii, and Legionella jordanis. Twelve predicted ORFs were L. pneumophila specific, including the gene encoding the dot/icm effector, lepB, as well as several genes predicted to play a role in lipopolysaccharide biosynthesis and cell wall synthesis and several sequences with similarity to virulence-associated determinants. A further nine predicted ORFs were in all L. pneumophila serotypes tested and an isolate of L. gormanii. These included icmD, the 5′ end of a pilMNOPQ locus, and two genes known to be upregulated during growth within macrophages, cadA2 and ceaA. Disruption of an L. pneumophila-specific gene (lpg2222 locus tag) encoding a putative protein with eight tetratricopeptide repeats resulted in reduced entry into the macrophage-like cell line, THP-1, and the type II alveolar epithelial cell line, A549. The gene was subsequently renamed lpnE, for “L. pneumophila entry.” In summary, this investigation has revealed important genetic differences between L. pneumophila and other Legionella species that may contribute to the phenotypic and clinical differences observed within this genus.
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Wood, Rebecca E., Patrice Newton, Eleanor A. Latomanski, and Hayley J. Newton. "Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires." Infection and Immunity 83, no. 10 (July 27, 2015): 4081–92. http://dx.doi.org/10.1128/iai.00461-15.
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Legionellaorganisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed theLegionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis ofLegionella pneumophila. Here, we have characterized theLegionella longbeachaereplicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV ofL. pneumophila(pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deletingdotBand demonstrated the inability of this mutant to replicate inside THP-1 cells.L. longbeachaedoes not enter THP-1 cells as efficiently asL. pneumophila, and this is reflected in the observation that translocation of BlaM-RalFLLO(where RalFLLOis theL. longbeachaehomologue of RalF) into THP-1 cells by theL. longbeachaeDot/Icm system is less efficient than that byL. pneumophila. This difference is negated in A549 cells whereL. longbeachaeandL. pneumophilainfect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, theRab-likeeffector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on thelongbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to thelongbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of theL. longbeachaereplicative vacuole, highlighting phenotypic similarities to the vacuole ofL. pneumophilaas well as unique aspects of LCV biology.
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Kowalczyk, Bożena, Agata Małek, and Marta Palusińska-Szysz. "Budowa IV systemu sekrecji Legionella pneumophilai jego znaczenie w patogenezie." Postępy Higieny i Medycyny Doświadczalnej 75, no. 1 (January 1, 2021): 548–62. http://dx.doi.org/10.2478/ahem-2021-0023.
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Abstrakt Bakterie Legionella pneumophila w środowisku naturalnym pasożytują wewnątrz komórek wybranych gatunków pierwotniaków, a po przedostaniu się do sztucznych systemów dystrybucji wody stają się ważnym czynnikiem etiologicznym zapalenia płuc u ludzi. Główną cechą determinującą patogenność tych bakterii jest zdolność do życia i replikacji w makrofagach płucnych, czyli w komórkach wyspecjalizowanych do fagocytozy, zabijania i trawienia mikroorganizmów. Warunkiem wstępnym rozwoju infekcji jest przełamanie mechanizmów bójczych makrofagów i utworzenie wakuoli replikacyjnej LCV (Legionella containing vacuole). Biogeneza wakuoli LCV jest możliwa dzięki sprawnemu funkcjonowaniu IV systemu sekrecji Dot/Icm, który jest wielobiałkowym, złożonym kompleksem umiejscowionym w wewnętrznej i zewnętrznej membranie osłony komórkowej bakterii. System Dot/Icm liczy 27 elementów, na które składają się m.in. kompleks rdzeniowo-transmembranowy, tworzący strukturalny szkielet całego systemu oraz kompleks białek sprzęgających. Geny kodujące komponenty systemu Dot/Icm są zorganizowane na dwóch regionach chromosomu bak-teryjnego. System sekrecji Dot/Icm umożliwia L. pneumophila wprowadzenie do cytozolu komórki gospodarza ponad 300 białek efektorowych, których skoordynowane działanie powoduje utrzymanie integralności błony wakuoli replikacyjnej oraz pozwala na manipulowanie różnymi procesami komórki. Ważnym elementem strategii wewnątrzkomórkowego namnażania się L. pneumophila jest modulowanie transportu pęcherzykowego, interakcja z retikulum endoplazmatycznym oraz zakłócenie biosyntezy białek, procesów autofagii i apoptozy komórki gospodarza. Poznanie złożonych mechanizmów regulacji i funkcji białek efektorowych systemu Dot/Icm ma decydujące znaczenie w zapobieganiu i leczeniu choroby legionistów.
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Matthews, Miguelina, and Craig R. Roy. "Identification and Subcellular Localization of the Legionella pneumophila IcmX Protein: a Factor Essential for Establishment of a Replicative Organelle in Eukaryotic Host Cells." Infection and Immunity 68, no. 7 (July 1, 2000): 3971–82. http://dx.doi.org/10.1128/iai.68.7.3971-3982.2000.
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ABSTRACT The gram-negative respiratory pathogen Legionella pneumophila infects and grows within mammalian macrophages and protozoan host cells. Upon uptake into macrophages, L. pneumophila establishes a replicative organelle that avoids fusion with endocytic vesicles. There are 24 dot/icm genes on the L. pneumophila chromosome required for biogenesis of this vacuole. Many of the Dot/Icm proteins are predicted to be components of a membrane-bound secretion apparatus similar to type IV conjugal transfer systems. We have been investigating the function ofL. pneumophila dot/icm gene products that do not have obvious orthologs in other type IV transfer systems, since these determinants could govern processes unique to phagosome biogenesis. TheicmX gene product falls into this category. To understand the role of the IcmX protein in pathogenesis, we have detailed interactions between an L. pneumophila icmX deletion mutant and murine bone marrow-derived macrophages. These data demonstrate thaticmX is required for biogenesis of the L. pneumophila replicative organelle. Immunoblot analysis indicates that the icmX gene product is a polypeptide with an estimated molecular mass of 50 kDa. The IcmX protein was localized to the bacterial periplasm, and periplasmic translocation was mediated by an N-terminal sec-dependent leader peptide. A truncated IcmX product was secreted into culture supernatants by wild-typeL. pneumophila growing extracellularly in liquid media; however, transport of the IcmX protein into eukaryotic host cells was not detected. Proteins similar in molecular weight to IcmX were identified in other Legionella species by immunoblot analysis using a monoclonal antibody specific for L. pneumophila IcmX protein. From these data, we conclude that the IcmX protein is an essential component of the dot/icmsecretion apparatus, and that a conserved mechanism of host cell parasitism exists for members of the Legionellaceae family.
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Losick, Vicki P., and Ralph R. Isberg. "NF-κB translocation prevents host cell death after low-dose challenge by Legionella pneumophila." Journal of Experimental Medicine 203, no. 9 (August 28, 2006): 2177–89. http://dx.doi.org/10.1084/jem.20060766.
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Legionella pneumophila, the causative agent of Legionnaires' disease, grows within macrophages and manipulates target cell signaling. Formation of a Legionella-containing replication vacuole requires the function of the bacterial type IV secretion system (Dot/Icm), which transfers protein substrates into the host cell cytoplasm. A global microarray analysis was used to examine the response of human macrophage-like U937 cells to low-dose infections with L. pneumophila. The most striking change in expression was the Dot/Icm-dependent up-regulation of antiapoptotic genes positively controlled by the transcriptional regulator nuclear factor κB (NF-κB). Consistent with this finding, L. pneumophila triggered nuclear localization of NF-κB in human and mouse macrophages in a Dot/Icm-dependent manner. The mechanism of activation at low-dose infections involved a signaling pathway that occurred independently of the Toll-like receptor adaptor MyD88 and the cytoplasmic sensor Nod1. In contrast, high multiplicity of infection conditions caused a host cell response that masked the unique Dot/Icm-dependent activation of NF-κB. Inhibition of NF-κB translocation into the nucleus resulted in premature host cell death and termination of bacterial replication. In the absence of one antiapoptotic protein, plasminogen activator inhibitor–2, host cell death increased in response to L. pneumophila infection, indicating that induction of antiapoptotic genes is critical for host cell survival.
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Oshiro, Robin K., Teresa Picone, and Betty H. Olson. "Modification of reagents in the EnviroAmp™ kit to increase recovery of Legionella organisms in water." Canadian Journal of Microbiology 40, no. 6 (June 1, 1994): 495–99. http://dx.doi.org/10.1139/m94-080.
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Organisms of the bacterial genus Legionella, commonly found in aqueous reservoirs, have been associated with Legionnaires' disease (legionella pneumonia, caused by Legionella pneumophila) and Pontiac fever (nonpneumonic legionellosis). EnviroAmp™ Legionella sample preparation, polymerase chain reaction amplification, and detection kits (Perkin-Elmer Corp.) were developed for rapid detection of DNA from organisms of the genus Legionella and the species L. pneumophila from environmental water samples. The kits are based on molecular techniques incorporating polymerase chain reaction amplification and detection by reverse dot blot hybridization to particular genus and species probes. The manufacturer states that the EnviroAmp™ Legionella sample preparation, polymerase chain reaction amplification, and detection kits can detect approximately 100 Legionella organisms/mL (10 000 organisms/100 mL) in the original water sample. The sensitivity of the kits was increased to 0.1 colony-forming units/mL (10 colony-forming units/100 mL), at least for cultured organisms, by modifying the EnviroAmp™ Legionella sample preparation kit protocol. Data obtained in this study indicated that sample volume could be increased from 100 to 1000 mL (in the absence of interfering substances such as humic acid) and DNA extraction volume could be decreased from 2 to 0.5 mL to increase the ability of the kit to detect lower numbers of Legionella spp. or L. pneumophila per volume.Key words: Legionella, environment, water, EnviroAmp™ kit.
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Sexton, Jessica A., Jennifer L. Miller, Aki Yoneda, Thomas E. Kehl-Fie, and Joseph P. Vogel. "Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex." Infection and Immunity 72, no. 10 (October 2004): 5983–92. http://dx.doi.org/10.1128/iai.72.10.5983-5992.2004.
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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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N'Guessan, Philippe Dje, Mirabelle O. Etouem, Bernd Schmeck, Andreas C. Hocke, Stefanie Scharf, Kremena Vardarova, Bastian Opitz, Antje Flieger, Norbert Suttorp, and Stefan Hippenstiel. "Legionella pneumophila-induced PKCα-, MAPK-, and NF-κB-dependent COX-2 expression in human lung epithelium." American Journal of Physiology-Lung Cellular and Molecular Physiology 292, no. 1 (January 2007): L267—L277. http://dx.doi.org/10.1152/ajplung.00100.2006.
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Legionella pneumophila causes community- and hospital-acquired pneumonia. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX) and microsomal PGE2synthase-1 (mPGES-1)-derived prostaglandins like prostaglandin E2(PGE2) are considered as important regulators of lung function. Herein we tested the hypothesis that L. pneumophila induced COX-2 and mPGES-1-dependent PGE2production in pulmonary epithelial cells. Legionella induced the release of PGE2in primary human small airway epithelial cells and A549 cells. This was accompanied by an increased expression of COX-2 and mPGES-1 as well as an increased PLA2activity in infected cells. Deletion of the type IV secretion system Dot/Icm did not impair Legionella-related COX-2 expression or PGE2release in A549 cells. L. pneumophila induced the degradation of IκBα and activated NF-κB. Inhibition of IKK blocked L. pneumophila-induced PGE2release and COX-2 expression. We noted activation of p38 and p42/44 MAP kinase in Legionella-infected A549 cells. Moreover, membrane translocation and activation of PKCα was observed in infected cells. PKCα and p38 and p42/44 MAP kinase inhibitors reduced PGE2release and COX-2 expression. In summary, PKCα and p38 and p42/44 MAP kinase controlled COX-2 expression and subsequent PGE2release by Legionella-infected lung epithelial cells. These pathways may significantly contribute to the host response in Legionnaires' disease.
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Watarai, Masahisa, Isabelle Derre, James Kirby, Joseph D. Growney, William F. Dietrich, and Ralph R. Isberg. "Legionella pneumophila Is Internalized by a Macropinocytotic Uptake Pathway Controlled by the Dot/Icm System and the Mouse Lgn1 Locus✪." Journal of Experimental Medicine 194, no. 8 (October 15, 2001): 1081–96. http://dx.doi.org/10.1084/jem.194.8.1081.
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The products of the Legionella pneumophila dot/icm genes enable the bacterium to replicate within a macrophage vacuole. This study demonstrates that the Dot/Icm machinery promotes macropinocytotic uptake of L. pneumophila into mouse macrophages. In mouse strains harboring a permissive Lgn1 allele, L. pneumophila promoted formation of vacuoles that were morphologically similar to macropinosomes and dependent on the presence of an intact Dot/Icm system. Macropinosome formation appeared to occur during, rather than after, the closure of the plasma membrane about the bacterium, since a fluid-phase marker preloaded into the macrophage endocytic path failed to label the bacterium-laden macropinosome. The resulting macropinosomes were rich in GM1 gangliosides and glycosylphosphatidylinositol-linked proteins. The Lgn1 allele restrictive for L. pneumophila intracellular replication prevented dot/icm-dependent macropinocytosis, with the result that phagosomes bearing the microorganism were targeted into the endocytic network. Analysis of macrophages from recombinant inbred mouse strains support the model that macropinocytotic uptake is controlled by the Lgn1 locus. These results indicate that the products of the dot/icm genes and Lgn1 are involved in controlling an internalization route initiated at the time of bacterial contact with the plasma membrane.
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de Felipe, Karim Suwwan, Sergey Pampou, Oliver S. Jovanovic, Christopher D. Pericone, Senna F. Ye, Sergey Kalachikov, and Howard A. Shuman. "Evidence for Acquisition of Legionella Type IV Secretion Substrates via Interdomain Horizontal Gene Transfer." Journal of Bacteriology 187, no. 22 (November 15, 2005): 7716–26. http://dx.doi.org/10.1128/jb.187.22.7716-7726.2005.
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ABSTRACT Intracellular pathogens exploit host cell functions to create a replication niche inside eukaryotic cells. The causative agent of Legionnaires' disease, the γ-proteobacterium Legionella pneumophila, resides and replicates within a modified vacuole of protozoan and mammalian cells. L. pneumophila translocates effector proteins into host cells through the Icm-Dot complex, a specialized type IVB secretion system that is required for intracellular growth. To find out if some effector proteins may have been acquired through interdomain horizontal gene transfer (HGT), we performed a bioinformatic screen that searched for eukaryotic motifs in all open reading frames of the L. pneumophila Philadelphia-1 genome. We found 44 uncharacterized genes with many distinct eukaryotic motifs. Most of these genes contain G+C biases compared to other L. pneumophila genes, supporting the theory that they were acquired through HGT. Furthermore, we found that several of them are expressed and up-regulated in stationary phase in an RpoS-dependent manner. In addition, at least seven of these gene products are translocated into host cells via the Icm-Dot complex, confirming their role in the intracellular environment. Reminiscent of the case with most Icm-Dot substrates, most of the strains containing mutations in these genes grew comparably to the parent strain intracellularly. Our findings suggest that in L. pneumophila, interdomain HGT may have been a major mechanism for the acquisition of determinants of infection.
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Zusman, Tal, Elena Degtyar, and Gil Segal. "Identification of a Hypervariable Region Containing New Legionella pneumophila Icm/Dot Translocated Substrates by Using the Conserved icmQ Regulatory Signature." Infection and Immunity 76, no. 10 (August 11, 2008): 4581–91. http://dx.doi.org/10.1128/iai.00337-08.
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ABSTRACT Legionella pneumophila is an intracellular pathogen that has been shown to utilize the Icm/Dot type IV secretion system for pathogenesis. This system was shown to be composed of Icm/Dot complex components, accessory proteins, and a large number of translocated substrates. In this study, comparison of the icmQ regulatory regions from many Legionella species revealed a conserved regulatory sequence that includes the icmQ −10 promoter element. Mutagenesis of this conserved regulatory element indicated that each of the nucleotides in it affects the level of expression of the icmQ gene but not in a uniform fashion. A genomic analysis discovered that four additional genes in L. pneumophila contain this conserved regulatory sequence, which was found to function similarly in these genes as well. Examination of these four genes indicated that they are dispensable for intracellular growth, but two of them were found to encode new Icm/Dot translocated substrates (IDTS). Comparison of the genomic regions encoding these two IDTS among the four available L. pneumophila genomic sequences indicated that one of these genes is located in a hypervariable genomic region, which was shown before to contain an IDTS-encoding gene. Translocation analysis that was performed for nine proteins encoded from this hypervariable genomic region indicated that six of them are new IDTS which are translocated into host cells in an Icm/Dot-dependent manner. Furthermore, a bioinformatic analysis indicated that additional L. pneumophila genomic regions that contain several neighboring IDTS-encoding genes are hypervariable in gene content.
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Schroeder, Gunnar N., Nicola K. Petty, Aurélie Mousnier, Clare R. Harding, Adam J. Vogrin, Bryan Wee, Norman K. Fry, et al. "Legionella pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins." Journal of Bacteriology 192, no. 22 (September 10, 2010): 6001–16. http://dx.doi.org/10.1128/jb.00778-10.
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ABSTRACT Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.
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Molmeret, Maëlle, Marina Santic’, Rexford Asare, Reynold A. Carabeo, and Yousef Abu Kwaik. "Rapid Escape of the dot/icm Mutants of Legionella pneumophila into the Cytosol of Mammalian and Protozoan Cells." Infection and Immunity 75, no. 7 (April 16, 2007): 3290–304. http://dx.doi.org/10.1128/iai.00292-07.
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ABSTRACT The Legionella pneumophila-containing phagosome evades endocytic fusion and intercepts endoplasmic reticulum (ER)-to-Golgi vesicle traffic, which is believed to be mediated by the Dot/Icm type IV secretion system. Although phagosomes harboring dot/icm mutants are thought to mature through the endosomal-lysosomal pathway, colocalization studies with lysosomal markers have reported contradictory results. In addition, phagosomes harboring the dot/icm mutants do not interact with endocytosed materials, which is inconsistent with maturation of the phagosomes in the endosomal-lysosomal pathway. Using multiple strategies, we show that the dot/icm mutants defective in the Dot/Icm structural apparatus are unable to maintain the integrity of their phagosomes and escape into the cytoplasm within minutes of entry into various mammalian and protozoan cells in a process independent of the type II secretion system. In contrast, mutants defective in cytoplasmic chaperones of Dot/Icm effectors and rpoS, letA/S, and letE regulatory mutants are all localized within intact phagosomes. Importantly, non-dot/icm L. pneumophila mutants whose phagosomes acquire late endosomal-lysosomal markers are all located within intact phagosomes. Using high-resolution electron microscopy, we show that phagosomes harboring the dot/icm transporter mutants do not fuse to lysosomes but are free in the cytoplasm. Inhibition of ER-to-Golgi vesicle traffic by brefeldin A does not affect the integrity of the phagosomes harboring the parental strain of L. pneumophila. We conclude that the Dot/Icm transporter is involved in maintaining the integrity of the L. pneumophila phagosome, independent of interception of ER-to-Golgi vesicle traffic, which is a novel function of type IV secretion systems.
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Wong, Kathy, Yinglu Zhang, Guennadi Kozlov, and Kalle Gehring. "Structural studies of Legionella effectors." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C583. http://dx.doi.org/10.1107/s2053273314094169.
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Legionella pneumophila is a gram-negative bacterium that causes Legionnaires' disease. It uses a Dot/Icm type IV secretion system to inject effector proteins into the host cell to manipulate host processes. Currently, about 300 Icm/Dot dependent effectors of L.pneumophila have been identified. Lpg1496 is an effector protein, which contains a conserved domain from the SidE family. To date, the middle domain and the conserved SidE domain have been crystallized and the structure solved at a resolution of 1.15Å and 2.3Å, respectively. A structural homology search using the middle domain suggested a similarity to phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase, an ATPase involved in purine nucleotide synthesis. We performed 1H–15N HSQC NMR titrations to show that this domain binds ATP, ADP and AMP, with the highest binding affinity for ADP. A structural homology search using the SidE domain showed a similarity to cyclic nucleotide phosphodiesterases. To further elucidate the function of lpg1496, other fragments have been cloned, expressed, and subjected to crystallization trials. Currently, we have successfully crystallized the N-terminal domain, with crystals diffracting to <2.0Å. Obtaining the crystal structure of lpg1496 and revealing its function will not only lead to a better understanding of the virulence of L. pneumophila, but also contribute to the development of novel therapeutic treatments of Legionnaires' disease.
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Silveira, Tatiana N., and Dario S. Zamboni. "Pore Formation Triggered by Legionella spp. Is an Nlrc4 Inflammasome-Dependent Host Cell Response That Precedes Pyroptosis." Infection and Immunity 78, no. 3 (January 4, 2010): 1403–13. http://dx.doi.org/10.1128/iai.00905-09.
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ABSTRACT Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with interleukin-1β secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication, and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis, and L. rubrilucens, and this trait correlated with flagellin expression by these species. Together, the results suggest that pore formation is neither L. pneumophila specific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.
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Hilbi, Hubert, Gil Segal, and Howard A. Shuman. "Icm/Dot-dependent upregulation of phagocytosis by Legionella pneumophila." Molecular Microbiology 42, no. 3 (July 7, 2008): 603–17. http://dx.doi.org/10.1046/j.1365-2958.2001.02645.x.
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Chen, Yang, and Matthias P. Machner. "Targeting of the Small GTPase Rab6A′ by the Legionella pneumophila Effector LidA." Infection and Immunity 81, no. 6 (April 8, 2013): 2226–35. http://dx.doi.org/10.1128/iai.00157-13.
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ABSTRACTWhen the bacteriumLegionella pneumophila, the causative agent of Legionnaires' disease, is phagocytosed by alveolar macrophages, it delivers a large number of effector proteins through its Dot/Icm type IV secretion system into the host cell cytosol. Among those proteins is LidA, an effector that interacts with several host GTPases of the Rab family, including Rab6A′, a regulator of retrograde vesicle trafficking within eukaryotic cells. The effect of LidA on Rab6A′ function and the role of Rab6A′ forL. pneumophilagrowth within host cells has been unclear. Here, we show that LidA preferentially binds Rab6A′ in the active GTP-bound conformation. Rab6 binding occurred through the central region of LidA and followed a stoichiometry for LidA and Rab6A′ of 1:2. LidA maintained Rab6A′ in the active conformation by efficiently blocking the hydrolysis of GTP by Rab6A′, even in the presence of cellular GTPase-activating proteins, suggesting that the function of Rab6A′ must be important for efficient intracellular replication ofL. pneumophila. Accordingly, we found that production of constitutively inactive Rab6A′(T27N) but not constitutively active Rab6A′(Q72L) significantly reduced the ability ofL. pneumophilato initiate intracellular replication in human macrophages. Thus, the presence of an active pool of Rab6 within host cells early during infection is required to support efficient intracellular growth ofL. pneumophila.
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Miyake, Masaki, Takurou Watanabe, Hitomi Koike, Maëlle Molmeret, Yasuyuki Imai, and Yousef Abu Kwaik. "Characterization of Legionella pneumophila pmiA, a Gene Essential for Infectivity of Protozoa and Macrophages." Infection and Immunity 73, no. 10 (October 2005): 6272–82. http://dx.doi.org/10.1128/iai.73.10.6272-6282.2005.
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ABSTRACT The ability of Legionella pneumophila to cause pneumonia is dependent on intracellular replication within alveolar macrophages. The Icm/Dot secretion apparatus is essential for the ability of L. pneumophila to evade endocytic fusion, to remodel the phagosome by the endoplasmic reticulum (ER), and to replicate intracellularly. Protozoan and macrophage infectivity (pmi) mutants of L. pneumophila, which include 11 dot/icm mutants, exhibit defects in intracellular growth and replication within both protozoa and macrophages. In this study we characterized one of the pmi loci, pmiA. In contrast to the parental strain, the pmiA mutant is defective in cytopathogenicity for protozoa and macrophages. This is a novel mutant that exhibits a partial defect in survival within U937 human macrophage-like cells but exhibits a severe growth defect within Acanthamoeba polyphaga, which results in elimination from this host. The intracellular defects of this mutant are complemented by the wild-type pmiA gene on a plasmid. In contrast to phagosomes harboring the wild-type strain, which exclude endosomal-lysosomal markers, the pmiA mutant-containing phagosomes acquire the late endosomal-lysosomal markers LAMP-1 and LAMP-2. In contrast to the parental strain-containing phagosomes that are remodeled by the ER, there was a decrease in the number of ER-remodeled phagosomes harboring the pmiA mutant. Among several Legionella species examined, the pmiA gene is specific for L. pneumophila. The predicted amino acid sequence of the PmiA protein suggests that it is a transmembrane protein with three membrane-spanning regions. PmiA is similar to several hypothetical proteins produced by bacteria with a type IV secretion apparatus. Importantly, the defect in pmiA abolishes the pore-forming activity, which has been attributed to the Icm/Dot type IV secretion system. However, the mutant is sensitive to NaCl, and this sensitivity is abrogated in the icm/dot mutants. These results suggest that PmiA is a novel virulence factor that is involved in intracellular survival and replication of L. pneumophila in macrophages and protozoan cells.
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Buscher, Benjamin A., Gloria M. Conover, Jennifer L. Miller, Sinae A. Vogel, Stacey N. Meyers, Ralph R. Isberg, and Joseph P. Vogel. "The DotL Protein, a Member of the TraG-Coupling Protein Family, Is Essential for Viability of Legionella pneumophila Strain Lp02." Journal of Bacteriology 187, no. 9 (May 1, 2005): 2927–38. http://dx.doi.org/10.1128/jb.187.9.2927-2938.2005.
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ABSTRACT Legionella pneumophila is able to survive inside phagocytic cells by an internalization route that bypasses fusion of the nascent phagosome with the endocytic pathway to allow formation of a replicative phagosome. The dot/icm genes, a major virulence system of L. pneumophila, encode a type IVB secretion system that is required for intracellular growth. One Dot protein, DotL, has sequence similarity to type IV secretion system coupling proteins (T4CPs). In other systems, coupling proteins are not required for viability of the organism. Here we report the first example of a strain, L. pneumophila Lp02, in which a putative T4CP is essential for viability of the organism on bacteriological media. This result is particularly surprising since the majority of the dot/icm genes in Lp02 are dispensable for growth outside of a host cell, a condition that does not require a functional Dot/Icm secretion complex. We were able to isolate suppressors of the ΔdotL lethality and found that many contained mutations in other components of the Dot/Icm secretion system. A systematic analysis of dot/icm deletion mutants revealed that the majority of them (20 of 26) suppressed the lethality phenotype, indicating a partially assembled secretion system may be the source of ΔdotL toxicity in the wild-type strain. These results are consistent with a model in which the DotL protein plays a role in regulating the activity of the L. pneumophila type IV secretion apparatus.
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Neild, Annie, Takahiro Murata, and Craig R. Roy. "Processing and Major Histocompatibility Complex Class II Presentation of Legionella pneumophila Antigens by Infected Macrophages." Infection and Immunity 73, no. 4 (April 2005): 2336–43. http://dx.doi.org/10.1128/iai.73.4.2336-2343.2005.
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ABSTRACT To better understand interactions between the intracellular pathogen Legionella pneumophila and macrophages (Mφs), host and bacterial determinants important for presentation of antigens on major histocompatibility complex class II molecules (MHC-II) were investigated. It was determined that immune CD4 T-cell responses to murine bone marrow-derived Mφs (BMφs) infected with wild-type L. pneumophila were higher than the responses to avirulent dotA mutant bacteria. Although this enhanced response by immune T cells required modulation of vacuole transport mediated by the Dot/Icm system, it did not require intracellular replication of L. pneumophila. Intracellular cytokine staining identified a population of immune CD4 T cells that produced gamma interferon upon incubation with BMφs infected with wild-type L. pneumophila that did not respond to Mφ infection with dotA mutant bacteria. Endocytic processing was required for presentation of L. pneumophila antigens on MHC-II as determined by a defect in CD4 T-cell responses when the pH of BMφ endosomes was neutralized with chloroquine. Investigation of MHC-II presentation of antigens by BMφs infected with L. pneumophila icmR, icmW, and icmS mutants indicated that these mutants have an intermediate presentation phenotype relative to those of wild-type and dotA mutant bacteria. In addition, it was found that antigens from dot and icm mutants are presented earlier than antigens from wild-type L. pneumophila. Although immune CD4 T-cell responses to proteins secreted by the L. pneumophila Lsp system were not detected, it was found that the Lsp system is important for priming L. pneumophila-specific T cells in vivo. These data indicate that optimal antigen processing and MHC-II presentation to immune CD4 T cells involves synthesis of L. pneumophila proteins in an endoplasmic reticulum-derived compartment followed by transport to lysosomes.
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Ensminger, Alexander W., and Ralph R. Isberg. "E3 Ubiquitin Ligase Activity and Targeting of BAT3 by Multiple Legionella pneumophila Translocated Substrates." Infection and Immunity 78, no. 9 (June 14, 2010): 3905–19. http://dx.doi.org/10.1128/iai.00344-10.
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ABSTRACT The intracellular bacterial pathogen Legionella pneumophila modulates a number of host processes during intracellular growth, including the eukaryotic ubiquitination machinery, which dictates the stability, activity, and/or localization of a large number of proteins. A number of L. pneumophila proteins contain eukaryotic-like motifs typically associated with ubiquitination. Central among these is a family of five F-box-domain-containing proteins of Legionella pneumophila. Each of these five proteins is translocated to the host cytosol by the Dot/Icm type IV protein translocation system during infection. We show that three of these proteins, LegU1, LegAU13, and LicA, interact with components of the host ubiquitination machinery in vivo. In addition, LegU1 and LegAU13 are integrated into functional Skp-Cullin-F-box (SCF) complexes that confer E3 ubiquitin ligase activity. LegU1 specifically interacts with and can direct the ubiquitination of the host chaperone protein BAT3. In a screen for additional L. pneumophila proteins that associate with LegU1 in mammalian cells, we identified the bacterial protein Lpg2160. We demonstrate that Lpg2160 also associates with BAT3 independently of LegU1. We show that Lpg2160 is a translocated substrate of the Dot/Icm system and contains a C-terminal translocation signal. We propose a model in which LegU1 and Lpg2160 may function redundantly or in concert to modulate BAT3 activity during the course of infection.
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Faulkner, Gary, Sharon G. Berk, Elizabeth Garduño, Marco A. Ortiz-Jiménez, and Rafael A. Garduño. "Passage through Tetrahymena tropicalis Triggers a Rapid Morphological Differentiation in Legionella pneumophila." Journal of Bacteriology 190, no. 23 (September 19, 2008): 7728–38. http://dx.doi.org/10.1128/jb.00751-08.
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ABSTRACT The intracellular bacterial pathogen Legionella pneumophila follows a developmental cycle in which replicative forms (RFs) differentiate into infectious stationary-phase forms (SPFs) in vitro and in vivo into highly infectious mature intracellular forms (MIFs). The potential relationships between SPFs and MIFs remain uncharacterized. Previously we determined that L. pneumophila survives, but does not replicate, while it transiently resides (for 1 to 2 h) in food vacuoles of the freshwater ciliate Tetrahymena tropicalis before being expelled as legionellae-laden pellets. We report here that SPFs have the ability to rapidly (<1 h) and directly (in the absence of bacterial replication) differentiate into MIFs while in transit through T. tropicalis, indicating that SPFs and MIFs constitute a differentiation continuum. Mutant RFs lacking the sigma factor gene rpoS, or the response regulator gene letA, were unable to produce normal SPFs in vitro and did not fully differentiate into MIFs in vivo, further supporting the existence of a common mechanism of differentiation shared by SPFs and MIFs. Mutants with a defective Dot/Icm system morphologically differentiated into MIFs while in transit through T. tropicalis. Therefore, T. tropicalis has allowed us to unequivocally conclude that SPFs can directly differentiate into MIFs and that the Dot/Icm system is not required for differentiation, two events that could not be experimentally addressed before. The Tetrahymena model can now be exploited to study the signals that trigger MIF development in vivo and is the only replication-independent model reported to date that allows the differentiation of Dot/Icm mutants into MIFs.
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Zusman, Tal, Michal Feldman, Einat Halperin, and Gil Segal. "Characterization of the icmH and icmF Genes Required for Legionella pneumophila Intracellular Growth, Genes That Are Present in Many Bacteria Associated with Eukaryotic Cells." Infection and Immunity 72, no. 6 (June 2004): 3398–409. http://dx.doi.org/10.1128/iai.72.6.3398-3409.2004.
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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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So, Ernest C., Corinna Mattheis, Edward W. Tate, Gad Frankel, and Gunnar N. Schroeder. "Creating a customized intracellular niche: subversion of host cell signaling byLegionellatype IV secretion system effectors." Canadian Journal of Microbiology 61, no. 9 (September 2015): 617–35. http://dx.doi.org/10.1139/cjm-2015-0166.
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The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.
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Zink, Steven D., Lisa Pedersen, Nicholas P. Cianciotto, and Yousef Abu Kwaik. "The Dot/Icm Type IV Secretion System of Legionella pneumophila Is Essential for the Induction of Apoptosis in Human Macrophages." Infection and Immunity 70, no. 3 (March 2002): 1657–63. http://dx.doi.org/10.1128/iai.70.3.1657-1663.2002.
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ABSTRACT We have previously shown that Legionella pneumophila induces caspase 3-dependent apoptosis in mammalian cells during early stages of infection. In this report, we show that nine L. pneumophila strains with mutations in the dotA, dotDCB, icmT, icmGCD, and icmJB loci are completely defective in the induction of apoptosis, in addition to their severe defects in intracellular replication and pore formation-mediated cytotoxicity. Importantly, all nine dot/icm mutants were complemented for all their defective phenotypes with the respective wild-type loci. We show that the role of the Dot/Icm type IV secretion system in the induction of apoptosis is independent of the RtxA toxin, the dot/icm-regulated pore-forming toxin, and the type II secretion system. However, the pore-forming toxin, which is triggered upon entry into the postexponential growth phase, enhances the ability of L. pneumophila to induce apoptosis. Our data provide the first example of the role of a type IV secretion system of a bacterial pathogen in the induction of apoptosis in the host cell.
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Bandyopadhyay, Purnima, Shuqing Liu, Carolina B. Gabbai, Zeah Venitelli, and Howard M. Steinman. "Environmental Mimics and the Lvh Type IVA Secretion System Contribute to Virulence-Related Phenotypes of Legionella pneumophila." Infection and Immunity 75, no. 2 (November 13, 2006): 723–35. http://dx.doi.org/10.1128/iai.00956-06.
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ABSTRACT Legionella pneumophila, the causative organism of Legionnaires' disease, is a fresh-water bacterium and intracellular parasite of amoebae. This study examined the effects of incubation in water and amoeba encystment on L. pneumophila strain JR32 and null mutants in dot/icm genes encoding a type IVB secretion system required for entry, delayed acidification of L. pneumophila-containing phagosomes, and intracellular multiplication when stationary-phase bacteria infect amoebae and macrophages. Following incubation of stationary-phase cultures in water, mutants in dotA and dotB, essential for function of the type IVB secretion system, exhibited entry and delay of phagosome acidification comparable to that of strain JR32. Following encystment in Acanthamoeba castellanii and reversion of cysts to amoeba trophozoites, dotA and dotB mutants exhibited intracellular multiplication in amoebae. The L. pneumophila Lvh locus, encoding a type IVA secretion system homologous to that in Agrobacterium tumefaciens, was required for restoration of entry and intracellular multiplication in dot/icm mutants following incubation in water and amoeba encystment and was required for delay of phagosome acidification in strain JR32. These data support a model in which the Dot/Icm type IVB secretion system is conditionally rather than absolutely required for L. pneumophila virulence-related phenotypes. The data suggest that the Lvh type IVA secretion system, previously thought to be dispensable, is involved in virulence-related phenotypes under conditions mimicking the spread of Legionnaires' disease from environmental niches. Since environmental amoebae are implicated as reservoirs for an increasing number of environmental pathogens and for drug-resistant bacteria, the environmental mimics developed here may be useful in virulence studies of other pathogens.
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Gal-Mor, Ohad, and Gil Segal. "Identification of CpxR as a Positive Regulator of icm and dot Virulence Genes of Legionella pneumophila." Journal of Bacteriology 185, no. 16 (August 15, 2003): 4908–19. http://dx.doi.org/10.1128/jb.185.16.4908-4919.2003.
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ABSTRACT To date, 24 Legionella pneumophila genes (icm and dot genes) have been shown to be required for intercellular growth and host cell killing. A previous report indicated that the regulation of these genes is complicated and probably involves several regulatory proteins. In this study, a genetic screen performed in Escherichia coli identified the CpxR response regulator as an activator of the L. pneumophila icmR gene. Construction of an L. pneumophila cpxR insertion mutant showed that the expression of icmR is regulated by CpxR. In addition, a conserved CpxR binding site (GTAAA) was identified in the icmR regulatory region and L. pneumophila His-tagged CpxR protein was shown to bind to the icmR regulatory region using a mobility shift assay. Besides its dramatic effect on the icmR level of expression, the CpxR regulator was also found to affect the expression of the icmV-dotA and icmW-icmX operons, but to a lesser extent. The role of CpxA, the cognate sensor kinase of CpxR, was also examined and its effect on the icmR level of expression was found to be less pronounced than the effect of CpxR. The RpoE sigma factor, which was shown to coregulate genes together with CpxR, was examined as well, but it did not influence icm and dot gene expression. In addition, when the cpxR mutant strain, in which the expression of the icmR gene was dramatically reduced, and the cpxA and rpoE mutant strains were examined for their ability to grow inside Acanthamoeba castellanii and HL-60-derived human macrophages, no intracellular growth defect was observed. This study presents the first evidence for a direct regulator (CpxR) of an icm-dot virulence gene (icmR). The CpxR regulator together with other regulatory factors probably concerts with the expression of icm and dot genes to result in successful infection.
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Ensminger, Alexander W., and Ralph R. Isberg. "Legionella pneumophila Dot/Icm translocated substrates: a sum of parts." Current Opinion in Microbiology 12, no. 1 (February 2009): 67–73. http://dx.doi.org/10.1016/j.mib.2008.12.004.
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Albers, Urs, Katrin Reus, Howard A. Shuman, and Hubert Hilbi. "The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii." Microbiology 151, no. 1 (January 1, 2005): 167–82. http://dx.doi.org/10.1099/mic.0.27563-0.
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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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Gal-Mor, Ohad, Tal Zusman, and Gil Segal. "Analysis of DNA Regulatory Elements Required for Expression of the Legionella pneumophilaicm and dot Virulence Genes." Journal of Bacteriology 184, no. 14 (July 15, 2002): 3823–33. http://dx.doi.org/10.1128/jb.184.14.3823-3833.2002.
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ABSTRACT To investigate the regulation of the Legionella pneumophila icm and dot genes required for intracellular growth, a series of nine icm::lacZ fusions were constructed. These icm::lacZ fusions were found to have different levels of expression in L. pneumophila, and five of them were more highly expressed at stationary phase than at exponential phase. When the expression of these fusions in Escherichia coli was tested, all of them were found to be expressed but three of them had dramatic changes in their levels of expression in comparison to those in L. pneumophila. Site-directed and PCR random mutagenesis with these icm::lacZ fusions was used to identify DNA regulatory elements of icm genes. Four icm genes (icmT, icmP, icmQ, and icmM) that had low levels of expression in L. pneumophila were found to contain a 6-bp sequence (TATACT) essential for their expression. This sequence was shown by primer extension to serve as their −10 promoter elements. A similar sequence, which constitutes the −10 promoter elements of the icmV, icmW, and icmR genes which had high levels of expression in L. pneumophila, was also identified. In addition, regulatory elements that probably serve as binding sites for transcription regulators were found in these genes. Altogether, 12 regulatory elements, 7 of which constitute the −10 promoter elements of the icm genes, were found. Even though all the icm and dot genes are part of one system required for L. pneumophila intracellular growth and even though their promoters are probably recognized by the vegetative sigma factor, it seems that they are subjected to different regulation mediated by several regulatory factors.
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Bandyopadhyay, Purnima, Huifang Xiao, Hope A. Coleman, Alexa Price-Whelan, and Howard M. Steinman. "Icm/Dot-Independent Entry of Legionella pneumophila into Amoeba and Macrophage Hosts." Infection and Immunity 72, no. 8 (August 2004): 4541–51. http://dx.doi.org/10.1128/iai.72.8.4541-4551.2004.
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ABSTRACT Legionella pneumophila, the causative agent of Legionnaires' disease, expresses a type IVB secretion apparatus that translocates bacterial proteins into amoeba and macrophage hosts. When stationary-phase cultures are used to infect hosts, the type IVB apparatus encoded by the icm/dot genes is required for entry, delay of phagosome-lysosome fusion, and intracellular multiplication within host cells. Null mutants with mutations in icm/dot genes are defective in these phenotypes. Here a new model is described in which hosts are infected with stationary-phase cultures that have been incubated overnight in pH 6.5 buffer. This model is called Ers treatment because it enhances the resistance to acid, hydrogen peroxide, and antibiotic stress beyond that of stationary-phase cultures. Following Ers treatment entry into amoeba and macrophage hosts does not require dotA, which is essential for Legionella virulence phenotypes when hosts are infected with stationary-phase cultures, dotB, icmF, icmV, or icmX. Defective host entry is also suppressed for null mutants with mutations in the KatA and KatB catalase-peroxidase enzymes, which are required for proper intracellular growth in amoeba and macrophage hosts. Ers treatment-induced suppression of defective entry is not associated with increased bacterial adhesion to host cells or with morphological changes in the bacterial envelope but is dependent on protein expression during Ers treatment. By using proteomic analysis, Ers treatment was shown to induce a protein predicted to contain eight tetratricopeptide repeats, a motif previously implicated in enhanced entry of L. pneumophila. Characterization of Ers treatment-dependent changes in expression is proposed as an avenue for identifying icm/dot-independent factors that function in the entry of Legionella into amoeba and macrophage hosts.
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Altman, Efrat, and Gil Segal. "The Response Regulator CpxR Directly Regulates Expression of Several Legionella pneumophila icm/dot Components as Well as New Translocated Substrates." Journal of Bacteriology 190, no. 6 (January 11, 2008): 1985–96. http://dx.doi.org/10.1128/jb.01493-07.
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ABSTRACT Legionella pneumophila has been shown to utilize the icm/dot type IV secretion system for pathogenesis. This system was shown to be composed of icm/dot complex components and accessory proteins, as well as a large number of translocated substrates. Bioinformatic analysis of the regulatory regions of all the genes revealed that several icm/dot genes, as well as two genes encoding icm/dot translocated substrates, contain the conserved CpxR regulatory element, a regulator that has been shown previously to control the expression of the icmR gene. An experimental analysis, which included a comparison of gene expression in a L. pneumophila wild-type strain and gene expression in a cpxR deletion mutant, construction of mutants with mutations in the CpxR conserved regulatory elements, controlled expression studies, and mobility shift assays, demonstrated the direct relationship between the CpxR regulator and the expression of the genes. Furthermore, genomic analysis identified nine additional genes that contain a putative CpxR regulatory element; five of these genes (two legA genes and three ceg genes) were suggested previously to be putative icm/dot translocated substrates. The three ceg genes identified, which were shown previously to contain a putative PmrA regulatory element, were found here to be regulated by both CpxR and PmrA. The other six genes (two legA genes and four new genes products were found to be regulated by CpxR. Moreover, using the CyaA translocation assay, these nine gene products were found to be translocated into host cells in an Icm/Dot-dependent manner. Our results establish that the CpxR regulator is a fundamental regulator of the icm/dot type IV secretion system in L. pneumophila.
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Abu-Zant, Alaeddin, Marina Santic, Maelle Molmeret, Snake Jones, Jürgen Helbig, and Yousef Abu Kwaik. "Incomplete Activation of Macrophage Apoptosis during Intracellular Replication of Legionella pneumophila." Infection and Immunity 73, no. 9 (September 2005): 5339–49. http://dx.doi.org/10.1128/iai.73.9.5339-5349.2005.
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ABSTRACT The ability of the intracellular bacterium Legionella pneumophila to cause disease is totally dependent on its ability to modulate the biogenesis of its phagosome and to replicate within alveolar cells. Upon invasion, L. pneumophila activates caspase-3 in macrophages, monocytes, and alveolar epithelial cells in a Dot/Icm-dependent manner that is independent of the extrinsic or intrinsic pathway of apoptosis, suggesting a novel mechanism of caspase-3 activation by this intracellular pathogen. We have shown that the inhibition of caspase-3 prior to infection results in altered biogenesis of the L. pneumophila-containing phagosome and in an inhibition of intracellular replication. In this report, we show that the preactivation of caspase-3 prior to infection does not rescue the intracellular replication of L. pneumophila icmS, icmR, and icmQ mutant strains. Interestingly, preactivation of caspase-3 through the intrinsic and extrinsic pathways of apoptosis in both human and mouse macrophages inhibits intracellular replication of the parental stain of L. pneumophila. Using single-cell analysis, we show that intracellular L. pneumophila induces a robust activation of caspase-3 during exponential replication. Surprisingly, despite this robust activation of caspase-3 in the infected cell, the host cell does not undergo apoptosis until late stages of infection. In sharp contrast, the activation of caspase-3 by apoptosis-inducing agents occurs concomitantly with the apoptotic death of all cells that exhibit caspase-3 activation. It is only at a later stage of infection, and concomitant with the termination of intracellular replication, that the L. pneumophila-infected cells undergo apoptotic death. We conclude that although a robust activation of caspase-3 is exhibited throughout the exponential intracellular replication of L. pneumophila, apoptotic cell death is not executed until late stages of the infection, concomitant with the termination of intracellular replication.
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Solomon, Jonathan M., Adam Rupper, James A. Cardelli, and Ralph R. Isberg. "Intracellular Growth of Legionella pneumophila in Dictyostelium discoideum, a System for Genetic Analysis of Host-Pathogen Interactions." Infection and Immunity 68, no. 5 (May 1, 2000): 2939–47. http://dx.doi.org/10.1128/iai.68.5.2939-2947.2000.
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ABSTRACT Conditions were established in which Legionella pneumophila, an intracellular bacterial pathogen, could replicate within the unicellular organism Dictyostelium discoideum. By several criteria, L. pneumophila grew by the same mechanism within D. discoideum as it does in amoebae and macrophages. Bacteria grew within membrane-bound vesicles associated with rough endoplasmic reticulum, and L. pneumophila dot/icm mutants, blocked for growth in macrophages and amoebae, also did not grow in D. discoideum. Internalized L. pneumophila avoided degradation by D. discoideum and showed evidence of reduced fusion with endocytic compartments. The ability of L. pneumophila to grow within D. discoideum depended on the growth state of the cells. D. discoideum grown as adherent monolayers was susceptible toL. pneumophila infection and to contact-dependent cytotoxicity during high-multiplicity infections, whereas D. discoideum grown in suspension was relatively resistant to cytotoxicity and did not support intracellular growth. Some knownD. discoideum mutants were examined for their effect on growth of L. pneumophila. The coronin mutant and themyoA/B double myosin I mutant were more permissive than wild-type strains for intracellular growth. Growth of L. pneumophila in a Gβ mutant was slightly reduced compared to the parent strain. This work demonstrates the usefulness of the L. pneumophila-D. discoideum system for genetic analysis of host-pathogen interactions.
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Tiaden, André, Thomas Spirig, Paula Carranza, Holger Brüggemann, Kathrin Riedel, Leo Eberl, Carmen Buchrieser, and Hubert Hilbi. "Synergistic Contribution of the Legionella pneumophila lqs Genes to Pathogen-Host Interactions." Journal of Bacteriology 190, no. 22 (September 19, 2008): 7532–47. http://dx.doi.org/10.1128/jb.01002-08.
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ABSTRACT The causative agent of Legionnaires' disease, Legionella pneumophila, is a natural parasite of environmental protozoa and employs a biphasic life style to switch between a replicative and a transmissive (virulent) phase. L. pneumophila harbors the lqs (Legionella quorum sensing) cluster, which includes genes encoding the autoinducer synthase LqsA, the sensor kinase LqsS, the response regulator LqsR, and a homologue of HdeD, which is involved in acid resistance in Escherichia coli. LqsR promotes host-cell interactions as an element of the stationary-phase virulence regulatory network. Here, we characterize L. pneumophila mutant strains lacking all four genes of the lqs cluster or only the hdeD gene. While an hdeD mutant strain did not have overt physiological or virulence phenotypes, an lqs mutant showed an aberrant morphology in stationary growth phase and was defective for intracellular growth, efficient phagocytosis, and cytotoxicity against host cells. Cytotoxicity was restored upon reintroduction of the lqs genes into the chromosome of an lqs mutant strain. The deletion of the lqs cluster caused more-severe phenotypes than deletion of only lqsR, suggesting a synergistic effect of the other lqs genes. A transcriptome analysis indicated that in the stationary phase more than 380 genes were differentially regulated in the lqs mutant and wild-type L. pneumophila. Genes involved in protein production, metabolism, and bioenergetics were upregulated in the lqs mutant, whereas genes encoding virulence factors, such as effectors secreted by the Icm/Dot type IV secretion system, were downregulated. A proteome analysis revealed that a set of Icm/Dot substrates is not produced in the absence of the lqs gene cluster, which confirms the findings from DNA microarray assays and mirrors the virulence phenotype of the lqs mutant strain.
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Feldman, Michal, and Gil Segal. "A Specific Genomic Location within the icm/dot Pathogenesis Region of Different Legionella Species Encodes Functionally Similar but Nonhomologous Virulence Proteins." Infection and Immunity 72, no. 8 (August 2004): 4503–11. http://dx.doi.org/10.1128/iai.72.8.4503-4511.2004.
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ABSTRACT Legionella pneumophila, the major causative agent of Legionnaires' disease, is a facultative intracellular pathogen that grows within human macrophages and amoebae. Intracellular growth involves the formation of a replicative phagosome that requires the Icm/Dot type IV secretion system. Part of the icm/dot region in L. pneumophila contains the icmTSRQPO genes. The proteins encoded by the icmR and icmQ genes were shown to exhibit a chaperone-substrate relationship. Analysis of this region from other pathogenic Legionella species, i.e., L. micdadei and L. longbeachae, indicated that the overall organization of this region is highly conserved, but it was found to contain a favorable site for gene variation. In the place where the icmR gene was expected to be located, other open reading frames that are nonhomologous to each other or to any entry in the GenBank database were found (migAB in L. micdadei and ligB in L. longbeachae). Examination of these unique genes revealed an outstanding phenomenon; by use of interspecies complementation, the icmR, migB, and ligB gene products were found to be functionally similar. In addition, the function of these proteins was usually dependent on the presence of the corresponding IcmQ proteins. Moreover, each of these proteins (IcmR, LigB, and MigB) was found to interact with the corresponding IcmQ proteins, and the genes encoding these proteins were found to be regulated by CpxR. This study reveals new evidence of gene variation occurring in the same genomic location within the icm/dot locus in various Legionella species. The genes found at this site were shown to be similarly regulated and to encode species-specific, nonhomologous, but functionally similar proteins.
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Hurtado-Guerrero, Ramon, Tal Zusman, Shalini Pathak, Adel F. M. Ibrahim, Sharon Shepherd, Alan Prescott, Gil Segal, and Daan M. F. van Aalten. "Molecular mechanism of elongation factor 1A inhibition by a Legionella pneumophila glycosyltransferase." Biochemical Journal 426, no. 3 (February 24, 2010): 281–92. http://dx.doi.org/10.1042/bj20091351.
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Legionnaires' disease is caused by a lethal colonization of alveolar macrophages with the Gram-negative bacterium Legionella pneumophila. LpGT (L. pneumophila glucosyltransferase; also known as Lgt1) has recently been identified as a virulence factor, shutting down protein synthesis in the human cell by specific glucosylation of EF1A (elongation factor 1A), using an unknown mode of substrate recognition and a retaining mechanism for glycosyl transfer. We have determined the crystal structure of LpGT in complex with substrates, revealing a GT-A fold with two unusual protruding domains. Through structure-guided mutagenesis of LpGT, several residues essential for binding of the UDP-glucose-donor and EF1A-acceptor substrates were identified, which also affected L. pneumophila virulence as demonstrated by microinjection studies. Together, these results suggested that a positively charged EF1A loop binds to a negatively charged conserved groove on the LpGT structure, and that two asparagine residues are essential for catalysis. Furthermore, we showed that two further L. pneumophila glycosyltransferases possessed the conserved UDP-glucose-binding sites and EF1A-binding grooves, and are, like LpGT, translocated into the macrophage through the Icm/Dot (intracellular multiplication/defect in organelle trafficking) system.
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Hojo, Fuhito, Daisuke Sato, Junji Matsuo, Masaki Miyake, Shinji Nakamura, Miyuki Kunichika, Yasuhiro Hayashi, et al. "Ciliates Expel Environmental Legionella-Laden Pellets To Stockpile Food." Applied and Environmental Microbiology 78, no. 15 (May 25, 2012): 5247–57. http://dx.doi.org/10.1128/aem.00421-12.
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ABSTRACTWhenTetrahymenaciliates are cultured withLegionella pneumophila, the ciliates expel bacteria packaged in free spherical pellets. Why the ciliates expel these pellets remains unclear. Hence, we determined the optimal conditions for pellet expulsion and assessed whether pellet expulsion contributes to the maintenance of growth and the survival of ciliates. When incubated with environmentalL. pneumophila, the ciliates expelled the pellets maximally at 2 days after infection. Heat-killed bacteria failed to produce pellets from ciliates, and there was no obvious difference in pellet production among the ciliates or bacterial strains. Morphological studies assessing lipid accumulation showed that pellets contained tightly packed bacteria with rapid lipid accumulation and were composed of the layers of membranes; bacterial culturability in the pellets rapidly decreased, in contrast to what was seen in ciliate-free culture, although the bacteria maintained membrane integrity in the pellets. Furthermore, ciliates newly cultured with pellets were maintained and grew vigorously compared with those without pellets. In contrast, a humanL. pneumophilaisolate killed ciliates 7 days postinfection in a Dot/Icm-dependent manner, and pellets harboring this strain did not support ciliate growth. Also, pellets harboring the human isolate were resuscitated by coculturing with amoebae, depending on Dot/Icm expression. Thus, while ciliates expel pellet-packaged environmentalL. pneumophilafor stockpiling food, the pellets packaging the human isolate are harmful to ciliate survival, which may be of clinical significance.
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Newton, Hayley J., Fiona M. Sansom, Jenny Dao, Adrian D. McAlister, Joan Sloan, Nicholas P. Cianciotto, and Elizabeth L. Hartland. "Sel1 Repeat Protein LpnE Is a Legionella pneumophila Virulence Determinant That Influences Vacuolar Trafficking." Infection and Immunity 75, no. 12 (September 24, 2007): 5575–85. http://dx.doi.org/10.1128/iai.00443-07.
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ABSTRACT The environmental pathogen Legionella pneumophila possesses five proteins with Sel1 repeats (SLRs) from the tetratricopeptide repeat protein family. Three of these proteins, LpnE, EnhC, and LidL, have been implicated in the ability of L. pneumophila to efficiently establish infection and/or manipulate host cell trafficking events. Previously, we showed that LpnE is important for L. pneumophila entry into macrophages and epithelial cells. In further virulence studies here, we show that LpnE is also required for efficient infection of Acanthamoeba castellanii by L. pneumophila and for replication of L. pneumophila in the lungs of A/J mice. In addition, we found that the role of LpnE in host cell invasion is dependent on the eight SLR regions of the protein. A truncated form of LpnE lacking the two C-terminal SLR domains was unable to complement the invasion defect of an lpnE mutant of L. pneumophila 130b in both the A549 and THP-1 cell lines. The lpnE mutant displayed impaired avoidance of LAMP-1 association, suggesting that LpnE influenced trafficking of the L. pneumophila vacuole, similar to the case for EnhC and LidL. We also found that LpnE was present in L. pneumophila culture supernatants and that its export was independent of both the Lsp type II secretion system and the Dot/Icm type IV secretion system. The fact that LpnE was exported suggested that the protein may interact with a eukaryotic protein. Using LpnE as bait, we screened a HeLa cell cDNA library for interacting partners, using the yeast two-hybrid system. Examination of the protein-protein interaction between LpnE and a eukaryotic protein, obscurin-like protein 1, suggested that LpnE can interact with eukaryotic proteins containing immunoglobulin-like folds via the SLR regions. This investigation has further characterized the contribution of LpnE to L. pneumophila virulence and, more specifically, the importance of the SLR regions to LpnE function.
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Harding, Clare R., Charlotte A. Stoneham, Ralf Schuelein, Hayley Newton, Clare V. Oates, Elizabeth L. Hartland, Gunnar N. Schroeder, and Gad Frankel. "The Dot/Icm Effector SdhA Is Necessary for Virulence of Legionella pneumophila in Galleria mellonella and A/J Mice." Infection and Immunity 81, no. 7 (May 6, 2013): 2598–605. http://dx.doi.org/10.1128/iai.00296-13.
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ABSTRACTLegionella pneumophilais an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed theLegionella-containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacterium. Here, we examined the role of two important virulence factors ofL. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in theGalleria mellonellainfection model. We demonstrate that flagellin expression does not contribute to virulence, replication, or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in hemocytes showed signs of instability and leakage. Furthermore, in contrast to wild-typeL. pneumophila, a ΔsdhAmutant caused a transient depletion of hemocytes and reduced mortality. Analysis of the ΔsdhAmutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.
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Miyamoto, Hiroshi, Shin-ichi Yoshida, Hatsumi Taniguchi, and Howard A. Shuman. "Virulence Conversion of Legionella pneumophila by Conjugal Transfer of Chromosomal DNA." Journal of Bacteriology 185, no. 22 (November 15, 2003): 6712–18. http://dx.doi.org/10.1128/jb.185.22.6712-6718.2003.
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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.
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Zusman, Tal, Ohad Gal-Mor, and Gil Segal. "Characterization of a Legionella pneumophila relA Insertion Mutant and Roles of RelA and RpoS in Virulence Gene Expression." Journal of Bacteriology 184, no. 1 (January 1, 2002): 67–75. http://dx.doi.org/10.1128/jb.184.1.67-75.2002.
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ABSTRACT To investigate the involvement of RelA in the regulation of Legionella pneumophila virulence, a deletion substitution was constructed in the relA gene. The relA knockout resulted in an undetectable level of ppGpp in the cells during the stationary phase, but the original level was restored when the relA gene product was supplied on a plasmid. The effect of the relA mutation was examined with two systems that are known to be expressed during the stationary phase in L. pneumophila. Pigment production was found to be dependent on the relA gene product, and only one-half as much pigment was produced by the relA mutant as by the wild-type strain. Flagellum gene expression was also found to be dependent on the relA gene product, as determined with a flaA::lacZ fusion. However, the relA gene product was found to be dispensable for intracellular growth both in HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii. To determine the involvement of the relA gene product in expression of L. pneumophila genes required for intracellular growth (icm/dot genes), nine icm::lacZ fusions were constructed, and expression of these fusions in the wild-type strain was compared with their expression in relA mutant strains. Expression of only one of the icm::lacZ fusions was moderately reduced in the relA mutant strain. Expression of the nine icm::lacZ fusions was also examined in a strain containing an insertion in the gene that codes for the stationary-phase sigma factor RpoS, and similar results were obtained. We concluded that RelA is dispensable for intracellular growth of L. pneumophila in the two hosts examined and that both RelA and RpoS play minor roles in L. pneumophila icm/dot gene expression.