scholarly journals Flagellum of Legionella pneumophilaPositively Affects the Early Phase of Infection of Eukaryotic Host Cells

2001 ◽  
Vol 69 (4) ◽  
pp. 2116-2122 ◽  
Author(s):  
Claudia Dietrich ◽  
Klaus Heuner ◽  
Bettina C. Brand ◽  
Jörg Hacker ◽  
Michael Steinert
Keyword(s):  
Electron Microscopy ◽  
Legionella Pneumophila ◽  
Kanamycin Resistance ◽  
Etiologic Agent ◽  
Polyclonal Antiserum ◽  
Host Cells ◽  
Kanamycin Resistance Cassette ◽  
Legionnaires Disease ◽  
Invasion Capacity

ABSTRACT Legionella pneumophila, the etiologic agent of Legionnaires' disease, contains a single, monopolar flagellum which is composed of one major subunit, the FlaA protein. To evaluate the role of the flagellum in the pathogenesis and ecology ofLegionella, the flaA gene of L. pneumophila Corby was mutagenized by introduction of a kanamycin resistance cassette. Immunoblots with antiflagellin-specific polyclonal antiserum, electron microscopy, and motility assays confirmed that the specific flagellar mutant L. pneumophila Corby KH3 was nonflagellated. The redelivery of the intact flaA gene into the chromosome (L. pneumophila Corby CD10) completely restored flagellation and motility. Coculture studies showed that the invasion efficiency of the flaA mutant was moderately reduced in amoebae and severely reduced in HL-60 cells. In contrast, adhesion and the intracellular rate of replication remained unaffected. Taking these results together, we have demonstrated that the flagellum of L. pneumophila positively affects the establishment of infection by facilitating the encounter of the host cell as well as by enhancing the invasion capacity.

scholarly journals Legionella pneumophila Entry GenertxA Is Involved in Virulence

2001 ◽  
Vol 69 (1) ◽  
pp. 508-517 ◽  
Author(s):  
Suat L. G. Cirillo ◽  
Luiz E. Bermudez ◽  
Sahar H. El-Etr ◽  
Gerald E. Duhamel ◽  
Jeffrey D. Cirillo
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Integrin Receptors ◽  
Legionnaires Disease ◽  
Cell Spread ◽  
Gain Access

ABSTRACT Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogen Legionella pneumophila. Currently, the mechanisms used by L. pneumophila to gain access to the intracellular environment are not well understood. We have recently isolated three loci, designated enh1,enh2, and enh3, that are involved in the ability of L. pneumophila to enter host cells. One of the genes present in the enh1 locus, rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to β2 integrin receptors. In the current study, we demonstrate that the L. pneumophila rtxA gene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, an rtxA mutant does not replicate as well as wild-type L. pneumophila in monocytes and is less virulent in mice. Thus, we conclude that the entry genertxA is an important virulence determinant in L. pneumophila and is likely to be critical for the production of Legionnaires' disease in humans.

scholarly journals Influence of the Alternative σ28 Factor on Virulence and Flagellum Expression of Legionella pneumophila

2002 ◽  
Vol 70 (3) ◽  
pp. 1604-1608 ◽  
Author(s):  
Klaus Heuner ◽  
Claudia Dietrich ◽  
Carina Skriwan ◽  
Michael Steinert ◽  
Jörg Hacker
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Mutant Strain ◽  
Legionella Pneumophila ◽  
Blot Analysis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Kanamycin Resistance Cassette

ABSTRACT The fliA gene of Legionella pneumophila encoding the alternative σ28 factor was inactivated by introducing a kanamycin resistance cassette. Electron microscopy and Western blot analysis revealed that the fliA mutant strain is aflagellate and expresses no flagellin. Reporter gene assays indicated that the flaA promoter is not active in the fliA mutant strain. The fliA mutant strain multiplied less effectively in coculture with amoebae than the wild-type strain and was not able to replicate in coculture with Dictyostelium discoideum.

scholarly journals Cyclic Diguanylate Signaling Proteins Control Intracellular Growth of Legionella pneumophila

mBio ◽  
2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Assaf Levi ◽  
Marc Folcher ◽  
Urs Jenal ◽  
Howard A. Shuman
Keyword(s):  
Causative Agent ◽  
Legionella Pneumophila ◽  
Second Messenger ◽  
Host Cells ◽  
Diguanylate Cyclase ◽  
Intracellular Growth ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Legionnaires Disease

ABSTRACTProteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle ofLegionella pneumophila, the causative agent of Legionnaires’ disease, has not previously been examined. TheL. pneumophilagenome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes ascdgS(cyclicdiguanylatesignaling) genes. Strains ofL. pneumophilacontaining deletions of all individualcdgSgenes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, severalcdgSgenes strongly decreased the ability ofL. pneumophilato grow inside host cells. Expression of thesecdgSgenes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth inL. pneumophila.L. pneumophilastrains overexpressing thesecdgSgenes were less cytotoxic to THP-1 macrophages than wild-typeL. pneumophilabut retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused bycdgSgene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of thecdgSgenes in aSalmonella entericaserovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that severalcdgSgenes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability ofL. pneumophilato grow in host cells.IMPORTANCEAll bacteria must sense and respond to environmental cues. Intracellular bacterial pathogens must detect and respond to host functions that limit their ability to carry out a successful infection. Small-molecule second messengers play key roles in transmitting signals from environmental receptors to the proteins and other components that respond to signals. Cyclic diguanylate is a ubiquitous bacterial second messenger known to play an important role in many sensing and signaling systems in bacteria. The causative agent of Legionnaires’ disease,Legionella pneumophila, is an intracellular pathogen that grows inside environmental protists and human macrophages by subverting the normal processes that these cells use to capture and destroy bacteria. We show that the several cyclic diguanylate signaling components inLegionellaplay a role in the ability to grow inside both kinds of host cells. This work highlights the role of cyclic diguanylate signaling during intracellular growth.

scholarly journals Legionella pneumophila CRISPR-Cas suggests recurrent encounters with one or more phages in the family Microviridae .

2021 ◽  
Author(s):  
Shayna R. Deecker ◽  
Malene L. Urbanus ◽  
Beth Nicholson ◽  
Alexander W. Ensminger
Keyword(s):  
Legionella Pneumophila ◽  
Sequence Similarity ◽  
Mobile Element ◽  
Current Data ◽  
Significant Sequence Similarity ◽  
Remediation Strategies ◽  
The Family ◽  
Legionnaires Disease ◽  
Outstanding Question

Legionella pneumophila is a ubiquitous freshwater pathogen and the causative agent of Legionnaires’ disease. L. pneumophila growth within protists provides a refuge from desiccation, disinfection, and other remediation strategies. One outstanding question has been whether this protection extends to phages. L. pneumophila isolates are remarkably devoid of prophages and to date no Legionella phages have been identified. Nevertheless, many L. pneumophila isolates maintain active CRISPR-Cas defenses. So far, the only known target of these systems is an episomal element that we previously named Legionella Mobile Element-1 (LME-1). The continued expansion of publicly available genomic data promises to further our understanding of the role of these systems. We now describe over 150 CRISPR-Cas systems across 600 isolates to establish the clearest picture yet of L. pneumophila ’s adaptive defenses. By searching for targets of 1,500 unique CRISPR-Cas spacers, LME-1 remains the only identified CRISPR-Cas targeted integrative element. We identified 3 additional LME-1 variants - all targeted by previously and newly identified CRISPR-Cas spacers - but no other similar elements. Notably, we also identified several spacers with significant sequence similarity to microviruses, specifically those within the subfamily Gokushovirinae . These spacers are found across several different CRISPR-Cas arrays isolated from geographically diverse isolates, indicating recurrent encounters with these phages. Our analysis of the extended Legionella CRISPR-Cas spacer catalog leads to two main conclusions: current data argue against CRISPR-Cas targeted integrative elements beyond LME-1, and the heretofore unknown L. pneumophila phages are most likely lytic gokushoviruses. IMPORTANCE Legionnaires’ disease is an often-fatal pneumonia caused by Legionella pneumophila , which normally grows inside amoebae and other freshwater protists. L. pneumophila trades diminished access to nutrients for the protection and isolation provided by the host. One outstanding question is whether L. pneumophila is susceptible to phages, given the protection provided by its intracellular lifestyle. In this work, we use Legionella CRISPR spacer sequences as a record of phage infection to predict that the “missing” L. pneumophila phages belong to the microvirus subfamily Gokushovirinae . Gokushoviruses are known to infect another intracellular pathogen, Chlamydia . How do gokushoviruses access L. pneumophila (and Chlamydia ) inside their “cozy niches”? Does exposure to phages happen during a transient extracellular period (during cell-to-cell spread) or is it indicative of a more complicated environmental lifestyle? One thing is clear, 100 years after their discovery, phages continue to hold important secrets about the bacteria upon which they prey.

The Legionella pneumophila effector RavY contributes to a replication-permissive vacuolar environment during infection

2021 ◽  
Author(s):  
Luying Liu ◽  
Craig R. Roy
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Effector Protein ◽  
Intracellular Replication ◽  
Phagocytic Cells ◽  
Host Molecules ◽  
Type Iv ◽  
Legionnaires Disease

Legionella pneumophila is the causative agent of Legionnaires’ Disease and is capable replicating inside phagocytic cells such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila , but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, nor does RavY contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild type L. pneumophila . Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.

scholarly journals Polar targeting and assembly of theLegionellaDot/Icm type IV secretion system (T4SS) by T6SS-related components

2018 ◽  
Author(s):  
KwangCheol C. Jeong ◽  
Jacob Gyore ◽  
Lin Teng ◽  
Debnath Ghosal ◽  
Grant J. Jensen ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Membrane Complex ◽  
Type Iv ◽  
Legionnaires Disease ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

SummaryLegionella pneumophila, the causative agent of Legionnaires’ disease, survives and replicates inside amoebae and macrophages by injecting a large number of protein effectors into the host cells’ cytoplasm via the Dot/Icm type IVB secretion system (T4BSS). Previously, we showed that the Dot/Icm T4BSS is localized to both poles of the bacterium and that polar secretion is necessary for the proper targeting of theLegionellacontaining vacuole (LCV). Here we show that polar targeting of the Dot/Icm core-transmembrane subcomplex (DotC, DotD, DotF, DotG and DotH) is mediated by two Dot/Icm proteins, DotU and IcmF, which are able to localize to the poles ofL. pneumophilaby themselves. Interestingly, DotU and IcmF are homologs of the T6SS components TssL and TssM, which are part of the T6SS membrane complex (MC). We propose thatLegionellaco-opted these T6SS components to a novel function that mediates subcellular localization and assembly of this T4SS. Finally, in depth examination of the biogenesis pathway revealed that polar targeting and assembly of theLegionellaT4BSS apparatus is mediated by an innovative “outside-inside” mechanism.

scholarly journals Coiling Phagocytosis of Trypanosomatids and Fungal Cells

1998 ◽  
Vol 66 (9) ◽  
pp. 4331-4339 ◽  
Author(s):  
M. G. Rittig ◽  
K. Schröppel ◽  
K.-H. Seack ◽  
U. Sander ◽  
E.-N. N’Diaye ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Legionella Pneumophila ◽  
Leishmania Major ◽  
Posterior Pole ◽  
Host Cells ◽  
Human Monocytes ◽  
Eukaryotic Microorganisms ◽  
Microscopic Studies

ABSTRACT Coiling phagocytosis has previously been studied only with the bacteria Legionella pneumophila and Borrelia burgdorferi, and the results were inconsistent. To learn more about this unconventional phagocytic mechanism, the uptake of various eukaryotic microorganisms by human monocytes, murine macrophages, and murine dendritic cells was investigated in vitro by video and electron microscopy. Unconventional phagocytosis of Leishmania spp. promastigotes, Trypanosoma cruzi trypomastigotes,Candida albicans hyphae, and zymosan particles fromSaccharomyces cerevisiae differed in (i) morphology (rotating unilateral pseudopods with the trypanosomatids, overlapping bilateral pseudopods with the fungi), (ii) frequency (high withLeishmania; occasional with the fungi; rare with T. cruzi), (iii) duration (rapid with zymosan; moderate with the trypanosomatids; slow with C. albicans), (iv) localization along the promastigotes (flagellum of Leishmania major andL. aethiopica; flagellum or posterior pole of L. donovani), and (v) dependence on complement (strong with L. major and L. donovani; moderate with the fungi; none with L. aethiopica). All of these various types of unconventional phagocytosis gave rise to similar pseudopod stacks which eventually transformed to a regular phagosome. Further video microscopic studies with L. major provided evidence for a cytosolic localization, synchronized replication, and exocytic release of the parasites, extending traditional concepts about leishmanial infection of host cells. It is concluded that coiling phagocytosis comprises phenotypically similar consequences of various disturbances in conventional phagocytosis rather than representing a single separate mechanism.

scholarly journals Humidifier-associated paediatric Legionnaires' disease, Israel, February 2012

2012 ◽  
Vol 17 (41) ◽  
Author(s):  
J Moran-Gilad ◽  
T Lazarovitch ◽  
M Mentasti ◽  
T Harrison ◽  
M Weinberger ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Cold Water ◽  
Tap Water ◽  
Fatal Case ◽  
Control Measures ◽  
Free Standing ◽  
Legionnaires Disease

We report a fatal case of community-acquired Legionnaires' disease in an infant aged under six months. Epidemiological and microbiological investigations suggested that a free-standing cold water humidifier using domestic tap water contaminated with Legionella pneumophila serogroup 1 served as a vehicle for infection. These findings were corroborated by sequence-based typing (SBT). Humidifier-associated Legionnaires' disease can be prevented by appropriate control measures. This case also illustrates the emerging role of SBT in the investigation of legionellosis.

scholarly journals Legionella Contamination in Hot Water of Italian Hotels

2005 ◽  
Vol 71 (10) ◽  
pp. 5805-5813 ◽  
Author(s):  
Paola Borella ◽  
Maria Teresa Montagna ◽  
Serena Stampi ◽  
Giovanna Stancanelli ◽  
Vincenzo Romano-Spica ◽  
...  
Keyword(s):  
Risk Factors ◽  
Legionella Pneumophila ◽  
Water Systems ◽  
Hot Water ◽  
Cross Sectional ◽  
Multicenter Survey ◽  
Multivariate Logistic Model ◽  
Legionnaires Disease ◽  
Building Characteristics

ABSTRACT A cross-sectional multicenter survey of Italian hotels was conducted to investigate Legionella spp. contamination of hot water. Chemical parameters (hardness, free chlorine concentration, and trace element concentrations), water systems, and building characteristics were evaluated to study risk factors for colonization. The hot water systems of Italian hotels were strongly colonized by Legionella; 75% of the buildings examined and 60% of the water samples were contaminated, mainly at levels of ≥103 CFU liter−1, and Legionella pneumophila was the most frequently isolated species (87%). L. pneumophila serogroup 1 was isolated from 45.8% of the contaminated sites and from 32.5% of the hotels examined. When a multivariate logistic model was used, only hotel age was associated with contamination, but the risk factors differed depending on the contaminating species and serogroup. Soft water with higher chlorine levels and higher temperatures were associated with L. pneumophila serogroup 1 colonization, whereas the opposite was observed for serogroups 2 to 14. In conclusion, Italian hotels, particularly those located in old buildings, represent a major source of risk for Legionnaires' disease due to the high frequency of Legionella contamination, high germ concentration, and major L. pneumophila serogroup 1 colonization. The possible role of chlorine in favoring the survival of Legionella species is discussed.

scholarly journals Identification of a Novel Adhesion Molecule Involved in the Virulence of Legionella pneumophila

2005 ◽  
Vol 73 (7) ◽  
pp. 4272-4280 ◽  
Author(s):  
Bin Chang ◽  
Fumiaki Kura ◽  
Junko Amemura-Maekawa ◽  
Nobuo Koizumi ◽  
Haruo Watanabe
Keyword(s):  
Cell Line ◽  
Adhesion Molecule ◽  
Legionella Pneumophila ◽  
Alveolar Epithelial Cell ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Human Macrophage ◽  
Convalescent Phase ◽  
Legionnaires Disease

ABSTRACT Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045 and may play a role in Legionnaires' disease in humans.

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