Importance of theicmNgene in the growth ofLegionella pneumophilain amoebic cells at low temperature

2012 ◽  
Vol 58 (4) ◽  
pp. 490-501
Author(s):  
Tian Qin ◽  
Ken-ichiro Iida ◽  
Zhenyu Piao ◽  
Susumu Shiota ◽  
Hongyu Ren ◽  
...  
Keyword(s):  
Low Temperature ◽  
Legionella Pneumophila ◽  
Gene Replacement ◽  
Gene Products ◽  
Intracellular Growth ◽  
Bacterial Proliferation ◽  
Intracellular Multiplication ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

Legionella pneumophila grows in amoebae and has achieved the ability to grow at various temperatures, although the mechanisms controlling this ability remain poorly understood. The Icm/Dot type IVB secretion system is composed of more than 25 proteins and is known to be essential for intracellular growth. The role of the icmN gene in intracellular multiplication and the effects of culture temperatures on it are not precisely understood. We conducted our investigation using an icmN mutant made by gene replacement mutagenesis. Intracellular growth of the mutant was impaired both in mammalian macrophages and amoeba at 37 °C. In particular, intracellular growth in amoebae was completely impaired at 25 °C. It was found that genes from icmN to icmC formed an operon, i.e., icmN, -M, -L, -E, -G, -C,, and the promoter activity of the icmN operon was stronger at 25 than at 37 °C. It was suggested that icmM and its downstream genes had a secondary promoter that enables icmN mutant grow in amoebae at lower temperatures and macrophages at 37 °C. These results show that the icmN promoter has a low temperature inducible nature, and gene products of the icmN operon require high expression for bacterial proliferation at low temperatures within amoeba.

scholarly journals Environmental Mimics and the Lvh Type IVA Secretion System Contribute to Virulence-Related Phenotypes of Legionella pneumophila

2006 ◽  
Vol 75 (2) ◽  
pp. 723-735 ◽  
Author(s):  
Purnima Bandyopadhyay ◽  
Shuqing Liu ◽  
Carolina B. Gabbai ◽  
Zeah Venitelli ◽  
Howard M. Steinman
Keyword(s):  
Stationary Phase ◽  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Secretion System ◽  
Resistant Bacteria ◽  
Causative Organism ◽  
Legionnaires Disease ◽  
Intracellular Multiplication ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

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.

scholarly journals The Dot/Icm Effector SdhA Is Necessary for Virulence of Legionella pneumophila in Galleria mellonella and A/J Mice

2013 ◽  
Vol 81 (7) ◽  
pp. 2598-2605 ◽  
Author(s):  
Clare R. Harding ◽  
Charlotte A. Stoneham ◽  
Ralf Schuelein ◽  
Hayley Newton ◽  
Clare V. Oates ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Galleria Mellonella ◽  
Model Organisms ◽  
Infection Model ◽  
Danger Signal ◽  
Wild Type ◽  
Content Type ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

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.

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 CD44-Regulated Intracellular Proliferation of Listeria monocytogenes

2003 ◽  
Vol 71 (7) ◽  
pp. 4102-4111 ◽  
Author(s):  
Emma Eriksson ◽  
Lone Dons ◽  
Antonio Gigliotti Rothfuchs ◽  
Paraskevi Heldin ◽  
Hans Wigzell ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Intracellular Growth ◽  
Murine Bone Marrow ◽  
Bacterial Proliferation ◽  
Serovar Typhimurium ◽  
Inflammatory Processes ◽  
And Control ◽  
Intracellular Proliferation ◽  
Hyaluronan Binding

ABSTRACT CD44 has been implicated in immune and inflammatory processes. We have analyzed the role of CD44 in the outcome of Listeria monocytogenes infection in murine bone marrow-derived macrophages (BMM). Surprisingly, a dramatically decreased intracellular survival of L. monocytogenes was observed in CD44−/− BMM. CD44−/− heart or lung fibroblast cultures also showed reduced bacterial levels. Moreover, livers from CD44−/−-infected mice showed diminished levels of L. monocytogenes. In contrast, intracellular growth of Salmonella enterica serovar Typhimurium was the same in CD44−/− and control BMM. The CD44-mediated increased bacterial proliferation was not linked to altered BMM differentiation or to secretion of soluble factors. CD44 did not mediate listerial uptake, and it played no role in bacterial escape from the primary phagosome or formation of actin tails. Furthermore, CD44-enhanced listerial proliferation occurred in the absence of intracellular bacterial spreading. Interestingly, coincubation of BMM with hyaluronidase or anti-CD44 antibodies that selectively inhibit hyaluronan binding increased intracellular listerial proliferation. Treatment of cells with hyaluronan, in contrast, diminished listerial growth and induced proinflammatory transcript levels. We suggest that L. monocytogenes takes advantage of the CD44-mediated signaling to proliferate intracellularly, although binding of CD44 to certain ligands will inhibit such response.

scholarly journals Structure and Function of Interacting IcmR-IcmQ Domains from a Type IVb Secretion System in Legionella pneumophila

Structure ◽  
2009 ◽  
Vol 17 (4) ◽  
pp. 590-601 ◽  
Author(s):  
Suchismita Raychaudhury ◽  
Jeremiah D. Farelli ◽  
Timothy P. Montminy ◽  
Miguelina Matthews ◽  
Jean-François Ménétret ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Secretion System ◽  
Structure And Function ◽  
Type Ivb Secretion ◽  
And Function ◽  
Type Ivb Secretion System

scholarly journals Plants in a cold climate

2002 ◽  
Vol 357 (1423) ◽  
pp. 831-847 ◽  
Author(s):  
Maggie Smallwood ◽  
Dianna J. Bowles
Keyword(s):  
Functional Analysis ◽  
Extracellular Matrix ◽  
Low Temperature ◽  
Cold Acclimation ◽  
Prolonged Exposure ◽  
Plant Cells ◽  
Cold Climate ◽  
Gene Products ◽  
Temperature Signal

Plants are able to survive prolonged exposure to sub–zero temperatures; this ability is enhanced by pre–exposure to low, but above–zero temperatures. This process, known as cold acclimation, is briefly reviewed from the perception of cold, through transduction of the low–temperature signal to functional analysis of cold–induced gene products. The stresses that freezing of apoplastic water imposes on plant cells is considered and what is understood about the mechanisms that plants use to combat those stresses discussed, with particular emphasis on the role of the extracellular matrix.

Structure of the Legionella Dot/Icm type IV secretion system in situ by electron cryotomography

2016 ◽  
Author(s):  
Debnath Ghosal ◽  
Yi-Wei Chang ◽  
Kwangcheol C. Jeong ◽  
Joseph P. Vogel ◽  
Grant J. Jensen
Keyword(s):  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Secretion Systems ◽  
Type Iv ◽  
Type Ivb Secretion ◽  
Structural Preservation ◽  
Electron Cryotomography ◽  
Type Ivb Secretion System

AbstractType IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila. This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ. While the Dot/Icm system shares almost no sequence homology with type IVA secretion systems (T4ASSs), its overall structure shows remarkable similarities to two previously imaged T4ASSs, suggesting shared aspects of mechanism. However, compared to one of these, the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, it is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ.

scholarly journals Evolution of the Arsenal ofLegionella pneumophilaEffectors To Modulate Protist Hosts

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Ashley Best ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Host Cells ◽  
Human Pathogen ◽  
Intracellular Growth ◽  
Content Type ◽  
Human Macrophages ◽  
Cellular Processes ◽  
Host Interaction

ABSTRACTWithin the human host,Legionella pneumophilareplicates within alveolar macrophages, leading to pneumonia. However,L. pneumophilais an aquatic generalist pathogen that replicates within a wide variety of protist hosts, including amoebozoa, percolozoa, and ciliophora. The intracellular lifestyles ofL. pneumophilawithin the two evolutionarily distant hosts macrophages and protists are remarkably similar. Coevolution with numerous protist hosts has shaped plasticity of the genome ofL. pneumophila, which harbors numerous proteins encoded by genes acquired from primitive eukaryotic hosts through interkingdom horizontal gene transfer. The Dot/Icm type IVb translocation system translocates ∼6,000 effectors amongLegionellaspecies and >320 effector proteins inL. pneumophilainto host cells to modulate a plethora of cellular processes to create proliferative niches. Since many of the effectors have likely evolved to modulate cellular processes of primitive eukaryotic hosts, it is not surprising that most of the effectors do not contribute to intracellular growth within human macrophages. Some of the effectors may modulate highly conserved eukaryotic processes, while others may target protist-specific processes that are absent in mammals. The lack of studies to determine the role of the effectors in adaptation ofL. pneumophilato various protists has hampered the progress to determine the function of most of these effectors, which are routinely studied in mouse or human macrophages. Since many protists restrictL. pneumophila, utilization of such hosts can also be instrumental in deciphering the mechanisms of failure ofL. pneumophilato overcome restriction of certain protist hosts. Here, we review the interaction ofL. pneumophilawith its permissive and restrictive protist environmental hosts and outline the accomplishments as well as gaps in our knowledge ofL. pneumophila-protist host interaction andL. pneumophila’s evolution to become a human pathogen.

scholarly journals Early Events in Phagosome Establishment Are Required for Intracellular Survival of Legionella pneumophila

1998 ◽  
Vol 66 (9) ◽  
pp. 4450-4460 ◽  
Author(s):  
Lawrence A. Wiater ◽  
Kenneth Dunn ◽  
Frederick R. Maxfield ◽  
Howard A. Shuman
Keyword(s):  
Legionella Pneumophila ◽  
Gene Products ◽  
Wild Type ◽  
Intracellular Replication ◽  
Early Step ◽  
Confocal Fluorescence ◽  
Legionnaires Disease ◽  
Organelle Trafficking ◽  
Intracellular Multiplication ◽  
Intracellular Fate

ABSTRACT During infection, the Legionnaires’ disease bacterium,Legionella pneumophila, survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes. In order to understand the molecular basis of this organism’s ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication. These mutations define a set of 20 genes (19 icm [for intracellular multiplication] genes and dotA [for defect in organelle trafficking]). In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L. pneumophila or one of several mutants defective in different icm genes ordotA. By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophilacolocalized with host lysosomes prelabeled with rhodamine-dextran. Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection. These results indicate that several, and possibly all, of the icmand dotA gene products act at an early step during phagosome establishment to determine whether L. pneumophila-containing phagosomes will fuse with lysosomes. Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication.

scholarly journals Role of Toll-Like Receptor 9 in Legionella pneumophila-Induced Interleukin-12 p40 Production in Bone Marrow-Derived Dendritic Cells and Macrophages from Permissive and Nonpermissive Mice

2006 ◽  
Vol 75 (1) ◽  
pp. 146-151 ◽  
Author(s):  
Cathy A. Newton ◽  
Izabella Perkins ◽  
Raymond H. Widen ◽  
Herman Friedman ◽  
Thomas W. Klein
Keyword(s):  
Dendritic Cells ◽  
Legionella Pneumophila ◽  
Gene Locus ◽  
Interleukin 12 ◽  
Mouse Strains ◽  
Common Mechanism ◽  
Toll Like Receptor ◽  
Intracellular Growth ◽  
In Cells

ABSTRACT The progression of Legionella pneumophila infection in macrophages is controlled by the Lgn1 gene locus, which expresses the nonpermissive phenotype in cells from BALB/c mice but the permissive phenotype in cells from A/J mice. Activation of dendritic cells and macrophages by L. pneumophila is mediated by the pathogen recognition receptor Toll-like receptor 2 (TLR2); furthermore, Legionella induces innate and adaptive immune cytokines by the MyD88-dependent pathway. TLR9 is coupled to MyD88 and mediates the production of interleukin-12 (IL-12) in dendritic cells infected with other facultatively intracellular pathogens. In the current study, L. pneumophila growth in dendritic cells from BALB/c and A/J mice was examined along with the role of TLR9 in the induction of IL-12 in these cells. Dendritic cells from both strains were nonpermissive for L. pneumophila intracellular growth, suggesting that the products of the Lgn1 gene locus that control intracellular growth in macrophages do not control the growth of Legionella in dendritic cells. In addition, chloroquine treatment suppressed IL-12 p40 production in response to Legionella treatment in dendritic cells and macrophages from BALB/c and A/J mice. Furthermore, the TLR9 inhibitor ODN2088 suppressed the Legionella-induced IL-12 production in dendritic cells from both mouse strains. These results suggest that L. pneumophila is similar to other intracellular bacteria in that it stimulates the production of immune-transitioning cytokines, such as IL-12, through activation of TLR9 and that this receptor provides a common mechanism for sensing these types of microbes and inducing innate and adaptive immunity.

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