scholarly journals The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes.

1983 ◽  
Vol 158 (6) ◽  
pp. 2108-2126 ◽  
Author(s):  
M A Horwitz
Keyword(s):  
Acid Phosphatase ◽  
Legionella Pneumophila ◽  
Thorium Dioxide ◽  
Live Bacteria ◽  
Phosphatase Cytochemistry ◽  
Legionnaires Disease ◽  
Intracellular Multiplication ◽  
Erythromycin A ◽  
Secondary Lysosomes ◽  
Bacterial Protein Synthesis

The interactions between the L. pneumophila phagosome and monocyte lysosomes were investigated by prelabeling the lysosomes with thorium dioxide, an electron-opaque colloidal marker, and by acid phosphatase cytochemistry. Phagosomes containing live L. pneumophila did not fuse with secondary lysosomes at 1 h after entry into monocytes or at 4 or 8 h after entry by which time the ribosome-lined L. pneumophila replicative vacuole had formed. In contrast, the majority of phagosomes containing formalin-killed L. pneumophila, live Streptococcus pneumoniae, and live Escherichia coli had fused with secondary lysosomes by 1 h after entry into monocytes. Erythromycin, a potent inhibitor of bacterial protein synthesis, at a concentration that completely inhibits L. pneumophila intracellular multiplication, had no influence on fusion of L. pneumophila phagosomes with secondary lysosomes. However, coating live L. pneumophila with antibody or with antibody and complement partially overcame the inhibition of fusion. Also activating the monocytes promoted fusion of a small proportion of phagosomes containing live L. pneumophila with secondary lysosomes. Acid phosphatase cytochemistry revealed that phagosomes containing live L. pneumophila did not fuse with either primary or secondary lysosomes. In contrast to phagosomes containing live bacteria, the majority of phagosomes containing formalin-killed L. pneumophila were fused with lysosomes by acid phosphatase cytochemistry. The capacity of L. pneumophila to inhibit phagosome-lysosome fusion may be a critical mechanism by which the bacterium resists monocyte microbicidal effects.

scholarly journals THE INTERACTION BETWEEN TOXOPLASMA GONDII AND MAMMALIAN CELLS

1972 ◽  
Vol 136 (5) ◽  
pp. 1173-1194 ◽  
Author(s):  
Thomas C. Jones ◽  
James G. Hirsch
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Acid Phosphatase ◽  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
Cell Function ◽  
Vacuolar Membrane ◽  
Thorium Dioxide ◽  
Phosphatase Cytochemistry ◽  
Secondary Lysosomes

Electron microscope methods have been used to study delivery of macrophage primary or secondary lysosomal contents to phagocytic vacuoles containing living or dead toxoplasmas. Secondary lysosomes were labeled by culturing the cells in colloidal thorium dioxide (thorotrast) or in ferritin. Acid phosphatase cytochemistry was employed for detection of primary as well as secondary lysosomal constituents. These various lysosomal labels were present in nearly all vacuoles containing toxoplasmas killed with glutaraldehyde, or in vacuoles containing those parasites undergoing degeneration 1 hr after the uptake of living toxoplasmas. In contrast, at times ranging from 1 to 20 hr after infection, no vacuoles containing morphologically normal, apparently viable toxoplasmas were thorotrast or ferritin positive, and only rarely did these vacuoles react for acid phosphatase. In many instances vacuoles containing viable toxoplasmas and no lysosomal markers were situated in the same cell nearby to vacuoles containing degenerating toxoplasmas and lysosomal constituents, thus indicating that the determinants of lysosomal fusion were operating locally in the immediate vicinity of the phagocytic vacuole, and not operating to influence general cell function. Thus, some toxoplasmas are able to prevent the delivery of lysosomal contents, and apparently the phagocytic vacuole provides for these parasites a sheltered microenvironment ideal for their growth. Morphologic evidence indicated that living toxoplasmas altered the phagocytic vacuolar membrane in macrophages, fibroblasts, and HeLa cells. Within minutes after phagocytosis, the vacuole became surrounded by closely apposed strips of endoplasmic reticulum and mitochondria; somewhat later, microvillous protrusions of the membrane into the vacuole were seen. These morphologic features of phagocytic vacuoles containing living toxoplasmas may be of importance in relation to the absence of lysosomal fusion, or they may serve some function in protecting the host cell or in nourishing the parasite.

Demonstration of Secondary Lysosomes in Bovine Megakaryocytes and Platelets Using Acid Phosphatase Cytochemistry with Cerium as a Trapping Agent

1990 ◽  
Vol 63 (01) ◽  
pp. 127-132 ◽  
Author(s):  
Michèle Ménard ◽  
Kenneth M Meyers ◽  
David J Prieur
Keyword(s):  
Acid Phosphatase ◽  
Electron Density ◽  
Golgi Complex ◽  
Initial Report ◽  
Cerium Phosphate ◽  
Virtual Absence ◽  
Phosphatase Cytochemistry ◽  
Secondary Lysosomes ◽  
Trapping Agent

SummaryThe ultrastructure of lysosomes from bovine megakaryocytes (MK) and platelets was characterized using acid phosphatase cytochemistry with beta-glycerophosphate as substrate and cerium as a trapping agent. The technique was easily reproducible; cerium-phosphate precipitates were uniform, readily visualized, and there was a virtual absence of nonspecific reaction product. Acid phosphatase was localized in the trans aspect of the Golgi complex and/or granules of less than 50 nm to 650 nm diameters in MK at all stages of maturation. Forty percent of the MK lysosomes contained inclusions of variable shapes, sizes and electron-density and were classified as secondary lysosomes. Twenty-four percent of the platelet sections contained acid phosphatase-positive granules. Fifty-four percent of these were secondary lysosomes. This is the initial report demonstrating secondary lysosomes in either resting MK or platelets using acid phosphatase cytochemistry. These findings suggest that MK and platelet lysosomes have an intracellular function in resting MK and platelets.

scholarly journals Molecular mechanism of elongation factor 1A inhibition by a Legionella pneumophila glycosyltransferase

2010 ◽  
Vol 426 (3) ◽  
pp. 281-292 ◽  
Author(s):  
Ramon Hurtado-Guerrero ◽  
Tal Zusman ◽  
Shalini Pathak ◽  
Adel F. M. Ibrahim ◽  
Sharon Shepherd ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Binding Sites ◽  
Substrate Recognition ◽  
Elongation Factor ◽  
Negative Bacterium ◽  
Glucose Binding ◽  
Legionnaires Disease ◽  
Organelle Trafficking ◽  
Intracellular Multiplication ◽  
Positively Charged

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.

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 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 Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes.

1983 ◽  
Vol 158 (4) ◽  
pp. 1319-1331 ◽  
Author(s):  
M A Horwitz
Keyword(s):  
Legionella Pneumophila ◽  
Vacuolar Membrane ◽  
Common Mechanism ◽  
Cytoplasmic Vacuole ◽  
Human Monocytes ◽  
Vacuole Formation ◽  
Cytoplasmic Organelles ◽  
Membrane Bound ◽  
Legionnaires Disease ◽  
Intracellular Multiplication

Previous studies have shown that L. pneumophila multiplies intracellularly in human monocytes and alveolar macrophages within a membrane-bound cytoplasmic vacuole studded with ribosomes. In this paper, the formation of this novel vacuole is examined. After entry into monocytes, L. pneumophila resides in a membrane-bound vacuole. During the first hour after entry, vacuoles containing L. pneumophila are found surrounded by smooth vesicles fusing with or budding off from the vacuolar membrane and by mitochondria closely apposed to the vacuolar membrane. By 4 h, vacuoles are found less frequently surrounded by these cytoplasmic organelles, but now ribosomes and rough vesicles are found gathered about the vacuole. By 8 h, the ribosome-lined vacuole has formed. Erythromycin, at concentrations that completely inhibit the intracellular multiplication of L. pneumophila, has no effect on vacuole formation. Formalin-killed L. pneumophila also reside in a membrane-bound vacuole after entry into monocytes. In contrast to the situation with live L. pneumophila, cytoplasmic organelles are not found surrounding vacuoles containing formalin-killed L. pneumophila at any time after entry. Formalin-killed bacteria are rapidly digested, and by 4 h, few remain intact. The L. pneumophila-containing vacuole has certain features in common with other intracellular organisms that inhibit phagosome-lysosome fusion; these organisms may share a common mechanism for vacuole formation and inhibition of phagosome-lysosome fusion.

scholarly journals Interaction of the legionnaires' disease bacterium (Legionella pneumophila) with human phagocytes. II. Antibody promotes binding of L. pneumophila to monocytes but does not inhibit intracellular multiplication.

1981 ◽  
Vol 153 (2) ◽  
pp. 398-406 ◽  
Author(s):  
M A Horwitz ◽  
S C Silverstein
Keyword(s):  
Host Defense ◽  
Humoral Immunity ◽  
Antibody Production ◽  
Legionella Pneumophila ◽  
Polymorphonuclear Leukocytes ◽  
Legionnaires Disease ◽  
Intracellular Multiplication ◽  
Human Polymorphonuclear Leukocytes

In an accompanying paper (13), we reported that human polymorphonuclear leukocytes kill only a limited proportion (0.5 log) of an inoculum of Legionella pneumophila (Philadelphia 1 strain) in the presence of human anti-L. pneumophila antibody and complement. We now report on the effect of anti-L. pneumophila antibody on L. pneumophila-monocyte interaction. The studies were carried out under antibiotic-free conditions. Monocytes bind more than three times as many viable L. pneumophila bacteria in the presence of both antibody and complement than in the presence of complement alone. Monocytes requires both antibody and complement to kill any L. pneumophila: however, even then, monocytes kill only a limited proportion (0.25 log) of an inoculum. The surviving bacteria multiply several logs in the monocytes and multiply as rapidly as when the bacteria enter monocytes in the absence of antibody. These findings suggest that humoral immunity may not be an effective host defense against L. pneumophila. Consequently, a vaccine that resulted only in antibody production against the Legionnaires' disease bacterium may not be efficacious.

scholarly journals Intracellular multiplication of Legionella pneumophila in cultured human embryonic lung fibroblasts

1980 ◽  
Vol 28 (3) ◽  
pp. 1014-1018 ◽  
Author(s):  
M C Wong ◽  
E P Ewing ◽  
C S Callaway ◽  
W L Peacock
Keyword(s):  
Legionella Pneumophila ◽  
Light And Electron Microscopy ◽  
Progressive Increase ◽  
Lung Fibroblasts ◽  
Embryonic Lung ◽  
Human Embryonic Lung ◽  
Bacterial Colony ◽  
Large Numbers ◽  
Legionnaires Disease ◽  
Intracellular Multiplication

Cell cultures of normal human embryonic lung fibroblasts were inoculated with two strains of Legionella pneumophila, the etiological agent of Legionnaires disease. Large numbers of intact organisms, including many dividing forms, were seen within fibroblasts by light and electron microscopy. Intracellular multiplication of organisms was demonstrated by the progressive increase in bacterial colony counts from plated extracts of infected fibroblast cultures in which extracellular multiplication had been eliminated by bactericidal concentrations of antibiotics. It is evident that L. pneumophila can thrive in an intracellular environment, and this property may be significant in the pathogenesis of Legionnaires disease.

scholarly journals The bacterial deubiquitinase Ceg23 regulates the association of Lys-63–linked polyubiquitin molecules on the Legionella phagosome

2020 ◽  
Vol 295 (6) ◽  
pp. 1646-1657 ◽  
Author(s):  
Kelong Ma ◽  
Xiangkai Zhen ◽  
Biao Zhou ◽  
Ninghai Gan ◽  
Yang Cao ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
High Specificity ◽  
Effector Proteins ◽  
Type Analysis ◽  
Polyubiquitin Chains ◽  
Legionnaires Disease ◽  
Organelle Trafficking ◽  
Intracellular Multiplication ◽  
Catalytic Motif ◽  
Chain Type

Legionella pneumophila is the causative agent of the lung malady Legionnaires' disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication. One important aspect of such modulation is the co-option of the host ubiquitin network with a panel of effector proteins. Here, using recombinantly expressed and purified proteins, analytic ultracentrifugation, structural analysis, and computational modeling, along with deubiquitinase (DUB), and bacterial infection assays, we found that the bacterial defective in organelle trafficking/intracellular multiplication effector Ceg23 is a member of the ovarian tumor (OTU) DUB family. We found that Ceg23 displays high specificity toward Lys-63–linked polyubiquitin chains and is localized on the LCV, where it removes ubiquitin moieties from proteins ubiquitinated by the Lys-63–chain type. Analysis of the crystal structure of a Ceg23 variant lacking two putative transmembrane domains at 2.80 Å resolution revealed that despite very limited homology to established members of the OTU family at the primary sequence level, Ceg23 harbors a catalytic motif resembling those associated with typical OTU-type DUBs. ceg23 deletion increased the association of Lys-63–linked polyubiquitin with the bacterial phagosome, indicating that Ceg23 regulates Lys-63–linked ubiquitin signaling on the LCV. In summary, our findings indicate that Ceg23 contributes to the regulation of the association of Lys-63 type polyubiquitin with the Legionella phagosome. Future identification of host substrates targeted by Ceg23 could clarify the roles of these polyubiquitin chains in the intracellular life cycle of L. pneumophila and Ceg23's role in bacterial virulence.

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