scholarly journals Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin.

1983 ◽  
Vol 71 (1) ◽  
pp. 15-26 ◽  
Author(s):  
M A Horwitz ◽  
S C Silverstein
Keyword(s):  
Legionella Pneumophila ◽  
Human Monocytes ◽  
Legionnaires Disease ◽  
Intracellular Multiplication

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 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 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 Legionella pneumophila inhibits acidification of its phagosome in human monocytes.

1984 ◽  
Vol 99 (6) ◽  
pp. 1936-1943 ◽  
Author(s):  
M A Horwitz ◽  
F R Maxfield
Keyword(s):  
Legionella Pneumophila ◽  
Sheep Erythrocyte ◽  
Bacterial Pathogen ◽  
Human Monocytes ◽  
E Coli ◽  
The Mean ◽  
Intracellular Multiplication ◽  
Fluorescence Images ◽  
Activated Monocytes ◽  
Quantitative Fluorescence

We used quantitative fluorescence microscopy to measure the pH of phagosomes in human monocytes that contain virulent Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in these phagocytes. The mean pH of phagosomes that contain live L. pneumophila was 6.1 in 14 experiments. In the same experiments, the mean pH of phagosomes containing dead L. pneumophila averaged 0.8 pH units lower than the mean pH of phagosomes containing live L. pneumophila, a difference that was highly significant (P less than 0.01 in all 14 experiments). In contrast, the mean pH of phagosomes initially containing live E. coli, which were then killed by monocytes, was the same as for phagosomes initially containing dead E. coli. The mean pH of L. pneumophila phagosomes in activated monocytes, which inhibit L. pneumophila intracellular multiplication, was the same as in nonactivated monocytes. To simultaneously measure the pH of different phagosomes within the same monocyte, we digitized and analyzed fluorescence images of monocytes that contained both live L. pneumophila and sheep erythrocytes. Within the same monocyte, live L. pneumophila phagosomes had a pH of approximately 6.1 and sheep erythrocyte phagosomes had a pH of approximately 5.0 or below. This study demonstrates that L. pneumophila is capable of modifying the pH of its phagocytic vacuole. This capability may be critical to the intracellular survival and multiplication of this and other intracellular pathogens.

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.

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