scholarly journals HtrA Homologue of Legionella pneumophila: an Indispensable Element for Intracellular Infection of Mammalian but Not Protozoan Cells

2001 ◽  
Vol 69 (4) ◽  
pp. 2569-2579 ◽  
Author(s):  
Lisa L. Pedersen ◽  
Marina Radulic ◽  
Miljenko Doric ◽  
Yousef Abu Kwaik
Keyword(s):  
Epithelial Cells ◽  
Confocal Laser Scanning Microscopy ◽  
Legionella Pneumophila ◽  
Parental Strain ◽  
Laser Scanning ◽  
Alveolar Epithelial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Intracellular Replication ◽  
Alveolar Epithelial

ABSTRACT Legionella pneumophila replicates within alveolar macrophages, and possibly, alveolar epithelial cells and also within protozoa in the aquatic environment. Here we characterize an L. pneumophila mutant defective in the HtrA/DegP stress-induced protease/chaperone homologue and show that HtrA is indispensable for intracellular replication within mammalian macrophages and alveolar epithelial cells and for intrapulmonary replication in A/J mice. Importantly, amino acid substitutions of two conserved residues in the catalytic domain of (H103➤R and S212➤A) and in-frame deletions of either or both of the two conserved PDZ domains of HtrA abolish its function. Interestingly, the htrAmutant exhibits a parental-type phenotype in intracellular replication within the protozoan host Acanthamoeba polyphaga. We used a promoterless lacZ fusion to the htrApromoter to probe the phagosomal microenvironment harboringL. pneumophila within macrophages and within A. polyphaga for the exposure to stress stimuli. The data show that expression through the htrA promoter is induced by 12,000- to 20,000-fold throughout the intracellular infection of macrophages but its induction is by 120- to 500-fold within protozoa compared to in vitro expression. Data derived from confocal laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the htrA mutant within U937 macrophages colocalize with the late endosomal-lysosomal marker LAMP-2, similar to killed L. pneumophila. Coinfection experiments examined by confocal laser scanning microscopy show that in communal phagosomes harboring both the parental strain and the htrA mutant, replication of the mutant is not rescued, while replication of a dotAmutant control, which is normally trafficked into a phagolysosome, is rescued by the parental strain. Our data show, for the first time, that the stress response by L. pneumophila (mediated, at least in part, by HtrA) is indispensable for intracellular replication within mammalian but not protozoan cells.

scholarly journals Temporal Pore Formation-Mediated Egress from Macrophages and Alveolar Epithelial Cells by Legionella pneumophila

2000 ◽  
Vol 68 (11) ◽  
pp. 6431-6440 ◽  
Author(s):  
O. A. Terry Alli ◽  
Lian-Yong Gao ◽  
Lisa L. Pedersen ◽  
Steven Zink ◽  
Marina Radulic ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
Pore Formation ◽  
Alveolar Epithelial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Intracellular Replication ◽  
Alveolar Epithelial ◽  
Bacterial Replication

ABSTRACT Legionella pneumophila does not induce apoptosis in the protozoan host, but induces pore formation-mediated cytolysis after termination of intracellular replication (L.-Y. Gao and Y. Abu Kwaik, Environ. Microbiol. 2:79–90, 2000). In contrast to this single mode of killing of protozoa, we have recently proposed a biphasic model by which L. pneumophila kills macrophages, in which the first phase is manifested through the induction of apoptosis during early stages of the infection, followed by an independent and temporal induction of necrosis during late stages of intracellular replication. Here we show that, similar to the protozoan host, the induction of necrosis and cytolysis of macrophages by L. pneumophila is mediated by the pore-forming toxin or activity. This activity is temporally and maximally expressed only upon termination of bacterial replication and correlates with cytolysis of macrophages and alveolar epithelial cells in vitro. We have identified five L. pneumophila mutants defective in the pore-forming activity. The phagosomes harboring the mutants do not colocalize with the late endosomal or lysosomal marker Lamp-1, and the mutants replicate intracellularly similar to the parental strain. Interestingly, despite their prolific intracellular replication, the mutants are defective in cytotoxicity and are “trapped” within and fail to lyse and egress from macrophages and alveolar epithelial cells upon termination of intracellular replication. However, the mutants are subsequently released from the host cell, most likely due to apoptotic death of the host cell. Data derived from cytotoxicity assays, confocal laser scanning microscopy, and electron microscopy confirm the defect in the mutants to induce necrosis of macrophages and the failure to egress from the host cell. Importantly, the mutants are completely defective in acute lethality (24 to 48 h) to intratracheally inoculated A/J mice. We conclude that the pore-forming activity of L. pneumophila is not required for phagosomal trafficking or for intracellular replication. This activity is expressed upon termination of bacterial replication and is essential to induce cytolysis of infected macrophages to allow egress of intracellular bacteria. In addition, this activity plays a major role in pulmonary immunopathology in vivo.

scholarly journals Essential Role for the Legionella pneumophila Rep Helicase Homologue in Intracellular Infection of Mammalian Cells

2000 ◽  
Vol 68 (12) ◽  
pp. 6970-6978 ◽  
Author(s):  
Omar S. Harb ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Alveolar Epithelial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Intracellular Replication ◽  
Simultaneous Exposure ◽  
U937 Macrophage

ABSTRACT We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar torep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in therep helicase homologue mutant, rendering it defective in intracellular replication.

scholarly journals Legionella dumoffii DjlA, a Member of the DnaJ Family, Is Required for Intracellular Growth

2004 ◽  
Vol 72 (6) ◽  
pp. 3592-3603 ◽  
Author(s):  
Hiroko Ohnishi ◽  
Yoshimitsu Mizunoe ◽  
Akemi Takade ◽  
Yoshitaka Tanaka ◽  
Hiroshi Miyamoto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Parental Strain ◽  
Laser Scanning ◽  
Bartonella Henselae ◽  
Alveolar Epithelial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Intracellular Growth ◽  
Transmission Electron

ABSTRACT Legionella dumoffii is one of the common causes of Legionnaires' disease and is capable of replicating in macrophages. To understand the mechanism of survival within macrophages, transposon mutagenesis was employed to isolate the genes necessary for intracellular growth. We identified four defective mutants after screening 790 transposon insertion mutants. Two transposon insertions were in genes homologous to icmB or dotC, within dot/icm loci, required for intracellular multiplication of L. pneumophila. The third was in a gene whose product is homologous to the 17-kDa antigen forming part of the VirB/VirD4 type IV secretion system of Bartonella henselae. The fourth was in the djlA (for “dnaj-like A”) gene. DjlA is a member of the DnaJ/Hsp40 family. Transcomplementation of the djlA mutant restored the parental phenotype in J774 macrophages, A549 human alveolar epithelial cells, and the amoeba Acanthamoeba culbertsoni. Using confocal laser-scanning microscopy and transmission electron microscopy, we revealed that in contrast to the wild-type strain, L. dumoffii djlA mutant-containing phagosomes were unable to inhibit phagosome-lysosome fusion. Transmission electron microscopy also showed that in contrast to the virulent parental strain, the djlA mutant was not able to recruit host cell rough endoplasmic reticulum. Furthermore, the stationary-phase L. dumoffii djlA mutants were more susceptible to H2O2, high osmolarity, high temperature, and low pH than was their parental strain. These results indicate that DjlA is required for intracellular growth and organelle trafficking, as well as bacterial resistance to environmental stress. This is the first report demonstrating that a single DjlA-deficient mutant exhibits a distinct phenotype.

ACTIVATED SLUDGE FLOC CHARACTERIZATION BY CONFOCAL LASER SCANNING MICROSCOPY

2012 ◽  
Vol 11 (3) ◽  
pp. 669-674 ◽  
Author(s):  
Szabolcs Szilveszter ◽  
Botond Raduly ◽  
Szilard Bucs ◽  
Beata Abraham ◽  
Szabolcs Lanyi ◽  
...  
Keyword(s):  
Activated Sludge ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser

Cuticular structures in the copulatory apparatus of Planorbis planorbis (Linnaeus, 1758) (Gastropoda: Pulmonata: Planorbidae)

2009 ◽  
Vol 18 (1) ◽  
pp. 11-16
Author(s):  
E.V. Soldatenko ◽  
A.A. Petrov
Keyword(s):  
Light Microscopy ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Taxonomic Status ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Copulatory Apparatus ◽  
The Family ◽  
Cuticular Structures

The morphology of the copulatory apparatus and associated cuticular structures in Planorbis planorbis was studied by light microscopy, SEM, TEM and confocal laser scanning microscopy. The significance of these cuticular structures for the taxonomic status of the species and for the systematics of the family Planorbidae in general is discussed.

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