scholarly journals Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila, plaB, Exhibiting Hemolytic Activity

2004 ◽  
Vol 72 (5) ◽  
pp. 2648-2658 ◽  
Author(s):  
Antje Flieger ◽  
Kerstin Rydzewski ◽  
Sangeeta Banerji ◽  
Markus Broich ◽  
Klaus Heuner
Keyword(s):  
Legionella Pneumophila ◽  
Hemolytic Activity ◽  
Genomic Library ◽  
Lipolytic Activity ◽  
Acanthamoeba Castellanii ◽  
Intracellular Survival ◽  
Phospholipase A ◽  
Wild Type ◽  
Gene Encoding ◽  
Human Red Blood Cells

ABSTRACT Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular pathogen of amoebae, macrophages, and epithelial cells. The pathology of Legionella infections involves alveolar cell destruction, and several proteins of L. pneumophila are known to contribute to this ability. By screening a genomic library of L. pneumophila, we found an additional L. pneumophila gene, plaB, which coded for a hemolytic activity and contained a lipase consensus motif in its deduced protein sequence. Moreover, Escherichia coli harboring the L. pneumophila plaB gene showed increased activity in releasing fatty acids predominantly from diacylphospho- and lysophospholipids, demonstrating that it encodes a phospholipase A. It has been reported that culture supernatants and cell lysates of L. pneumophila possess phospholipase A activity; however, only the major secreted lysophospholipase A PlaA has been investigated on the molecular level. We therefore generated isogenic L. pneumophila plaB mutants and tested those for hemolysis, lipolytic activities, and intracellular survival in amoebae and macrophages. Compared to wild-type L. pneumophila, the plaB mutant showed reduced hemolysis of human red blood cells and almost completely lost its cell-associated lipolytic activity. We conclude that L. pneumophila plaB is the gene encoding the major cell-associated phospholipase A, possibly contributing to bacterial cytotoxicity due to its hemolytic activity. On the other hand, in view of the fact that the plaB mutant multiplied like the wild type both in U937 macrophages and in Acanthamoeba castellanii amoebae, plaB is not essential for intracellular survival of the pathogen.

scholarly journals Characterization of the Gene Encoding the Major Secreted Lysophospholipase A of Legionella pneumophila and Its Role in Detoxification of Lysophosphatidylcholine

2002 ◽  
Vol 70 (11) ◽  
pp. 6094-6106 ◽  
Author(s):  
Antje Flieger ◽  
Birgid Neumeister ◽  
Nicholas P. Cianciotto
Keyword(s):  
Fatty Acids ◽  
Legionella Pneumophila ◽  
Lipolytic Activity ◽  
Cholesterol Acyltransferase ◽  
Phospholipase A ◽  
Wild Type ◽  
Gene Encoding ◽  
Acyltransferase Activity ◽  
Lipolytic Enzymes ◽  
Cloned Gene

ABSTRACT We previously showed that Legionella pneumophila secretes, via its type II secretion system, phospholipase A activities that are distinguished by their specificity for certain phospholipids. In this study, we identified and characterized plaA, a gene encoding a phospholipase A that cleaves fatty acids from lysophospholipids. The plaA gene encoded a 309-amino-acid protein (PlaA) which had homology to a group of lipolytic enzymes containing the catalytic signature GDSL. In Escherichia coli, the cloned gene conferred trypsin-resistant hydrolysis of lysophosphatidylcholine and lysophosphatidylglycerol. An L. pneumophila plaA mutant was generated by allelic exchange. Although the mutant grew normally in standard buffered yeast extract broth, its culture supernatants lost greater than 80% of their ability to release fatty acids from lysophosphatidylcholine and lysophosphatidylglycerol, implying that PlaA is the major secreted lysophospholipase A of L. pneumophila. The mutant's reduced lipolytic activity was confirmed by growth on egg yolk agar and thin layer chromatography and was complemented by reintroduction of an intact copy of plaA. Overexpression of plaA completely protected L. pneumophila from the toxic effects of lysophosphatidylcholine, suggesting a role for PlaA in bacterial detoxification of lysophospholipids. The plaA mutant grew like the wild type in U937 cell macrophages and Hartmannella vermiformis amoebae, indicating that PlaA is not essential for intracellular infection of L. pneumophila. In the course of characterizing plaA, we discovered that wild-type legionellae secrete a phospholipid cholesterol acyltransferase activity, highlighting the spectrum of lipolytic enzymes produced by L. pneumophila.

The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 167-182 ◽  
Author(s):  
Urs Albers ◽  
Katrin Reus ◽  
Howard A. Shuman ◽  
Hubert Hilbi
Keyword(s):  
Legionella Pneumophila ◽  
Lipid A ◽  
Sequence Similarity ◽  
Acanthamoeba Castellanii ◽  
Growth Defect ◽  
Wild Type ◽  
Intracellular Growth ◽  
Plate Test ◽  
Raw264.7 Macrophages ◽  
Mutant Strains

Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.

scholarly journals The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii

1999 ◽  
Vol 181 (16) ◽  
pp. 4879-4889 ◽  
Author(s):  
Laura M. Hales ◽  
Howard A. Shuman
Keyword(s):  
Stationary Phase ◽  
Mutant Strain ◽  
Legionella Pneumophila ◽  
Regulatory Networks ◽  
Acanthamoeba Castellanii ◽  
Logarithmic Phase ◽  
Insertion Mutation ◽  
Wild Type ◽  
E Coli ◽  
Resistance To Stress

ABSTRACT To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of theEscherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoSmutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in therpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host,Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS inL. pneumophila is very different from what has previously been reported for E. coli rpoS.

scholarly journals Characterization of the Major Secreted Zinc Metalloprotease- Dependent Glycerophospholipid:Cholesterol Acyltransferase, PlaC, of Legionella pneumophila

2005 ◽  
Vol 73 (5) ◽  
pp. 2899-2909 ◽  
Author(s):  
Sangeeta Banerji ◽  
Mayte Bewersdorff ◽  
Björn Hermes ◽  
Nicholas P. Cianciotto ◽  
Antje Flieger
Keyword(s):  
Legionella Pneumophila ◽  
Hydrolytic Enzymes ◽  
Acanthamoeba Castellanii ◽  
Severe Pneumonia ◽  
Phospholipase A ◽  
Type Ii Secretion ◽  
Lipolytic Enzyme ◽  
Acyltransferase Activity ◽  
Zinc Metalloprotease ◽  
Bacterial Culture Supernatant

ABSTRACT Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.

scholarly journals l-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae Are Caused by a Mutation in the PRO1 Gene Encoding γ-Glutamyl Kinase

2003 ◽  
Vol 69 (1) ◽  
pp. 212-219 ◽  
Author(s):  
Yuko Morita ◽  
Shigeru Nakamori ◽  
Hiroshi Takagi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Library ◽  
Reductase Activity ◽  
Freeze Tolerance ◽  
Proline Accumulation ◽  
Yeast Cells ◽  
Single Amino Acid ◽  
Wild Type ◽  
Gene Encoding

ABSTRACT We previously isolated a mutant which showed a high tolerance to freezing that correlated with higher levels of intracellular l-proline derived from l-proline analogue-resistant mutants. The mutation responsible for the analogue resistance and l-proline accumulation was a single nuclear dominant mutation. By introducing the mutant-derived genomic library into a non-l-proline-utilizing strain, the mutant was found to carry an allele of the wild-type PRO1 gene encoding γ-glutamyl kinase, which resulted in a single amino acid replacement; Asp (GAC) at position 154 was replaced by Asn (AAC). Interestingly, the allele of PRO1 was shown to enhance the activities of γ-glutamyl kinase and γ-glutamyl phosphate reductase, both of which catalyze the first two steps of l-proline synthesis from l-glutamate and which together may form a complex in vivo. When cultured in liquid minimal medium, yeast cells expressing the mutated γ-glutamyl kinase were found to accumulate intracellular l-proline and showed a prominent increase in cell viability after freezing at −20°C compared to the viability of cells harboring the wild-type PRO1 gene. These results suggest that the altered γ-glutamyl kinase results in stabilization of the complex or has an indirect effect on γ-glutamyl phosphate reductase activity, which leads to an increase in l-proline production in Saccharomyces cerevisiae. The approach described in this paper could be a practical method for breeding novel freeze-tolerant yeast strains.

scholarly journals Legionella pneumophila Catalase-Peroxidases Are Required for Proper Trafficking and Growth in Primary Macrophages

2003 ◽  
Vol 71 (8) ◽  
pp. 4526-4535 ◽  
Author(s):  
Purnima Bandyopadhyay ◽  
Brenda Byrne ◽  
Yolande Chan ◽  
Michele S. Swanson ◽  
Howard M. Steinman
Keyword(s):  
Legionella Pneumophila ◽  
Type Strain ◽  
Wild Type Strain ◽  
Acanthamoeba Castellanii ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Wild Type ◽  
Type Iv ◽  
Virulence Traits

ABSTRACT Legionella pneumophila, a parasite of aquatic amoebae and pathogen of pulmonary macrophages, replicates intracellularly, utilizing a type IV secretion system to subvert the trafficking of Legionella-containing phagosomes. Defense against host-derived reactive oxygen species has been proposed as critical for intracellular replication. Virulence traits of null mutants in katA and katB, encoding the two Legionella catalase-peroxidases, were analyzed to evaluate the hypothesis that L. pneumophila must decompose hydrogen peroxide to establish a replication niche in macrophages. Phagosomes containing katA or katB mutant Legionella colocalize with LAMP-1, a late endosomal-lysosomal marker, at twice the frequency of those of wild-type strain JR32 and show a decreased frequency of bacterial replication, in similarity to phenotypes of mutants with mutations in dotA and dotB, encoding components of the Type IV secretion system. Quantitative similarity of the katA/B phenotypes indicates that each contributes to virulence traits largely independently of intracellular compartmentalization (KatA in the periplasm and KatB in the cytosol). These data support a model in which KatA and KatB maintain a critically low level of H2O2 compatible with proper phagosome trafficking mediated by the type IV secretion apparatus. During these studies, we observed that dotA and dotB mutations in wild-type strain Lp02 had no effect on intracellular multiplication in the amoeba Acanthamoeba castellanii, indicating that certain dotA/B functions in Lp02 are dispensable in that experimental model. We also observed that wild-type JR32, unlike Lp02, shows minimal contact-dependent cytotoxicity, suggesting that cytotoxicity of JR32 is not a prerequisite for formation of replication-competent Legionella phagosomes in macrophages.

scholarly journals MfLIP1, a gene encoding an extracellular lipase of the lipid-dependent fungus Malassezia furfur

Microbiology ◽  
2006 ◽  
Vol 152 (2) ◽  
pp. 547-554 ◽  
Author(s):  
Sascha Brunke ◽  
Bernhard Hube
Keyword(s):  
Fatty Acids ◽  
Skin Diseases ◽  
Genomic Library ◽  
Amine Oxidase ◽  
Lipolytic Activity ◽  
Malassezia Furfur ◽  
Dimorphic Fungus ◽  
Gene Encoding ◽  
Wide Range ◽  
Secreted Lipase

Malassezia furfur is a dimorphic fungus and a member of the normal cutaneous microflora of humans. However, it is also a facultative pathogen, associated with a wide range of skin diseases. One unusual feature of M. furfur is an absolute dependency on externally provided lipids which the fungus hydrolyses by lipolytic activity to release fatty acids necessary for both growth and pathogenicity. In this study, the cloning and characterization of the first gene encoding a secreted lipase of M. furfur possibly associated with this activity are reported. The gene, MfLIP1, shows high sequence similarity to other known extracellular lipases, but is not a member of a lipase gene family in M. furfur. MfLIP1 consists of 1464 bp, encoding a protein with a molecular mass of 54·3 kDa, a conserved lipase motif and an N-terminal signal peptide of 26 aa. By using a genomic library, two other genes were identified flanking MfLIP1, one of them encoding a putative secreted catalase, the other a putative amine oxidase. The cDNA of MfLIP1 was expressed in Pichia pastoris and the biochemical properties of the recombinant lipase were analysed. MfLip1 is most active at 40 °C and the pH optimum was found to be 5·8. The lipase hydrolysed lipids, such as Tweens, frequently used as the source of fatty acids in M. furfur media, and had minor esterase activity. Furthermore, the lipase is inhibited by different bivalent metal ions. This is the first molecular description of a secreted lipase from M. furfur.

scholarly journals Significant Role for ladC in Initiation of Legionella pneumophila Infection

2008 ◽  
Vol 76 (7) ◽  
pp. 3075-3085 ◽  
Author(s):  
Hayley J. Newton ◽  
Fiona M. Sansom ◽  
Jenny Dao ◽  
Christel Cazalet ◽  
Holger Bruggemann ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Virulence Gene ◽  
Early Time ◽  
Dominant Negative ◽  
Wild Type ◽  
Virulence Gene Expression ◽  
Mammalian Cell Lines

ABSTRACT Previously, we identified ladC in a cohort of genes that were present in Legionella pneumophila but absent in other Legionella species. Here we constructed a ladC mutant of L. pneumophila and assessed its ability to replicate in mammalian cell lines and Acanthamoeba castellanii. The ladC mutant was recovered in significantly lower numbers than wild-type L. pneumophila at early time points, which was reversed upon transcomplementation with ladC but not ladC N430A/R434A, encoding a putative catalytically inactive derivative of the protein. In fact, complementation of ladC::Km with ladC N430A/R434A resulted in a severe replication defect within human and amoeba cell models of infection, which did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the ladC mutant exhibited a 10-fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the ladC mutant dropped fivefold over 72 h, indicating a significant attenuation compared to wild-type L. pneumophila. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that ladC does not influence the transcriptional profile of L. pneumophila in vitro or during A. castellanii infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in L. pneumophila infection.

scholarly journals Role of Catalase in Campylobacter jejuniIntracellular Survival

2000 ◽  
Vol 68 (11) ◽  
pp. 6337-6345 ◽  
Author(s):  
William A. Day ◽  
Jaime L. Sajecki ◽  
Todd M. Pitts ◽  
Lynn A. Joens
Keyword(s):  
Cell Survival ◽  
Kinetic Studies ◽  
Intracellular Survival ◽  
Peritoneal Macrophages ◽  
Host Cells ◽  
Wild Type ◽  
Gene Encoding ◽  
Zymographic Analysis ◽  
Oxide Synthase

ABSTRACT The ability of Campylobacter jejuni to penetrate normally nonphagocytic host cells is believed to be a key virulence determinant. Recently, kinetics of C. jejuni intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies conducted by Pesci et al. indicate that superoxide dismutase contributes to intraepithelial cell survival, as isogenicsod mutants are 12-fold more sensitive to intracellular killing than wild-type strains. These findings suggest that bacterial factors that combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. Zymographic analysis indicated that C. jejuniexpresses a single catalase enzyme. The gene encoding catalase (katA) was cloned via functional complementation, and an isogenic katA mutant strain was constructed. Kinetic studies indicate that catalase provides resistance to hydrogen peroxide in vitro but does not play a role in intraepithelial cell survival. Catalase does however contribute to intramacrophage survival. Kinetic studies of C. jejuni growth in murine and porcine peritoneal macrophages demonstrated extensive killing of both wild-type and katA mutant strains shortly following internalization. Long-term cultures (72 h postinfection) of infected phagocytes permitted recovery of viable wild-typeC. jejuni; in contrast, no viable katA mutant bacteria were recovered. Accordingly, inhibition of macrophage nitric oxide synthase or NADPH oxidase permitted recovery of katAmutant C. jejuni. These observations indicate that catalase is essential for C. jejuni intramacrophage persistence and growth and suggest a novel mechanism of intracellular survival.

scholarly journals In Vitro Activity of Acanthamoeba castellanii on Human Platelets and Erythrocytes

2008 ◽  
Vol 77 (2) ◽  
pp. 733-738 ◽  
Author(s):  
A. Mattana ◽  
L. Alberti ◽  
G. Delogu ◽  
P. L. Fiori ◽  
P. Cappuccinelli
Keyword(s):  
Hemolytic Activity ◽  
Acanthamoeba Castellanii ◽  
Human Platelets ◽  
Free Calcium ◽  
Plasma Membrane Permeability ◽  
Human Red Blood Cells ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration

ABSTRACT The effect of Acanthamoeba on human platelets and erythrocytes has not been fully elucidated. This paper reports that cell-free supernatants prepared from A. castellanii can activate human platelets, causing both a significant increase in the cytosolic free-calcium concentration and platelet aggregation. In addition, we demonstrated that platelet activation depends on the activity of ADP constitutively secreted into the medium by trophozoites. This study also showed that A. castellanii can affect human red blood cells, causing hemolysis, and provided evidence that hemolysis occurs in both contact-dependent and contact-independent ways; there are differences in kinetics, hemolytic activity, and calcium dependency between the contact-dependent and contact-independent mechanisms. Partial characterization of contact-independent hemolysis indicated that ADP does not affect the plasma membrane permeability of erythrocytes and that heat treatment of amoebic cell-free supernatant abolishes its hemolytic activity. These findings suggest that some heat-labile molecules released by A. castellanii trophozoites are involved in this phenomenon. Finally, our data suggest that human platelets and erythrocytes may be potential cell targets during Acanthamoeba infection.

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