scholarly journals Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes.

1987 ◽  
Vol 166 (5) ◽  
pp. 1310-1328 ◽  
Author(s):  
M A Horwitz
Keyword(s):  
Legionella Pneumophila ◽  
High Titer ◽  
Serial Passage ◽  
Secretory Protein ◽  
Mononuclear Phagocyte ◽  
Human Monocytes ◽  
Wild Type ◽  
Artificial Medium ◽  
Legionnaires Disease

Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve as prototypes in the development of safe and effective vaccines against intracellular pathogens.

scholarly journals Secreted Enzymatic Activities of Wild-Type andpilD-Deficient Legionella pneumophila

2000 ◽  
Vol 68 (4) ◽  
pp. 1855-1863 ◽  
Author(s):  
Virginia Aragon ◽  
Sherry Kurtz ◽  
Antje Flieger ◽  
Birgid Neumeister ◽  
Nicholas P. Cianciotto
Keyword(s):  
Fatty Acids ◽  
Free Fatty Acids ◽  
Legionella Pneumophila ◽  
Wild Type ◽  
Intracellular Infection ◽  
Type Iv ◽  
Nitrophenyl Phosphate ◽  
Pilus Biogenesis ◽  
Legionnaires Disease

ABSTRACT Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by apilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release ofp-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.

scholarly journals Expression of Multiple Pili by Legionella pneumophila: Identification and Characterization of a Type IV Pilin Gene and Its Role in Adherence to Mammalian and Protozoan Cells

1998 ◽  
Vol 66 (4) ◽  
pp. 1768-1775 ◽  
Author(s):  
Barbara J. Stone ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Insertion Mutation ◽  
Wild Type ◽  
Type Iv ◽  
Transmission Electron ◽  
Wild Type Phenotype ◽  
Type Iv Pilin ◽  
Legionnaires Disease ◽  
Pilin Gene

ABSTRACT Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 μm) or short (0.1 to 0.6 μm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL ). An insertion mutation was constructed in pilEL and introduced into theL. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that thepilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires’ disease and invasion of amoebas in the environment.

scholarly journals Legionella pneumophila Entry GenertxA Is Involved in Virulence

2001 ◽  
Vol 69 (1) ◽  
pp. 508-517 ◽  
Author(s):  
Suat L. G. Cirillo ◽  
Luiz E. Bermudez ◽  
Sahar H. El-Etr ◽  
Gerald E. Duhamel ◽  
Jeffrey D. Cirillo
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Integrin Receptors ◽  
Legionnaires Disease ◽  
Cell Spread ◽  
Gain Access

ABSTRACT Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogen Legionella pneumophila. Currently, the mechanisms used by L. pneumophila to gain access to the intracellular environment are not well understood. We have recently isolated three loci, designated enh1,enh2, and enh3, that are involved in the ability of L. pneumophila to enter host cells. One of the genes present in the enh1 locus, rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to β2 integrin receptors. In the current study, we demonstrate that the L. pneumophila rtxA gene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, an rtxA mutant does not replicate as well as wild-type L. pneumophila in monocytes and is less virulent in mice. Thus, we conclude that the entry genertxA is an important virulence determinant in L. pneumophila and is likely to be critical for the production of Legionnaires' disease in humans.

scholarly journals Phylogenomic and Biochemical Characterization of Three Legionella pneumophila Polypeptide Deformylases

2006 ◽  
Vol 188 (14) ◽  
pp. 5249-5257 ◽  
Author(s):  
Jianzhong Huang ◽  
Glenn S. Van Aller ◽  
Amy N. Taylor ◽  
John J. Kerrigan ◽  
Wu-Schyong Liu ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Biochemical Characterization ◽  
Enzymatic Characterization ◽  
Genome Sequences ◽  
Catalytic Activities ◽  
Synthetic Polypeptides ◽  
Catalytically Active ◽  
Legionnaires Disease ◽  
Antibacterial Target

ABSTRACT Legionella pneumophila is a gram-negative facultative intracellular human pathogen that can cause fatal Legionnaires' disease. Polypeptide deformylase (PDF) is a novel broad-spectrum antibacterial target, and reports of inhibitors of PDF with potent activities against L. pneumophila have been published previously. Here, we report the identification of not one but three putative pdf genes, pdfA, pdfB, and pdfC, in the complete genome sequences of three strains of L. pneumophila. Phylogenetic analysis showed that L. pneumophila PdfA is most closely related to the commonly known γ-proteobacterial PDFs encoded by the gene def. PdfB and PdfC are more divergent and do not cluster with any specific bacterial or eukaryotic PDF. All three putative pdf genes from L. pneumophila strain Philadelphia 1 have been cloned, and their encoded products have been overexpressed in Escherichia coli and purified. Enzymatic characterization shows that the purified PDFs with Ni2+ substituted are catalytically active and able to remove the N-formyl group from several synthetic polypeptides, although they appear to have different substrate specificities. Surprisingly, while PdfA and PdfB with Zn2+ substituted are much less active than the Ni2+ forms of each enzyme, PdfC with Zn2+ substituted was as active as the Ni2+ form for the fMA substrate and exhibited substrate specificity different from that of Ni2+ PdfC. Furthermore, the catalytic activities of these enzymes are potently inhibited by a known small-molecule PDF inhibitor, BB-3497, which also inhibits the extracellular growth of L. pneumophila. These results indicate that even though L. pneumophila has three PDFs, they can be effectively inhibited by PDF inhibitors which can, therefore, have potent anti-L. pneumophila activity.

scholarly journals Genome Characterization of Yellow Fever Virus Wild-Type Strain Asibi, Parent to Live-Attenuated 17D Vaccine, from Three Different Sources

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1383
Author(s):  
Emily H. Davis ◽  
Jill K. Thompson ◽  
Steven G. Widen ◽  
Alan D. T. Barrett
Keyword(s):  
Yellow Fever ◽  
Wild Type Strain ◽  
Yellow Fever Virus ◽  
Serial Passage ◽  
Vaccine Virus ◽  
Fever Virus ◽  
Wild Type ◽  
Chicken Tissue ◽  
Different Sources

The yellow fever virus vaccine, 17D, was derived through the serial passage of the wild-type (WT) strain Asibi virus in mouse and chicken tissue. Since its derivation, the mechanism of attenuation of 17D virus has been investigated using three 17D substrains and WT Asibi virus. Although all three substrains of 17D have been sequenced, only one isolate of Asibi has been examined genetically and all interpretation of attenuation is based on this one isolate. Here, we sequenced the genome of Asibi virus from three different laboratories and show that the WT strain is genetically homogenous at the amino acids that distinguish Asibi from 17D vaccine virus.

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 Structural and Functional Characterization of Legionella pneumophila Effector MavL

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1802
Author(s):  
Kevin Voth ◽  
Shivani Pasricha ◽  
Ivy Yeuk Wah Chung ◽  
Rachelia R. Wibawa ◽  
Engku Nuraishah Huda E. Zainudin ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Functional Characterization ◽  
Effector Proteins ◽  
Type Iv ◽  
Ubiquitin Conjugating Enzyme ◽  
Legionnaires Disease ◽  
Organelle Trafficking ◽  
Binding Cleft ◽  
Adp Ribosyltransferase

Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires' disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose “reader” proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitin-conjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila.

scholarly journals Identification of the Aspartate-β-Semialdehyde Dehydrogenase Gene of Legionella pneumophila and Characterization of a Null Mutant

1998 ◽  
Vol 66 (5) ◽  
pp. 1898-1903 ◽  
Author(s):  
Omar S. Harb ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Culture Media ◽  
Host Cells ◽  
Diaminopimelic Acid ◽  
Contaminated Water ◽  
Intracellular Environment ◽  
Semialdehyde Dehydrogenase ◽  
Dehydrogenase Gene ◽  
Legionnaires Disease

ABSTRACT The ability of Legionella pneumophila to cause Legionnaires’ disease is dependent on its capacity to survive in the intracellular environment of its host cells. Furthermore, outbreaks of this disease have been associated with contaminated water sources whereL. pneumophila survives as a parasite of protozoa. In this study, we determined the effect of nutritional auxotrophy on the ability of L. pneumophila to survive in the intracellular environment of its host cells. We generated a diaminopimelic acid (DAP) auxotroph (AA400) of L. pneumophila by disruption of the aspartate-β-semialdehyde (asd) gene. The ability of AA400 to survive within macrophages and protozoa was found to be defective. This defect was due solely to the asd disruption since complementation of the mutant with the wild-type asd gene restored its capacity for intracellular survival. Furthermore, the defect was not completely complemented by DAP supplementation to the culture media. Thus, our results suggest that disruption of theasd gene may prove to be useful in the design of attenuated vaccines against Legionnaires’ disease.

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