scholarly journals In Vitro and Intracellular Activities of LBM415 (NVP PDF-713) against Legionella pneumophila

2005 ◽  
Vol 49 (6) ◽  
pp. 2533-2535 ◽  
Author(s):  
Paul H. Edelstein ◽  
Baofeng Hu ◽  
Martha A. C. Edelstein
Keyword(s):  
Legionella Pneumophila ◽  
Intracellular Growth ◽  
Intracellular Activities

ABSTRACT LBM415 activity against extracellular and intracellular Legionella pneumophila was studied. The LBM415 MIC50 for 20 Legionella sp. strains was 4 μg/ml, versus 0.06, 0.25, and ≤ 0.03 μg/ml for azithromycin, erythromycin, and levofloxacin, respectively. LBM415 (0.5 and 16 μg/ml) reversibly prevented intracellular growth of two L. pneumophila strains and was less active than erythromycin.

scholarly journals Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

1998 ◽  
Vol 36 (5) ◽  
pp. 1392-1398 ◽  
Author(s):  
Futoshi Higa ◽  
Nobuchika Kusano ◽  
Masao Tateyama ◽  
Takashi Shinzato ◽  
Noriko Arakaki ◽  
...  
Keyword(s):  
Yeast Extract ◽  
Legionella Pneumophila ◽  
Colorimetric Assay ◽  
Assay System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intracellular Growth ◽  
Cell Monolayers ◽  
Cell Counts ◽  
Efficient Processing ◽  
Intracellular Activities

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.

scholarly journals Glucose Metabolism in Legionella pneumophila: Dependence on the Entner-Doudoroff Pathway and Connection with Intracellular Bacterial Growth

2010 ◽  
Vol 192 (11) ◽  
pp. 2892-2899 ◽  
Author(s):  
Eiji Harada ◽  
Ken-Ichiro Iida ◽  
Susumu Shiota ◽  
Hiroaki Nakayama ◽  
Shin-Ichi Yoshida
Keyword(s):  
Glucose Metabolism ◽  
Legionella Pneumophila ◽  
Glucose Transporter ◽  
Intracellular Growth ◽  
Gene Encoding ◽  
Permeabilized Cell ◽  
Appreciable Change ◽  
Exogenous Glucose ◽  
Formed Part

ABSTRACT Glucose metabolism in Legionella pneumophila was studied by focusing on the Entner-Doudoroff (ED) pathway with a combined genetic and biochemical approach. The bacterium utilized exogenous glucose for synthesis of acid-insoluble cell components but manifested no discernible increase in the growth rate. Assays with permeabilized cell preparations revealed the activities of three enzymes involved in the pathway, i.e., glucokinase, phosphogluconate dehydratase, and 2-dehydro-3-deoxy-phosphogluconate aldolase, presumed to be encoded by the glk, edd, and eda genes, respectively. Gene-disrupted mutants for the three genes and the ywtG gene encoding a putative sugar transporter were devoid of the ability to metabolize exogenous glucose, indicating that the pathway is almost exclusively responsible for glucose metabolism and that the ywtG gene product is the glucose transporter. It was also established that these four genes formed part of an operon in which the gene order was edd-glk-eda-ywtG, as predicted by genomic information. Intriguingly, while the mutants exhibited no appreciable change in growth characteristics in vitro, they were defective in multiplication within eukaryotic cells, strongly indicating that the ED pathway must be functional for the intracellular growth of the bacterium to occur. Curiously, while the deficient glucose metabolism of the ywtG mutant was successfully complemented by the ywtG + gene supplied in trans via plasmid, its defect in intracellular growth was not. However, the latter defect was also manifested in wild-type cells when a plasmid carrying the mutant ywtG gene was introduced. This phenomenon, resembling so-called dominant negativity, awaits further investigation.

scholarly journals Legionella pneumophila OxyR Is a Redundant Transcriptional Regulator That Contributes to Expression Control of the Two-Component CpxRA System

2016 ◽  
Vol 199 (5) ◽  
Author(s):  
Jennifer R. Tanner ◽  
Palak G. Patel ◽  
Jacqueline R. Hellinga ◽  
Lynda J. Donald ◽  
Celine Jimenez ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Mutational Analysis ◽  
Transcriptional Regulator ◽  
Intracellular Growth ◽  
Content Type ◽  
Expression Control ◽  
Two Component ◽  
Legionnaires Disease

ABSTRACT Nominally an environmental organism, Legionella pneumophila is an intracellular parasite of protozoa but is also the causative agent of the pneumonia termed Legionnaires' disease, which results from inhalation of aerosolized bacteria by susceptible humans. Coordination of gene expression by a number of identified regulatory factors, including OxyR, assists L. pneumophila in adapting to the stresses of changing environments. L. pneumophila OxyR (OxyRLp) is an ortholog of Escherichia coli OxyR; however, OxyRLp was shown elsewhere to be functionally divergent, such that it acts as a transcription regulator independently of the oxidative stress response. In this study, the use of improved gene deletion methods has enabled us to generate an unmarked in-frame deletion of oxyR in L. pneumophila. Lack of OxyRLp did not affect in vitro growth or intracellular growth in Acanthamoeba castellanii protozoa and U937-derived macrophages. The expression of OxyRLp does not appear to be regulated by CpxR, even though purified recombinant CpxR bound a DNA sequence similar to that reported for CpxR elsewhere. Surprisingly, a lack of OxyRLp resulted in elevated activity of the promoters located upstream of icmR and the lpg1441-cpxA operon, and OxyRLp directly bound to these promoter regions, suggesting that OxyRLp is a direct repressor. Interestingly, a strain overexpressing OxyRLp demonstrated reduced intracellular growth in A. castellanii but not in U937-derived macrophages, suggesting that balanced expression control of the two-component CpxRA system is necessary for survival in protozoa. Taken together, this study suggests that OxyRLp is a functionally redundant transcriptional regulator in L. pneumophila under the conditions evaluated herein. IMPORTANCE Legionella pneumophila is an environmental pathogen, with its transmission to the human host dependent upon its ability to replicate in protozoa and survive within its aquatic niche. Understanding the genetic factors that contribute to L. pneumophila survival within each of these unique environments will be key to limiting future point-source outbreaks of Legionnaires' disease. The transcriptional regulator L. pneumophila OxyR (OxyRLp) has been previously identified as a potential regulator of virulence traits warranting further investigation. This study demonstrated that oxyR is nonessential for L. pneumophila survival in vitro and in vivo via mutational analysis. While the mechanisms of how OxyRLp expression is regulated remain elusive, this study shows that OxyRLp negatively regulates the expression of the cpxRA two-component system necessary for intracellular survival in protozoa.

scholarly journals In Vitro and Intracellular Activities of Omadacycline against Legionella pneumophila

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Jacques Dubois ◽  
Maïtée Dubois ◽  
Jean-François Martel
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Pneumonia ◽  
Respiratory Infections ◽  
Content Type ◽  
Intracellular Activities ◽  
Extracellular Activity

ABSTRACT Omadacycline is an aminomethylcycline antibiotic with in vitro activity against pathogens causing community-acquired bacterial pneumonia (CABP). This study investigated the activity of omadacycline against Legionella pneumophila strains isolated between 1995 and 2014 from nosocomial or community-acquired respiratory infections. Omadacycline exhibited extracellular activity similar to comparator antibiotics; intracellular penetrance was found by day 3 of omadacycline exposure. These results support the utility of omadacycline as an effective antibiotic for the treatment of CABP caused by L. pneumophila.

scholarly journals Iron depletion limits intracellular bacterial growth in macrophages

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 866-874 ◽  
Author(s):  
Prasad N. Paradkar ◽  
Ivana De Domenico ◽  
Nina Durchfort ◽  
Irene Zohn ◽  
Jerry Kaplan ◽  
...  
Keyword(s):  
Bacterial Growth ◽  
Legionella Pneumophila ◽  
Bacterial Infections ◽  
Iron Chelator ◽  
Chlamydia Psittaci ◽  
Loss Of Function ◽  
Intracellular Growth ◽  
Intracellular Iron ◽  
Cellular Iron

AbstractMany intracellular pathogens infect macrophages and these pathogens require iron for growth. Here we demonstrate in vitro that the intracellular growth of Chlamydia psittaci, trachomatis, and Legionella pneumophila is regulated by the levels of intracellular iron. Macrophages that express cell surface ferroportin, the only known cellular iron exporter, limit the intracellular growth of these bacteria. Hepcidin is an antimicrobial peptide secreted by the liver in response to inflammation. Hepcidin binds to ferroportin mediating its internalization and degradation. Addition of hepcidin to infected macrophages enhanced the intracellular growth of these pathogens. Macrophages from flatiron mice, a strain heterozygous for a loss-of-function ferroportin mutation, showed enhanced intracellular bacterial growth independent of the presence of exogenous hepcidin. Macrophages, from wild-type or flatiron mice, incubated with the oral iron chelator deferriprone or desferasirox showed reduced intracellular bacterial growth suggesting that these chelators might be therapeutic in chronic intracellular bacterial infections.

scholarly journals In vitro and intracellular activities of frog skin temporins against Legionella pneumophila and its eukaryotic hosts

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Alexandre Crépin ◽  
Jean-François Jégou ◽  
Sonia André ◽  
Florine Ecale ◽  
Anastasia Croitoru ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Frog Skin ◽  
Intracellular Activities

scholarly journals The Legionella pneumophila Incomplete Phosphotransferase System Is Required for Optimal Intracellular Growth and Maximal Expression of PmrA-Regulated Effectors

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Yariv Speiser ◽  
Tal Zusman ◽  
Anna Pasechnek ◽  
Gil Segal
Keyword(s):  
Legionella Pneumophila ◽  
Intracellular Growth ◽  
Phosphotransferase System ◽  
Content Type ◽  
Double Deletion ◽  
Regulatory Cascade ◽  
Encoding Genes ◽  
Enzyme I ◽  
Maximal Expression

ABSTRACT The nitrogen phosphotransferase system (PTSNtr) is a regulatory cascade present in many bacteria, where it controls different functions. This system is usually composed of three basic components: enzyme INtr (EINtr), NPr, and EIIANtr (encoded by the ptsP, ptsO, and ptsN genes, respectively). In Legionella pneumophila, as well as in many other Legionella species, the EIIANtr component is missing. However, we found that deletion mutations in both ptsP and ptsO are partially attenuated for intracellular growth. Furthermore, these two PTSNtr components were found to be required for maximal expression of effector-encoding genes regulated by the transcriptional activator PmrA. Genetic analyses which include the construction of single and double deletion mutants and overexpression of wild-type and mutated forms of EINtr, NPr, and PmrA indicated that the PTSNtr components affect the expression of PmrA-regulated genes via PmrA and independently from PmrB and that EINtr and NPr are part of the same cascade and require their conserved histidine residues in order to function. Furthermore, expression of the Legionella micdadei EIINtr component in L. pneumophila resulted in a reduction in the levels of expression of PmrA-regulated genes which was completely dependent on the L. pneumophila PTS components and the L. micdadei EIINtr conserved histidine residue. Moreover, reconstruction of the L. pneumophila PTS in vitro indicated that EINtr is phosphorylated by phosphoenolpyruvate (PEP) and transfers its phosphate to NPr. Our results demonstrate that the L. pneumophila incomplete PTSNtr is functional and involved in the expression of effector-encoding genes regulated by PmrA.

scholarly journals In Vitroand Intracellular Activities of Peptide Deformylase Inhibitor GSK1322322 against Legionella pneumophila Isolates

2014 ◽  
Vol 59 (1) ◽  
pp. 707-710
Author(s):  
Jacques Dubois ◽  
Maïtée Dubois ◽  
Jean-François Martel ◽  
Kelly Aubart ◽  
Deborah Butler
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Pneumonia ◽  
Peptide Deformylase ◽  
Content Type ◽  
Intracellular Activity ◽  
Intracellular Activities ◽  
Intracellular Proliferation

ABSTRACTGSK1322322, a novel peptide deformylase inhibitor currently in development as an oral and intravenous agent for the treatment of hospitalized community-acquired bacterial pneumonia, showed poorin vitroactivity against a panel of 50Legionella pneumophilastrains, with MICs ranging from 1 to 16 μg/ml and an MIC90of 16 μg/ml, but very potent intracellular activity, with the minimum extracellular concentrations capable of inhibiting intracellular proliferation (MIECs) ranging from 0.12 to 2 μg/ml and 98% of the strains being inhibited by concentrations of ≤1 μg/ml.

scholarly journals Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

2004 ◽  
Vol 72 (10) ◽  
pp. 5983-5992 ◽  
Author(s):  
Jessica A. Sexton ◽  
Jennifer L. Miller ◽  
Aki Yoneda ◽  
Thomas E. Kehl-Fie ◽  
Joseph P. Vogel
Keyword(s):  
Cell Surface ◽  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Wide Distribution ◽  
Host Cells ◽  
Growth Defect ◽  
Intracellular Growth ◽  
Homologous Proteins ◽  
Type Iv ◽  
Cell Surface Structure

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

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