Role of the LuxR family transcriptional regulator Lpg2524 in the survival ofLegionella pneumophilain water

2017 ◽  
Vol 63 (6) ◽  
pp. 535-545 ◽  
Author(s):  
Laam Li ◽  
Sébastien P. Faucher
Keyword(s):  
Cell Density ◽  
Legionella Pneumophila ◽  
High Cell Density ◽  
Acanthamoeba Castellanii ◽  
Transcriptional Regulator ◽  
Human Macrophage ◽  
High Cell ◽  
Legionnaires Disease

The water-borne Gram-negative bacterium Legionella pneumophila (Lp) is the causative agent of Legionnaires’ disease. Lp is typically transmitted to humans from water systems, where it grows inside amoebae. Survival of Lp in water is central to its transmission to humans. A transcriptomic study previously identified many genes induced by Lp in water. One such gene, lpg2524, encodes a putative LuxR family transcriptional regulator. It was hypothesized that this gene could be involved in the survival of Lp in water. Deletion of lpg2524 does not affect the growth of Lp in rich medium, in the amoeba Acanthamoeba castellanii, or in human macrophage-like THP-1 cells, showing that Lpg2524 is not required for growth in vitro and in vivo. Nevertheless, deletion of lpg2524 results in a faster colony-forming unit (CFU) reduction in an artificial freshwater medium, Fraquil, indicating that Lpg2524 is important for Lp to survive in water. Overexpression of Lpg2524 also results in a survival defect, suggesting that a precise level of this transcriptional regulator is essential for its function. However, our result shows that Lpg2524 is dispensable for survival in water when Lp is at a high cell density (109CFU/mL), suggesting that its regulon is regulated by another regulator activated at high cell density.

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 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.

scholarly journals Identification of a Novel Adhesion Molecule Involved in the Virulence of Legionella pneumophila

2005 ◽  
Vol 73 (7) ◽  
pp. 4272-4280 ◽  
Author(s):  
Bin Chang ◽  
Fumiaki Kura ◽  
Junko Amemura-Maekawa ◽  
Nobuo Koizumi ◽  
Haruo Watanabe
Keyword(s):  
Cell Line ◽  
Adhesion Molecule ◽  
Legionella Pneumophila ◽  
Alveolar Epithelial Cell ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Human Macrophage ◽  
Convalescent Phase ◽  
Legionnaires Disease

ABSTRACT Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045 and may play a role in Legionnaires' disease in humans.

scholarly journals 3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Andrew C. Daly ◽  
Matthew D. Davidson ◽  
Jason A. Burdick
Keyword(s):  
Cell Density ◽  
Spatial Organization ◽  
High Cell Density ◽  
Self Healing ◽  
High Cell ◽  
Cellular Models ◽  
Functional Features ◽  
Cell Densities ◽  
Tissue Models

AbstractCellular models are needed to study human development and disease in vitro, and to screen drugs for toxicity and efficacy. Current approaches are limited in the engineering of functional tissue models with requisite cell densities and heterogeneity to appropriately model cell and tissue behaviors. Here, we develop a bioprinting approach to transfer spheroids into self-healing support hydrogels at high resolution, which enables their patterning and fusion into high-cell density microtissues of prescribed spatial organization. As an example application, we bioprint induced pluripotent stem cell-derived cardiac microtissue models with spatially controlled cardiomyocyte and fibroblast cell ratios to replicate the structural and functional features of scarred cardiac tissue that arise following myocardial infarction, including reduced contractility and irregular electrical activity. The bioprinted in vitro model is combined with functional readouts to probe how various pro-regenerative microRNA treatment regimes influence tissue regeneration and recovery of function as a result of cardiomyocyte proliferation. This method is useful for a range of biomedical applications, including the development of precision models to mimic diseases and the screening of drugs, particularly where high cell densities and heterogeneity are important.

scholarly journals Intracellular Growth of Legionella pneumophila Gives Rise to a Differentiated Form Dissimilar to Stationary-Phase Forms

2002 ◽  
Vol 70 (11) ◽  
pp. 6273-6283 ◽  
Author(s):  
Rafael A. Garduño ◽  
Elizabeth Garduño ◽  
Margot Hiltz ◽  
Paul S. Hoffman
Keyword(s):  
Stationary Phase ◽  
Hela Cells ◽  
Legionella Pneumophila ◽  
Protein A ◽  
Plaque Assay ◽  
Growth Cycle ◽  
Potential Marker ◽  
Legionnaires Disease

ABSTRACT When Legionella pneumophila grows in HeLa cells, it alternates between a replicative form and a morphologically distinct “cyst-like” form termed MIF (mature intracellular form). MIFs are also formed in natural amoebic hosts and to a lesser extent in macrophages, but they do not develop in vitro. Since MIFs accumulate at the end of each growth cycle, we investigated the possibility that they are in vivo equivalents of stationary-phase (SP) bacteria, which are enriched for virulence traits. By electron microscopy, MIFs appeared as short, stubby rods with an electron-dense, laminar outer membrane layer and a cytoplasm largely occupied by inclusions of poly-β-hydroxybutyrate and laminations of internal membranes originating from the cytoplasmic membrane. These features may be responsible for the bright red appearance of MIFs by light microscopy following staining with the phenolic Giménez stain. In contrast, SP bacteria appeared as dull red rods after Giménez staining and displayed a typical gram-negative cell wall ultrastructure. Outer membranes from MIFs and SP bacteria were equivalent in terms of the content of the peptidoglycan-bound and disulfide bond cross-linked OmpS porin, although additional proteins, including Hsp60 (which acts as an invasin for HeLa cells), were detected only in preparations from MIFs. Proteomic analysis revealed differences between MIFs and SP forms; in particular, MIFs were enriched for an ∼20-kDa protein, a potential marker of development. Compared with SP bacteria, MIFs were 10-fold more infectious by plaque assay, displayed increased resistance to rifampin (3- to 5-fold) and gentamicin (10- to 1,000-fold), resisted detergent-mediated lysis, and tolerated high pH. Finally, MIFs had a very low respiration rate, consistent with a decreased metabolic activity. Collectively, these results suggest that intracellular L. pneumophila differentiates into a cyst-like, environmentally resilient, highly infectious, post-SP form that is distinct from in vitro SP bacteria. Therefore, MIFs may represent the transmissible environmental forms associated with Legionnaires' disease.

scholarly journals Expression of putative pathogenicity-related genes inXylella fastidiosagrown at low and high cell density conditions in vitro

2003 ◽  
Vol 222 (1) ◽  
pp. 83-92 ◽  
Author(s):  
Leandra M. Scarpari ◽  
Marcio R. Lambais ◽  
Denise S. Silva ◽  
Dirce M. Carraro ◽  
Helaine Carrer
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
High Cell

scholarly journals 3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels

2020 ◽  
Author(s):  
Andrew C. Daly ◽  
Matthew D. Davidson ◽  
Jason A. Burdick
Keyword(s):  
Cell Density ◽  
Spatial Organization ◽  
High Cell Density ◽  
Self Healing ◽  
High Cell ◽  
Cellular Models ◽  
Functional Features ◽  
Cell Densities ◽  
Tissue Models

AbstractCellular models are needed to study human development and disease in vitro, including the screening of drugs for toxicity and efficacy. However, current approaches are limited in the engineering of functional tissue models with requisite cell densities and heterogeneity to appropriately model cell and tissue behaviors. Here, we develop a new bioprinting approach to transfer spheroids into self-healing support hydrogels at high resolution, which enables their patterning and fusion into high-cell density microtissues of prescribed spatial organization. As an example application, we bioprint induced pluripotent stem cell-derived cardiac microtissue models with spatially controlled cardiomyocyte and fibroblast cell ratios to replicate the structural and functional features of scarred cardiac tissue that arise following myocardial infarction, including reduced contractility and irregular electrical activity. The bioprinted in vitro model is combined with functional readouts to probe how various pro-regenerative microRNA treatment regimes influence tissue regeneration and recovery of function as a result of cardiomyocyte proliferation. This method is useful for a range of biomedical applications, including the development of precision models to mimic diseases and for the screening of drugs, particularly where high cell densities and heterogeneity are important.

Sign in / Sign up

Export Citation Format

Share Document