The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform

David R Murdoch - One of the best experts on this subject based on the ideXlab platform.

  • diagnosis of Legionella Infection
    Clinical Infectious Diseases, 2003
    Co-Authors: Barth L Reller, Melvin P Weinstein, David R Murdoch
    Abstract:

    Legionellae, which are important causes of pneumonia in humans, continue to be incorrectly labeled as exotic pathogens. The ability to diagnose Legionella Infection is limited by the nonspecific nature of clinical features and the shortcomings of diagnostic tests. Despite recent improvements, existing diagnostic tests for Legionella Infection either lack sensitivity for detecting all clinically important Legionellae or are unable to provide results within a clinically useful time frame. Understanding local Legionella epidemiology is important for making decisions about whether to test for Legionella Infection and which diagnostic tests to use. In most situations, the use of both the urinary antigen test plus sputum culture is the best diagnostic combination. Polymerase chain reaction (PCR) is a promising tool, but standardized assays are not commercially available. Further work needs to focus on the development of urinary antigen tests assays that detect a wider range of pathogenic Legionellae and on the development of standardized PCR assays.

Barry M Farr - One of the best experts on this subject based on the ideXlab platform.

  • ultraviolet light disInfection of hospital water for preventing nosocomial Legionella Infection a 13 year follow up
    Infection Control and Hospital Epidemiology, 2003
    Co-Authors: Keri K Hall, Eve T Giannetta, Sandra I Getchellwhite, Lisa J Durbin, Barry M Farr
    Abstract:

    BACKGROUND AND OBJECTIVE: CDC has estimated that 23% of Legionella Infections are nosocomial. When a new hospital was being constructed and a substantial increase in transplantation was anticipated, an ultraviolet light apparatus was installed in the water main of the new building because 27% of water samples from taps in the old hospital contained Legionella. This study reports the rate of nosocomial Legionella Infection and water contamination since opening the new hospital. METHODS: Charts of all patients with positive Legionella cultures, direct immunofluorescent antibody (DFA), or urine antigen between April 1989 and November 2001 were reviewed. Frequencies of DFAs and urine antigens were obtained from the laboratory. RESULTS: None of the 930 cultures of hospital water have been positive since moving into the new building. Fifty-three (0.02%) of 219,521 patients had a positive Legionella test; 41 had pneumonia (40 community acquired). One definite L. pneumophila pneumonia confirmed by culture and DFA in August 1994 was nosocomial (0.0005%) by dates. This patient was transferred after prolonged hospitalization in another country, was transplanted 11 days after admission, and developed symptoms 5 days after liver transplant. However, tap water from the patient's room did not grow Legionella. Seventeen (2.5%) of 670 urine antigens were positive for Legionella (none nosocomial). Thirty-three (1.2%) of 2,671 DFAs were positive, including 7 patients (21%) without evidence of pneumonia and 6 (18%) who had an alternative diagnosis. CONCLUSION: Ultraviolet light usage was associated with negative water cultures and lack of clearly documented nosocomial Legionella Infection for 13 years at this hospital.

  • Ultraviolet light disInfection of hospital water for preventing nosocomial Legionella Infection: a 13-year follow-up.
    Infection control and hospital epidemiology, 2003
    Co-Authors: Keri K Hall, Eve T Giannetta, Lisa J Durbin, Sandra I. Getchell-white, Barry M Farr
    Abstract:

    BACKGROUND AND OBJECTIVE: CDC has estimated that 23% of Legionella Infections are nosocomial. When a new hospital was being constructed and a substantial increase in transplantation was anticipated, an ultraviolet light apparatus was installed in the water main of the new building because 27% of water samples from taps in the old hospital contained Legionella. This study reports the rate of nosocomial Legionella Infection and water contamination since opening the new hospital. METHODS: Charts of all patients with positive Legionella cultures, direct immunofluorescent antibody (DFA), or urine antigen between April 1989 and November 2001 were reviewed. Frequencies of DFAs and urine antigens were obtained from the laboratory. RESULTS: None of the 930 cultures of hospital water have been positive since moving into the new building. Fifty-three (0.02%) of 219,521 patients had a positive Legionella test; 41 had pneumonia (40 community acquired). One definite L. pneumophila pneumonia confirmed by culture and DFA in August 1994 was nosocomial (0.0005%) by dates. This patient was transferred after prolonged hospitalization in another country, was transplanted 11 days after admission, and developed symptoms 5 days after liver transplant. However, tap water from the patient's room did not grow Legionella. Seventeen (2.5%) of 670 urine antigens were positive for Legionella (none nosocomial). Thirty-three (1.2%) of 2,671 DFAs were positive, including 7 patients (21%) without evidence of pneumonia and 6 (18%) who had an alternative diagnosis. CONCLUSION: Ultraviolet light usage was associated with negative water cultures and lack of clearly documented nosocomial Legionella Infection for 13 years at this hospital.

Ivan Dikic - One of the best experts on this subject based on the ideXlab platform.

  • bacterial otu deubiquitinases regulate substrate ubiquitination upon Legionella Infection
    eLife, 2020
    Co-Authors: Donghyuk Shin, Anshu Bhattacharya, Yilin Cheng, Marta Campos Alonso, Ahmad Reza Mehdipour, Gerbrand J Van Der Heden Van Noort, Huib Ovaa, Gerhard Hummer, Ivan Dikic
    Abstract:

    Legionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the Infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two L egionella OT U (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 A. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1'), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host-pathogen interactions.

  • serine ubiquitination regulates golgi morphology and the secretory pathway upon Legionella Infection
    bioRxiv, 2020
    Co-Authors: Yaobin Liu, Rukmini Mukherjee, Florian Bonn, Thomas Colby, Ivan Matic, Ivan Dikic
    Abstract:

    Abstract SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial Infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA induces fragmentation of the Golgi stacks due to its ubiquitin ligase activity. The Golgi tethering factors GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi fragmentation is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the secretory pathway, including cytokine release in cells. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial Infection.

  • novel class of otu deubiquitinases regulate substrate ubiquitination upon Legionella Infection
    bioRxiv, 2020
    Co-Authors: Donghyuk Shin, Anshu Bhattacharya, Yilin Cheng, Marta Campos Alonso, Gerbrand J Van Der Heden Van Noort, Huib Ovaa, Gerhard Hummer, Ahnad Reza Medipour, Ivan Dikic
    Abstract:

    Legionella pneumophila is a gram-negative pathogenic bacterium that causes Legionaries′ disease. The Legionella genome codes more than 300 effector proteins able to modulate host-pathogen interactions during Infection. Among them are also enzymes altering the host-ubiquitination system including bacterial ligases and deubiquitinases. In this study, based on homology-detection screening on 305 Legionella effector proteins, we identified two Legionella OTU-like deubiquitinases (LOT; LotB (Lpg1621/Ceg23) and LotC (Lpg2529), LotA (Lpg2248/Lem21) is already known). A crystal structure of LotC catalytic core (LotC14-310) was determined at 2.4A and compared with other OTU deubiquitinases, including LotB. Unlike the classical OTU-family, the structures of Legionella OTU-family (LotB and LotC) shows an extended helical lobe between the Cys-loop and the variable loop, which define a novel class of OTU-deubiquitinase. Despite structural differences in their helical lobes, both LotB and LotC interact with ubiquitin. LotB has an additional ubiquitin-binding site (S19) enabling specific cleavage of Lys63-linked poly-ubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of catalytically inactive LotB and LotC identified different categories of host-substrates for these two related DUBs. Together, our results provide new structural insights of bacterial OTU deubiquitinases and indicate distinct roles of bacterial deubiquitinases in host-pathogen interactions.

Hubert Hilbi - One of the best experts on this subject based on the ideXlab platform.

  • acanthamoeba and dictyostelium as cellular models for Legionella Infection
    Frontiers in Cellular and Infection Microbiology, 2018
    Co-Authors: Leoni A Swart, Michael Steinert, Ludwig Eichinger, Christopher F Harrison, Hubert Hilbi
    Abstract:

    Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of "effector" proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.

  • Live Cell Imaging of Phosphoinositide Dynamics During Legionella Infection
    Methods in molecular biology (Clifton N.J.), 2014
    Co-Authors: Stephen Weber, Hubert Hilbi
    Abstract:

    The "accidental" pathogen Legionella pneumophila replicates intracellularly in a distinct compartment, the Legionella-containing vacuole (LCV). To form this specific pathogen vacuole, the bacteria translocate via the Icm/Dot type IV secretion system approximately 300 different effector proteins into the host cell. Several of these secreted effectors anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. L. pneumophila thus largely controls the localization of secreted bacterial effectors and the recruitment of host factors to the LCV through the modulation of the vacuole membrane PI pattern. The LCV PI pattern and its dynamics can be studied in real-time using fluorescently labeled protein probes stably produced by the soil amoeba Dictyostelium discoideum. In this chapter, we describe a protocol to (1) construct and handle amoeba model systems as a tool for observing PIs in live cell imaging, (2) capture rapid changes in membrane PI patterning during uptake events, and (3) observe the dynamics of LCV PIs over the course of a Legionella Infection.

  • live cell imaging of phosphoinositide dynamics and membrane architecture during Legionella Infection
    Mbio, 2014
    Co-Authors: Stephen Weber, Maria A Wagner, Hubert Hilbi
    Abstract:

    ABSTRACT The causative agent of Legionnaires’ disease, Legionella pneumophila, replicates in amoebae and macrophages in a distinct membrane-bound compartment, the Legionella -containing vacuole (LCV). LCV formation is governed by the bacterial Icm/Dot type IV secretion system that translocates ~300 different “effector” proteins into host cells. Some of the translocated effectors anchor to the LCV membrane via phosphoinositide (PI) lipids. Here, we use the soil amoeba Dictyostelium discoideum, producing fluorescent PI probes, to analyze the LCV PI dynamics by live-cell imaging. Upon uptake of wild-type or Icm/Dot-deficient L. pneumophila, PtdIns(3,4,5) P 3 transiently accumulated for an average of 40 s on early phagosomes, which acquired PtdIns(3) P within 1 min after uptake. Whereas phagosomes containing Δ icmT mutant bacteria remained decorated with PtdIns(3) P , more than 80% of wild-type LCVs gradually lost this PI within 2 h. The process was accompanied by a major rearrangement of PtdIns(3) P -positive membranes condensing to the cell center. PtdIns(4) P transiently localized to early phagosomes harboring wild-type or Δ icmT L. pneumophila and was cleared within minutes after uptake. During the following 2 h, PtdIns(4) P steadily accumulated only on wild-type LCVs, which maintained a discrete PtdIns(4) P identity spatially separated from calnexin-positive endoplasmic reticulum (ER) for at least 8 h. The separation of PtdIns(4) P -positive and ER membranes was even more pronounced for LCVs harboring Δ sidC - sdcA mutant bacteria defective for ER recruitment, without affecting initial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI lipids implicated in LCV formation and provide insight into host cell membrane and effector protein interactions. IMPORTANCE The environmental bacterium Legionella pneumophila is the causative agent of Legionnaires’ pneumonia. The bacteria form in free-living amoebae and mammalian immune cells a replication-permissive compartment, the Legionella -containing vacuole (LCV). To subvert host cell processes, the bacteria secrete the amazing number of ~300 different proteins into host cells. Some of these proteins bind phosphoinositide (PI) lipids to decorate the LCV. PI lipids are crucial factors involved in host cell membrane dynamics and LCV formation. Using Dictyostelium amoebae producing one or two distinct fluorescent probes, we elucidated the dynamic LCV PI pattern in high temporal and spatial resolution. Notably, the endocytic PI lipid PtdIns(3) P was slowly cleared from LCVs, thus incapacitating the host cell’s digestive machinery, while PtdIns(4) P gradually accumulated on the LCV, enabling critical interactions with host organelles. The LCV PI pattern underlies the spatiotemporal configuration of bacterial effector proteins and therefore represents a crucial aspect of LCV formation.

  • the natural alternative protozoa as cellular models for Legionella Infection
    Cellular Microbiology, 2014
    Co-Authors: Christine Hoffmann, Christopher F Harrison, Hubert Hilbi
    Abstract:

    Summary The severe pneumonia known as Legionnaires' disease occurs following Infection by the Gram-negative bacterium Legionella pneumophila. Normally resident in fresh-water sources, Legionella are subject to predation by eukaryotic phagocytes such as amoeba and ciliates. To counter this, L. pneumophila has evolved a complex system of effector proteins which allow the bacteria to hijack the phagocytic vacuole, hiding and replicating within their erstwhile killers. These same mechanisms allow L. pneumophila to hijack another phagocyte, lung-based macrophages, which thus avoids a vital part of the immune system and leads to Infection. The course of Infection can be divided into five main categories: pathogen uptake, formation of the replication-permissive vacuole, intracellular replication, host cell response, and bacterial exit. L. pneumophila effector proteins target every stage of this process, interacting with secretory, endosomal, lysosomal, retrograde and autophagy pathways, as well as with mitochondria. Each of these steps can be studied in protozoa or mammalian cells, and the knowledge gained can be readily applied to human pathogenicity. Here we describe the manner whereby L. pneumophila infects host protozoa, the various techniques which are available to analyse these processes and the implications of this model for Legionella virulence and the pathogenesis of Legionnaires' disease.

  • Analysis of Legionella Infection by flow cytometry.
    Methods in molecular biology (Clifton N.J.), 2012
    Co-Authors: André N. Tiaden, Aline Kessler, Hubert Hilbi
    Abstract:

    Legionella pneumophila infects and replicates in environmental protozoa and metazoan macrophages within a specific vacuole. The Infection of phagocytes by L. pneumophila can be assessed by an agar plating assay or by fluorescence microscopy. Here, we describe the analysis of Legionella Infection by automated flow cytometry using wild-type and mutant bacteria that constitutively produce the green fluorescent protein (GFP). Advantages of the flow cytometry technique include (1) a software-assisted multiple parameter analysis of Legionella Infections in real-time at distinct stages of the Infection cycle, (2) the simultaneous and fast acquisition of a high number of data points, and (3) a characterization of the infecting bacteria in parallel with the infected host cells.

Pernille L. Elverdal - One of the best experts on this subject based on the ideXlab platform.