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      High-Throughput Screen for Inhibitors of Klebsiella pneumoniae Virulence Using a Tetrahymena pyriformis Co-Culture Surrogate Host Model

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          Abstract

          The continuing emergence of antibacterial resistance reduces the effectiveness of antibiotics and drives an ongoing search for effective replacements. Screening compound libraries for antibacterial activity in standard growth media has been extensively explored and may be showing diminishing returns. Inhibition of bacterial targets that are selectively important under in vivo (infection) conditions and, therefore, would be missed by conventional in vitro screens might be an alternative. Surrogate host models of infection, however, are often not suitable for high-throughput screens. Here, we adapted a medium-throughput Tetrahymena pyriformis surrogate host model that was successfully used to identify inhibitors of a hyperviscous Klebsiella pneumoniae strain to a high-throughput format and screened circa 1.2 million compounds. The screen was robust and identified confirmed hits from different chemical classes with potent inhibition of K. pneumoniae growth in the presence of T. pyriformis that lacked any appreciable direct antibacterial activity. Several of these appeared to inhibit capsule/mucoidy, which are key virulence factors in hypervirulent K. pneumoniae . A weakly antibacterial inhibitor of LpxC (essential for the synthesis of the lipid A moiety of lipopolysaccharides) also appeared to be more active in the presence of T. pyriformis, which is consistent with the role of LPS in virulence as well as viability in K. pneumoniae.

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          A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.

          The ability to identify active compounds (³hits²) from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the ³suitability² of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called ³Z-factor,² is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.
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            Hypervirulent (hypermucoviscous) Klebsiella pneumoniae

            A new hypervirulent (hypermucoviscous) variant of Klebsiella pneumoniae has emerged. First described in the Asian Pacific Rim, it now increasingly recognized in Western countries. Defining clinical features are the ability to cause serious, life-threatening community-acquired infection in younger healthy hosts, including liver abscess, pneumonia, meningitis and endophthalmitis and the ability to metastatically spread, an unusual feature for enteric Gram-negative bacilli in the non-immunocompromised. Despite infecting a healthier population, significant morbidity and mortality occurs. Although epidemiologic features are still being defined, colonization, particularly intestinal colonization, appears to be a critical step leading to infection. However the route of entry remains unclear. The majority of cases described to date are in Asians, raising the issue of a genetic predisposition vs. geospecific strain acquisition. The traits that enhance its virulence when compared with “classical” K. pneumoniae are the ability to more efficiently acquire iron and perhaps an increase in capsule production, which confers the hypermucoviscous phenotype. An objective diagnostic test suitable for routine use in the clinical microbiology laboratory is needed. If/when these strains become increasingly resistant to antimicrobials, we will be faced with a frightening clinical scenario.
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              Lipopolysaccharide endotoxins.

              Bacterial lipopolysaccharides (LPS) typically consist of a hydrophobic domain known as lipid A (or endotoxin), a nonrepeating "core" oligosaccharide, and a distal polysaccharide (or O-antigen). Recent genomic data have facilitated study of LPS assembly in diverse Gram-negative bacteria, many of which are human or plant pathogens, and have established the importance of lateral gene transfer in generating structural diversity of O-antigens. Many enzymes of lipid A biosynthesis like LpxC have been validated as targets for development of new antibiotics. Key genes for lipid A biosynthesis have unexpectedly also been found in higher plants, indicating that eukaryotic lipid A-like molecules may exist. Most significant has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells. TLR4 belongs to a family of innate immunity receptors that possess a large extracellular domain of leucine-rich repeats, a single trans-membrane segment, and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88. The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking inflammation associated with infection.
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                Author and article information

                Journal
                ACS Omega
                ACS Omega
                ao
                acsodf
                ACS Omega
                American Chemical Society
                2470-1343
                01 February 2022
                15 February 2022
                : 7
                : 6
                : 5401-5414
                Affiliations
                []Infectious Diseases, Novartis Institutes for Biomedical Research Inc , 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
                []Global Discovery Chemistry, Novartis Institutes for Biomedical Research Inc , 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
                [§ ]Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research Inc , 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
                []Global Discovery Chemistry, Novartis Institutes for Biomedical Research Inc , Emeryville California 94608-2916, United States
                Author notes
                [* ]Email: thomasm.smith@ 123456novartis.com . Tel.: +1 617 871 4275.
                Author information
                https://orcid.org/0000-0003-3937-6243
                https://orcid.org/0000-0001-6343-2490
                Article
                10.1021/acsomega.1c06633
                8851646
                029a0dd9-5dc4-4cf5-bcd6-4fa3b9441c4d
                © 2022 The Authors. Published by American Chemical Society

                Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works ( https://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 23 November 2021
                : 08 December 2021
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                ao1c06633

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