high-throughput-real-time-dual-readout-testing-intracellular

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Beth Israel Deaconess Medical Center

Systemic anthrax infection is usually fatal even with optimal medical care. Further insights into anthrax pathogenesis are therefore urgently needed to develop more effective therapies. Animal models that reproduce human disease will facilitate this research. Here, we describe the detailed histopathology of systemic anthrax infection in A/J mice infected with Bacillus anthracis Sterne, a strain with reduced virulence for humans. Subcutaneous infection leads to systemic disease with multiple pathologies including oedema, haemorrhage, secondary pneumonia and lymphocytolysis. These pathologies bear marked similarity to primary pathologies observed during human disease. Therefore, this simple, small animal model will allow researchers to study the major pathologies observed in humans, while permitting experimentation in more widely available Biosafety Level 2 facilities.

Histopathology in a murine model of anthrax.

Legionella pneumophila is a gram-negative pathogen that causes a severe pneumonia known as Legionnaires' disease. Here, we demonstrate for the first time that L. pneumophila infects and grows within cultured human endothelial cells. Endothelial infection may contribute to lung damage observed during Legionnaires' disease and to systemic spread of this organism.

Infection of cultured human endothelial cells by Legionella pneumophila.

Bartonella are ubiquitous gram-negative pathogens that cause chronic blood stream infections in mammals. Two species most often responsible for human infection, B. henselae and B. quintana, cause prolonged febrile illness in immunocompetent hosts, known as cat scratch disease and trench fever, respectively. Fascinatingly, in immunocompromised hosts, these organisms also induce new blood vessel formation leading to the formation of angioproliferative tumors, a disease process named bacillary angiomatosis. In addition, they cause an endothelial-lined cystic disease in the liver known as bacillary peliosis. Unfortunately, there are as yet no completely satisfying small animal models for exploring these unique human pathologies, as neither species appears able to sustain infection in small animal models. Therefore, we investigated the potential use of other Bartonella species for their ability to recapitulate human pathologies in an immunodeficient murine host. Here, we demonstrate the ability of Bartonella taylorii to cause chronic infection in SCID/BEIGE mice. In this model, Bartonella grows in extracellular aggregates, embedded within collagen matrix, similar to previous observations in cat scratch disease, bacillary peliosis, and bacillary angiomatosis. Interestingly, despite overwhelming infection later in disease, evidence for significant intracellular replication in endothelial or other cell types was not evident. We believe that this new model will provide an important new tool for investigation of Bartonella-host interaction.

An immunocompromised murine model of chronic Bartonella infection.

Interpretation of high throughput screening (HTS) data in cell-based assays may be confounded by cytotoxic properties of screening compounds. Therefore, assessing cell toxicity in real time during the HTS process itself would be highly advantageous. Here, we investigate the potential of putatively impermeant, fluorescent, DNA-binding dyes to give cell toxicity readout during HTS. Amongst 19 DNA-binding dyes examined, three classes were identified that were (1) permeant, (2) cytotoxic, or (3) neither permeant nor cytotoxic during 3-day incubation with a macrophage cell line. In the last class, four dyes (SYTOX Green, CellTox Green, GelGreen, and EvaGreen) gave highly robust cytotoxicity data in 384-well screening plates. As proof of principle, successful combination with a luminescence-based assay in HTS format was demonstrated. Here, both intracellular growth of Legionella pneumophila (luminescence) and host cell viability (SYTOX Green exclusion) were assayed in the same screening well. Incorporation of membrane-impermeant, DNA-binding, fluorescent dyes in HTS assays should prove useful by allowing evaluation of cytotoxicity in real time, eliminating reagent addition steps and effort associated with endpoint cell viability analysis, and reducing the need for follow-up cytotoxicity screening.

Evaluation of impermeant, DNA-binding dye fluorescence as a real-time readout of eukaryotic cell toxicity in a high throughput screening format.

Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche.

Article	Year
Histopathology in a murine model of anthrax. International journal of experimental pathology\| PubMed ID: 16623757	2006
Infection of cultured human endothelial cells by Legionella pneumophila. PloS one\| PubMed ID: 18431493	2008
An immunocompromised murine model of chronic Bartonella infection. The American journal of pathology\| PubMed ID: 20395436	2010
Evaluation of impermeant, DNA-binding dye fluorescence as a real-time readout of eukaryotic cell toxicity in a high throughput screening format. Assay and drug development technologies\| PubMed ID: 24831788	2014
High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila. Antimicrobial agents and chemotherapy\| PubMed ID: 26392509	2015

Lucius Chiaraviglio

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