Name: Video: Antimicrobial Activity and Toxicity Assessment of Compounds Targeting Mycobacterium abscessus in Macrophage Infection Assays
Uploaded: 2024-10-25T13:50:00
Duration: 5 min 37 s
Description: 313 Views. Antimicrobial Activity and Toxicity Assessment of Compounds Targeting Mycobacterium abscessus in Macrophage Infection Assays

antimicrobial activity and toxicity assessment of compounds targeting mycobacterium abscessus in macrophage infection assays

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

Mycobacterium abscessus (Mab) is an opportunistic pathogen responsible for pulmonary infections, especially in people with cystic fibrosis and other lung disorders. Infections caused by Mab are infamously challenging to treat due to exquisite intrinsic drug resistance, comparable to multidrug-resistant tuberculosis1. The available drugs are largely ineffective due to the highly impermeable mycobacterial cell envelope and a genome that encodes several enzymes that deactivate antibiotics2. Thus, treatment includes the combination of several drugs taking months to years. This challenging multi-drug regimen combined with low patient compliance results in an average cure rate of 30% to 50%3. Furthermore, the prevalence of pulmonary infections caused by non-tuberculous mycobacteria has increased over the past few decades, including the ones caused by Mab1,4. Consequently, the scientific community is racing to develop new compounds to treat Mab infections.
One of the strategies pursued with this aim is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs. This circumvents the biggest challenge accompanying a new drug&#39;s discovery and development pipeline - time5. This simple concept takes advantage of the already established pharmacokinetic and safety profiles of several drugs to reduce developmental costs and shorten the timescale that it takes to get a drug from bench to bedside6. Thus, libraries that combine hundreds to thousands of such compounds have been compiled, allowing researchers to quickly test the possibility of repurposing drugs against their pathogen of interest.
Most studies on drug development against Mab are based around a gold-standard yet traditional assay that evaluates a compound&#39;s in vitro activity against mycobacteria - colony-forming units counting7. Despite its accuracy, this procedure is extremely time-consuming, quickly becoming non-feasible when someone aims to test libraries containing thousands of compounds. To this end, high-throughput screenings (HTS) have been integrated into drug development - robust assays that take advantage of robotics and liquid handling devices, allowing thousands of compounds to be quickly screened in parallel8. This is typically done by testing a single concentration initially, using microtiter plates of 96-, 384-, 1536-, or 3456- well formats, acting as a starting point to identify hits and further optimize them down the pipeline to be clinically used.
Reporter-based assays provide a significant advantage for the robustness of HTS due to their simplicity and sensitivity compared to other dye and absorbance-based assays7,9. However, to our knowledge, only a few studies have optimized a high-throughput screening against Mab9.
Recently, our lab has developed double reporter strains capable of concomitantly emitting luminescence and fluorescence10. Mab operon_mScarlet is one of those strains. It is auto-luminescent due to the expression of the LuxABCDE operon, which includes a bacterial luciferase (by expression of the luxAB genes) and a long-chain aldehyde substrate (by expression of the luxCDE genes). On the other hand, the fluorescent readout is obtained through the expression of a recently developed red fluorescent protein, mScarlet, which outperforms the more typically used eGFP and mCherry proteins, providing a more potent signal11. Using this strain enables us to assess the bacterial viability in liquid culture by measuring the luminescent signal in a microplate reader without adding reagents or performing additional steps. In terms of detection, intrinsic fluorescence allows for microscope visualization in live or fixed cells without using dyes or antibodies. Having one single strain with both readouts provides researchers with a significant advantage when using it in HTS assays - the reduced variability between assays with different readouts, as there is no need to exchange strains based on the nature of the assay.
Thus, this work developed a high-throughput assay against Mab using an in house engineered double reporter strain (Figure 1). This allowed for a quick assessment of the in vitro activity&#160;1280 drugs intended for repurposing using a commercial library (see Table of Materials). Firstly, the activities were assessed in broth culture assay using luminescence, and secondly, by using Mab-infected macrophages taking advantage of the fluorescent signal, better emulating the infection process seen in vivo12.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66860/66860fig01v2.jpg" /> 
Figure 1:&#160;Graphical abstract of the established protocol. The key player in this screening is an in house developed double reporter mycobacterial strain, which is used throughout all experiments. Firstly, using a reagent dispenser and planktonic bacteria, an initial screening is performed by testing the compounds at a single concentration. The identified hits carry on to the validation assay, where three different concentrations are tested. Both experiments are done using the luminescent readout. The validated hits carry on to the infection assay, where three different concentrations are also tested, and bone marrow-derived macrophages are infected at MOI 1. A high-content imaging system acquires the bacterial fluorescent signal, and analysis software is used to assess the intracellular load through the Mycoload formula. The number of compounds reduces as assay complexity increases. Created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/66860/66860fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Antimicrobial Activity and Toxicity Assessment of Compounds Targeting Mycobacterium abscessus in Macrophage Infection Assays

antimicrobial-activity-toxicity-assessment-compounds-targeting

Research

JoVE Journal

Immunology and Infection

313 Views. Antimicrobial Activity and Toxicity Assessment of Compounds Targeting Mycobacterium abscessus in Macrophage Infection Assays

Video: Antimicrobial Activity and Toxicity Assessment of Compounds Targeting Mycobacterium abscessus in Macrophage Infection Assays

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...