Morphological profiling of tubercule bacilli identifies drug pathways of action

Abstract

34 35 36 Keywords 37 tuberculosis, drug discovery, cell morphology, high throughput 38 39 40 41 42 43 2 Abstract 44Morphological profiling is a method to classify target pathways of antibacterials based on how 45 bacteria respond to treatment through changes to cellular shape and spatial organization. Here, we utilized 46 the cell-to-cell variation in morphological features of Mycobacterium tuberculosis bacilli to develop a rapid 47 profiling platform called Morphological Evaluation and Understanding of Stress (MorphEUS). MorphEUS 48 classified 94% of tested drugs correctly into broad categories according to modes of action previously 49 identified in the literature. In the other 6%, MorphEUS pointed to key off-target or secondary bactericidal 50 activities. We observed cell-wall damaging activity induced by bedaquiline and moxifloxacin through 51 secondary effects downstream from their main target pathways. We implemented MorphEUS to correctly 52 classify three compounds in a blinded study and identified an off-target effect for one compound that was 53 not readily apparent in previous studies. We anticipate that the ability of MorphEUS to rapidly identify 54 pathways of drug action and the proximal cause of bactericidal activity in tubercule bacilli will make it 55 applicable to other pathogens and cell types where morphological responses are subtle and 56 heterogeneous. 58 Significance Statement 59Tuberculosis is a leading cause of death in the world and requires treatment with an arduous 60 multidrug regimen. Many new tuberculosis drugs are in development, and the drug development pipeline 61 would benefit from more rapid methods to learn drug mechanism of action and off-target effects. Here, we 62 describe a high throughput imaging method for rapidly classifying drugs into categories based on the 63 primary and secondary cellular damage called Morphological Evaluation and Understanding of drug-Stress 64 (MorphEUS). We anticipate that MorphEUS will assist in rapidly pinpointing causes of cellular death in 65 response to drug treatment in tuberculosis and other bacterial pathogens. 66 67 68 69 3 Main Text 70 Introduction 71Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), causes more deaths 72 annually than any other infectious agent (1). Tuberculosis treatment is lengthy, lasting between four months 73 to over a year (1). The difficult regimen, rate of relapse, and incidence of drug resistant Mtb has motivated 74 a significant effort to develop new antibacterial compounds that are effective in sterilizing Mtb infection (2). 75Many new drug classes and derivative compounds have been developed (2), but rapidly identifying the 76 primary and secondary pathways of action of each compound is often a protracted process due to the 77 difficulty in generating resistant mutants, and dissecting the broad-reaching metabolic effects of drug 78 treatment leading to death (3). Furthermore, drug action on bacterial cells can elicit dynamic responses in 79 multiple pathways both on-and off-target, s...

Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status

“…Mycobacteria, including Mtb , demonstrate morphological and metabolic plasticity in response to environmental and antibiotic stresses ( 16 – 18 ). We reasoned that if Mtb bacilli were exposed to antibiotics before aerosolization, a discernible change in bacterial morphology and/or metabolic state might be detectable between treatment groups.…”

Identification of cell wall synthesis inhibitors active against Mycobacterium tuberculosis by competitive activity-based protein profiling

“…In replicating Mtb pretomanid has been shown to act in part by inhibiting the biosynthesis of essential mycolic acids (Stover et al, 2000). Although bedaquiline is an ATP synthesis inhibitor, we and others have shown that the resulting downstream metabolic perturbation produces morphological changes that resemble those from cell wall-acting inhibitors (Mackenzie et al, 2020;Smith et al, 2020).…”

“…Compound-treated Mtb cultures were fixed with 4% paraformaldehyde (Alfa Aesar, 43368) for 1 h, washed twice with 100 mL PBS containing 0.2% Tween 80 (PBST), and resuspended in 100 mL PBST. Staining and imaging were as described previously (Smith et al, 2020). In brief, 50 mL of fixed Mtb cells was diluted with 50 mL PBST and stained with 0.6 mg of FM4-64-FX (Thermo Fisher Scientific; F34653) and 15 mL of 0.1 mM SYTO 24 (Thermo Fisher Scientific; S7559) at room temperature in the dark for 30 min.…”

“…The morphological changes for cells treated with AA691, AA692, AA701, or AA702 were then processed and analyzed using the MorphEUS analysis pipeline and an existing reference drug set (Smith et al, 2020). The profile for each compound at a designated time point was individually applied onto the morphological space, constructed using 34 compounds with known molecular targets.…”

“…To better understand the mode of action underlying AA692's antibacterial activity, we used a recently developed morphological profiling platform called MorphEUS (Smith et al, 2020). MorphEUS is based on the principle that drugs with similar mechanisms of action will induce similar changes in bacterial morphologies.…”