A High-Throughput Screen for Tuberculosis Progression

Abstract

One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo.

The Proprotein Convertase Subtilisin/Kexin FurinA Regulates Zebrafish Host Response against Mycobacterium marinum

“…Since furinA regulated the granulopoiesis, we addressed its role in innate immunity by inhibiting the expressions of furinA and furinB in the embryonic M. marinum infection model (27,32,33). Infecting either control (random control MO injected [RC]) or the double furin gene knockdown embryos with M. marinum (131 Ϯ 125 CFU) resulted in substantial lethality by 7 days postinfection (dpi; 93 and 100%, respectively, Fig.…”

“…The injection volume was set to 2 nl, and 0.25 pmol of both furinA and furinB MOs or 0.5 pmol of RC MO was used. M. marinum was simultaneously coinjected into the yolk sac, and 2% polyvinylpyrrolidone was used as a carrier solution in the suspension (27,33). Survival was analyzed daily with a visual inspection with an Olympus IX71 microscope.…”

“…Consequently, an M. marinum infection in fish is considered a relevant, cost-effective and ethical tool for studying the human mycobacterial disease. Both embryo and adult zebrafish infection models are now well established; while embryos can be used to specifically investigate innate immune responses (27,28), the adult model enables the study of a chronic progressive mycobacterial infection, as well as spontaneous latency (25,29).…”

Research and Development of Biotechnologies Using Zebrafish and Its Application on Drug Discovery

“…One advantage of zebrafish transgenics having compared with the mammalian counterpart technology is that reproduction involves external fertilization and embryo development, eliminating the need for surgical intervention. Nowadays, the zebrafish are becoming a useful genetic model and starting to be employed in various researches such as infection desiease 17 , cancer research 18 , chemical genetic screening 19 , toxicology 20 , and proteome 21 . Some researchers noted on zebrafish as an in vivo protein expression system, which can be applied for useful protein production 22 , while they used for genetic model are spreading.…”

Mycobacterium aurum is Unable to Survive Mycobacterium tuberculosis Latency Associated Stress Conditions: Implications as Non-suitable Model Organism

“…M. smegmatis remains most widely used organism for rapid investigation of genes under M. tuberculosis associated in vitro latency conditions [5,6] as well as for drug screening. However, M. marinum [7,8] and M. avium [9] have also been used as surrogates for the same, yet none of them is completely able to mimic M. tuberculosis human latent infection under in vitro conditions.…”