A role for the ESX-4 Type VII secretion system in interbacterial competition in the opportunistic pathogen Mycobacterium abscessus?
Final Report Abstract
Mycobacterium abscessus is known to be a troublesome pathogen especially for patients with cystic fibrosis. Once infected outcomes are often incurable due to its multi drug resistance and this is a barrier to lung transplantation. These infections occur in late childhood and therefore M. abscessus must have effective mechanisms to overcome the resident lung microbiota. One strategy could be the release of antibacterial toxins via the Type 7 secretion system (T7SS). It has been shown that gram-positive bacteria use the T7SS to release toxins like DNases and lipases for bacterial competition. However, this function has not been assigned to the mycobacterial T7SS. Mycobacteria can contain up to five T7SS called ESX1 to ESX5. ESX4 is the most ancient one and no specific function could be assigned to it in M. tuberculosis. M. abscessus only encodes ESX3 and ESX4 and research claims that these systems are used for immune modulation and survival and replication in the host, respectively. My bioinformatic survey assigns another role to ESX4. I detected genes linked to the ESX4 secretion system encompassing a typical T7- secretion motif and a toxin domain. About 40 of these genes have been identified in the M. abscessus ATCC19977 strain. Their antibacterial toxin domains are predicted to be DNases, RNases, peptidoglycan hydrolases etc. Therefore, this project aims to show that M abscessus releases antibacterial toxins via the T7SS-ESX4 for bacterial competition. To test my hypothesis, I chose the MAB_1832 protein of M. abscessus ATCC19977. This protein has a N-terminal T7-secretion signal and a toxin domain matching a peptidoglycan hydrolase. I generated mutants of the T7SS ESX3 and ESX4 in M. abscessus ATCC19977 and performed secretion assays by expressing MAB_1832 under the regulation of a L-arabinose promoter. My results suggest secretion of the antibacterial toxin via ESX4. Additionally, I expressed, purified and crystallized MAB_1832 in E. coli and showed interaction with its small WxG partner and its immunity protein to protect the bacteria from self-intoxication. Unfortunately, crystals do not diffract well enough to determine a structure and crystallography experiments have to be optimized. Furthermore, I conducted experiments regarding activity and toxicity of MAB_1832. So far, I could not show activity of MAB_1832 using purified peptidoglycan from Mycobacterium smegmatis. However, toxicity assays in M. smegmatis introducing MAB_1832 under the regulation of a L-arabinose promoter showed toxicity. My results support my project proposal although more experiments have to be done to validate and strengthen my hypothesis. Altogether this project assigns a new role to the mycobacterial T7SS and indicates how M. abscessus has a competitive advantage over other microbes.
Publications
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A type VII-secreted lipase toxin with reverse domain arrangement. Nature Communications, 14(1).
Garrett, Stephen R.; Mietrach, Nicole; Deme, Justin; Bitzer, Alina; Yang, Yaping; Ulhuq, Fatima R.; Kretschmer, Dorothee; Heilbronner, Simon; Smith, Terry K.; Lea, Susan M. & Palmer, Tracy
