GIBH Created a Resistance Expression Cassette for Effective Construction of Selection-Marker Free Recombinant Mycobacteria

March 24, of every year is designated the "The World Tuberculosis Day". The event is marked to build public awareness that tuberculosis (TB) still remains at epidemic level in most parts of the world, causing deaths to nearly 1.5 million people each year, mostly in developing countries.

This year's event was commemorated under the theme “Unite to End Tuberculosis” which underscores the joint social efforts to eliminate the TB hazard. The lack of effective drugs capable of clearing the pathogen from the host, is making the fight against TB difficult. Furthermore, low efficiency and inaccurate drug screening models have been hampering the research and development efforts of new drugs.

Coincidentally, on the same date the Tuberculosis Research Laboratory team lead by Professor Tianyu Zhang at State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health (GIBH), the Chinese Academy of Sciences, had their latest research findings published by the journal Frontiers in Microbiology.

The work going under the title“A Cassette Containing Thiostrepton, Gentamicin Resistance Genes and dif sequences is Effective in Construction of Recombinant Mycobacteria” is a new invention showing the potential of the cassette in the genetic manipulation of mycobacteria.

The first author is Julius Mugweru, an international PhD student of GIBH and University of Chinese Academy of Sciences. Since Mycobacterium tuberculosis genetic manipulation is an onerous task that have limited choices of selection markers, the team under the guidance of Dr. Zhang decided to explore for alternative selection marker.

The use of kanamycin for selection to construct recombinant strains is prone to spontaneous mutations leading false positive colonies, besides kanamycin is among the aminoglycosides drugs used in TB treatment. Still, a lot of clinical strains are resistant kanamycin. So for these strains, Kan^r can't be used as a resistance genetic marker. Another marker used is hygromycin resistance gene which is relatively expensive, requires large concentrations besides being unstable.

The team for the first time found that thiostreptone’s resistance gene (tsr) can be used as a genetic resistance marker for M. tuberculosis and M. bovis. Thiostreptone is relatively cheap, requires less concentrations, and the mutation rate of M. tuberculosis emergence resistance to thiostreptone is extremely low. Being a drug that is not used in the treatment of TB, we think it is better for genetic manipulation in clinical M. tuberculosis isolates without the risk of producing the drug-resistant TB.

The gentamicin resistance gene (aacC1) can be used for constructing fast growing recombinant mycobacterial strains as well as in E. coli selection. Therefore, the research group utilized the two genes combined with suitable promoter and dif sequences that can be identified and automatically disassembled by the mycobacterial Xer system. They constituted the resistance expression cassette of dif - hsp60- aacC1- tsr – dif as an E.coli- mycobacterium shuttle plasmid that can effectively construct selection-marker free recombinant mycobacteria and filed for a patent (2016101801161450).

The study was supported by The National Natural Science Foundation of China, The Chinese Academy of Sciences, UCAS scholarship, CAS – TWAS scholarship, Guangzhou Science and Technology Projects and National Key Laboratory of Respiratory Disease.