Biochemical and Genetic Characterization of Lysin A from Mycobacteriophage TM4 and its Application as a Potential Enzybiotic

PAYASLIAN, FLORENCIA; MARTÍ, MARCELO; URDÁNIZ, ESTEFANÍA; PIURI, MARIANA; MARTÍN, MARIANO

Congreso; 23 rd Biennial Evergreen International Phage Meeting; 2019

Tuberculosis is an infectious disease caused by M. tuberculosis. The emergence of multi-resistant and extensively drug resistant strains is one of the most difficult challenges regarding its control.One promising approach to treat multi-drug resistant infections is the use of phage derivative proteins that can act as antibiotics. The bacteriophage TM4 has a broad host range infecting several mycobacteria, including M. smegmatis and M. tuberculosis. TM4 genome encodes two proteins with potential lytic activity: gp29 has been identified in silico as lysin A (LysA), with putative N-acetylmuramoil-L-alanine amidase activity and Gp30 as a putative mycolic acidesterase. The aim of this study was to characterize the mechanism of action of LysA on peptidoglycan (PG) and its lytic activity from within and without in several bacteria. A bioinformatic analysis confirmed the presence of three domains, which had been previously described as peptidase, amidase and a PG recognition domain in the C-terminal region. We determined the putative key amino acids involved in the catalytic reaction in the amidase domain,located in positions E290, H226, H335 and D347, and based on this we constructed the H226S, E290Q, H335S and D347N recombinant mutant proteins. The sequence of lysA was cloned under the control of an inducible promoter. M. smegmatis cells transformed with this construction were induced and permeated with chloroform to allow access of the protein to the PG through the plasmatic membrane. After induction, a decrease in the optical density was observed, corroboratingthe lytic effect of Gp29 from within this bacterium. When a similar assay was performed in E.coli, the same results were obtained, while no lytic activity was detected for the tested mutants. Then, the lytic activity of LysA was tested using a zymogram assay containing Micrococcus lysodeikticus ATCC 4698 (0.2% m/v) as a substrate. Lytic activity was detected by a colourless halo for the WTprotein but not for the mutant derivatives, indicating that LysA is able to degrade the PG cell wall from without, but the mutants are defective. To complete the biochemical analysis of the protein, using a muramoyl-dipeptide (MDP) synthetic substrate in combination with analysis of the products of the reaction by mass spectrometry, we were able to confirm that LysA is an N-acetylmuramoyl amidase. In order to evaluate if the tested mutations in LysA could affect the lytic capacity of TM4, we replaced lysA for the mutated versions E290Q or H226S in the phage genome using BRED (Bacteriophage Recombineering of Electroporated DNA). Unexpectedly, isolated phages carrying these mutations could still replicate and complete the lytic cycle equally in M. smegmatis mc2155 or a derivative strain expressing the lysin A gene (Gp69) from Corndogbacteriophage. Altogether, our results confirm that LysA can degrade the PG cell wall, and it was possible to identify at least key four residues of the catalytic site. However, the phage lytic capacity was not affected when carrying the mutated LysA, indicating that the enzyme may not be essential for progeny release or suggesting the presence of a second Lysin A in the TM4 genome.