Biochemical and genetic characterization of Lysin A from mycobacteriophage TM4 and its application as a potential enzybiotic

PAYASLIAN FLORENCIA; PIURI *, MARIANA

Congreso; 23rd Biennial Evergreen International Phage Meeting; 2019

Tuberculosis is an infectious disease caused by M. tuberculosis. Despite being treatable, 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 bacteriophage therapy or the use of phage derivative proteins that can act as enzybiotics. 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 acid esterase.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. By the use of PDBSum and PFAM 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 y D347N recombinant mutant proteins.The activity of LysA from within was assessed in M. smegmatis. The sequence of lysA was cloned under 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 could be observed corroborating the lytic effect of Gp29 from within in 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 substrate. Lytic activity could be detected by a colourless halo only for the WT protein but not for the mutant derivatives, indicating that LysA is able to degrade the peptidoglycan cell wall from without but the mutants are defective. No differences were obtained between the wt and mutants variants by circular dichroism supporting that lack of activity in the mutants is due to alteration of the catalytic site and not because of missfolding of the proteins. No lysis could be detected in M. smegmatis when LysA was applied from without, probably due to lack of permeation of the lytic enzyme through the mycolic-arabinogalactan outside layer present in mycobacteria. 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 a N-acetyl muramoyl amidase. Unfortunately, our efforts to obtain crystals of the entire protein were fruitless and we are currently working in the expression and crystallization of the amidase domain alone. 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 Corndog bacteriophage.Altogether, our results confirm that LysA can degrade the peptidoglycan 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.