Biochemical characterization of Lysin A activity of Mycobacteriophage TM4

PAYASLIAN FLORENCIA; ESTEFANIA URDANIZ; MARIANO MARTIN; MARIANA PIURI

Congreso; Viruses of Microbes 2022; 2022

ISVM

Bacteriophage endolysins are crucial for progeny release at the end of the lytic cycle. In TM4, a bacteriophage that infects mycobacteria including M. tuberculosis, a cassette encoding putative LysA, LysB and holin (gp29-30-31) was identified.Through bioinformatic analysis we were able to establish that LysA consists of 3 modules; a C-terminal domain that probably binds to the cell wall, a central domain with high similarity to an amidase-2 and an N-terminal domain proposed to encode for a peptidase. In the amidase domain, the catalytic Zn ion is coordinated by His226, His335, and Asp347 and we also identified the amino acid Glu290 as the catalytic residue.Four derivatives of the protein containing a mutation on each of these key residues were constructed.Purified fractions of LysA were incubated with MDP, a synthetic molecule that emulates the bonds on peptidoglycan. The products of the reaction were analyzed by HPLC-MS, and N-acetyl-muramic-acid and L-Ala-D-isoGlutamine dipeptide were detected, confirming that LysA has an amidase activity, as predicted in silico.We also assessed the ability of LysA to lyse E. coli or M. smegmatis from within, monitoring the optical density of cultures transformed with a plasmid expressing either LysA WT or the mutants. There was a significant decrease in optical density of the cultures expressing the LysA WT version but no lysis was observed in any of the mutants. These results indicate that the four predicted residues are essential for the function of the protein in vitro and when expressed in a homologous or heterologous host.We also generated phages either carrying the E290 or the H266S mutations. Interestingly, no difference in the efficiency of plating was found between the mutated versions of the phage and the WT. This could indicate that either these mutations are not sufficient to abrogate lysis in vivo, or that a cryptic endolysin gene is encoded in TM4 genome