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Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Published on: June 11, 2015

Functional characterization of the endolysins derived from mycobacteriophage PDRPxv

Kandasamy Eniyan¹, Avni Sinha¹, Shazeb Ahmad¹

¹Department of Biomedical Science, Acharya Narendra Dev College (University of Delhi), Govindpuri, New Delhi, 110019, India.

World Journal of Microbiology & Biotechnology

|May 30, 2020

Abstract:

Bacteriophage-derived endolysin enzymes play a critical role in disintegration of the host bacterial cell wall and hence have gained considerable attention as possible therapeutics for the treatment of drug-resistant infections. Endolysins can target both dividing and non-dividing cells and given the vital role peptidoglycan plays in bacterial survival, bacteria are less likely to modify it even if continuously exposed to lysins. Hence, probability of bacteria developing resistance to lysins appear bleak. Endolysins from mycobacteriophages offer great potential as alternative therapeutics for the drug-resistant TB. However, considering that a large number of mycobacteriophages have been discovered so far, the information on endolysins come from only a few mycobacteriophages. In this study, we report the structural and functional characterization of endolysins (LysinA and LysinB) encoded by mycobacteriophage PDRPxv which belongs to B1 sub cluster. On in silico analysis, we found LysinA to be a modular protein having peptidase domain at the N-terminal (104 aa), a central amidase domain (174 aa) and the peptidoglycan binding domain (62 aa) at the C-terminal. Additionally, 'H-X-H', which is a conserved motif and characteristic of peptidase domains, and the conserved residues His-His-Asp, which are characteristic of amidase domain were also observed. In LysinB enzyme, a single α/β hydrolase domain having a catalytic triad (Ser-Asp-His) and G-X-S-X-G motif, which are characteristic of the serine esterase enzymes were predicted to be present. Both the enzymes were purified as recombinant proteins and their antimycobacterial activity against M. smegmatis was demonstrated through turbidimetric experiments and biochemical assay. Interesting observation in this study is the secretory nature of LysinA evident by its periplasmic expression in E.coli, which might explain the ability of PDRPxv to lyse the bacterial host in the absence of transmembrane Holin protein.

Insights

Bacteriophage endolysins, particularly LysinA and LysinB from mycobacteriophage PDRPxv, show promise as therapeutics against drug-resistant tuberculosis. Their structural characterization and demonstrated antimycobacterial activity against M. smegmatis highlight their potential.

Area of Science:

Microbiology and Molecular Biology
Biochemistry and Enzymology
Drug Discovery and Development

Background:

Bacteriophage-derived endolysins are crucial for bacterial cell wall lysis and are investigated as therapeutics against drug-resistant infections.
Mycobacteriophage endolysins present a promising avenue for treating drug-resistant tuberculosis (TB), yet their characterization remains limited.
The low probability of bacteria developing resistance to endolysins due to the essential nature of peptidoglycan is a significant advantage.

Purpose of the Study:

To structurally and functionally characterize endolysins LysinA and LysinB from mycobacteriophage PDRPxv.
To evaluate the antimycobacterial activity of these endolysins against Mycobacterium smegmatis.
To investigate the potential of these endolysins as therapeutic agents for drug-resistant tuberculosis.

Main Methods:

In silico analysis to predict the structural domains and conserved motifs of LysinA and LysinB.
Recombinant protein purification of LysinA and LysinB.
Turbidimetric experiments and biochemical assays to assess antimycobacterial activity against M. smegmatis.
Periplasmic expression in E. coli to investigate the secretory nature of LysinA.

Main Results:

LysinA was identified as a modular protein with N-terminal peptidase, central amidase, and C-terminal peptidoglycan binding domains, featuring characteristic conserved motifs.
LysinB was predicted to possess a single α/β hydrolase domain with a catalytic triad and a serine esterase motif.
Both purified recombinant LysinA and LysinB demonstrated significant antimycobacterial activity against M. smegmatis.
LysinA exhibited periplasmic expression in E. coli, suggesting a secretory nature that may facilitate host lysis independently of Holin proteins.

Conclusions:

Endolysins LysinA and LysinB from mycobacteriophage PDRPxv possess distinct structural features and exhibit potent antimycobacterial activity.
The characterized endolysins are potential candidates for developing novel therapeutics against drug-resistant tuberculosis.
The secretory nature of LysinA offers a unique mechanism for bacterial lysis, enhancing its therapeutic potential.

Read full analysis

Keywords:

Endolysins Esterase Mycobacteriophage PDRPxv Periplasmic expression

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