Research Projects

Infectious diseases remain the second leading cause of mortality worldwide. Among the major pathogens, Staphylococcus aureus, a Gram-positive bacterium, is a key agent responsible for both hospital- and community-acquired infections, ranging from mild skin and soft tissue infections to life-threatening conditions such as pneumonia. According to the World Health Organization (WHO), methicillin-resistant S. aureus (MRSA) poses a significant global health threat, exacerbated by bacterial evolution and the overuse of antibiotics. Hospital-acquired MRSA (HA-MRSA) frequently leads to severe infections—including pneumonia, endocarditis, and septicemia—especially in surgical sites, ICUs, and inpatient wards, complicating treatment and recovery. This underscores the urgent need for novel antibacterial strategies. Bacteriophage-derived endolysins represent promising biotherapeutic alternatives due to their ability to selectively degrade bacterial peptidoglycan, leading to bacterial cell death. The therapeutic concept is based on “lysis from without,” which involves the external application of recombinant endolysins to lyse bacterial cells without producing phage particles. Endolysins have garnered significant interest in recent years owing to their remarkable therapeutic potential against multidrug-resistant bacteria. PlyGRCS, an endolysin encoded by the recently identified S. aureus GRCS bacteriophage isolated from raw sewage in India, has demonstrated specific antimicrobial activity against S. aureus. We recently determined the full-length tertiary structure of PlyGRCS, comprising the CHAP and SH3_5 domains, at a resolution of 2.1 Å and also showed the importance of Ca2+ ions for the catalytic function (Krishnappa et al., 2023, 2024; SERB Project Ref No: CRG/2019/002603). Interestingly, a serendipitous discovery revealed PlyGRCS’s interaction with Escherichia coli cold-shock protein C (CspC), which functions as an RNA chaperone and stress regulator. PlyGRCS also recognizes S. aureus CspC (unpublished results). Endolysins specifically recognize and cleave covalent bonds within the peptidoglycan, a mesh-like polymer composed of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), cross-linked by short peptide chains. Although the biosynthesis and structure of peptidoglycan (PG) are fairly well known (Fig. 1), our understanding of PG maintenance and hydrolysis remains incomplete. PlyGRCS is believed to exhibit both amidase activity (cleaving the bond between MurNAc and the peptide stem) and endopeptidase activity (targeting peptide cross-bridges) targeting S. aureus PG. However, the detailed functional mechanisms remain elusive. Addressing this knowledge gap will shed light on how PlyGRCS’s cell-wall binding domain (SH3_5b) recognizes specific PG regions and facilitates degradation via its catalytic domain (CHAP). This proposal aims to elucidate the molecular mechanism by which PlyGRCS mediates cell-wall lysis in S. aureus through structural and functional analyses of PlyGRCS with various lengths of peptidoglycans and the peptidoglycan whole extract. Additionally, we also carry out screening and evaluating PlyGRCS’s lytic specificity across various clinical S. aureus isolates—including MRSA, CA-MRSA, HA-MRSA, and LA-MRSA—sourced from NIMHANS, contributing toward translational research in antimicrobial therapeutics. The proposal involves a multifaceted approach to molecular biology, protein biochemistry, protein crystallography, and antimicrobial research studies. Collectively, this research will deepen our insight into the functional mechanisms of PlyGRCS and support its advancement as a promising biotherapeutic candidate against MRSA infections.