Assessing the synergistic potential of bacteriophage endolysins and antimicrobial peptides for eradicating bacterial biofilms.

Phage-encoded endolysins have emerged as a potential substitute to conventional antibiotics due to their exceptional benefits including host specificity, rapid host killing, least risk of resistance. In addition to their antibacterial potency and biofilm eradication properties, endolysins are reported to exhibit synergism with other antimicrobial agents. In this study, the synergistic potency of endolysins was dissected with antimicrobial peptides to enhance their therapeutic effectiveness. Recombinantly expressed and purified bacteriophage endolysin [T7 endolysin (T7L); and T4 endolysin (T4L)] proteins have been used to evaluate the broad-spectrum antibacterial efficacy using different bacterial strains. Antibacterial/biofilm eradication studies were performed in combination with different antimicrobial peptides (AMPs) such as colistin, nisin, and polymyxin B (PMB) to assess the endolysin's antimicrobial efficacy and their synergy with AMPs. In combination with T7L, polymyxin B and colistin effectively eradicated the biofilm of Pseudomonas aeruginosa and exhibited a synergistic effect. Further, a combination of T4L and nisin displayed a synergistic effect against Staphylococcus aureus biofilms. In summary, the obtained results endorse the theme of combinational therapy consisting of endolysins and AMPs as an effective remedy against the drug-resistant bacterial biofilms that are a serious concern in healthcare settings.

Engineering of a T7 Bacteriophage Endolysin Variant with Enhanced Amidase Activity.

The therapeutic use of bacteriophage-encoded endolysins as enzybiotics has increased significantly in recent years due to the emergence of antibiotic resistant bacteria. Phage endolysins lyse the bacteria by targeting their cell wall. Various engineering strategies are commonly used to modulate or enhance the utility of therapeutic enzymes. This study employed a structure-guided mutagenesis approach to engineer a T7 bacteriophage endolysin (T7L) with enhanced amidase activity and lysis potency via replacement of a noncatalytic gating residue (His 37). Two H37 variants (H37A and H37K) were designed and characterized comprehensively using integrated biophysical and biochemical techniques to provide mechanistic insights into their structure-stability-dynamics-activity paradigms. Among the studied proteins, cell lysis data suggested that the obtained H37A variant exhibits amidase activity (∼35%) enhanced compared to that of wild-type T7 endolysin (T7L-WT). In contrast to this, the H37K variant is highly unstable, prone to aggregation, and less active. Comparison of the structure and dynamics of the H37A variant to those of T7L-WT evidenced that the alteration at the site of H37 resulted in long-range structural perturbations, attenuated the conformational heterogeneity, and quenched the microsecond to millisecond time scale motions. Stability analysis confirmed the altered stability of H37A compared to that of its WT counterpart. All of the obtained results established that the H37A variant enhances the lysis activity by regulating the stability-activity trade-off. This study provided deeper atomic level insights into the structure-function relationships of endolysin proteins, thus aiding researchers in the rational design of engineered endolysins with enhanced therapeutic properties.

Quantifying bacterial cell lysis using GFP based fluorimetric assay.

Quantitative measurement of cell lysis against a given microbial strain is essential to calculate the antimicrobial potency of protein/peptide/nanomaterial based formulations. Fluorescence spectroscopy based measurements offer precise quantification of a process via selected flurophore emission profile. In this context, we elucidate a reliable and robust green fluorescent protein (GFP) based fluorescence spectroscopy protocol to evaluate the antimicrobial activity of proteins. The technique is based on the fact that the intensity of the GFP emission released from cells correlates with cell lysis and henceforth the antimicrobial potential of the chosen agent. The technique was demonstrated with two different families of bacteriophage endolysins (T7 and T4 endolysins) using GFP expressing E. coli cells. The GFP based method allowed the absolute quantification of T4 and T7 endolysins cell lysis characteristics at different pH, salt concentrations, and metal ions. The results obtained from GFP based fluorimetric assay were substantiated with turbidimetric assay and fluorescence microscopy. This fluorimetric method in conjugation with different GFP expressing microbial strains and antimicrobial agents can be efficiently applied as a quantification technique to precisely measure cell lysis.