Characterization of holins, the membrane proteins of coliphage ASEC2201: a genomewide in silico approach

Abstract

Drug-resistant Escherichia coli poses a significant healthcare burden, driving the search for novel antimicrobials. We have previously done the isolation and whole-genome sequencing of ASEC2201, a novel coliphage derived from multidrug-resistant clinical E. coli strains. Here, we report the identification and characterization of phage enzyme, holin by in silico approaches. Genome annotation using Prokka identified three putative holin genes (PROKKA_03659, PROKKA_04292, and PROKKA_04422) belonging to the Phage_holin_2_1 superfamily. Upstream promoter prediction revealed active regulatory elements at positions 112, 177, and 186 for these genes, indicating robust transcriptional activity. Transmembrane topology analysis using DeepTMHMM confirmed the presence of two to three α-helical membrane-spanning domains in each holin, essential for pore formation. Homology modeling with SWISS-MODEL yielded high-confidence three-dimensional structures characterized by conserved membrane-anchoring motifs, as supported by QMEAN and GMQE quality scores. In silico identification of cell-penetrating peptide motifs within the holin sequences suggests potential for enhanced intracellular delivery in CPP-fusion therapeutic constructs. Overall, our in-depth analysis elucidates the structural and functional properties of ASEC2201 holins, underscoring their biotechnological significance as scaffolds for developing novel antimicrobial strategies against MDR E. coli. It gives us an understanding on how the holins, with their inherent membrane-disrupting functions, can be explored in detail for future use as lysis modules in programmable bacterial systems, while their identified CPP motifs offer additional potential for engineering targeted therapeutic delivery vehicles. This study also demonstrates the potential of integrative in silico approaches in developing a comprehensive foundation for future experimental validation for proteins with no prior functional annotation.

Keywords: ASEC2201; bacteriophage; bacteriophage-encoded enzymes; functional annotation; holins; phage-mediated lysis; protein 3D structure; transmembrane domain.

Figure 1

Transport of endolysin mediated by holin or the transmembrane domain (TMD), which results in different morphological changes in host cells.

Figure 2

Phylogenetic analysis of PROKKA_03659, PROKKA_04292 and PROKKA_04422 by using neighbor joining method in MegaXII.

Figure 3

Tertiary structure of PROKKA_03659 (A), PROKKA_04292 (B) and PROKKA_04422 (C) determined via the Swiss model and the HHpred tool.

Figure 4

The PROCHECK program validated the Ramachandran plot statistics of the three-dimensional protein structure predicted by Swiss model. The graph’s red areas represent the locations that are most permissible. Green, light green, and white fields are, respectively, demarcated as additional allowed, generously allowed, and restricted regions [in the figure above, (A) is PROKKA_03659, (B) is PROKKA_04292 and (C) is PROKKA_04422].

Figure 5

Catalytic triad sequence similarity in PROKKA_03659, 04292, and 04422 (shown here with an arrow sign).

Figure 6

Protein transmembrane domain recognition by deep transmembrane helices hidden Markov models (DeepTMHMM), where (A) represents the result of PROKKA_03659, (B) for PROKKA_04292 and (C) for PROKKA_04422, respectively.

Figure 7

Predicted transmembrane N-terminal regions of annotated proteins PROKKA_03659 (A); PROKKA_04292 (B); PROKKA_04422 (C), via Phyre2 Modeling.