Decoding the Molecular Properties of Mycobacteriophage D29 Holin Provides Insights into Holin Engineering

Abstract

ABSTRACT Holins are bacteriophage-encoded small transmembrane proteins that determine the phage infection cycle duration by forming nonspecific holes in the host cell membrane at a specific time postinfection. Thus, holins are also termed “protein clocks.” Holins have one or more transmembrane domains, and a charged C-terminal region, which, although conserved among holins, has not yet been examined in detail. Here, we characterize the molecular properties of the mycobacteriophage D29 holin C-terminal region, and investigate the significance of the charged residues and coiled-coil (CC) domain present therein. We show that the CC domain is indispensable for holin-mediated efficient bacterial cell lysis. We further demonstrate that, out of the positively- and negatively-charged residues present in the C-terminal region, substituting the former, and not the latter, with serine, renders holin nontoxic. Moreover, the basic residues present between the 59th and the 79th amino acids are the most crucial for holin-mediated toxicity. We also constructed an engineered holin, HolHC, by duplicating the C-terminal region. Compared to the wild type, the HolHC protein shows higher toxicity in both Escherichia coli and Mycobacterium smegmatis and causes rapid killing of both bacteria upon expression. An oligomerization property of HolHC similar to that of the wild-type holin allows us to propose that the C-terminal region of the D29 holin determines the timing, and not the extent, of oligomerization and, thereby, hole formation. Such knowledge-based engineering of mycobacteriophage holin will help in developing novel phage-based therapeutics to kill pathogenic mycobacteria, including Mycobacterium tuberculosis. IMPORTANCE Holins are bacteriophage-encoded small membrane perforators that play an important role in determining the timing of host cell lysis toward the end of the phage infection cycle. The ability of holin to precisely time the hole formation in the cell membrane, ensuring cell lysis, is both interesting and intriguing. Here, we examined the molecular properties of the mycobacteriophage D29 holin C-terminal region, which harbors several polar charged residues and a coiled-coil domain. Our data allowed us to engineer holin with an ability to rapidly kill bacteria and to show higher toxicity than the wild-type protein. Due to their ability to kill host bacteria by membrane disruption, it becomes important to explore the molecular properties of holins that allow them to function in a timely and efficient manner. Understanding these details can help us modulate holin activity and engineer bacteriophages with superior lytic properties to kill pathogenic bacteria, curtail infections, and combat antimicrobial resistance.