Division of Biological Sciences, Department of Molecular Biology, University of California at San Diego La Jolla, CA, USA
Received date: 21/12/2017; Accepted date: 11/01/2018; Published date: 18/01/2018
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Holins, cell catabolism, endolysin, homooligomers
Holins are conserved viral and cellular proteins that elicit a variety of effects and serve various functions . Their best studied function involves bacterial cell lysis, a process used by double stranded DNA phages to form open or tightly sealed channels in the host bacterial cell membrane, releasing an autolysin and ultimately, following lysis of the cell, releasing the phage progeny [2-4]. At a precise, highly regulated time during phage development, these hydrophobic membrane proteins spontaneously oligomerize in the bacterial cytoplasmic membrane, forming large, nonspecific, transmembrane “holes” that reduce the membrane potential and release endolysins that hydrolyze specific bonds in the peptidoglycan cell wall . This allows the release of completely synthesized virions following viral multiplication at the expense of host bacterial cell catabolism after the cell has been parasitized by the phage.
Not all phage holins function in the same mechanistic way. For example, pinholins, such as the one identified in lambdoid phage 21, depolarizes the membrane by forming small holes, activating a signal that regulates release of the endolysin . Holins form transmembrane pores as solo agents, rarely fusing to or functioning with other proteins, and the holes formed are homooligomeric transmembrane structures with variable numbers of subunits, depending on the size of the hole required for export of the endolysin (Figure 1). Moreover, these proteins exhibit variable numbers of transmembrane alpha-helical segments (TMSs), (between 1 and 4 TMSs), depending on the holin (Figure 2). Some holins have originated as small 2 TMS proteins and duplicated internally during evolutionary time to yield 4 TMS proteins that are about twice as large . It is thought that the pore adds and eliminates subunits while shaping itself around the endolysin as it passes through the membrane [8,9].
Figure 1: Schematic depiction of the proteins involved in Gram-negative bacterial cell envelope disruption by holin-type lysis systems. Pinholins form small heptameric pores that collapse the membrane potential (the PMF) across the inner membrane, while the more conventional holins form large multi-subunit pores of variable sizes that allow release of fully folded endolysins from the cytoplasm, which in the periplasm hydrolyze specific bonds in the peptidoglycan cell wall, depending on the lysin type. One- or two-component spanins disrupt the outer membrane by an unknown mechanism, possibly involving fusion of the outer membrane with the inner membrane. C+, a cation.
Figure 2: Schematic view of the topologies of members of the seven holin superfamilies (I to VII). Solid lines represent TMSs that are present in all known members of the superfamily. Dashed lines represent TMSs present in some but not all members. # TMSs, the number of transmembrane α-helical segments (TMSs) predicted for the proteins of a superfamily. Family numbers refer to the families in subclass 1.E of the Transporter Classification Database.
Despite their large sequence and topological diversity, all holins have an N-terminal transmembrane region and a highly charged C-terminal region . Fifty-nine sequence divergent families of holins and putative holins are tabulated, and twenty-two of these families cluster into seven apparently phylogenetically unrelated groups or superfamilies as categorized in the Transporter Classification Database (TCDB; www.tcdb.org) [11,12]. Thus, Superfamilies I - VII include 1, 5, 7, 4, 2, 2 and 1 families, respectively, and the two superfamilies with only one family include multiple sequence divergent sub-families (3 and 6 subfamilies for superfamilies I (family with TC#1.E.11) and VII (family with TC#1.E.36), respectively .
Holins have been identified in numerous free living and pathogenic bacterial species. These include members of numerous phyla including Proteobacteria, Firmicutes, Spirochetes, Bacteroidetes, Actinobacteria, Chloroflexi, Cyanobacteria, Deinococcus/ Thermus, Fusobacteria, Planctomycetes, Tenericutes, Thermatogae and Verrucomicrobia , with the best characterized deriving from Proteobacteria and Firmicutes. Pathogenic bacterial species such as those found in the genuses (and their phage) of Lepto-spira , Bacillus , Clostridium , Staphylococcus  and Streptococcus  are often effective as anti-bacterial agents. These holins vary from one to four TMSs and sizes that are roughly proportional to the numbers of TMSs (see Figure 2) . It has been noted that holins are very rarely fused to other protein domains, suggesting that they form transmembrane pores as homooligomers without the participation of other proteins or protein domains . This conclusion has been established for several of the best-characterized holins.
Holins are not only found encoded in phage genomes; they are also encoded by bacterial, archaeal and eukaryotic genes . Saier and Reddy reviewed the various functions apparently served by these proteins . These functions include  intercellular gene transfer,  biofilm formation,  spore morphogenesis,  DNA release  toxin export  programmed cell death,  oxidative stress adaptation, and  possibly, control of acetate metabolism. Some of these functions, such as programmed cell death, have probably been transmitted to eukaryotes. See Saier and Reddy, 2015 for references to the original research articles describing the roles of holins and potentially related proteins in these cellular processes.
Phage and bacterial cell holins have been reported to exert antibacterial activity against heterogenous pathogens [16,18-20] as well as antiviral activity . Holins alone, without endolysins, compromise the cell membrane but are unable to destabilize the cell wall. Thus, peptidoglycan destruction, mediated by the holin-exported endolysin, is necessary for complete cell lysis.
Additional proteins, called spanins (Figure 1), are necessary for cell lysis in osmotically stabilizing environments [22,23]. E. coli cells infected with λ phage without an endolysin gene do not lyse, but cells lose their membrane potential and their ability to respire; they become “leaky” to normally impermeant molecules . A holin-like protein, Tmp 1, has been reported to have bacteriostatic and membrane damaging properties . The ability of Tmp 1 to damage the cell membrane is associated with its N-terminal hydrophobic transmembrane domain, typical of holins . Such studies provide strong evidence that holin-mediated destruction of cell membranes, alone or together with autolysins and spanins, can be used to cause antibacterial and bacteriostatic effects, serving as an alternative to the use of antibiotics .
The combined use of holins and endolysins may be an effective strategy to cure infections and prevent bacterial resistance to antibiotics. Common bacteriophages , virophages , mycophages , zymophages  and cyanophages  provide successful and promising alternative therapies against infections caused by bacteria. They can also function in food safety against fungal infections, prevent resistance against zoonotic infections and be used to protect our aquatic environment [32,33].
To summarize, holins, with or without their companion autolysins and spanins, serve useful functions to bacterial cells and are required for the successful propagation of numerous viruses in bacterial hosts. They additionally have tremendous potential in combating bacterial drug resistance and have been invaluable in the development of biotechnological advances . We are likely to discover many additional functions and uses for these proteins as further research on these amazing proteins advances