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Insights into the regulation of bacteriophage endolysin: Multiple means to the same end
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Amol Arunrao Pohane*, Vikas Jain*
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Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian
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Institute of Science Education and Research (IISER), Bhopal – 462023, India
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Running title: Regulation of endolysin during phage infection
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*Corresponding author
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Address correspondence to:
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Microbiology and Molecular Biology Laboratory, Indian Institute of Science Education and
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Research Bhopal, Transit campus: ITI (Gas Rahat) Building, Govindpura, Bhopal – 462023,
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Madhya Pradesh, India
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Tel: +91-755-4092318
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Fax: +91-755-4092392
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E-mail: [email protected] (VJ); [email protected] (AAP)
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Keywords
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Endolysin; peptidoglycan hydrolases; autoregulation; protein translocation; post-translational
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modification; bacteriophages.
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Subject category
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Minireview
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Worde count
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Abstract = 207
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Main text (excluding abstract, figure legend, and references) = 3,873
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Abstract
Antibiotics are the molecules of choice to treat bacterial infections. However, because of
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the rapid emergence of drug-resistant bacteria, alternative modes of combating infections are
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being envisaged. Bacteriophages, which infect and lyse bacterial cells, may function as effective
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antimicrobial agents. Most bacteriophages produce their own peptidoglycan hydrolase called
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endolysin or lysin, which breaks down the cell wall of bacteria and aids in the release of newly
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assembled virions. In this article, we have discussed several findings that help us in
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understanding how endolysins are regulated. We observe that there is no common mechanism
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that is followed in all the cases. Many different modes of activity regulation have been observed
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in endolysins, including regulation of protein expression, translocation across the cell membrane,
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and post-translational modifications. These processes not only demonstrate how endolysins are
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made dependent on other accessory proteins and non-protein factors for their synthesis,
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translocation across the cytoplasmic membrane, and activity, but also show how autoregulation
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helps in maintaining the enzyme in an inactive form. Various regulatory mechanisms discussed
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in this paper are particularly applicable to endolysins. Nevertheless, a detailed study of these
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methods opens new avenues of investigation in the area of protein translocation system and the
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novel ways of enzyme activation and regulation in bacteria.
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Introduction
Bacteriophages, since their discovery, have always fascinated microbiologists,
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biochemists, biophysicists, and geneticists for the peculiar characteristics that they display
48
(Duckworth, 1976; Wittebole et al., 2014). The bacteriophages are able to infect and lyse
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bacterial cells and it is this feature that makes them important antimicrobial agents (Sulakvelidze
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et al., 2001; Hermoso et al., 2007), especially because of the rapid emergence of multidrug-
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resistant bacteria (Loeffler et al., 2001; Schuch et al., 2002; O'Flaherty et al., 2009; Fischetti,
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2010). Bacteriophages use two independent mechanisms to lyse host cells. Whereas some single
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strand DNA and RNA viruses use a peptidoglycan (PG) synthesis inhibitor (Young et al., 2000;
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Zheng et al., 2009; Tanaka & Clemons, 2012), all of the double strand DNA viruses follow a
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lysin-mediated breakdown of PG (Young et al., 2000; Young, 2014; Lood et al., 2015).
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Endolysin is a PG hydrolysing enzyme that carries out enzymatic digestion of the cell wall PG at
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the end of the phage infection cycle, which ensures the release of newly packed phage particles.
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Endolysins produced by bacteriophages infecting Gram-positive and Gram-negative
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bacteria differ from each other. The phages infecting Gram-negative bacteria (G– phages)
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produce endolysins that are mostly uni-domain in nature, with a few exceptions (Zhang &
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Studier, 2004; Lukacik et al., 2012; Walmagh et al., 2012). On the other hand, endolysins from
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the phages infecting Gram-positive bacteria (G+ phage) are modular in nature (Sudiarta et al.,
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2010). The G+ phage endolysin consists of one or two domains at the N-terminus and a C-
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terminal cell wall-binding domain (CBD) (Loessner, 2005). Depending on the requirement of the
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CBD for the enzyme activity, endolysins can be further classified as CBD-dependent and CBD-
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independent (Porter et al., 2007; Low et al., 2011). Endolysin is produced in the cytoplasm and
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is translocated to the periplasmic space where it acts upon the cell wall PG. In this review, we
4
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have summarized several findings that have demonstrated how the activity of endolysin is
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regulated in various systems. The processes by which endolysin activity is regulated include
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endolysin synthesis, export, activation, and substrate specificity, and are depicted in Figure 1.
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Regulation of endolysin expression
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The bacteriophage endolysin is required at the final stage of the phage infection cycle.
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Therefore, it is expected that expression of endolysin occurs only towards the end of the
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infection cycle. Interestingly, several studies have shown that the transcription of endolysin gene
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occurs at the early stage of phage infection. A northern blotting experiment with temperate
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Streptococcus thermophilus phage Sfi21 suggests that the transcription of both holin and
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endolysin occurs at the early stage of the lytic cycle. Additionally, the amount of transcript
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increases progressively, and it is maintained until the last stage of the cycle (Ventura et al.,
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2002). In one of the most characterized lytic phages of E. coli, the T4 phage, DNA microarray
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data showed that the transcription of lytic genes starts at the early stage but the proteins are
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synthesized only at the late stage (Luke et al., 2002); suppression of mRNA translation is
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achieved by means of a stable hairpin structure adopted by endolysin mRNA that inhibits
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translation (McPheeters et al., 1986). It is, however, not clear why translation of mRNA is
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suppressed since the endolysin, even if synthesized early, will accumulate in the cytoplasm and
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will remain physically separated from the substrate (PG).
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The T7 phage, another well-studied bacteriophage of E. coli, shows an early transcription
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of its endolysin gene coding for T7 lysozyme, and holin (an integral membrane protein required
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for membrane perforation and passive diffusion of endolysin into the periplasmic space;
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discussed later). In T7, the mRNA for both lysozyme and holin could be observed just after 3
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minutes of infection (Nguyen & Kang, 2014). DNA microarray studies carried out with the
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Gram-positive Streptococcus thermophilus phages, cos-type DT1 and pac-type 2972, also
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revealed that their endolysin genes show gradual increase in the transcript level from early stage
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and reach maximum at late stage (Duplessis et al., 2005). Similarly, RNAseq studies of the
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mycobacteriophage Giles demonstrated that the transcription of lytic genes started at an early
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stage of infection, although it reached its highest levels at the late stage of infection (Dedrick et
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al., 2013). Early transcription clearly suggests that endolysin can be transcribed by the host RNA
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polymerase, preferably using the housekeeping sigma factor. Indeed, in order to identify the
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promoter for the endolysin gene of mycobacteriophage Ms6, the desired region was fused to the
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lacZ reporter. It was then demonstrated that lacZ was expressed from two σ70-like promoters,
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providing evidence that transcription of endolysin genes can be carried out by host transcription
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factors (Garcia et al., 2002). It would be of immense interest to explore the involvement of
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various transcription factors in the expression of endolysin genes. For example, in Thermus
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thermophilus HB8, it has been shown that gp39 and gp76 of bacteriophage P23-45 can recognize
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host RNA polymerase and regulate phage gene transcription (Berdygulova et al., 2011).
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Regulation during translocation
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The site of action of endolysin is the PG layer of the bacterial cell wall. In order to reach
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the target, endolysin must travel from the cytoplasm to the periplasm. To accomplish this,
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endolysin follows different methods. One of the most common and well studied mechanisms
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involves holin and forms what is known as the holin-endolysin cell lysis system. Holin is a small
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transmembrane protein, which possesses 1–3 transmembrane regions (Blasi & Young, 1996;
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Young, 2002; Reddy & Saier, 2013; Savva et al., 2014). Upon oligomerisation at a specific time,
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holin forms holes in the cytoplasmic membrane of bacteria and allows the passive diffusion of
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endolysin from the cytoplasm to the periplasm thus triggering the cell lysis event (Park et al.,
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2006; Wang et al., 2008; Pang et al., 2010; Catalao et al., 2011a; White et al., 2011; Pang et al.,
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2013).
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However, the discovery of holin-independent export of endolysin in many bacteriophages
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suggested that the holin-endolysin system may not be the only method of choice. In several
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bacteriophages, the endolysin is produced with an N-terminally located signal-arrest-release
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(SAR) sequence that is responsible for the transport of endolysin. Therefore, in these
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bacteriophages, endolysin is secreted with the help of SAR sequence. The first secretory
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endolysin Lys44 was reported from Oenococcus oeni phage fOg44 (Sao-Jose et al., 2000). Upon
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expression in E. coli cells, Lys44 showed two bands on SDS-PAGE with apparent molecular
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weights of 51 and 43 kDa, belonging to the primary and the mature forms, respectively. Upon
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expression, endolysin crosses the cytoplasmic membrane and, with the help of the proteolytic
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activity of periplasmic protease LepB, is released into the periplasm (Sao-Jose et al., 2000).
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Furthermore, the work also showed that processing of endolysin is necessary for its lytic activity.
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Although the processing of endolysin was observed in many other bacteriophages, there are
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some exceptions wherein SAR sequence is not cleaved by the periplasmic protease. E. coli
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bacteriophage P1 lysin, for example, showed the presence of N-terminal SAR sequence when its
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periplasmic and cytoplasmic fractions were tested on SDS-PAGE, suggesting that the SAR
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sequence remains attached to the endolysin even after export (Xu et al., 2004). Similar
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observations were made for Erwinia amylovora phage ERA103 and other bacteriophages (Kuty
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et al., 2010; Briers et al., 2011).
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Recently, two reports showed very different modes of translocation of endolysin to the
periplasmic space. The pneumococcal phage SV1 lysin (Svl) was found to contain no signal
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sequence at its N-terminus, could be transported to the periplasmic space in a holin-deficient
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phage, and carried out holin-independent killing (Frias et al., 2013). Interestingly, the choline-
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containing teichoic acid (TA) was found to be responsible for endolysin transport. Svl was not
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translocated to the periplasmic space when the culture was grown in ethanolamine-containing
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medium, which is the blocking agent for the loading of TA on choline precursor in the
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cytoplasm.
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Another interesting mode of transport of endolysin was shown in mycobacteriophage
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Ms6 lysin. The deletion of holin in mycobacteriophage Ms6 did not result in the loss of viability
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of the phage. This suggested that Ms6 lysin could translocate in a holin-independent manner
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(Catalao et al., 2010). Expression of Ms6 lysin along with Gp1 showed a lysis phenotype.
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Interestingly, Gp1 does not belong to the holin class of proteins. Instead, the biophysical analysis
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of Gp1 suggested that it belongs to the chaperones of type III secretion system. It was suggested
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that the N-terminal region of Gp1 physically interacts with lysin and transports it using the
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SecA-dependent pathway (Catalao et al., 2011b). Mycobacteriophages L5, D29, and Kostya
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have also shown holin-independent killing of mycobacteria (Payne & Hatfull, 2012; Pohane et
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al., 2014). However, the mechanism of transport of endolysin in these cases is yet to be
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elucidated. Nevertheless, these studies suggest that, apart from the canonical mode of transport
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through holin, bacteriophages also use other strategies for the translocation of endolysins.
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Regulation by inactivation
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It is interesting to note that although endolysin in several bacteriophages can be
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translocated in a holin-independent manner, many of these phages do produce holin during their
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infection cycle. So, why is holin required, if endolysin can be translocated without it? Several
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studies have shown that endolysin remains in an inactive form after localisation to the
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periplasmic space. Various processes then either activate the inactive endolysin or enhance its
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catalytic activity for the rapid lysis of the host cells. One of the most common modes of
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activation of endolysin in the periplasmic space is the depolarisation of the cytoplasmic
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membrane that occurs by the activity of holin or pinholin (Young, 2002). Pinholins, like holins,
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form holes in the cytoplasmic membrane of the host. However, unlike holins, the holes formed
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by pinholins are not of sufficient dimension to allow for the diffusion of endolysin, and yet are
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large enough to cause leakage of protons leading to the disruption of proton motive force (PMF),
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thus resulting in the depolarization of membrane (Park et al., 2007; Pang et al., 2010; Pang et al.,
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2013). As mentioned previously, the Oenococcus oeni phage endolysin Lys44 is transported by a
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cleavable SAR signal sequence (Sao-Jose et al., 2000). When the purified endolysin from phage
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fOg44 was added externally to O. oeni culture, the protein was unable to carry out lysis of the
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bacterial cells. However, an addition of a membrane-disrupting agent such as nisin along with
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the endolysin resulted in a rapid decline in the culture optical density (Nascimento et al., 2008).
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Furthermore, when endolysin was expressed within the cells, lysis occurred only when nisin was
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supplied externally (Nascimento et al., 2008). Although the study verifies the requirement of the
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disruption of PMF in endolysin activation, a direct involvement of fOg44 holin in the phage’s
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endolysin activation has currently not been examined. In another report, the expression of
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Pseudomonas aeruginosa phage ɸKMV endolysin resulted in the lysis of E. coli 60–90 minutes
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after induction, whereas the co-expression of endolysin and holin led to the bacterial cell lysis
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within 15 minutes of induction (Briers et al., 2011).
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So far, we have noted that the phage-encoded endolysin, upon activation due to the
disruption of PMF, causes cell wall PG degradation resulting in phage release. However,
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interestingly, bacteriophages have also been shown to manipulate bacteria-encoded autolysins
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with the help of holin for their own benefit. Autolysins are the bacteria-encoded PG hydrolases
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that are required for the cellular processes such as cell growth, cell division, and pathogenesis
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among others (Shockman et al., 1996; Smith et al., 1996). In the case of phage SV1 of
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Streptococcus pneumoniae, the autolysin LytA was able to lyse the host cells after induction with
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holin and, consequently, SV1 phage was shown to form plaques in the absence of its endolysin.
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This observation underlines the importance of LytA for efficient release of phage particles (Frias
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et al., 2009). Similar property of autolysin induction by phage was previously reported in the
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case of Micrococcus lysodeikticus infected with phage N1 (Goepfert & Naylor, 1967). When the
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holin of pneumococcal bacteriophage EJ-1 was expressed in E. coli, it also activated LytA
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autolysin and caused cell death (Diaz et al., 1996). These experiments suggest that the bacterial
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cell death after phage infection, occurring due to the interference in cell wall biosynthesis or the
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cell wall disruption, requires depolarisation of the cell membrane (Bayles, 2000; Martinez-
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Cuesta et al., 2000; Bayles, 2003; Clementi et al., 2012; Sadykov & Bayles, 2012).
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A very interesting mechanism of regulation of endolysin was identified in bacteriophage
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P1. The P1 endolysin is transported by the SAR domain sequence present at the N-terminus (Xu
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et al., 2005). The active site of P1 endolysin consists of Glu42, Thr57, and Cys51 and the
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regulation of the activity is achieved by the involvement of two other cysteines, Cys44 and Cys13.
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By carrying out chemical cleavage using cyanylated cysteines and ammonium hydroxide, P1
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endolysin was found to have two isoforms: one in which Cys13 had a free sulfhydryl group, and
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the other in which Cys51 had a reduced thiol group (Cys-SH). Based on cell lysis and X-ray
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crystallography data, it was suggested that the formation of a disulphide bond between Cys44 and
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Cys51 results in the inactivation of endolysin, whereas a disulphide bond between Cys13 and
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Cys44 activates the protein (Xu et al., 2005). A similar mechanism of regulation was observed in
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the case of Erwinia phage ERA103 endolysin (Kuty et al., 2010). Here, the N-terminal catalytic
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domain of endolysin contains three Cys residues viz. Cys12, Cys42, and Cys45. The Cys12 is
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present in the SAR domain of ERA103 endolysin, Cys12Ser mutation results in a catalytically
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inactive protein. The genetic and biochemical studies suggested that a disulphide bond between
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Cys42 and Cys45 forms a cage that traps the catalytic Glu43 residue and thus blocks the enzyme
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activity. Shifting of the disulphide bond from Cys42-Cys45 to Cys42- Cys12 leads to removal of the
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block, thus making the enzyme catalytically active.
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Such endolysin activity regulation by the disulphide bond isomerization to trap a catalytic
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residue is an interesting way of inhibiting enzyme activity in SAR endolysins. However, not all
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endolysins contain cysteine residues in their SAR domain. How do these endolysins block their
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active site? In the case of coliphage 21 endolysin (R21), although a SAR domain is present at the
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N-terminus of endolysin, it lacks a Cys residue (Xu et al., 2004). When the SAR-truncated
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derivative of R21 was prepared, it showed no activity in vitro. Although the addition of a
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cleavable secretory signal sequence from PelB (an Erwinia carotovora pectate lyase) resulted in
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the transport of endolysin, the protein remained catalytically inactive. These experiments
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demonstrated that SAR has an important role in active site regulation. The structural studies
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suggested that the conformation of the SAR domain in the inactive form of endolysin differs
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from that in the active form of protein. While the SAR domain conformation in the active form
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allows the formation of the catalytic triad, the SAR domain remains associated with the
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membrane in the inactive form (Sun et al., 2009). However, the mechanism by which the SAR
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domain is released from the membrane to activate endolysin needs to be elucidated.
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Lysin activity regulation by trapping the active site residue in a loop has been reported in
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Gp5 of T4 bacteriophage. Gp5 is a tail protein that is required for the phage infection (Arisaka et
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al., 2003). In Gp5, the enzyme inactivation occurs by the formation of an inactivation loop that
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spans from Pro346 to Ser360. The enzyme is activated by an autoproteolytic cleavage of the protein
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between Ser351 and Ala352 within the loop (Kanamaru et al., 2005).
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The activity of endolysin has also been shown to be regulated by means of
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oligomerization. One of the best studied examples includes PlyC, an endolysin produced by
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streptococcal C1 phage. PlyC oligomer is formed by the association of one molecule of PlyCA,
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which bears the catalytic domains, with eight molecules of PlyCB, which has the cell wall
235
binding ability. Interestingly, PlyCA shows no lytic activity in the absence of PlyCB suggesting
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that an oligomeric assembly is required for the functioning of the protein (McGowan et al.,
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2012). The CTP1L endolysin of CTP1 phage that infects Clostridium tyrobutyricum is known to
238
form dimer. The biochemical and biophysical examination of CTP1L suggested that the side-by-
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side dimerization of the protein leads to an autocleavage in the molecule that enhances endolysin
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activity (Dunne et al., 2014). Dimerization-based endolysin activity regulation has been reported
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in the case of Cpl-1 endolysin produced by Cp-1 bacteriophage that infects Streptococcus
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pneumoniae. Cpl-1 endolysin shows enhanced activity upon dimerization that is a result of the
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interaction between its cell wall binding domain and the bacterial cell wall component choline
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(Sanz et al., 1992; Buey et al., 2007; Monterroso et al., 2008). It is worth adding here that an
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introduction of a disulphide bond leading to the stabilization of the dimeric form of Cpl-1
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resulted in higher activity of the endolysin and reduced plasma clearance (Resch et al., 2011).
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This report thus also demonstrates that the bioengineering of endolysins for therapeutic purposes
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can be carried out. Although in some studied cases as noted here, endolysin activity has been
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shown to be affected by multimerization, it is not clear how phage utilizes the oligomerization
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property of endolysin to regulate the protein’s activity during host cell lysis.
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Regulation by specificity
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The bacteriophages infecting Gram-positive bacteria harbour endolysins that are modular
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in nature, and contain at least one catalytic and one cell wall binding domain (Fischetti, 2005;
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Fischetti, 2010; Sudiarta et al., 2010). On the other hand, endolysins of phages infecting Gram-
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negative bacteria contain only a catalytic domain (Cheng et al., 1994). It was found that the cell
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wall binding domain of endolysin binds specifically to the host cell wall. Interestingly, however,
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most of the bacterial autolysins do not display PG binding specificity (Steen et al., 2003; Nelson
258
et al., 2006; Fischetti, 2008). The G+ phage endolysins have a very narrow range of substrate
259
specificity, i.e. their ability to degrade the cell wall is confined to specific host species or strains
260
(Fischetti, 2008). For example, the Dp-1 bacteriophage lytic enzyme, Pal, shows lytic activity
261
specifically on Streptococcus pneumoniae. However, it has either very low or no activity on
262
other species of streptococci (Loeffler et al., 2001). Similar observations were made for C1 phage
263
endolysin of streptococci, where the enzyme showed very specific lytic activity on Group A
264
Streptococcus but not on B, D, F, G, L, and other species of streptococci (Nelson et al., 2001).
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This is an intriguing property of G+ phage endolysin that makes the protein very useful not only
266
for the diagnostic purposes, but also for the targeted elimination of pathogenic Gram-positive
267
bacteria.
268
Many studies have shown that the species-specific cell lysis property of G+ phage
269
endolysins is attributed to the cell wall binding domain (Low et al., 2005), and it has also been
270
studied in the case of Mycobacterium phage D29 lysin A (Pohane et al., 2014). Lysin A of phage
13
271
D29 contains two N-terminal catalytic domains and a C-terminal cell wall binding domain
272
(Payne & Hatfull, 2012; Pohane et al., 2014). While the full-length lysin A was toxic to
273
mycobacterial cells, it failed to kill E. coli. Interestingly, however, the individual catalytic
274
domains could kill E. coli cells. It has been proposed that the ability of D29 lysin A to kill M.
275
smegmatis but not E. coli is a result of interaction between the catalytic and the cell wall binding
276
domains. Furthermore, the cell wall binding domain of D29 lysin A binds only to the
277
mycobacterial cell wall (Pohane et al., 2014; Pohane et al., 2015). The data thus suggest that the
278
endolysin stays in a ‘lock’ conformation in the non-host species, owing to an interaction between
279
the catalytic and the cell wall binding domains. In the canonical host, this interaction is
280
abrogated by the interference from the cell wall, thus allowing the catalytic domain to act on the
281
PG. The interaction of the cell wall binding domain with the cell wall has been studied in the
282
Listeria monocytogenes phage endolysins Ply118 and Ply500 (Loessner et al., 2002), and
283
Bacillus anthracis bacteriophage endolysins PlyL and PlyG (Mo et al., 2012). In these examples,
284
the affinity of the cell wall binding domains with the cell wall components was found to be in
285
nanomolar range. However, the affinity between the catalytic and the cell-wall binding domains
286
of D29 lysin A was reported to be in micromolar range (Pohane et al., 2014). Conceivably, the
287
opening of ‘lock’ conformation to release the catalytic site in some of the endolysins would be a
288
result of the higher affinity between its cell wall binding domain and the cell wall PG.
289
A similar mode of autoregulation was proposed for the cell wall hydrolases required for
290
cell separation during bacterial cell division (Bublitz et al., 2009; Ruggiero et al., 2010; Yang et
291
al., 2012). Although endolysins of some G+ phages show broad substrate preference, their
292
activity was increased when the cell wall binding component was added. For example, the
293
endolysin of pneumococcal bacteriophage EJ-1 that infects Gram-positive bacteria could act on
14
294
E. coli cell wall. Interestingly, a two-fold increase in the activity was observed when a cell wall
295
binding domain substrate, choline, was added in the reaction mixture (Diaz et al., 1996).
296
It was hypothesized that cell wall binding domain of endolysin helps in avoiding the
297
killing of uninfected bacterial cells by perturbing the free diffusion of endolysin thus resulting in
298
the availability of more host cells to be infected by the phages (Loessner et al., 2002; Fischetti,
299
2008). Additionally, the interaction between the catalytic and the cell wall binding domains of
300
endolysin may not allow the enzyme to lyse other cells, in a case where an endolysins does get
301
released from an infected cell. Together, these regulatory mechanisms in the bacteriophage
302
endolysins will help to maintain balance in microbial ecosystems.
303
Conclusions and future prospects
304
Endolysins are regulated not only at the transcription level, but also post-translationally.
305
Many of them rely on either holin-mediated transport or a signal sequence to reach the
306
periplasmic space. However, the involvement of teichoic acids and chaperones in the
307
transportation of endolysins opens new avenues for exploring the protein transport systems in
308
bacteria. Upon reaching the periplasmic space, in some studied cases, endolysin stays in an
309
inactive form and the activation requires significant changes in the protein conformation.
310
However, it is unclear how these intramolecular conformational changes occur. Furthermore, the
311
host specificity of phages infecting Gram-positive bacteria is an interesting observation in the
312
field of bacteriophage research. The interaction between the catalytic and the cell wall binding
313
domains will allow us to further explore it in the light of enzyme autoregulation. However, this
314
may not be the only mechanism; another important aspect such as substrate choice also needs
315
exploration. Moreover, the mechanism employed for the disruption of the interaction between
15
316
the catalytic and the cell wall binding domains is yet to be studied in more detail. The species-
317
specific substrate selection in the G+ phage endolysin will allow for the development of novel
318
therapeutics to control the diseases caused by pathogenic Gram-positive bacteria. Further
319
research in this area will certainly help in combating bacterial infections.
16
320
Acknowledgements
321
A.A.P. thanks the Council of Scientific and Industrial Research, Govt. of India for the senior
322
research fellowship. The work is supported by a grant from the Department of Biotechnology,
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Govt. of India.
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References
325
326
Arisaka, F., Kanamaru, S., Leiman, P. & Rossmann, M. G. (2003). The tail lysozyme complex of
bacteriophage T4. Int J Biochem Cell Biol 35, 16-21.
327
328
329
Bayles, K. W. (2000). The bactericidal action of penicillin: new clues to an unsolved mystery. Trends
Microbiol 8, 274-278.
330
331
332
Bayles, K. W. (2003). Are the molecular strategies that control apoptosis conserved in bacteria? Trends
Microbiol 11, 306-311.
333
334
335
Berdygulova, Z., Westblade, L. F., Florens, L. & other authors (2011). Temporal regulation of gene
expression of the Thermus thermophilus bacteriophage P23-45. J Mol Biol 405, 125-142.
336
337
338
Blasi, U. & Young, R. (1996). Two beginnings for a single purpose: The dual-start holins in the regulation
of phage lysis. Mol Microbiol 21, 675-682.
339
340
341
Briers, Y., Peeters, L. M., Volckaert, G. & Lavigne, R. (2011). The lysis cassette of bacteriophage KMV
encodes a signal-arrest-release endolysin and a pinholin. Bacteriophage 1, 25-30.
342
343
344
345
Bublitz, M., Polle, L., Holland, C., Heinz, D. W., Nimtz, M. & Schubert, W. D. (2009). Structural basis for
autoinhibition and activation of Auto, a virulence-associated peptidoglycan hydrolase of Listeria
monocytogenes. Mol Microbiol 71, 1509-1522.
346
347
348
349
Buey, R. M., Monterroso, B., Menendez, M., Diakun, G., Chacon, P., Hermoso, J. A. & Diaz, J. F. (2007).
Insights into molecular plasticity of choline binding proteins (pneumococcal surface proteins) by SAXS. J
Mol Biol 365, 411-424.
350
351
352
353
Catalao, M. J., Gil, F., Moniz-Pereira, J. & Pimentel, M. (2010). The mycobacteriophage Ms6 encodes a
chaperone-like protein involved in the endolysin delivery to the peptidoglycan. Mol Microbiol 77, 672686.
354
355
356
Catalao, M. J., Gil, F., Moniz-Pereira, J. & Pimentel, M. (2011a). Functional analysis of the holin-like
proteins of mycobacteriophage Ms6. J Bacteriol 193, 2793-2803.
357
358
359
360
Catalao, M. J., Gil, F., Moniz-Pereira, J. & Pimentel, M. (2011b). The endolysin-binding domain
encompasses the N-terminal region of the mycobacteriophage Ms6 Gp1 chaperone. J Bacteriol 193,
5002-5006.
361
18
362
363
Cheng, X., Zhang, X., Pflugrath, J. W. & Studier, F. W. (1994). The structure of bacteriophage T7
lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase. Proc Natl Acad Sci USA 91, 4034-4038.
364
365
366
367
Clementi, E. A., Marks, L. R., Duffey, M. E. & Hakansson, A. P. (2012). A novel initiation mechanism of
death in Streptococcus pneumoniae induced by the human milk protein-lipid complex HAMLET and
activated during physiological death. J Biol Chem 287, 27168-27182.
368
369
370
371
Dedrick, R. M., Marinelli, L. J., Newton, G. L., Pogliano, K., Pogliano, J. & Hatfull, G. F. (2013).
Functional requirements for bacteriophage growth: gene essentiality and expression in
mycobacteriophage Giles. Mol Microbiol 88, 577-589.
372
373
374
375
Diaz, E., Munthali, M., Lunsdorf, H., Holtje, J. V. & Timmis, K. N. (1996). The two-step lysis system of
pneumococcal bacteriophage EJ-1 is functional in gram-negative bacteria: triggering of the major
pneumococcal autolysin in Escherichia coli. Mol Microbiol 19, 667-681.
376
377
Duckworth, D. H. (1976). "Who discovered bacteriophage?". Bacteriological Reviews 40, 793-802.
378
379
380
Dunne, M., Mertens, H. D., Garefalaki, V. & other authors (2014). The CD27L and CTP1L endolysins
targeting Clostridia contain a built-in trigger and release factor. PLoS Pathog 10, e1004228.
381
382
383
Duplessis, M., Russell, W. M., Romero, D. A. & Moineau, S. (2005). Global gene expression analysis of
two Streptococcus thermophilus bacteriophages using DNA microarray. Virology 340, 192-208.
384
385
Fischetti, V. A. (2005). Bacteriophage lytic enzymes: novel anti-infectives. Trends Microbiol 13, 491-496.
386
387
Fischetti, V. A. (2008). Bacteriophage lysins as effective antibacterials. Curr Opin Microbiol 11, 393-400.
388
389
390
Fischetti, V. A. (2010). Bacteriophage endolysins: a novel anti-infective to control Gram-positive
pathogens. Int J Med Microbiol 300, 357-362.
391
392
393
Frias, M. J., Melo-Cristino, J. & Ramirez, M. (2009). The autolysin LytA contributes to efficient
bacteriophage progeny release in Streptococcus pneumoniae. J Bacteriol 191, 5428-5440.
394
395
396
Frias, M. J., Melo-Cristino, J. & Ramirez, M. (2013). Export of the pneumococcal phage SV1 lysin
requires choline-containing teichoic acids and is holin-independent. Mol Microbiol 87, 430-445.
397
398
399
400
Garcia, M., Pimentel, M. & Moniz-Pereira, J. (2002). Expression of Mycobacteriophage Ms6 lysis genes
is driven by two sigma(70)-like promoters and is dependent on a transcription termination signal
present in the leader RNA. J Bacteriol 184, 3034-3043.
19
401
402
403
Goepfert, J. M. & Naylor, H. B. (1967). Characteristics of a lytic enzyme induced by bacteriophage
infection of Micrococcus lysodeikticus. J Virol 1, 701-710.
404
405
406
Hermoso, J. A., Garcia, J. L. & Garcia, P. (2007). Taking aim on bacterial pathogens: from phage therapy
to enzybiotics. Curr Opin Microbiol 10, 461-472.
407
408
409
Kanamaru, S., Ishiwata, Y., Suzuki, T., Rossmann, M. G. & Arisaka, F. (2005). Control of bacteriophage
T4 tail lysozyme activity during the infection process. J Mol Biol 346, 1013-1020.
410
411
412
Kuty, G. F., Xu, M., Struck, D. K., Summer, E. J. & Young, R. (2010). Regulation of a phage endolysin by
disulfide caging. J Bacteriol 192, 5682-5687.
413
414
415
Loeffler, J. M., Nelson, D. & Fischetti, V. A. (2001). Rapid killing of Streptococcus pneumoniae with a
bacteriophage cell wall hydrolase. Science 294, 2170-2172.
416
417
418
419
Loessner, M. J., Kramer, K., Ebel, F. & Scherer, S. (2002). C-terminal domains of Listeria monocytogenes
bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial
cell wall carbohydrates. Mol Microbiol 44, 335-349.
420
421
422
Loessner, M. J. (2005). Bacteriophage endolysins--current state of research and applications. Curr Opin
Microbiol 8, 480-487.
423
424
425
426
Lood, R., Winer, B. Y., Pelzek, A. J., Diez-Martinez, R., Thandar, M., Euler, C. W., Schuch, R. & Fischetti,
V. A. (2015). Novel phage lysins capable of killing the multidrug resistant Gram-negative bacterium
Acinetobacter baumannii in a mouse sepsis model. Antimicrob Agents Chemother 59, 1983-1991.
427
428
429
Low, L. Y., Yang, C., Perego, M., Osterman, A. & Liddington, R. C. (2005). Structure and lytic activity of a
Bacillus anthracis prophage endolysin. J Biol Chem 280, 35433-35439.
430
431
432
433
Low, L. Y., Yang, C., Perego, M., Osterman, A. & Liddington, R. (2011). Role of net charge on catalytic
domain and influence of cell wall binding domain on bactericidal activity, specificity, and host range of
phage lysins. J Biol Chem 286, 34391-34403.
434
435
436
Lukacik, P., Barnard, T. J., Keller, P. W. & other authors (2012). Structural engineering of a phage lysin
that targets gram-negative pathogens. Proc Natl Acad Sci USA 109, 9857-9862.
437
438
439
Luke, K., Radek, A., Liu, X. P., Campbell, J., Uzan, M., Haselkorn, R. & Kogan, Y. (2002). Microarray
analysis of gene expression during bacteriophage T4 infection. Virology 299, 182-191.
20
440
441
442
Martinez-Cuesta, M. C., Kok, J., Herranz, E., Pelaez, C., Requena, T. & Buist, G. (2000). Requirement of
autolytic activity for bacteriocin-induced lysis. Appl Environ Microbiol 66, 3174-3179.
443
444
445
McGowan, S., Buckle, A. M., Mitchell, M. S. & other authors (2012). X-ray crystal structure of the
streptococcal specific phage lysin PlyC. Proc Natl Acad Sci USA 109, 12752-12757.
446
447
448
McPheeters, D. S., Christensen, A., Young, E. T., Stormo, G. & Gold, L. (1986). Translational regulation
of expression of the bacteriophage T4 lysozyme gene. Nucleic Acids Res 14, 5813-5826.
449
450
451
452
Mo, K. F., Li, X., Li, H., Low, L. Y., Quinn, C. P. & Boons, G. J. (2012). Endolysins of Bacillus anthracis
bacteriophages recognize unique carbohydrate epitopes of vegetative cell wall polysaccharides with
high affinity and selectivity. J Am Chem Soc 134, 15556-15562.
453
454
455
456
Monterroso, B., Saiz, J. L., Garcia, P., Garcia, J. L. & Menendez, M. (2008). Insights into the structurefunction relationships of pneumococcal cell wall lysozymes, LytC and Cpl-1. J Biol Chem 283, 2861828628.
457
458
459
460
Nascimento, J. G., Guerreiro-Pereira, M. C., Costa, S. F., Sao-Jose, C. & Santos, M. A. (2008). Nisintriggered activity of Lys44, the secreted endolysin from Oenococcus oeni phage fOg44. J Bacteriol 190,
457-461.
461
462
463
464
Nelson, D., Loomis, L. & Fischetti, V. A. (2001). Prevention and elimination of upper respiratory
colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc Natl Acad Sci
USA 98, 4107-4112.
465
466
467
Nelson, D., Schuch, R., Chahales, P., Zhu, S. & Fischetti, V. A. (2006). PlyC: a multimeric bacteriophage
lysin. Proc Natl Acad Sci USA 103, 10765-10770.
468
469
470
Nguyen, H. M. & Kang, C. (2014). Lysis delay and burst shrinkage of coliphage T7 by deletion of
terminator Tphi reversed by deletion of early genes. J Virol 88, 2107-2115.
471
472
473
O'Flaherty, S., Ross, R. P. & Coffey, A. (2009). Bacteriophage and their lysins for elimination of
infectious bacteria. FEMS Microbiol Rev 33, 801-819.
474
475
Pang, T., Park, T. & Young, R. (2010). Mutational analysis of the S21 pinholin. Mol Microbiol 76, 68-77.
476
477
478
Pang, T., Fleming, T. C., Pogliano, K. & Young, R. (2013). Visualization of pinholin lesions in vivo. Proc
Natl Acad Sci USA 110, E2054-2063.
21
479
480
481
Park, T., Struck, D. K., Deaton, J. F. & Young, R. (2006). Topological dynamics of holins in programmed
bacterial lysis. Proc Natl Acad Sci USA 103, 19713-19718.
482
483
484
Park, T., Struck, D. K., Dankenbring, C. A. & Young, R. (2007). The pinholin of lambdoid phage 21:
control of lysis by membrane depolarization. J Bacteriol 189, 9135-9139.
485
486
487
Payne, K. M. & Hatfull, G. F. (2012). Mycobacteriophage endolysins: diverse and modular enzymes with
multiple catalytic activities. PLoS One 7, e34052.
488
489
490
491
Pohane, A. A., Joshi, H. & Jain, V. (2014). Molecular dissection of phage endolysin: an interdomain
interaction confers host specificity in Lysin A of Mycobacterium phage D29. J Biol Chem 289, 1208512095.
492
493
494
Pohane, A. A., Patidar, N. D. & Jain, V. (2015). Modulation of domain-domain interaction and protein
function by a charged linker: A case study of mycobacteriophage D29 endolysin. FEBS Lett 589, 695-701.
495
496
497
Porter, C. J., Schuch, R., Pelzek, A. J. & other authors (2007). The 1.6 A crystal structure of the catalytic
domain of PlyB, a bacteriophage lysin active against Bacillus anthracis. J Mol Biol 366, 540-550.
498
499
500
Reddy, B. L. & Saier, M. H., Jr. (2013). Topological and phylogenetic analyses of bacterial holin families
and superfamilies. Biochim Biophys Acta 1828, 2654-2671.
501
502
503
Resch, G., Moreillon, P. & Fischetti, V. A. (2011). A stable phage lysin (Cpl-1) dimer with increased
antipneumococcal activity and decreased plasma clearance. Int J Antimicrob Agents 38, 516-521.
504
505
506
507
Ruggiero, A., Marasco, D., Squeglia, F., Soldini, S., Pedone, E., Pedone, C. & Berisio, R. (2010). Structure
and functional regulation of RipA, a mycobacterial enzyme essential for daughter cell separation.
Structure 18, 1184-1190.
508
509
510
Sadykov, M. R. & Bayles, K. W. (2012). The control of death and lysis in staphylococcal biofilms: a
coordination of physiological signals. Curr Opin Microbiol 15, 211-215.
511
512
513
Sanz, J. M., Diaz, E. & Garcia, J. L. (1992). Studies on the structure and function of the N-terminal
domain of the pneumococcal murein hydrolases. Mol Microbiol 6, 921-931.
514
515
516
517
Sao-Jose, C., Parreira, R., Vieira, G. & Santos, M. A. (2000). The N-terminal region of the Oenococcus
oeni bacteriophage fOg44 lysin behaves as a bona fide signal peptide in Escherichia coli and as a cisinhibitory element, preventing lytic activity on oenococcal cells. J Bacteriol 182, 5823-5831.
22
518
519
520
Savva, C. G., Dewey, J. S., Moussa, S. H., To, K. H., Holzenburg, A. & Young, R. (2014). Stable micronscale holes are a general feature of canonical holins. Mol Microbiol 91, 57-65.
521
522
523
Schuch, R., Nelson, D. & Fischetti, V. A. (2002). A bacteriolytic agent that detects and kills Bacillus
anthracis. Nature 418, 884-889.
524
525
526
Shockman, G. D., Daneo-Moore, L., Kariyama, R. & Massidda, O. (1996). Bacterial walls, peptidoglycan
hydrolases, autolysins, and autolysis. Microb Drug Resist 2, 95-98.
527
528
529
Smith, T. J., Blackman, S. A. & Foster, S. J. (1996). Peptidoglycan hydrolases of Bacillus subtilis 168.
Microb Drug Resist 2, 113-118.
530
531
532
533
Steen, A., Buist, G., Leenhouts, K. J., El Khattabi, M., Grijpstra, F., Zomer, A. L., Venema, G., Kuipers, O.
P. & Kok, J. (2003). Cell wall attachment of a widely distributed peptidoglycan binding domain is
hindered by cell wall constituents. J Biol Chem 278, 23874-23881.
534
535
536
537
Sudiarta, I. P., Fukushima, T. & Sekiguchi, J. (2010). Bacillus subtilis CwlP of the SP-β prophage has two
novel peptidoglycan hydrolase domains, muramidase and cross-linkage digesting DD-endopeptidase. J
Biol Chem 285, 41232-41243.
538
539
540
Sulakvelidze, A., Alavidze, Z. & Morris, J. G., Jr. (2001). Bacteriophage therapy. Antimicrob Agents
Chemother 45, 649-659.
541
542
543
Sun, Q., Kuty, G. F., Arockiasamy, A., Xu, M., Young, R. & Sacchettini, J. C. (2009). Regulation of a
muralytic enzyme by dynamic membrane topology. Nat Struct Mol Biol 16, 1192-1194.
544
545
546
Tanaka, S. & Clemons, W. M., Jr. (2012). Minimal requirements for inhibition of MraY by lysis protein E
from bacteriophage PhiX174. Mol Microbiol 85, 975-985.
547
548
549
550
Ventura, M., Foley, S., Bruttin, A., Chennoufi, S. C., Canchaya, C. & Brussow, H. (2002). Transcription
mapping as a tool in phage genomics: the case of the temperate Streptococcus thermophilus phage
Sfi21. Virology 296, 62-76.
551
552
553
Walmagh, M., Briers, Y., dos Santos, S. B., Azeredo, J. & Lavigne, R. (2012). Characterization of modular
bacteriophage endolysins from Myoviridae phages OBP, 201phi2-1 and PVP-SE1. PLoS One 7, e36991.
554
555
556
557
Wang, S., Kong, J. & Zhang, X. (2008). Identification and characterization of the two-component cell
lysis cassette encoded by temperate bacteriophage phiPYB5 of Lactobacillus fermentum. J Appl
Microbiol 105, 1939-1944.
23
558
559
560
White, R., Chiba, S., Pang, T., Dewey, J. S., Savva, C. G., Holzenburg, A., Pogliano, K. & Young, R. (2011).
Holin triggering in real time. Proc Natl Acad Sci USA 108, 798-803.
561
562
563
Wittebole, X., De Roock, S. & Opal, S. M. (2014). A historical overview of bacteriophage therapy as an
alternative to antibiotics for the treatment of bacterial pathogens. Virulence 5, 226-235.
564
565
566
Xu, M., Struck, D. K., Deaton, J., Wang, I. N. & Young, R. (2004). A signal-arrest-release sequence
mediates export and control of the phage P1 endolysin. Proc Natl Acad Sci USA 101, 6415-6420.
567
568
569
Xu, M., Arulandu, A., Struck, D. K., Swanson, S., Sacchettini, J. C. & Young, R. (2005). Disulfide
isomerization after membrane release of its SAR domain activates P1 lysozyme. Science 307, 113-117.
570
571
572
Yang, D. C., Tan, K., Joachimiak, A. & Bernhardt, T. G. (2012). A conformational switch controls cell wallremodelling enzymes required for bacterial cell division. Mol Microbiol 85, 768-781.
573
574
575
Young, I., Wang, I. & Roof, W. D. (2000). Phages will out: strategies of host cell lysis. Trends Microbiol 8,
120-128.
576
577
Young, R. (2002). Bacteriophage holins: deadly diversity. J Mol Microbiol Biotechnol 4, 21-36.
578
579
Young, R. (2014). Phage lysis: three steps, three choices, one outcome. J Microbiol 52, 243-258.
580
581
582
Zhang, X. & Studier, F. W. (2004). Multiple roles of T7 RNA polymerase and T7 lysozyme during
bacteriophage T7 infection. J Mol Biol 340, 707-730.
583
584
585
Zheng, Y., Struck, D. K. & Young, R. (2009). Purification and functional characterization of phiX174 lysis
protein E. Biochemistry 48, 4999-5006.
586
587
588
24
589
Figure Legend
590
Figure 1. Schematic representation of the regulation of bacteriophage endolysin. The
591
various stages at which the regulation of endolysin activity is achieved are: (1) expression, (2)
592
translocation, (3) activation, and (4) specificity. The first step of regulation of endolysin is its
593
expression in the host cell. For example, in some studied cases, endolysin gene is expressed at an
594
early stage of infection, whereas the mRNAs are translated only at the late stage. The second
595
regulatory step of endolysin occurs at its translocation from the site of synthesis (cytoplasm) to
596
the site of action (cell wall). The most common mechanism followed by endolysin is the passive
597
diffusion through the holes formed by holin. Some endolysins, however, use other transport
598
systems for their export. After reaching the periplasmic space, endolysin stays in a catalytically
599
inactive form and requires activation, which represents the third regulatory step. A well-studied
600
example includes the activation of endolysin by the disruption of the proton motive force using
601
integral membrane protein such as pinholin. Finally, the activity of endolysin depends on the
602
substrate specificity. Although only four modes of regulation are depicted here, we do not rule
603
out the possibility of other mechanisms that are yet to be discovered. Furthermore, a
604
bacteriophage may follow more than one mechanism for the regulation of endolysin activity. PG:
605
Peptidoglycan layer, PS: Periplasmic space, CM: Cell membrane, H: Holin, L: Endolysin,
606
RNAP: RNA polymerase.
25