The Holin of Staphylococcus Aureus Bacteriophage GH15

Journal of General Virology (2016), 97, 1272–1281 DOI 10.1099/jgv.0.000428

Identiﬁcation and characterization of HolGH15: the holin of Staphylococcus aureus bacteriophage GH15 Jun Song,1 Feifei Xia,1 Haiyan Jiang,2 Xinwei Li,1 Liyuan Hu,1 Pengjuan Gong,1 Liancheng Lei,1 Xin Feng,1 Changjiang Sun,1 Jingmin Gu1 and Wenyu Han1,3

Correspondence 1College of Veterinary Medicine, Jilin University, Changchun 130062, PR China Jingmin Gu 2The First Afﬁliated Hospital to Changchun University of Chinese Medicine, Changchun 130021, [email protected] PR China Wenyu Han 3Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious [email protected] Disease and Zoonoses, Yangzhou 225009, PR China

Holins are phage-encoded hydrophobic membrane proteins that spontaneously and non- specifically accumulate and form lesions in the cytoplasmic membrane. The ORF72 gene (also designated HolGH15) derived from the genome of the Staphylococcus aureus phage GH15 was predicted to encode a membrane protein. An analysis indicated that the protein encoded by HolGH15 potentially consisted of two hydrophobic transmembrane helices. This protein exhibited the structural characteristics of class II holins and belonged to the phage_holin_1 superfamily. Expression of HolGH15 in Escherichia coli BL21 cells resulted in growth retardation of the host cells, which was triggered prematurely by the addition of 2,4-dinitrophenol. The expression of HolGH15 caused morphological alterations in engineered E. coli cells, including loss of the cell wall and cytoplasmic membrane integrity and release of intracellular components, which were visualized by transmission electron microscopy. HolGH15 exerted efficient antibacterial activity at 37 8C and pH 5.2. Mutation analysis indicated that the two transmembrane domains of HolGH15 were indispensable for the activity of the full-length protein. HolGH15 showed a broad antibacterial range: it not only inhibited Staphylococcus aureus, but also demonstrated antibacterial activity against other species, including Listeria monocytogenes, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli. At the minimal inhibitory concentration, HolGH15 evoked the release of cellular contents and resulted in the shrinkage and death of Staphylococcus aureus and Listeria monocytogenes Received 7 October 2015 cells. To the best of our knowledge, this study is the first report of a Staphylococcus aureus Accepted 11 February 2016 phage holin that exerts antibacterial activity against heterogeneous pathogens.

INTRODUCTION infected bacterial cells. At least two proteins, including a holin and one or more cell-wall-degrading enzymes, are Bacteriophages (phages) are natural enemies of bacteria involved in phage lysis. Holins are phage-encoded hydro- and are essentially ubiquitous, with an estimated 1031 phobic membrane proteins that cause bacterial membrane phage particles present on Earth (Bergh et al., 1989). lysis during late protein synthesis. Holins spontaneously Phage therapy was developed because phages can specific- assemble into oligomers, resulting in the formation of ally infect and lyse bacteria (Hudson et al., 2005). Recently, non-specific pores in the cytoplasmic membrane. Holin phage therapy has been resurrected, primarily because of proteins play an important role in the phage infection the continuing increase in antibiotic resistance worldwide cycle by permitting the release of endolysin and regulating (Fischetti, 2001; Merril et al., 2003). bacterial lysis at precise time points, as a ‘clock’ function The dsDNA phages of the order Caudovirales usually utilize (Chang et al., 1995; Wang et al., 2000). multiple strategies to release viral progeny from phage- Holins have been identified in a wide variety of bacterio- Four supplementary figures and two supplementary tables are available phages (Catalaõ et al., 2010; Donovan, 2007; Wang et al., with the online Supplementary Material. 2000). To date, 52 families of holins have been identified,

Downloaded from www.microbiologyresearch.org by 1272 000428 G 2016 The Authors Printed in Great Britain IP: 128.172.32.33 On: Wed, 22 Mar 2017 15:50:46 HolGH15: the holin of S. aureus phage GH15 and representative well-characterized members, as well as that HolGH15 has two putative hydrophobic transmem- distant homologues with unknown functions, have been brane domains (TMDs), and its N terminus and C terminus entered into the Transporter Classification Database are cytoplasmic (Fig. 1b). HolGH15 has a hydro-philic C (Reddy & Saier, 2013). Although the holin family is large, terminus that contains several charged amino acids. The and it evolved independently in the National Center for N terminus of HolGH15 contains five positively charged Biotechnology Information protein database, only a few amino acids and only one negatively charged residue. recognized members have been verified. Holins possess As HolGH15 shares structural characteristics with class II certain common characteristics (Shi et al., 2012b). (1) An holins, HolGH15 should be a member of the class II analysis of topological features has shown that holins pos- family (Reddy & Saier, 2013). HolGH15 consists of sess at least one transmembrane a-helical sequence (Reddy 167 aa and exhibits high sequence identity with some & Saier, 2013). (2) Most holins are encoded by a gene adja- members that belong to the phage_holin_1 superfamily cent to the endolysin gene. (3) Although holins do not (PF04531) in the Pfam sequence database (http://pfam. share sequence similarity, they possess highly charged and xfam.org/family/PF04531) (Fig. S1, available in the online hydrophilic C-terminal domains. Supplementary Material). The amino acid sequence of HolGH15 was also compared with other published holins The basic functions of most phage-encoded holins are to of Gram-positive phages, such as the Staphylococcus, Strep- permit the release of endolysins and to dissipate the tococcus suis and Lactobacillus fermentum phages (Fig. 2). proton motive force (Saier & Reddy, 2015). The typical The sequence alignment demonstrated that HolGH15 holin forms very large pores (large enough for 500 kDa showed different levels of identity with the putative proteins) that allow the non-specific release of endolysins holins derived from Staphylococcus phage K (95% identity and other proteins into the cytoplasm (Bla¨si & Young, at the amino acid level), Staphylococcus phage Twort 1996; Wang et al., 2003). However, the holin protein of (60%), Staphylococcus phage vB_SepS_SEP9 (58%), lambda phage 21 forms pinholes that only depolarize the Staphylococcus phage 11 (42%), Staphylococcus phage membrane (Pang et al., 2009, 2010a, b, 2013). Holins are phiWMY (39%), Lactobacillus fermentum phage phiPYB5 also important for many activities, such as classification, (10.8%) and Streptococcus phage SMP (10.1%). gene transfer (Lang & Beatty, 2001), biofilm formation (Ranjit et al., 2011) and biotechnology material production (Gao et al., 2013). In contrast to the holins derived from Gram-negative bacterial phages (particularly the lambda, Expression and purification of HolGH15 T4 and 21 phages) (Golec et al., 2010; Krupovic & Bamford, The whole putative HolGH15 holin gene of phage GH15 2008; Rydman & Bamford, 2003; Ziedaite et al., 2005), was amplified using the primers holF and holR. HolGH15 holins encoded by Gram-positive bacterial phages have was cloned into the pET-28a(+) prokaryotic expression not been subjected to systematic and in-depth research. vector. The constructed vector pET-28a-HolGH15 was We have previously described the staphylococcal phage verified using double restriction enzyme digestion GH15 (Gu et al., 2011). The complete genome (GenBank (Eco RI/Sal I). To verify the expression of the predicted accession number JQ686190.1) was sequenced and ana- GH15 holin gene from the plasmid pET-28a-HolGH15, lysed (Gu et al., 2012), revealing that a putative lysis total cellular protein samples were prepared at 10 min b module exists in the GH15 phage genome (Gu et al., intervals after induction with isopropyl -D-1-thiogalacto- 2013). The lysin of GH15 (LysGH15) has been identified pyranoside (IPTG). The presence of HolGH15 in the col- and the molecular mechanism of its lytic activity has lected samples was determined by Western blotting. The been elucidated in previous studies (Gu et al., 2011, OD600 value decreased from 0.578 to 0.496 (Fig. 3a), 2014). However, the putative holin of GH15, as well as suggesting that the expression of HolGH15 might evoke other homologous staphylococcal phage holins, has not growth inhibition of host Escherichia coli BL21-CodonPlus been characterized. Hence, in this report, the holin of cells. Additionally, transmission electron microscopy GH15 (HolGH15) was identified, and the function and (TEM) observations indicated cytological alterations in characteristics of HolGH15 were studied. the induced E. coli cells. The cell membranes and cell walls were disrupted and damaged, resulting in the release of cellular contents and an appearance similar to a bacterial RESULTS ghost (Fig. 3b). Western blotting showed that HolGH15 was localized at the cell membrane (Fig. 3c). 2,4-Dinitro- Gene and protein sequence analysis of HolGH15 phenol (DNP; 1 mM) was added to further investigate HolGH15 function. HolGH15 expression was triggered The 504 bp ORF72 of phage GH15 was located next to the 10 min prematurely by the addition of DNP (Fig. 3a). LysGH15 gene. The analysis indicated that ORF72 probably encodes a holin protein (designated HolGH15) (Fig. 1a). Western blotting analysis demonstrated the presence of a There was only a 1 bp overlap between HolGH15 and band of *21 kDa (Fig. 3d). The size was consistent with LysGH15. The results of the PredictProtein, TMHMM and the predicted mass of HolGH15 and this band could be SOSUI analyses suggested that HolGH15 is a membrane detected after a 10 min induction with IPTG (Fig. 3d). protein with typical holin traits. The prediction indicated Based on the kinetics of HolGH15 expression, samples

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(a) 37341 bp 35351 bp

GH15 DNA 139806 bp

35351 bp 36838 bp 37341 bp

Lysin (ORF71) Holin (ORF72)

Lysis module

(b) Periplasm (outside) +++++++++++++++

1–23 inside 24–46 TMD 47–54 outside Cytoplasmic membrane 55–73 TMD 74–167 inside

––––––––––––––– Cytoplasm (inside) + – + – – NH - – ––+ 2 –––––+– -COOH – – – –

Fig. 1. Analysis of ORF72 from the GH15 phage genome. (a) Location of LysGH15 (ORF71) and HolGH15 (ORF72). These two genes are likely to compose the putative lysis module of phage GH15. (b) Topological model of HolGH15. Black

box, TMD1; grey box, TMD2. were collected at 60 min. HolGH15 was purified and con- most susceptible to HolGH15 at 37 uC. The greatest antimi- centrated, and SDS-PAGE analysis was performed to exam- crobial activity of HolGH15 was detected at pH 5.2. ine the purified target proteins. A band of *21 kDa was detected; the size was consistent with the Western blotting The antimicrobial spectrum of HolGH15 was tested. results and the predicted mass of HolGH15 (Fig. 3e). The As shown in Table 1, 31 of the 36 tested Staphylococcus 2 total yield of HolGH15 was *0.5 mg (l culture) 1. aureus isolates showed sensitivity to HolGH15. The addition of HolGH15 resulted in the inhibition of other Gram-positive Construction of HolGH15 mutants and functional bacteria, including Listeria monocytogenes and B. subtilis. Sur- analysis prisingly, three of the tested Gram-negative bacteria (E. coli, Pseudomonas aeruginosa and Klebsiella pneumoniae)also To identify the function of the holin TMDs, HolGH15- exhibited sensitivity to HolGH15. HolGH15 showed the TMD1(–) and HolGH15-TMD2(–) were constructed by greatest antimicrobial activity to Staphylococcus aureus and fusion PCR. As shown in Fig. S2, the expression levels of Listeria monocytogenes (Fig. S4). HolGH15-TMD1(–) and HolGH15-TMD2(–) were significantly higher than that of HolGH15 (Fig. S2). Neither The minimal inhibitory concentration (MIC) values for mutant resulted in growth retardation or damage to HolGH15 against different sensitive strains are listed in E. coli BL21-CodonPlus cells compared with full-length Table S2. The results indicated that different strains exhib- HolGH15 (Fig. 4a). In contrast to the negative control, ited differential sensitivity to HolGH15. Staphylococcus the TMD1 and TMD2 deletion mutants had almost com- aureus ATCC 25923 and Listeria monocytogenes ATCC 2 pletely lost their antibacterial activity (Fig. 4b). 19112 had the same MIC (120 mgml 1). K. pneumoniae NDM-1 and B. subtilis ATCC 14579 were inhibited by 2 Characteristics of HolGH15 180 mg HolGH15 ml 1. Moreover, the haemolytic activity 2 The optimum temperature and pH conditions for HolGH15 detection assay demonstrated that HolGH15 (15–480 mgml 1) were determined, as shown in Fig. S3. Bacterial cells were had no effect on defibrinated rabbit blood (Fig. 5).

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S_aureus_phage_GH15_HolGH15 S_aureus_phage_K_Holin S_aureus_phage_Twort_HolTW S_aureus_phage_vB_SepS_SEP9_hol S_aureus_phage_phiWMY_holWMY S_aureus_phage_phill_holin Lb_fermentum_phage_phiPYB5_Hyb S_suis_phage_SMP_HolSMP

Fig. 2. Sequence alignment of HolGH15 and homologous proteins. Sequence alignment of HolGH15 with several reported holins from Staphylococcus aureus, Streptococcus suis and Lactobacillus fermentum. Strictly conserved residues are boxed in white on a red background and highly conserved residues are boxed in red on a white background. All the alignments were generated using CLUSTAL W (http://www.ch.embnet.org/software/ClustalW.html). The ﬁgure was generated using ESPript (http://espript.ibcp.fr/ESPript/ESPript/index.php).

Morphology of Staphylococcus aureus and resulting in obvious cell shrinkage and rupture. The con- Listeria monocytogenes treated with HolGH15 tents of Listeria monocytogenes cells almost vanished. TEM and scanning electron microscopy (SEM) images of bacterial cells exposed to HolGH15 showed ultrastructural 21 DISCUSSION and morphological changes (Fig. 6). At the MIC (120 mgml ), HolGH15 provoked the rough texture and shrinkage of Holins are membrane proteins that are encoded by phages. Staphylococcus aureus, according to our SEM observations. Holins accumulate non-speciﬁcally and cause damage to Additionally, cell membranes and cell walls were disrupted the cytoplasmic membrane. The function of the holin pro- and damaged, resulting in the release or condensation of tein is to regulate lysine access to targets and to degrade the cellular contents, which was visible by TEM (Fig. 6). Lis- peptidoglycans of the host cell wall when it is time to teria monocytogenes was more sensitive to HolGH15. Com- release progeny phages (Wang et al., 2000). HolGH15, pared with untreated Listeria monocytogenes cells, bacterial the putative holin of the staphylococcal phage GH15, is cells exposed to HolGH15 exhibited remarkable damage, found in the lysis module (Gu et al., 2013), consists of

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(a) (b) pET-25a(+) IPTG(+)DNP(–) pET-28a(+) IPTG(+)DNP(+) 1.3 pET-HolGH15 IPTG(–)DNP(–) pET-HolGH15 IPTG(+)DNP(–) 1.2 pET-HolGH15 IPTG(+)DNP(+) 1.1 )

600 1.0 0.9 0.8 0.7 Growth (OD 0.6 0.5 0.4 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time after induction (min)

pET-28a pET-HolGH15 30

(d) 23 M– 0 10 20 30 40 50 60 70

17 Time after induction (min)

Fig. 3. Expression and purification of HolGH15. (a) Influence of 2,4-dinitrophenol (DNP) on the growth of E. coli BL21-CodonPlus cells harbouring the pET-28a-HolGH15 plasmid. The bacterial cultures were induced with IPTG (final concentration 1 mM). DNP was added at a final concentration of 1 mM. Non-induced E. coli BL21-CodonPlus cells containing pET-28a-HolGH15 and pET-28a(+) were monitored as controls. (b) Morphological observations of Escherichia coli expressing HolGH15. Data represent mean¡SD, n53. Bar, 1 mm. (c) Localization of HolGH15 in E. coli. (d) Expression of HolGH15 at 10 min intervals. Western blotting was performed with total cellular proteins from IPTG-induced E. coli BL21-CodonPlus cells harbouring pET-28a-HolGH15 to detect HolGH15 expression. E. coli BL21-CodonPlus cells harbouring pET-28a(+) cultures were collected 60 min after induction as a negative control (–). (e) SDS-PAGE analysis of purified HolGH15. Lane M, molecular mass marker; lane 1, supernatant from induced E. coli BL21-CodonPlus cells harbouring the pET-28a-HolGH15 construct after cellular disruption; lane 2, purified HolGH15 fraction eluted from the Ni-NTA His?Bind slurry.

167 aa and belongs to the phage_holin_1 superfamily. identity with the HolGH15 amino acid sequence, similar A bioinformatics analysis suggested that HolGH15 consists to the phage K holin protein (data not shown). However, of two putative hydrophobic TMDs, and both the N there are no reports on the structure, function and activity terminus and C terminus of HolGH15 exist in the cyto- of the holins from these Staphylococcus aureus phages. plasm, which is a typical trait of holins. Furthermore, Therefore, HolGH15 might represent a new type of phage HolGH15 shares structural characteristics with class II holin protein. holins. Sequence alignment demonstrated that HolGH15 Prokaryotic expression systems (particularly E. coli exhibits sequence identity with the phage_holin_1 superfamily. Amino acid residues 13–95, which contain the TMDs, expression systems) are effective and convenient tools for are relatively conserved domains. HolGH15 showed high the expression of holin proteins derived from phages that sequence identity with only the putative holin protein infect Gram-positive bacteria (Shi et al., 2012a; Steiner & of Staphylococcus aureus phage K (95% identity at the Bla¨si, 1993; Taka´c et al., 2005). The expression analysis of amino acid level). The putative holins of Staphylococcus the putative holin protein HolGH15 was performed using aureus phages Sb-1, A5W, ISP and G1 all displayed high E. coli BL21-CodonPlus cells, and the HolGH15 protein

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(a) Table 1. Bacterial strains used to determine the antibacterial 1.3 pET-28a(+) spectrum of HolGH15 1.2 pET-HolGH15 HolGH15-TMD1(–) ) 1.1 Organism Strain Source* Bacteriostasis HolGH15-TMD2(–)

600 1.0 + 0.9 Meticillin-susceptible ATCC 25923 1 Staphylococcus aureus + 0.8 ATCC 19685 1 + 0.7 ATCC 29213 1 2

Growth (OD CVCC 2661 2 0.6 R6166 3 + 0.5 W1 4 + 0.4 W4661 3 + 0 10 20 30 40 50 60 70 80 90 + 100110 120 130 W903 3 Time after induction (min) N14 3 + N25 3 + (b) + 5 N26 3 N3 3 + N38 3 + 4 N4 3 + +

–1 N5 3 3 N6 3 + Meticillin-resistant 208 3 + 2 Staphylococcus aureus R196 3 +

Log c.f.u. ml R23 3 + + 1 R238 3 R3659 3 + R3784 3 + 0 R3790 3 + + (–) (–) R5886 3 1 2 + Control R6012 3 HolGH15 R6040 3 + R6046 3 2 HolGH15-TMDHolGH15-TMD R6186 3 2 R6199 3 + R6491 3 2 Fig. 4. Influence of TMDs on HolGH15 activity. (a) Growth R6994 3 + assays with E. coli BL21-CodonPlus cells harbouring different R75 3 2 vector constructs. The growth of different cultures was observed R89 3 + after the addition of IPTG. Data represent mean¡SD, n53. N10 3 + + (b) Antibacterial activity of HolGH15-TMD1(–) and HolGH15- W11 3 + TMD2(–). Exponential-phase cultures of Staphylococcus aureus W25 3 2 ATCC 25923 (105 c.f.u. ml 1) were exposed to HolGH15, W3275 3 + + HolGH15-TMD1(–) and HolGH15-TMD2(–) (final concentration YB57 3 2 + 300 mgml 1), respectively. The results are based on the number Listeria monocytogenes ATCC 19112 1 + of viable bacteria after buffer treatment divided by the number of ATCC 19115 1 + viable bacteria after exposure. Means of triplicate samples for ATCC 15313 1 Streptococcus suis CVCC 606 2 2 each assay were used for the calculations. Error bars, SD. CVCC 607 2 2 Enterococcus faecalis ATCC 14506 1 2 was successfully expressed and purified. Some studies have Bacillus subtilis ATCC 14579 1 + Escherichia coli ATCC 25922 1 2 reported that holin proteins exhibit toxicity towards E. coli a + cells (Garrett et al., 1990; Shi et al., 2012a); in our study, the DH5 4 BL21(DE3)pLysS 4 + expression of HolGH15 caused growth inhibition of host Pseudomonas aeruginosa ATCC 27853 1 + E. coli cells. TEM showed the disruption of and damage Salmonella typhimurium CVCC 541 2 2 to the cell membranes and cell walls, resulting in the release Salmonella enterica ATCC 12011 1 2 of cell contents. Additionally, HolGH15 was localized on Klebsiella pneumoniae NDM-1 1 + the cell membrane, suggesting that it is indeed a membrane protein. HolGH15 was sensitive to energy poisoning with *1, Purchased from American Type Culture Collection (ATCC); DNP and could be triggered prematurely, which are 2, purchased from China Veterinary Culture Collection (CVCC); characteristics of other holins (Gru¨ndling et al., 2001; 3, isolated from the First Hospital of Jilin University, Changchun, Takać et al., 2005). These data suggest that HolGH15 can Jilin Province; 4, laboratory strain.

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the hypothesis that this protein functions as a holin. 450

A 80 TMDs are major components of holins and play an 70 important role in exerting the active functions of this 60 type of protein. Previous studies have shown that the 50 holin from lambda bacteriophage 21 has two TMDs, and 40 that TMD1 is dispensable for pore formation and lysis 30 (Pang et al., 2009). However, our results showed that 20 both TMD1 and TMD2 of HolGH15 are indispensable for Rabbit red cell haemolysis 10 the function of the full-length protein. The expression (percentage of maximum 0 levels of HolGH15-TMD1(–) and HolGH15-TMD2(–) NT -HL MIC were significantly higher, but they lacked the ability to a 4MIC 2MIC 1/2MIC 1/4MIC 1/8MIC inhibit E. coli and other bacteria. Therefore, the two HolGH15 concentration TMDs are essential parts of HolGH15 and the lack of either TMD leads to a loss of activity. Fig. 5. Haemolytic activity of HolGH15 at graded concentrations. We found that exogenous HolGH15 displayed antibacterial Haemolysis assays were performed using defibrinated rabbit activity against Staphylococcus aureus. Interestingly, blood cells in PBS treated with different concentrations of HolGH15 also displayed antibacterial activity against other purified HolGH15. The cell supernatants were analysed by quan- species, including both Gram-negative and Gram-positive tifying the A450 and comparing the values with those for the species, such as Listeria monocytogenes, B. subtilis, E. coli, a-haemolysin (a-HL)-induced haemolysis group. Data represent K. pneumoniae and P. aeruginosa. Different strains exhibited mean¡SD, n53. NT, No treatment. differential sensitivities to HolGH15. Therefore, the antibacterial activity of HolGH15 may be caused by the spontaneous accumulation and formation of pores in a non-specific membranes causes the release of cellular contents, and manner, similar to HolSMP (Shi et al., 2012a). Nevertheless, results in cell shrinkage and death (Santhana Raj et al., HolGH15 did not induce erythrocyte rupture. 2007). These results suggested that HolGH15 possesses great potential as an antibacterial agent against Staphylococ- The antibacterial activity experiment and electron cus aureus and Listeria monocytogenes. microscopy observations demonstrated that Staphylococcus aureus and Listeria monocytogenes were sensitive to In this study, HolGH15, the holin derived from Staphylo- HolGH15. HolGH15 caused obvious damage to these two coccus aureus bacteriophage GH15, was identified and bacteria. HolGH15 was targeted to the membrane and characterized. HolGH15 showed a number of features: destroyed the membrane integrity. The difference in (1) HolGH15 belongs to the phage_holin_1 superfamily, osmotic pressure between the outer and inner bacterial class II holins, (2) HolGH15 consists of two hydrophobic

Staphylococcus aureus Listeria monocytogenes SEM TEM SEM TEM

HolGH15 treated

Fig. 6. Effects of HolGH15 on Staphylococcus aureus and Listeria monocytogenes as observed by TEM and SEM. 2 Staphylococcus aureus and Listeria monocytogenes cells were exposed to 120 mg HolGH15 ml 1 (HolGH15 treated). Cells treated with an equivalent amount of PBS served as controls (NT, no treatment). Bar, 1 mm.

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TMDs that are indispensable for the function of the full- phosphate, pH 8.0) and sonicated (5 s on/20 s off cycles) in an ice length protein and (3) HolGH15 possesses a broad antibac- bath at 400 W for 30 min. The cell debris was ultracentrifuged at terial range. 100 000 g for 1 h at 4 uC to separate the membrane fraction. The membrane proteins were isolated using 5 ml ME buffer (1% Triton X-100, 10% glycerine, 0.5 M NaCl, 35 mM MgCl2 and 220 mM Tris/ HCl, pH 8.0) as described previously (Shi et al., 2012b). The whole- METHODS cell, cytoplasmic fraction and membrane fraction extracts were mixed 2 with SDS buffer [50 mmol Tris/HCl l 1, 2% (w/v) SDS, 0.1% (w/v) Bacterial strains, phages, plasmids and growth conditions. The Bromophenol blue, 10% (v/v) glycerol and 1% (w/v) thioglycol] and bacterial strains used in this study are listed in Table 1. Staphylococcus boiled. The samples were analysed by SDS-PAGE. aureus, B. subtilis, E. coli, P. aeruginosa, Salmonella typhimurium, Salmonella enterica and K. pneumoniae were cultured in standard The antibacterial activity of and optimal conditions for Luria–Bertani medium (LB) at 37 uC. Listeria monocytogenes, Strep- HolGH15. To determine the antibacterial activity of HolGH15, tococcus suis and Enterococcus faecalis were cultured in BHI broth Staphylococcus aureus ATCC 25923 was used as an indicator strain. The (BD) at 37 uC. E. coli BL21-CodonPlus cells containing plasmids were 2 antibacterial activity was expressed as the c.f.u. reduction following cultured in LB supplemented with kanamycin (50 mgml1). All exposure. The strain culture was grown to mid-exponential phase (OD bacterial strains were stored in 30% glycerol at 280 uC. The GH15 600 0.5), collected (10 000 g for 15 min at 4 uC) and resuspended in PBS bacteriophage was prepared as described previously (Gu et al., 2011). (pH 7.4), and the initial colony count was determined. HolGH15 was The pET-28a(+) plasmid was purchased from Novagen. 21 added to the bacterial suspension (final concentration 300 mgml )and the mixture was incubated for 8 h at 37 uC. The colony count was then Bioinformatics analysis. DNA and protein sequences of the puta- determined again. As a negative control, the bacterial strains were treated tive holin protein (HolGH15) in the phage GH15 were analysed using with PBS under the same conditions. BLAST tools. The TMHMM (http://www.cbs.dtu.dk/services/TMHMM/), SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/) and PredictProtein The optimal conditions for HolGH15 were tested at various pH values servers (http://www.predictprotein.org) were used to predict the in 50 mM sodium acetate (pH 4.0 and 5.2) or 20 mM PBS (pH 6.8, transmembrane helices in HolGH15. Sequence alignment and analysis 7.2 and 8.5) at different temperatures (4, 16, 25, 37 and 42 uC). of HolGH15 were performed with BLAST (http://blast.ncbi.nlm.nih. Experiments testing each condition (media, pH and temperature) gov/Blast.cgi). All alignments were generated using CLUSTAL W were performed in triplicate. (http://www.ch.embnet.org/software/ClustalW.html). To determine the antibacterial spectrum, the sensitivity of Staphylo- Cloning, expression and purification of HolGH15. The HolGH15 coccus aureus (36 strains), Listeria monocytogenes, Streptococcus suis, gene was amplified by PCR from purified GH15 phage genomic DNA Enterococcus faecalis, B. subtilis, E. coli, P. aeruginosa, Salmonella and using the holF and holR primers (Table S1). The primers were K. pneumoniae to HolGH15 was tested. designed based on the putative full-length HolGH15 gene sequence. Construction of HolGH15 mutants. To verify the functions of the The amplified product was sequenced and a BLAST search was performed. The PCR product containing 59 Eco RI and 39 Sal I restriction TMDs, HolGH15-TMD1(–) and HolGH15-TMD2(–) were constructed. sites was digested with Eco RI/Sal I and ligated into the pET-28a(+) The strategy for the deletion of TMD1 (60 bp) and TMD2 (51 bp) vector. The full expression vector pET-HolGH15 was transformed involved fusion PCR (Ninomiya et al., 2004). The primers p-1, p-2, p-3 and p-4 were used to construct the TMD deletion mutants (Table S1). into competent E. coli BL21-CodonPlus cells (Stratagene). Exponen- 9 9 tially growing cultures were induced with IPTG at 25 uC and The 5 flanking DNA (holF and p-1) and 3 flanking DNA (p-2 and 180 r.p.m. Cell growth was monitored by measuring the change in holR) were mixed and used as a template for fusion PCR using the OD at 10 min intervals after induction. primers holF and holR under the following conditions: initial dena- 600 turation at 94 uC for 2 min and 10 cycles of 94 uC for 15 s, annealing at Bacterial cells were washed with 20 mM PBS, disrupted with an 50 uC for 30 s and extension at 72 uC for 30 s. Then, the following PCR ultrasonic disintegrator and centrifuged at 4 uC (10 000 g; 15 min). conditions were used: 94 uC for 30 s; 25 cycles of 58 uC for 30 s and The supernatants were analysed by 15% SDS-PAGE. Recombinant 72 uC for 30 s, and a final extension at 72 uC for 7 min. HolGH15- HolGH15 in solution was purified using HisPur Ni-NTA Resin TMD2(–) was constructed using the primers p-3, p-4, holF and holR as (Pierce Biotechnology). All of the buffers used for purification described earlier. The fusion PCR products were analysed on a 1.5% contained 20 mM sodium phosphate, 300 mM NaCl and different agarose gel. The expression and activity of HolGH15-TMD1(–) and concentrations of imidazole with 0.1% Triton X-100. The purification HolGH15-TMD2(–) were determined as described earlier. processes were performed according to the manufacturer’s instructions. MIC determination of HolGH15. The MIC values for HolGH15 against sensitive strains were assessed in triplicate using a standard Western blot analysis of HolGH15. HolGH15 was analysed by microdilution method as recommended by the Clinical and Labora- Western blotting. After electrophoresis, the proteins were directly tory Standards Institute (CLSI, 2012). Fresh cultures of the tested transferred from the gel onto a PVDF membrane (Millipore) by strains were inoculated in 96-well plates (Corning) at a final inoculum 2 electroblotting. For the immunodetection of HolGH15, anti-His- of 105 c.f.u. ml 1. Serial twofold dilutions of HolGH15 were made. tagged mAb (1:1000 dilution) and HRP-labelled goat anti-mouse IgG As a negative control, the bacterial strains were treated with PBS alone (1:5000 dilution; Immunology Consultants Laboratory) were used as under the same conditions. After a 24 h incubation at 37 uC, the the primary and secondary antibodies, respectively. The bands were presence or absence of growth was observed in each well. The MIC detected with Pierce ECL Plus Western blotting Substrate (Thermo was defined as the lowest concentration of HolGH15 that yielded no Fisher Scientific). growth or a marked change in growth compared with the growth control plate. HolGH15 localization in E. coli. A 1 l culture of E. coli BL21- CodonPlus cells expressing pET-HolGH15 was induced for 40 min Morphological changes in cells caused by HolGH15. Morpho- with IPTG and then harvested by centrifugation at 13 000 g for 5 min logical changes in E. coli BL21-CodonPlus cells expressing HolGH15 at 4 uC. To prepare the cytoplasmic fraction, harvested cells were were determined by TEM. Morphological and ultrastructural changes suspended in 5 ml lysis buffer (300 mM NaCl and 50 mM sodium in Staphylococcus aureus and Listeria monocytogenes cells were visu-

Downloaded from www.microbiologyresearch.org by http://jgv.microbiologyresearch.org 1279 IP: 128.172.32.33 On: Wed, 22 Mar 2017 15:50:46 J. Song and others alized after exposure to HolGH15 by TEM (H-7650; Hitachi) and Gu, J., Xu, W., Lei, L., Huang, J., Feng, X., Sun, C., Du, C., Zuo, J., Li, Y. SEM (XL30ESEM-FEG; FEI). The samples were prepared and fixed as & other authors (2011). LysGH15, a novel bacteriophage lysin, described previously (Diao et al., 2012). protects a murine bacteremia model efficiently against lethal methicillin-resistant Staphylococcus aureus infection. J Clin Microbiol Haemolysis assays. Haemolytic activity was assessed as described 49, 111–117. previously with some modifications (Diao et al., 2012). Briefly, 25 ml Gu, J., Liu, X., Lu, R., Li, Y., Song, J., Lei, L., Sun, C., Feng, X., Du, C. & defibrinated rabbit blood (Biotech) and 375 ml PBS were added to other authors (2012). Complete genome sequence of Staphylococcus 96-well plates (Corning). Then, 100 ml serial dilutions of HolGH15 aureus bacteriophage GH15. J Virol 86, 8914–8915. were added to the wells containing blood. Additionally, a-haemolysin Gu, J., Liu, X., Yang, M., Li, Y., Sun, C., Lu, R., Song, J., Zhang, Q., was used as a positive control and PBS served as a negative control. Lei, L. & other authors (2013). Genomic characterization of lytic The plates were incubated at 37 uC for 30 min. Following centrifu- Staphylococcus aureus phage GH15: providing new clues to intron gation, the A values of the supernatants were determined using 450 shift in phages. J Gen Virol 94, 906–915. an ELX800 reader (BioTek Instruments). Each measurement was performed in triplicate. Gu, J., Feng, Y., Feng, X., Sun, C., Lei, L., Ding, W., Niu, F., Jiao, L., Yang, M. & other authors (2014). Structural and biochemical Statistical analysis. Prism 5 software (GraphPad) was used for all characterization reveals LysGH15 as an unprecedented ‘‘EF-hand- the statistical analyses. like’’ calcium-binding phage lysin. PLoS Pathog 10, e1004109. Hudson, J. A., Billington, C., Carey-Smith, G. & Greening, G. (2005). Bacteriophages as biocontrol agents in food. J Food Prot 68, ACKNOWLEDGEMENTS 426–437. This study was supported by the National Natural Science Foundation Krupovic, M. & Bamford, D. H. (2008). Holin of bacteriophage of China (31130072, 31502103 and 31572553). lambda: structural insights into a membrane lesion. Mol Microbiol 69, 781–783. Lang, A. S. & Beatty, J. T. (2001). The gene transfer agent of REFERENCES Rhodobacter capsulatus and ‘‘constitutive transduction’’ in prokaryotes. Arch Microbiol 175, 241–249. Bergh, O., Børsheim, K. Y., Bratbak, G. & Heldal, M. (1989). High Merril, C. R., Scholl, D. & Adhya, S. L. (2003). The prospect for abundance of viruses found in aquatic environments. 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