Staphylococcal Pathogenicity Island Interference with Helper Phage Reproduction Is a Paradigm of Molecular Parasitism

Staphylococcal pathogenicity island interference with helper phage reproduction is a paradigm of molecular parasitism Geeta Rama, John Chena, Krishan Kumara, Hope F. Rossa, Carles Ubedaa,1, Priyadarshan K. Damleb, Kristin D. Laneb, José R. Penadésc, Gail E. Christieb, and Richard P. Novicka,2 aSkirball Institute Program in Molecular Pathogenesis and Departments of Microbiology and Medicine, New York University Medical Center, New York, NY 10016; bDepartment of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298-0678; and cDepartment of Genomics and Proteomics, Instituto de Biomedicina de Valencia (IBV-CSIC), 46010 Valencia, Spain Edited* by Sankar Adhya, National Institutes of Health, National Cancer Institute, Bethesda, MD, and approved August 3, 2012 (received for review March 19, 2012) Staphylococcal pathogenicity islands (SaPIs) carry superantigen burst size was greatly reduced, and survival of phage-infected cells and resistance genes and are extremely widespread in Staphylo- was increased (11). It was observed at that time that SaPI1 was coccus aureus and in other Gram-positive bacteria. SaPIs represent packaged in small infectious particles, and it was soon shown that a major source of intrageneric horizontal gene transfer and a many SaPIs contain two or more capsid morphogenesis (cpm) stealth conduit for intergeneric gene transfer; they are phage sat- genes (2, 12, 13) responsible for diverting phage virion proteins ellites that exploit the life cycle of their temperate helper phages to small capsid formation. It was hypothesized that the diversion with elegant precision to enable their rapid replication and pro- of phage virion proteins to the formation of these particles was responsible for interference (8). miscuous spread. SaPIs also interfere with helper phage reproduc- fi tion, blocking plaque formation, sharply reducing burst size and In this report, we con rm this hypothesis and go beyond it to demonstrate that there are other mechanisms of phage in- enhancing the survival of host cells following phage infection. Here, terference. Some SaPIs use one mechanism, some use a second, we show that SaPIs use several different strategies for phage in- some use both, and some add a third. We suggest that inter- terference, presumably the result of convergent evolution. One ference is not simply a consequence of competition between MICROBIOLOGY strategy, not described previously in the bacteriophage microcosm, SaPI and phage for “goods and services,” but is a key feature of fi involves a SaPI-encoded protein that directly and speci cally inter- the SaPI lifestyle. feres with phage DNA packaging by blocking the phage terminase small subunit. Another strategy involves interference with phage Results reproduction by diversion of the vast majority of virion proteins To begin these studies, we questioned whether the SaPI1 inter- to the formation of SaPI-specific small infectious particles. Several ference mechanism was universal, and immediately realized that SaPIs use both of these strategies, and at least one uses neither it could not be, because several of the known SaPIs, including but possesses a third. Our studies illuminate a key feature of the SaPIbov2 (1) and SaPIbov5 (see below), do not contain homo- evolutionary strategy of these mobile genetic elements, in addi- logs of the cpm genes or produce small capsids. SaPIbov2 is 27 kb tion to their carriage of important genes—interference with helper in length, owing to the presence of a 12-kb transposon, and could phage reproduction, which could ensure their transferability and not be accommodated by small capsids, whose size is presumably long-term persistence. determined by capsomere geometry. Nevertheless, SaPIbov2 strongly interferes with helper phage reproduction, blocking pla- temperate phage | small terminase | capsid morphogenesis que formation and causing a 500-fold reduction in helper phage plaque-forming titer (Table 1 and Fig. 1A; note that interference is assessed as a reduction in phage titer or as a reduction in taphylococcal pathogenicity islands (SaPIs) are prototypes plaque size or number, and that these two parameters do not of a large family of phage-inducible chromosomal islands S always match precisely). An earlier study hinted that gene 12 of (PICIs; formerly referred to as PRCIs) in Gram-positive bacteria the closely related 15-kb SaPIbov1 might be involved in phage (1), and their genomes are organized to take optimal advantage interference (9). We originally designated this gene pif (phage of the temperate phages that they parasitize to carry out their life fi fi interference function) (9), but because pif had been used pre- cycle (1). Speci cally, SaPIs reside at speci c sites in the chro- viously (14), we now use ppi for phage packaging interference mosome of their host and remain quiescent under the control of (see below) and add a subscript to indicate its origin, e.g., ppispb2 a master repressor (2). Upon superinfection by certain phages or denotes the SaPIbov2 version. We cloned ppi gene and tested fi spb2 upon SOS induction of a resident prophage, SaPIs use speci c its role in phage interference. As shown in Fig. 1A, the cloned nonessential phage proteins to lift the repression, enabling the gene completely blocked 80α plaque formation, and its deletion expression of their integrase and excision genes (3–5); following fully restored plaque formation and increased the 80α plaque excision, they initiate replication at a SaPI-specific replication titer nearly to that seen with the host strain lacking any SaPI origin using SaPI-coded initiator and primase, and continue replication using the host cell replication machinery (6, 7). SaPIs reorganize the phage virion proteins to form small capsids Author contributions: G.R., C.U., J.R.P., G.E.C., and R.P.N. designed research; G.R. and K.K. commensurate with their genomes (usually about one-third the performed research; J.C., P.K.D., and K.D.L. contributed new reagents/analytic tools; G.R., size of the phage genome), and they use a SaPI-coded terminase J.C., K.K., H.F.R., C.U., P.K.D., K.D.L., and R.P.N. analyzed data; and H.F.R. and R.P.N. wrote small subunit to package their DNA preferentially (8, 9). This the paper. process results in the efficient production of SaPI-specific in- The authors declare no conflict of interest. fectious particles by means of which they are spread not only *This Direct Submission article had a prearranged editor. among staphylococci but also to other bacteria, such as Listeria 1Present address: Department of Genomics and Health, Center for Advanced Research in monocytogenes (10), at very high frequency. Public Health, Valencia 46020, Spain. Upon discovery of the first SaPI, SaPI1, it was immediately 2To whom correspondence should be addressed. E-mail: [email protected]. α realized that reproduction of its inducing phage, 80 , was greatly This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. diminished. Indeed, plaque formation was blocked and the phage 1073/pnas.1204615109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1204615109 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Table 1. Effect of wild-type SaPIs, SaPI mutations, and the mutations were at the same site, Q132, and in each of these, overexpressed SaPI genes on phage 80α and SaPI titers the glutamine was replaced by either an arginine or a lysine (Fig. S1B). The 10th mutant had an isoleucine substitution for thre- Phage titer onine 106 in the same gene. To confirm that the TerS mutations SaPI (pfu/mL) × SaPI titer, P † ‡ were necessary and sufficient to confer the observed phenotype, SaPI mutation Plasmid 108 TU/mL we replaced the native TerSP gene with the mutated one (Q132K) α 520 ± 10 in an 80 prophage and found that the mutant phage was Ppispb2 pCN51 440 ± 60 resistant (Fig. S2A). We next performed a bacterial two-hybrid α pCN51-ppispb2 2 ± 0.2 test, comparing the WT 80 TerSP with a Ppispb2-resistant TerSP cpmAB ± for any interaction with Ppispb2. As shown in Fig. 2A, Ppispb2 pCN51- sp1 15 2 α SaPIbov2 – 1 ± 0.1 1.1 ± 0.1 × 106 bound strongly to the WT 80 TerSP but not to the mutant Δppi 360 ± 10 0.5 ± 0.1 × 106 protein, and it did not interact with the cognate SaPI TerSS. Interaction between Ppi and TerS was confirmed by a pull- Δppi pCN51 230 ± 30 4.2 ± 0.4 × 106 spb2 P Δppi ppi ± ± × 6 down assay using S-tagged Ppispb2 (Fig. 2 B and C), suggesting pCN51- spb2 1 0.5 3.6 0.4 10 that Ppi directly and specifically interacts with TerS and SaPI1 – 40 ± 6 2.3 ± 0.4 × 108 spb2 P Δ ± ± × 8 hence blocks the packaging of phage DNA. Because the muta- cpmAB 250 30 3.4 0.6 10 tions were in the C-terminal region of the protein, which, in other Δ ± ± × 8 cpmAB pCN51 210 20 1.0 0.2 10 phages, is the region of binding to TerL (15, 16), we suggest that ΔcpmAB cpmAB ± ± × 7 pCN51- sp1 8 1.4 6.3 0.3 10 Ppi acts by interfering with the interaction of TerS with TerL, 8 spb2 P SaPIbov1 – 49 ± 7 4.2 ± 0.1 × 10 and thus blocks cleavage and packaging of phage DNA. Δppi 150 ± 20 1.1 ± 0.1 × 108 The TerSP proteins fall into seven superfamilies (18). Four ΔcpmAB ± ± × 9 36 7 1.6 0.3 10 tested phages were sensitive to Ppispb2 (Table S1); all of these Δ ± ± × 9 ppi, 210 80 1.2 0.2 10 belong to the terminase_2 superfamily, as do the SaPI TerSS ΔcpmAB (Fig. S2B). We tested four other staphylococcal phages that – ± ± × 8 SaPI2 3 0.4 2.9 0.3 10 encode TerSP proteins not belonging to the terminase_2 super- 8 Δppi 3 ± 0.1 5.8 ± 0.6 × 10 family, and all four were resistant to Ppispb2 (Table S1), and were ΔcpmAB{ 13 ± 1 2.0 ± 0.3 × 109 quite dissimilar in the region surrounding position 132 (Fig.

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