Staphylococcal pathogenicity island interference with helper phage reproduction is a paradigm of molecular parasitism

Abstract

Staphylococcal pathogenicity islands (SaPIs) carry superantigen and resistance genes and are extremely widespread in Staphylococcus aureus and in other Gram-positive bacteria. SaPIs represent a major source of intrageneric horizontal gene transfer and a stealth conduit for intergeneric gene transfer; they are phage satellites that exploit the life cycle of their temperate helper phages with elegant precision to enable their rapid replication and promiscuous spread. SaPIs also interfere with helper phage reproduction, blocking plaque formation, sharply reducing burst size and enhancing the survival of host cells following phage infection. Here, we show that SaPIs use several different strategies for phage interference, presumably the result of convergent evolution. One strategy, not described previously in the bacteriophage microcosm, involves a SaPI-encoded protein that directly and specifically interferes with phage DNA packaging by blocking the phage terminase small subunit. Another strategy involves interference with phage reproduction by diversion of the vast majority of virion proteins to the formation of SaPI-specific small infectious particles. Several SaPIs use both of these strategies, and at least one uses neither but possesses a third. Our studies illuminate a key feature of the evolutionary strategy of these mobile genetic elements, in addition to their carriage of important genes-interference with helper phage reproduction, which could ensure their transferability and long-term persistence.

Fig. 1.

Ppi interference with phage 80α. (A) Approximately 10⁸ bacteria were infected with phage 80α, plated on phage bottom agar, and incubated 36–48 h at 32 °C. Plates were stained with 0.1% TTC in TSB and photographed. Genes cloned into pCN51 were induced with 1.0 μM CdCl₂. (B and C) Electron microscopic analysis of sedimented particles from (B) phage 80α lysate of RN4220 + pCN51 and from a (C) 80α lysate of RN4220 + pCN51 – ppi_spb2. CP, complete phage 80α particles; FT, free phage tails; PH, phage proheads.

Fig. 2.

Interactions between Ppi_spb2 and TerS_P (80α). (A) BACTH analysis. Spots in each row represent three independent colonies. Plasmid combinations are numbered as follows: 1, pKT25-zip + pUT18C-zip (positive control); 2, pKT25-TerS_P (WT80α) + pUT18-Ppi_spb2; 3, pKT25-TerS_P (Ppi_spb2-resistant 80α) + pUT18-Ppi_spb2; 4, pKT25-TerS_S (SaPIbov2) + pUT18-Ppi_spb2; and 5, pKT25 + pUT18 (negative control). Blue color indicates cAMP-dependent lacZ expression following reconstitution of adenlyate cyclase activity by interaction of fusion proteins. (B and C) Pull-down of His-tag–TerS_P (80α) coexpressed with Ppi_spb2–S-tag using S-protein agarose column: lane 1, protein eluted from S-protein agarose beads after binding and washing of cell lysate obtained from cells coexpressing both the proteins; lane 2, prestained markers; lane 3, eluate from S-protein agarose beads after binding and washing of cell lysate obtained from cells expressing His-TerS_P (80α); lane 4, cell lysate obtained from cells expressing His-TerS_P (80α). (B) Coomassie-stained gel. (C) Western blot of the gel shown in B using Penta-His HRP conjugate and S-protein HRP conjugate antibodies.

Fig. 3.

Effect of SaPI interference genes on phage 80α. Phage platings were as in Fig. 1A. (A) Effect of SaPI1 CpmAB. (B) Effects of SaPI2 and SaPIbov1 CpmAB and Ppi. Note that ORF13 was always included in deletions of SaPI2 cpmAB because a deletion of the two genes alone could not be constructed. ORF13 does not affect interference. (C) Agarose gel analysis of DNA extracted from sedimented phage particles. Phage 80α was grown on RN4220 and various derivatives of SaPI2 (i; Upper) or SaPI1 (ii; Lower). The resulting lysates were treated with DNase, PEG-precipitated, and phenol extracted. Equivalent amounts of DNA were separated on 0.8% agarose gel for 13 h at 60 V, strained with ethidium bromide, and photographed.

Fig. 4.

SaPI2 interference with different phages. SaPI2 uses three mechanisms of phage interference: with phage 80 (Left) it uses ORF17, and with phage 80α (Right) Ppi and CpmAB.