Staphylococcus aureus Prophage-Encoded Protein Causes Abortive Infection and Provides Population Immunity against Kayviruses

Abstract

Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated Pdp_Sau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdp_Sau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted Pdp_Sau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of Pdp_Sau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding Pdp_Sau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.

Keywords: Kayvirus; Staphylococcus aureus; abortive infection; bacteriophage evolution; bacteriophage therapy; bacteriophages; cell death; lysogeny; phage resistance; phage therapy.

FIG 1

Impact of pdp_Sau gene expression on Kayvirus lytic action demonstrated by drop assay performed with phages K, 812, and 812a on S. aureus RN4220 derivatives. Testing was performed at four phage concentrations, nondiluted phage with a titer of 10⁹ PFU/mL (N) and dilutions of 10⁻², 10⁻⁴, and 10⁻⁶. Four types of resulting lytic zones were distinguished—confluent lysis, semiconfluent lysis, single plaques, growth inhibition, or no lysis. If single plaques appeared at any dilution, the strain was considered susceptible. (A) Phage-sensitive control strain RN4220 (pCN51) compared to wild-type phage resistant RN4220 (53⁺) (pCN51) and RN4220 (pCN51-pdp_Sau) harboring pdp_Sau gene on prophage 53 or plasmid pCN51, respectively. Phage 812a replicates effectively on strains with the pdp_Sau gene. (B) S. aureus strains RN4220 (53⁺) (pCN51-ORF812a_191) and RN4220 (pCN51-pdp_Sau-ORF812a_191) coexpressing pdp_Sau and ORF 812a_191, which restores the sensitive phenotype. The restoration of the phage-sensitive phenotype occurs both in the lysogenic strain with prophage-encoded pdp_Sau and the strain with coexpressed pdp_Sau and ORF 812a_191 from a common promoter.

FIG 2

Phylogenetic insights into prophages harboring pdp_Sau gene identified in whole-genome sequences of Staphylococcaceae family and their characteristics. (A) Bacteriophage and prophage genomes extracted from whole-genome sequences that matched pdp_Sau were compared using the Genome-BLAST Distance Phylogeny method with the settings recommended for prokaryotic viruses (90). The resulting intergenomic distances were used to infer a balanced minimum evolution tree with branch support via FASTME including Subtree Pruning and Regrafting (SPR) postprocessing (91). The branch lengths of the resulting tree are scaled in terms of the respective distance formula used. Phage genomes were characterized by phage type corresponding to serological group (65), integrase gene type (44), and amidase gene type (42). The nucleotide identity of pdp_Sau homologs to ORF016 of phage 53 is shown. Multilocus sequence type and staphylococcal protein A (spa) type were derived from the genome assemblies. NR, not relevant; NT, not typeable. (B) Nucleotide sequence alignment showing the gene structure of lytic modules and accessory genes in the genomes of four S. aureus prophages as follows: (i) phi PVL (92), (ii) phi 53 (39), (iii) prophage vB_StaphS-IVBph354 (93), and (iv) prophage from strain PS/BAC/169/17/W (94). ORFs with proven or predicted functions are depicted as colored boxes. Nucleotide identity between genomic regions is indicated by blue-shaded regions. Putative promoters and terminators are depicted as blue flags and red pins, respectively.

FIG 3

Structure prediction of immunity protein Pdp_Sau and new fusion protein restoring phage sensitivity encoded by phage mutant 812a. (A) 3D structure prediction of Pdp_Sau (blue) with N-terminal transmembrane helix (lime green) and oligosaccharide-binding fold-like region (purple) highlighted. The structure is superimposed with the predicted structure of characterized immunity protein AbiP (A2RIX6) from Lactococcus (gray). (B) EMBOSS Needle pairwise alignment (BLOSUM62) of oligosaccharide-binding (OB) domains of RPA replication protein (UniProtKB accession no. Q24492) with OB_fold hit in Pdp_Sau protein (score, 25; similarity, 40.5%). (C) Sequence alignment of genomic loci encoding ORF 812_189 and ORF 812_192 in phage 812 and fusion ORF 812a_191 in the genome of host-range mutant phage 812a, depicting the emerging deletion. (D) 3D structure prediction of fusion protein ORF812a_191, superimposed with the solved structure of the antiactivator Aqs1 (PDB accession no. 6V7U; gray). (E) Structural alignment of gene products encoded by ORF 812_189 (cyan), ORF 812_192 (magenta), and fusion ORF 812a_191 (yellow).

FIG 4

Changes in S. aureus membrane permeability through Pdp_Sau-based phage defense mechanism illustrated by LIVE/DEAD staining. (A) Fluorescence microscopy of S. aureus infected with phages 812 or 812a and no phage control sample, visualized by fluorescence microscopy at time points 0, 10, 20, and 40 min after the onset of infection. Propidium iodide, a red-fluorescent nucleic acid-binding dye, which cannot enter cells with intact membranes, was used as a marker for the loss of membrane integrity and cell death. Dead cells are represented by red dots and live cells by green dots. (B) Percentage of dead bacterial cells 10 min after the onset of infection. S. aureus cultures were infected with phages 812 or 812a and compared with noninfected strains. (C) Changes in membrane polarity examined by BacLight bacterial membrane potential kit assay on S. aureus RN4220, RN4220 (53⁺), RN4220 (53⁺) (pCN51-orf812a_191), and clinical MRSA strain E48 treated with phages 812 or 812a at an MOI_input of 10. Treatment with the ionophore carbonyl cyanide 3-chlorophenylhydrazone, component B (CCCP) at a final concentration of 50 mM was used as a positive control of depolarization. The decrease in membrane potential was measured as a loss of red fluorescence emitted by carbocyanine dye DiOC₂(3) (3,3′-diethyloxa-carbocyanine iodide).