Elsevier

Gene

Volume 268, Issues 1–2, 2 May 2001, Pages 195-206
Gene

Phage conversion of Panton-Valentine leukocidin in Staphylococcus aureus: molecular analysis of a PVL-converting phage, φSLT

https://doi.org/10.1016/S0378-1119(01)00390-0Get rights and content

Abstract

Staphylococcal Panton-Valentine leukocidin (PVL) is an important virulence factor, which causes leukocytolysis and tissue necrosis. Our previous report on the existence of the PVL genes (lukS-PV and lukF-PV) on the genome of prophage φPVL in the Staphylococcus aureus strain V8 suggested the horizontal transmission of PVL genes by temperate bacteriophage among S. aureus (Kaneko, et al., 1998. Gene 215, 57–67). Here, we demonstrated the phage conversion of S. aureus leading to the production of PVL by discovery of a novel PVL-carrying phage, φSLT (Staphylococcal Leukocytolytic Toxin) from a clinical isolate of S. aureus. φSLT was able to lysogenize several clinical isolates of PVL-negative S. aureus strains as well as strain RN4220 at the conserved 29-bp sequence (attB) and all the lysogenized S. aureus strains had the ability to produce PVL. φSLT had an elongated head of about 100×50 nm and a flexible tail of 400 nm long, that was quite different from φPVL which had an isometric hexagonal head of about 60 nm diameter. The linear double-stranded φSLT genome comprised 42,942 bp with 29-bp attachment core sequences and contained 62 open reading frames. Only 6.4 kbp region containing lysis cassette, PVL genes, attP, integrase, and orf204 of φSLT was identical to that of φPVL, while other regions were different from those of φPVL. Thus, it can be concluded that PVL genes are carried by different temperate phages, which have the same attachment site.

Introduction

Several bacterial virulence factors are carried on mobile genetic elements such as phages, plasmids, or transposons. Genes for diphtheria toxin in Corynebacterium diphtheriae (Uchida et al., 1971), pyrogenic toxins A and C in group A Streptococcus (Johnson et al., 1986), neurotoxin in Clostridium botulinum (Inoue and Iida, 1970), Shiga-like toxins in Escherichia coli (Scotland et al., 1983, O'Brien et al., 1984), cytolysin in Pseudomonas aeruginosa (Hayashi et al., 1990) and cholera toxin in Vibrio cholerae (Walor and Mekalanos, 1996) are known to be carried by phages. Genome projects on pathogenic bacteria accelerate the discovery of phages-related virulence genes.

Staphylococcus aureus is a community-acquired and nosocomial pathogen, which produces a variety of extracellular virulence factors. Lysogenic conversion leading to the synthesis of virulence factors in S. aureus has been demonstrated for enterotoxin A (Betley and Mekalanos, 1985) and lipase (Lee and Iandolo, 1985). Phages φ13 and φ42 share the same attachment site and integrase gene, and the production of β-hemolysin is negatively regulated by insertion of these phages into the gene of β-hemolysin. φ13 has staphylokinase gene (sak) and is therefore a double-converting phage. Phage φ42, in addition to sak, mediates the positive conversion for enterotoxin A (Carroll et al., 1995).

γ-Hemolysin (Hlg) and leukocidin (Luk) have been isolated as bi-component cytotoxins from S. aureus. γ-Hemolysin shares a common component with leukocidin (LukF, class F component) and the target cell specificities of these bi-component toxins were mainly determined by the class S (Hlg2 for γ-hemolysin and LukS for leukocidin) proteins (see review, Tomita and Kamio, 1997). The class S protein, co-operatively with the class F protein, forms the membrane pores on the target cells (Sugawara et al., 1997). We demonstrated that in addition to these polypeptides for Hlg/Luk, S. aureus strains V8 (ATCC 49775) and P83 (ATCC 31890) produce two polypeptides for another bi-component pore-forming cytotoxin: one hand LukF-PV and LukS-PV for strain V8 forming the panton-valentine leukocidin (PVL), and on the other hand LukF-PV and LukM for strain P83. In each of these two strains, genes for those toxin components belong to two genetic clusters, i.e. Hlg/Luk cluster and PVL cluster. The former consists of hlg2, lukS, and lukF and the latter consists of lukS-PV (or lukM) and lukF-PV (see review, Tomita and Kamio, 1997). PVL has been believed to be an important virulence factor of S. aureus infection, because of its high cytolytic specificity against human polymorphonuclear cells and macrophages, whereas Luk shows slight hemolytic activity besides leukocytolytic activity. In France, PVL-producing strains are isolated from 2% of clinical isolates of S. aureus, whereas Luk/Hlg is produced by almost all (≥99%) S. aureus clinical isolates (Prévost et al., 1995). However, in this country, most of cutaneous necrotic lesions, such as furuncles or primary abscesses, and severe necrotic skin infection (Prévost et al., 1995, Lina et al., 1999) as well as severe necrotic hemorrhagic pneumonia (all community- acquired) (Lina et al., 1999) are associated with PVL-positive S. aureus strains. Vijver et al. previously reported evidence of lysogenic conversion in S. aureus by a group A phage leading to an increase of ‘leukocidin’ production (van der Vijver et al., 1972). However, it is not clear which leukocidin was concerned, Luk or PVL, because the assay system used could not distinguish PVL from Luk, and the direct evidence of the phage conversion for ‘PVL genes’ was not available. In 1997, we isolated a temperate phage, φPVL, carrying the PVL genes from a lysate of mitomycin C-treated S. aureus V8. We determined the complete nucleotide sequence of φPVL and also identified the structure of attachment sites on the chromosome of the V8 strain (Kaneko et al., 1997, Kaneko et al., 1998). The linear double-stranded DNA forming the φPVL genome comprised 41,401 bp with 3′- staggered cohesive ends (cos) of nine bases. The genes of lytic enzyme (lyt) (peptidoglycan hydrolase), [lukS-PV-lukF-PV] cluster, and the phage-bacterial junction (attR) of φPVL prophage on the V8 chromosome were all located very close to each other in the given order. The existence of φPVL carrying PVL genes strongly suggested horizontal transmission of the PVL genes cluster via the bacteriophage. However, we could not demonstrate phage conversion by the PVL genes because we failed to find an indicator strain of S. aureus able to be lysogenized by φPVL (Kaneko et al., 1997).

In another work, we also found that PVL-like genes in strain P83 were carried by a prophage which was designated as φPV83-pro (Zou et al., 2000). The precise genome size was 45,636 bp with att core sequence of 10 base pairs. The lukM-lukF-PV cluster was located 2.1 kb upstream of the attL site. Furthermore, two insertion sequence, ISSA1 and ISSA2 were integrated into attL site and orf44, respectively. φPV83-pro was not induced as phage particle from P83 regardless of its treatment with mitomycin C. The insertion of ISSA1 into attL of φPV83-pro was one of the reasons for the excision failure of the phage genome from the P83 chromosome and this fact prompted us to assume the fixation of these exogenous virulence genes in the host genome.

Since φPVL and φPV83-pro were found to be defective phages, other PVL-carrying phages having maintained their infectious ability were desired to evidence the phage conversion leading to the production of PVL. In this study, we presented direct evidence showing that PVL-negative strains of S. aureus can be converted into PVL producers by infection of a novel temperate phage, φSLT, which was isolated from a PVL-producing clinical isolate A980470. We analyzed the complete nucleotide sequence of the φSLT genome and its structure was compared with those of φPVL and φPV83-pro.

Section snippets

Bacterial strains and media

Staphylococcus aureus strain A980470 is an original lysogen of φSLT, which was screened from a set of 28 PVL-producing strains from the collections of the Strasbourg University General Hospital (Strasbourg, France) and of the Centre National de Référence des Toxémies a Staphylocoques (Lyon, France). S. aureus restriction-negative strain RN4220 was used as an indicator strain for routine bacterial infection by phages. One hundred and thirty seven S. aureus strains from food poisoning origin,

Isolation of infectious PVL-carrying phage from clinical isolates

The cells of 28 clinical isolates of S. aureus harboring PVL genes were lyzed by adding mitomycin C (1 μg/ml) to the liquid culture medium. The phage particles from seven strains were found to contain PVL genes and one phage preparation from A980470 strain formed plaques on the lawn of S. aureus RN4220. The restriction patterns of the 42-kbp genome of phage obtained from A980470 were distinct from those of φPVL genome (Kaneko et al., 1997) (summarized in Fig. 3). Sequencing of two XbaI

Comparative studies of phage genomes

The lysogeny module in each of these three PVL-carrying phages from S. aureus is flanked by the attachment sites and the replication module. This organization in modules is common among the temperate Siphoviridae of low G±C content from gram-positive bacteria, such as Sfi21 from Streptococcus thermophilus (Lucchini et al., 1999a). Multiple alignment among φSLT, φPVL, and φPV83-pro revealed the chimeric and mosaic structures of these phage genomes. These structural, differences were mainly

Acknowledgements

We thank to T. Okubo of Gunma University School of Medicine and Dr H. Shiraishi of Miyagi Prefectural Institute of Public Health and Environment for the gift of indicator strain RN4220 and PVL-negative strains, respectively. This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture, Japan (11460034, 11694191, and 11760050) and Uehara Memorial Foundation.

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