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Infection and Immunity, June 2002, p. 3227-3233, Vol. 70, No. 6
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.6.3227-3233.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Kumiko Sugama,2 Rieko Suzuki,3 Daisuke Tanaka,4 Aki Tamaru,5 Yoshihiro Fujinaga,6 Yoshiaki Abe,7 Yoshikata Shimizu,8 Haruo Watanabe,1 and the Working Group for Group A Streptococci in Japan
Department of Bacteriology, National Institute of Infectious Diseases, Tokyo,1 Department of Bacteriology, Fukushima Prefectural Institute of Public Health, Fukushima,2 Department of Bacteriology and Pathology, Kanagawa Prefectural Public Health Laboratory, Yokohama,3 Department of Bacteriology, Toyama Institute of Health, Toyama,4 Department of Microbiology, Osaka Prefectural Institute of Public Health, Osaka,5 Division of Biological Medicine, Yamaguchi Prefectural Research Institute of Public Health, Yamaguchi,6 Department of Bacteriology, The Oita Prefectural Institute of Health and Environment, Oita,7 Department of Anaesthesia, Asahi General Hospital, Asahi, Japan8
Received 26 December 2001/ Returned for modification 18 February 2002/ Accepted 21 March 2002
| ABSTRACT |
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NIH1.1, containing a new superantigen gene speL near its right attachment site. The C-terminal part of the deduced amino acid sequence of speL has 48 and 46% similarity with well-characterized erythrogenic toxin SpeC and the most potent superantigen, SmeZ-2, respectively. None of 10 T3 isolates recovered during or before 1973 has speL, whereas all of 18 M3/T3 isolates recovered during or after 1992 and, surprisingly, Streptococcus equi subsp. equi ATCC 9527 do have this gene. Though plaques could not be obtained from
NIH1.1, its DNA became detectable from the phage particle fraction upon mitomycin C induction, showing that this phage is not defective. A horizontal transfer of the phage carrying speL may explain the observed change in M3/T3 S. pyogenes isolates in Japan. | INTRODUCTION |
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| MATERIALS AND METHODS |
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Lambda library construction. Genomic DNAs of NIH1 were partially digested by Sau3AI (Roche Diagnostics) and coligated with BamHI (Roche Diagnostics)-digested Lambda DASH II DNA (Stratagene, La Jolla, Calif.). XL1-Blue MRA (P2) (Stratagene) was used as a host strain to make a phage library.
PCR.
PCRs were performed by using AmpliTaq Gold (Applied Biosystems, Foster City, Calif.) with PCR Gold Buffer. For amplifying speL, steps consisting of denaturing at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min were repeated 30 times after initially activating the enzyme at 95°C for 10 min. Primers used were speL-fwrd (5'-GTCATATCATGTTGTATGCAA-3') and speL-rev (5'-GTTTAAGTGAACATCAAAGTG-3'). The same PCR condition as above was applied for amplifying the downstream junctional region of temperate phage
NIH1.1 with primers #5-T7-R1 (5'-AACACTTTTAAGAAAGTCATATCT-3') and K23rj (5'-CAATTAACAATGAGTACACACGGT-3'). Primers p7-T3-2 (5'-GATAGGCATTAGAAAAAAGGA-3') and p5-T3-2 (5'-CATCTATTCAAGATAGCCTACTTC-3') were also used under the same PCR condition as above. Location and direction of each primer are shown (see Fig. 2).
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Nucleotide sequencing.
The nucleotide sequence of
NIH1.1 was determined by automated sequencers ABI PRISM 377 DNA Sequencer (Applied Biosystems) and ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Direct genome sequencing was performed according to a previous description (31) by using primer p7-T3-3 (5'-AGATAAGAGTTTCAATATGACGAG-3'), whose location in NIH1 is shown (see Fig. 2).
Nucleotide sequence accession number.
The nucleotide sequence of
NIH1.1 has been deposited in GenBank under accession number AY050245.
| RESULTS |
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NIH1.1. Like other streptococcal temperate phages (15),
NIH1.1 has a putative hyaluronidase gene (Fig. 2). Interestingly, at a 3' part of
NIH1.1, there is an ORF whose amino acid sequence has 48 and 46% similarity with the C-terminal part of erythrogenic toxin SpeC and with that of the most potent superantigen, SmeZ-2 (24), respectively. Figure 3 shows multiple amino acid sequence alignments of streptococcal superantigens with this ORF. The C-terminal half of the ORF shares the well-conserved amino acids with the other superantigens; from this part, three zinc-binding ligands, histidine or aspartic acid, are provided, and the fourth is from the ß-chain of the major histocompatibility class II (MHC-II) molecule (1, 24). Sequence homologies and experimental superantigenic characteristics (see Discussion) of the ORF led us to name this reading frame speL.
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NIH1.1.
We examined the inducibility of
NIH1.1 by using mitomycin C. In spite of multiple attempts, we could not obtain plaques with a variety of old indicator strains shown in Table 1. Instead, when induced, a 40- to 42-kb DNA fragment became detectable from the phage particle fraction by ethidium bromide staining (Fig. 5, lane 2). And, this band reacted with a speL probe (lane 4), which was prepared from the PCR product from NIH1 shown in Fig. 4. On lane 3, a faint hybridization signal was also detected from the phage particle fraction owing to leakage, i.e., a low grade of induction without inducer, of
NIH1.1 (lane 3).
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| DISCUSSION |
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NIH1.1 comprising 41,796-bp nucleotides. The profiles of the erythrogenic toxin genes speA and speC, the sequence data of the emm genes (Table 1), and the PFGE data (Fig. 1) strongly suggest that recent M3/T3 isolates in Japan have originated from the same ancestor. The acquired sequence has characteristics of lambdoid temperate phages in low-GC gram-positive organisms (6). It contains a putative hyaluronidase gene and the superantigen gene speL. Several streptococcal superantigens, SmeZ, SmeZ-2, SpeC, SpeJ and SpeG, make a superantigen subfamily (24). In addition to these members, SpeH and SpeI were already shown to possess superantigenic activities with different potentials toward different T-cell subsets (19, 21, 23, 24). Besides, SpeL does have superantigenic properties: a recombinant fusion protein between glutathione S-transferase and SpeL stimulates T cells with particular Vßs with a dependence on MHC-II molecules (Imanishi et al., unpublished data).
The notion that SpeL may play roles for severe streptococcal infections is an attractive hypothesis, but the distribution of speL is almost specific to M3/T3 isolates, and no M1/T1 TSLS isolate in Japan examined has this gene. Another hypothesis is that regulatory gene(s) in
NIH1.1 would alter expression patterns of multiple toxins and superantigens, whose genes had been present in genomes of old M3/T3 strains before acquisition of the phage sequence. M1 S. pyogenes strain SF370, whose genome sequence has been determined by Ferretti et al. (9), has three complete temperate phage sequences and five remnant phage sequences (5) accompanied by multiple toxin, superantigen, or mitogenic-factor genes near the attachment sites of each phage sequence. The locations of these toxin genes in temperate phages are also true for the speA in phages T12,
270, and
49 (33) and the speC in phage CS112 (12, 18). Broudy et al. have reported that SpeC is induced along with phage CS112 induction by factors released from human cells (2), suggesting the speC is transcribed from internal promoter(s) of phage CS112. Similarly, other phage-associated virulence genes are likely to be controlled by one or more regulators within these phage sequences. In the case of the recent Japanese M3/T3 isolates, along with induction of
NIH1.1, multiple virulence genes would be activated simultaneously to cause various severe and sometimes fatal symptoms. Acquisition of large genomic fragments was also observed in recent M1 (3, 22), M12 (T. Murase, personal communication), and M22 (our unpublished observation) isolates, including those of TSLS origin. Thirdly, additional change(s) in M3/T3 genomes not detectable by PFGE may be essential for causing TSLS or severe infection. Tanaka observed the sudden change in PFGE patterns of Japanese T3 isolates between 1984 and 1985 (30); the pattern after the change corresponds to that observed in our recent M3/T3 isolates. In another report (22), similar changes in PFGE patterns of Japanese M3/T3 isolates seem to have occurred more gradually, but actually, the period was between 1983 and 1985 or earlier (T. Murase, personal communication). Since then, almost 10 years had passed until the first TSLS case was presented in Japan in 1992 (27). The speL gene or other gene(s) in
NIH1.1 must have conferred the advantage on the M3/T3 S. pyogenes clones for surviving and disseminating in human populations in the recent antibiotic era, but the event or events that occurred during this lag period remain veiled.
We could not obtain plaques from
NIH1.1, but the increased hybridization signal from mitomycin C-induced phage DNA with the speL probe proves that the phage is not defective. In agreement with our observation, Broudy et al. reported that the speC-carrying phage is detectable by electron microscopy but does not form plaques on a wide range of indicators (2). Goshorn also reported similar results (12). Interestingly, a contig in the ongoing genome sequencing project of S. equi subsp. equi in the Sanger Center contains a phage-associated nucleotide sequence whose deduced amino acid sequence has 98% similarity to SpeL. Another S. equi subsp. equi strain ATCC 9527 is also positive for PCR detecting speL (Fig. 5). Because this strain was isolated early in the 20th century, a horizontal transfer of the phage-associated speL from S. equi subsp. equi to S. pyogenes in 1980s may explain the observed change in M3/T3 S. pyogenes isolates in Japan. Analysis of protein profiles affected by phage gene products and the inter- and intraspecies transducing experiments will provide clues to solving the enigmatic changes in S. pyogenes infections in recent years.
| ACKNOWLEDGMENTS |
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This study was partially supported by a grant from the Ministry of Health, Labour and Welfare (H12-Shinkou-27).
| FOOTNOTES |
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Present address: Laboratory of Plant Pathology and Genetic Engineering, Faculty of Agriculture, Okayama University, Tsushima-naka 1-1-1, Okayama 700-8530, Japan. ![]()
The members of this group are Shoko Murayama, Shirou Yamai, Yotaku Gyobu, Chihiro Katsukawa, Atsushi Katayama, and Kikuo Hoashi. ![]()
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