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Identification of the third type of PVL phage in ST59 methicillin-resistant Staphylococcus aureus (MRSA) strains

Meng Zhang , Teruyo Ito , Shanshuang Li , Jingxun Jin , Fumihiko Takeuchi , Tsai-Ling Yang Lauderdale , Masato Higashide , Keiichi Hiramatsu
DOI: http://dx.doi.org/10.1111/j.1574-6968.2011.02355.x 20-28 First published online: 1 October 2011

Abstract

The genes lukS-PV and lukF-PV for Panton–Valentine leukocidin (PVL) that confers high virulence to Staphylococcus aureus are located on the prophages (PVL phages) which have been classified into group 1 and 2 sfi21-like Siphoviridae. We report novel PVL phages lysogenized in ST59 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Japan (JCSC7247) and Taiwan (JCSC5967). The genomes of φ7247PVL and φ5967PVL showed more than 99% identity, and the regions containing the five genes located at both ends of the prophages, int (integrase), hol (holin), ami (amidase), lukS-PV, and lukF-PV, are highly homologous to extant PVL phages. The genes for the structural module are less homologous to these phages, but are highly homologous to non-PVL phages belonging to group 3 Sfi21-like Siphoviridae, for example φN315. Subsequent PCR identification and nucleotide sequencing of an additional 11 Taiwanese ST59 MRSA isolates suggested they all carry the same phage as φ5967PVL, which differed from φ7247PVL by a single base. This study adds evidence to the notion that novel PVL phages would be generated through illegitimate recombination events by acquiring the region at which hol, ami, luk, and int genes would line up upon lytic growth, and suggests that the PVL-positive MRSA clones that have emerged worldwide may carry distinct phages.

Keywords
  • methicillin-resistant Staphylococcus aureus
  • Panton–Valentine leukocidin
  • lukS-PV
  • lukF-PV
  • bacteriophage
  • ST59

Introduction

Panton–Valentine leukocidin (PVL) is a two-component and hetero-oligomeric pore-forming cytolytic toxin identified in 1932 by Panton and Valentine (Panton & Valentine, 1932). Most of the community-associated methicillin-resistantStaphylococcus aureus (CA-MRSA) strains that have emerged in recent years carry the genes encoding PVL, lukS-PV and lukF-PV, and cause a spectrum of infections (CDC, 1999; Baba et al., 2002; Diep et al., 2006). The role of PVL in the pathogenicity was re-evaluated, and PVL has been shown to play a key role in the pathogenesis of necrotizing pneumonia (Labandeira-Rey et al., 2007; Cremieux et al., 2009).

PVL-positive S. aureus strains are lysogens of PVL phages, which belonged to Siphoviridae, a family of double-stranded DNA viruses that share a long noncontractile tail and capsid with an isometric or an elongated shape (Kaneko et al., 1998; Narita et al., 2001; Baba et al., 2002; Kaneko & Kamio, 2004; Diep et al., 2006; Ma et al., 2008).Canchaya et al. (2003) classified S. aureus prophages into five groups based on differences in structural module, for example tail and capsid: groups 1–3 Sfi21-like cos-site Siphoviridae, and groups 1 and 2 sfi11-like pac-site Siphoviridae. PVL phages reported to date belong to either group 1 (isometric head type) or group 2 (elongated head type) of Sfi21-like cos-site Siphoviridae (Canchaya et al., 2003; Kaneko & Kamio, 2004). However, considerable differences exist in the DNA replication/transcriptional regulation region of PVL phages. We developed a PCR system to classify PVL phages based on differences in this region (Ma et al., 2008).

To date, many PVL-positive MRSA and methicillin-susceptible S. aureus (MSSA) clones have been reported (Vandenesch et al., 2003; Rasigade et al., 2010) but there are few reports describing the correlations between the structure of prophage and genetic background of host cells. The representative CA-MRSA strains in the United States belong to CC1 [USA400 in pulsed-field type (PFT)] and CC8 (USA300 in PFT) (McDougal et al., 2003). These strains are presumed to carry prophages similar to φSa2mw carried by MW2 (a CC1 clone) or φSa2USA carried by FPR3757 (a CC8 clone) (Baba et al., 2002; Diep et al., 2006).Boakes et al. (2011) reported that the majority of CC22 strains disseminated in England carry PVL phages belonging to group 1 Siphoviridae (Boakes et al., 2011). However, the structure of PVL phages carried by other CA-MRSA clones, for example CC80 MRSA strains, the major CA-MRSA clone in Europe (Faria et al., 2005; Holmes et al., 2005), CC59 MRSA strains that are mostly identified in Asia, for example Taiwan, and CC30 MRSA strains disseminating in Oceania and other countries (Holmes et al., 2005; Cha et al., 2006; Hsu et al., 2006) has not been reported.

When we examined the types of PVL phages carried by Japanese PVL-positive MRSA strains isolated in the 2000s with the PCRs identifying PVL phages, we noticed that a ST59 strain carried an untypeable PVL phage (Ma et al., 2008). Aspvl-positive ST59 MRSA strains are prevalent in Taiwan, we carried out a study to characterize the PVL phages isolated from our Japanese strain and those of Taiwanese strains. We report here the novel PVL phages carried by ST59 strains isolated from Japan and Taiwan.

Materials and methods

Strains used in this study

Thirteen PVL-positive ST59 MRSA strains were studied. JCSC7247 was isolated in Japan in 2007 (Ma et al., 2008). The 12 strains from Taiwan include TSGH17 (kindly provided by Dr Chih-Chien Wang from Tri-Service General Hospital in Taiwan) (Boyle-Vavra et al., 2005) and 11 strains isolated in 2002 (Chen et al., 2005). A PVL-positive MRSA strain, MW2, was used as reference. To identify phages induced from lysogenized bacteria, an MSSA strain 1039 (kindly provided by Yukio Yoshizawa, Jikei University, Japan) was used as the indicator strain (Yoshizawa, 1985).

Determination of the entire nucleotide sequence of φ7247PVL and φ5967PVL

The fosmid library on the genomic DNA of JCSC7247 was constructed using the CopyControl fosmid library production kit (Epicentre Biotechnologies, Madison, WI). A total of 1500 colonies were screened by PCR forlukS-PV andlukF-PV. Plasmid DNA of p7247-1 was extracted from positive clones and used as templates for nucleotide sequencing. DNA fragments were amplified by long-range PCR on JCSC7247 and JCSC5967 using primers indicated inFig. 1 and listed in Supporting Information, Table S1. Nucleotide sequences were subsequently determined by primer walking.

1

Structural comparisons of three PVL phages. Structures of three PVL phages are illustrated based on the nucleotide sequences deposited in databases DDBJ/EMBL/GenBank under accession nos BA000033, AB009866, and AP011956 for φSa2mw, φPVL, and φ7247PVL, respectively. Red arrowhead indicates the location ofatt, core, and attB. The nucleotide sequences of the core are indicated above the arrow heads. P. Black bars indicate the locus of amplified DNA fragments using six sets of primers and the locus of a fosmid clone p7247-1. Green bars indicate the locus of amplified DNA identifying the carriage of gene linkages in φ7247PVL. ORFs are colored as follows: orange, ORFs related to lysogeny; red, ORFs in DNA replication/recombination region with assigned functions; bright green, ORFs related to capsid formation; yellowish orange, ORFs related to head formation; yellow, ORFs related to tail formation; blue, ORFs related to cell lysis; black,lukS-PV andlukF-PV.

PCRs identifying type V(5C2&5) SCCmec elements and φ7247PVL

Chromosomal DNAs were extracted fromS. aureus strains as described previously (Ito et al., 2001). PCRs were performed to identify type V(5C2&5) SCCmec elements and φ7247PVL using primers listed in Table S1.

Induction of prophages fromS. aureus cells and identification of PVL phages by plaque hybridization

Prophages were induced by mitomycin C treatment, and infected byS. aureus 1039. PVL phages were identified by plaque hybridization with digoxigenin-labelled probe as described previously (Ma et al., 2008).

Electron microscopy

Formvar membrane-coated grids were placed on a drop of bacteriophage diluted with distilled water. The grid was then placed on a drop of 2% uranylacetate for 10–20s, lifted and dried with filter paper, then dried in air. The grid was examined using a Hitachi 7100 transmission electron microscope (Hitachi High-Technology Co., Katsuta, Japan). Photos were taken using Advantage-HR (Advanced Microscopy Techniques, Danvers, MA).

Computer analysis

Pairwise comparison of the homologous regions of the phage genome was made using theblast bl2seq program (http://blast.ncbi.nlm.nih.gov) and drawn as a dot plot usingmathematica software (http://www.wolfram.com/products/mathematica/index.html). To view the similarity among 16 phages simultaneously, we concatenated the genome sequences and performed the bl2seq comparison.

Accession numbers

Nucleotide sequences of two PVL phages of strains JCSC7247 and JCSC5967 have been deposited in DDBJ/EMBL/GenBank, accession nos AP011956 and AP011955, respectively.

Results

Overall organization of φ7247PVL

The nucleotide sequence of φ7247PVL identified in a Japanese ST59 MRSA was determined and compared with those of six PVL phages (φPVL, φ108PVL, φ2958PVL, φSa2mw, φSLT, and φSa2usa) identified in MSSA and MRSA strains of distinct genetic backgrounds (Table 1). φ7247PVL was 42142bp in length from the rightmost phage attachment site (attP-R) to the leftmost site (attP-L), in which 42 predicted ORFs of larger than 99bp were identified. The core sequence of 29 nucleotides is located at both ends of φ7247PVL (Fig. 1). The G+C content of φ7247PVL was 33.3%, which was comparable to other staphylococcal phages. The overall organization of φ7247PVL is the same as the other six PVL phages listed inTable 1 and consists of five regions related to (1) lysogeny, (2) DNA replication/transcriptional regulation, (3) structural module (the packaging/head and tail), (4) the lysis module, and (5)lukS-PV andlukF-PV (Fig. 1).

View this table:
1

Characteristics of PVL phages and their hosts

Among ORFs in the φ7247PVL genome, those relating to lysogeny, cell lysis and toxin production are highly homologous to those of the six PVL phages.Table 2 lists the nucleotide identities of representative genes in φ7247PVL with two PVL phages φ108PVL and φSa2mw that belonged to group 1 and 2 of Sfi21-likeSiphoviridae, respectively, and a non-PVL phage φN315 (Table 2 and Table S2). Theint (integrase) of φ7247PVL is highly homologous (>98% identities) toint genes carried by the other six PVL phages. This is consistent with the fact that all phages are integrated at the same locus on the chromosome corresponding to the bacterial attachment sites flanking the left and right ends of the phage (attB-L andattB-R). Two ORFs related to lysis of host cells,hol encoding holin protein andami encoding amidase (Grundling et al., 2001; Zou & Hou, 2010), are highly homologous with nucleotide identities ranging from 91.4% to 100%.lukS-PV andlukF-PV genes are highly conserved (>99.8% identities) among the seven PVL phages. In contrast, genes in the modules for DNA replication/transcriptional regulation and phage structure are less homologous.

View this table:
2

Nucleotide identities of representative ORFs in φ7247PVL to φSa2mw, φ108PVL and φN315ß

Characterization of Taiwanese ST59 MRSA strains

As PVL-positive ST59 MRSA strains are mostly isolated from Taiwan, we compared the characteristics of JCSC7247 with 12 PVL-positive ST59 MRSA strains from Taiwan (Table 3). All 13 strains carried the type V(5C2&5) SCCmec element. To determine whether Taiwanese isolates carried the same PVL phage as φ7247PVL, PCRs were performed using two sets of primers targeting the gene linkages betweenint andrep encoding repressor protein (primer set A), and between tail length tape major protein andlukS-PV (primer set B) (Fig. 1 and Table S1). Amplicons of expected sizes were obtained from all 12 Taiwanese strains (Table 3), suggesting that they carried the phage similar to φ7247PVL based on PCR results.

View this table:
3

Identification of PVL-carrying phages induced by mitomycin C treatment

We then examined whether infective PVL phages could be induced from these strains using MW2 as a positive control. Prophages were induced by mitomycin C treatment from all 13 strains. Subsequent plaque hybridization experiments with a probe identifyinglukS-PV andlukF-PV confirmed that PVL-positive plaques were generated in all but two strains, JCSC7247 and JCSC5982 (Table 3). We then conducted further hybridization experiments on 1630 plaques from JCSC7247 and 1052 plaques from JCSC5982; no plaques for PVL phage were identified.

We then chose a Taiwanese strain, JCSC5967, and determined its prophage nucleotide sequence to compare with φ7247PVL. φ5967PVL and φ7247PVL are identical except for a base difference in ORFs FP32 and TP32, resulting in a change at the 69th amino acid, glutamic acid (FP32 in φ7247PVL) and glycine (TP32 in φ5967PVL). PCRs and subsequent sequencing of amplified DNA fragments showed that all 12 Taiwanese strains carried the same TP32 ORFs, indicating that the other Taiwanese MRSA strains carried φ5967PVL. The phage particles of φ5967PVL were viewed by electron microscopy (Fig. S1). The phages showed isometric heads (approximately 54nm in diameter) and noncontractile flexible tails (approximately 200nm in length).

Two phages are classified into group 3 sfi21-likeSiphoviridae

The long region of 19.2kb in φ7247PVL and φ5967PVL carries 15 ORFs that encode proteins essential for phage structure, for example packaging of phage DNA (terL,por, andpro), capsid (four ORFs), and tail formation (seven ORFs including tail tape measure protein). These ORFs are less homologous to those carried by the other six PVL phages but they are highly homologous to those of φN315 (Table 2). Three dot plot pairwise comparisons are shown inFig. 2: φ7247PVL vs. φPVL (group 1 Sfi21-likeSiphoviridae); φ7247PVL vs. φSa2mw (group 2 Sfi21-likeSiphoviridae); and φ7247PVL vs. φN315 (group 3 Sfi21-likeSiphoviridae). φ7247PVL shares homologouslukS-PV- andlukF-PV-containing regions of 4.4 and 6.6kb with φSa2mw and φPVL, respectively. However, other regions are less homologous, although several short regions having >90% identities were identified. In contrast, the long region of 13.0kb containing genes related to the structural module of φ7247PVL was highly homologous to the module of φN315, and was less homologous to the modules of φPVL and φSa2mw. The data indicated that φ7247PVL should be classified into the third type of PVL phage that belonged to a distinct group (group 3) of Sfi21-likeSiphoviridae.

2

Pairwise comparisons among PVL phages with dot plot analysis. The genome sequence of φ7247PVL was compared with those of φSa2mw (group 2cos-siteSiphoviridae), φPVL (group 1cos-siteSiphoviridae), and φN315 (group 3cos-siteSiphoviridae) in the plot. Ordinate indicates the genome φ7247PVL. Abscissa indicates the genomes of three phages, φSa2mw, φPVL, and φN315, in plots (a), (b), and (c), respectively. The regions that showed >90% nucleotide identities between φ7247PVL, φSa2mw, and φPVL are: φSa2mw and φ7247PVL, 1.31kb (from core to nt. 1318), 1.19kb (from nt. 14084 to nt. 15273), 0.42kb (from nt. 15623 to nt. 16042) from the left extremity of φ7247PVL; φPVL and φ7247PVL, 2.2kb (from core to nt. 2229), 1.83kb (from nt. 15719 to nt. 17548), 1.28kb (from nt. 29000 to nt. 30281), and 2.19kb (from nt. 33204 to nt. 35395). The nucleotide positions counted from the core sequence of φ7247PVL located at left end were indicated.

Comparisons of predicted gene linkages ofint-lukS-PV-lukF-PV-ami-hol

The region carrying the gene linkage ofint-lukS-PV-lukF-PV-ami-hol in φ7247PVL was compared with six PVL phages (Fig. 3). This five-gene linkage is predicted to be formed when phage PVL is circularly permuted. The 83-bp region fromattP-L toint is highly homologous (>99% identities) in all six PVL phages. In φSa2mw and φ108PVL, the homologous regions ended atint. In the other four phages, the homologous region contained an ORF followingint (FP02). The regions fromhol toattP-R (excluding IS256 in the corresponding region of φ108PVL) are highly homologous except for thehol of φSLT, which was slightly less homologous (92% identity). As the sizes of the homologous regions varied due to differences in the left- and right-flanking regions, it could be presumed that PVL phage acquired the region encodinglukS-PV,lukF-PV, andint by non-site-specific illegitimate recombination events.

3

Comparison of the regions with five genes,hol, ami, lukS-PV,lukF-PV, andint. These arrangements can be seen upon the propagation of phages. The regions having nucleotide identities of >90% are shown with the following colored bars: orange bar, fromattP-L throughint to the end of homologous region (>98.4% identities); thick black bar, fromlukS toattP-R (>99.8% identities); thin black bar, intergenic region betweenami andlukS (>97.4% identities); blue, betweenhol andami (>91.5% identities); purple, the homologous regions upstream ofhol (>95.4% identities). The red triangle indicates the positions ofattP-R andattP-L.

Variation among PVL phages

The 12.4-kb region afterant and beforeter in φ7247PVL carried 17 ORFs (FP07–FP23) related to DNA replication/transcriptional regulation. Among these 17 ORFs, the functions of only three ORFs could be predicted. FP13 encodes a single-strand DNA-binding protein (ssb), FP15 encodes a protein related to DNA replication, and FP20 encodes dUTPase (dut). FP13 (ssb) is highly homologous (98.9% identity) only to that of φSLT. FP15 has 100% identity with φSLT and 80.5% identity with φ108PVL. FP20 (dut) has the highest identity (77.3%) with φSa2usa.

Discussion

Identification of novel structured PVL phages

The two PVL phages (φ7247PVL and φ5967PVL) identified in this study shared several characteristics in common with previously reported PVL phages: (1) the same integration site; (2) carriage of a 29-bp core sequence at both ends of the prophage; (3) the same structural organization; and (4) carriage of five (or six) genes that are highly homologous to those of extant PVL phages. However, the regions encoding genes for the structure module and DNA replication/transcriptional regulation in these two PVL phages differed greatly from those of extant PVL phages.

The genomes of 15 phages, the aforementioned six PVL phages and nine representative prophages, were compared by dot plot (data not shown). Dot plots showed that φ7247PVL belonged to group 3 Sfi21-likecos-siteSiphoviridae. Electron microscopic observation of φ5967PVL indicated that the phage shared an isometric head similar to that of group 1 phage (Fig. S1). These data indicate that the phages identified in ST59 strains are distinct from previously reported PVL phages and should be regarded as a novel third type of PVL-carrying phage.

Dissemination of ST59 MRSA strains carrying the third type of PVL phage

In this study, we also demonstrated that ST59 MRSA strains isolated from Japan and Taiwan are lysogens of the same novel third type of PVL phage. PVL-positive phage particles were induced from 11 of 12 Taiwanese MRSA strains. The sequences of φ5967PVL, chosen as a representative of the inducible Taiwanese strains, and φ7247PVL from a Japanese strain, are identical except for one nucleotide, resulting in a difference of amino acid in ORFs, glutamic acid in FP32 of φ7247PVL and glycine in TP32 of φ5967PVL. All 13 MRSA strains carried the same type V(5C2&5) SCCmec. Moreover, their pulsed-field gel electrophoresis banding patterns were closely similar (data not shown). As PVL-positive ST59 MRSA strains have rarely been identified in Japan, whereas they are the predominant Taiwanese CA-MRSA (Chen et al., 2005, 2009; Takizawa et al., 2005; Ma et al., 2006), JCSC7247 may have originated from Taiwan. PVL-positive ST59 MSSA strains have also been isolated in Taiwan (Chen et al., 2009). Further studies on the PVL phage type carried by these MRSA and MSSA strains will help to elucidate the evolution of PVL-positive ST59 MRSA strains.

Infective PVL phages which might play a role in the dissemination of PVL phage were generated from 11 of 13 tested ST59 MRSA strains. We have conducted PCRs identifying PVL phages using primer pairs identifyinglukS-PV, andluk F-PV andint (Goerke et al., 2009) on template DNAs extracted from DNase-1-treated lysate after mitomycin C treatment.lukS, F-PV, andint(φSa2) were positive with extracts from JCSC7247 and JCSC5982. The data showed that PVL phages might be induced from the cells but the number of induced PVL phages might be too small to be identified by plaque hybridization experiments, although there the possibility that induced phage could not create plaques on lawn of cells of strain 1039. As all 12 Taiwanese strains carried the same TP32 ORF, the possibility that the amino-acid difference in the ORFs was associated with inducibility of the prophage or the infectivity of the induced phage is small.

Characteristics of PVL phages in CA-MRSA strains

Among the six extant PVL phages, two phages, φPVL and φ108PVL belonging to group 1 Sfi-21-likeSiphoviridae, carried some truncated genes related to tail formation of φPVL and φ108PVL, and no infective PVL phages were induced from these two strains. In contrast, infective PVL-carrying phages were produced from three group 2 Sfi21-likeSiphoviridae PVL phages, φSLT, φ2958PVL, and φSa2mw, which shared intact head and tail genes (Kaneko et al., 1998; Baba et al., 2002; Ma et al., 2008). These data suggest that intact structural module is necessary for the generation of infective PVL phage. In the cases of PVL phages where we could not obtain positive results in plaque hybridization experiments, for example φSa2 in FPR3757 (X.X. Ma, unpublished data), PCR identification of the PVL phage in the mitomycin-treated cell lysate will be a useful method to infer whether the phage is induced from the cells.

When we compared the structures of PVL phages from earlier years with the novel PVL phages from this study, we noticed that the mosaic structure of the phage genome can be roughly classified into three regions: (1) the region common to all PVL phages, which contains five genes,int, lukS, lukF, hol, andami; (2) the region encoding the phage structural module, which is the essential region for the grouping of phages; (3) the region that is distinct in each PVL phage, albeit some homologous sections exist. As the third region, which is composed mostly of genes for DNA replication/transcriptional regulation and part of the region encoding lysogeny, differed in these PVL phages, it could be said that PVL phages carried by CA-MRSA strains that have emerged in recent years are not descendants of PVL phages that spread in earlier years. Therefore, we speculate that these novel PVL phages were generated through some recombination events and that PVL phages carrying CA-MRSA strains were formed rather recently.

Generation of PVL phages

The fact thatlukS-PV andlukF-PV genes were found in three morphologically distinct bacteriophages suggested that PVL phages might be generated by insertion of the virulence determinants, the geneslukS-PV andlukF-PV. Of note is that the integrase gene carried by the third type of PVL phage in the present study was nearly identical to those of extant PVL phages but differed greatly from those of group 3 non-PVL phages, φN315, φMu50A, and φNM3, among which theint genes are highly homologous (Kuroda et al., 2001; Bae et al., 2006). The difference in the types of integrase correlated very well with integration sites of phages. All PVL phages are integrated at the same position as φSa2 in the chromosome, indicating that all PVL phages belonged to the φSa2 family based on the integrase-based classification, whereas the other three group 3 phages integrated at the same position as φSa3 in the chromosome (Goerke et al., 2009).

Although the life cycle of staphylococcal phages has not been elucidated, we can infer it from the life cycle of coliphage lambda, which also belongs toSiphoviridae. After the bacteriophage DNA is injected into the cells, a circular form of phage DNA would be generated in the cytoplasm to prevent phage DNA degradation by host restriction enzymes. In the case of phages having acos-site, the circular form as well as the linear concatenated DNA would be formed by ligation at thecos-site upon propagation of phage. The five-gene linkage ofint-lukS-PV-lukF-PV-ami-hol would be formed in both forms. Therefore, it is presumed thatlukS-PV andlukF-PV genes originating elsewhere were integrated into a phage and converted it to a PVL-carrying phage. Once the PVL phage was established, novel PVL phages were further generated by acquiring the region containing genesint, lukS-PV,lukF-PV,ami, andhol, through illegitimate recombination events. Our data adds evidence in support of the hypothesis and show again that phages play an important role as carriers of virulent genes and a novel virulent strain will be generated by the acquisition of virulent phages.

In conclusion, we have identified two novel PVL phages from SCCmec V(5C2&5):ST59 MRSA strains in Japan and Taiwan. The PVL phages carried by these ST59 MRSA strains are distinct from previously reported PVL phages. Our data suggest that representative CA-MRSA strains disseminating worldwide may carry distinct PVL phages.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Table S1. List of primers used in this experiment.

Table S2. ORFs in and around φ7247PVL and their similarities to φSa2mw and φ108PVL.

Figure S1. Transmission electron microscopy images of bacteriophage φ5967PVL.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Acknowledgements

We thank Mitutaka Yoshida, Division of Ultrastructural Research, for taking electron microscopy photos. This work was supported by a Grant-in-Aid for Scientific Research C19590456 from the Ministry of Education, Science, Sports, Culture and Technology of Japan and a Grant-in-Aid (S0991013) for the Foundation of Strategic Research Projects in Private Universities from the Ministry of Education, Science, Sports, Culture and Technology of Japan.

Footnotes

  • Editor: Ross Fitzgerald

References

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