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ABSTRACT: Mycoplasma pneumoniae is an important causative pathogen in community-acquired pneumonia, particularly among children. Two major groups of this bacterium (subtypes 1 and 2) are known, and they are distinguished by variations in the gene encoding P1 cytadhesin. In this study, we describe the first report of subtype 2b variants of M. pneumoniae from Japanese clinical strains. We have sequenced full-length of P1 genes of these strains.
Journal of Medical Microbiology 08/2012; 61(Pt 11):1633-5. · 2.50 Impact Factor
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ABSTRACT: Mycoplasma pneumoniae strain 309, a type 2a (subtype 2 variant) strain of this bacterium, has variations in the P1 protein, which is responsible for attachment of the bacterium to host cells. Here, we report the complete genome sequence of M. pneumoniae strain 309 isolated from a pneumonia patient in Japan.
Journal of bacteriology 03/2012; 194(5):1253-4. · 3.94 Impact Factor
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ABSTRACT: Mycoplasma pneumoniae, a pathogen causing human pneumonia, binds to solid surfaces at its membrane protrusion and glides by a unique mechanism. In this study, P1 adhesin, which functions as a "leg" in gliding, was isolated from mycoplasma culture and characterized. Using gel filtration, blue-native polyacrylamide gel electrophoresis (BN-PAGE), and chemical cross-linking, the isolated P1 adhesin was shown to form a complex with an accessory protein named P90. The complex included two molecules each of P1 adhesin and P90 (protein B), had a molecular mass of about 480 kDa, and was observed by electron microscopy to form 20-nm-diameter spheres. Partial digestion of isolated P1 adhesin by trypsin showed that the P1 adhesin molecule can be divided into three domains, consistent with the results from trypsin treatment of the cell surface. Sequence analysis of P1 adhesin and its orthologs showed that domain I is well conserved and that a transmembrane segment exists near the link between domains II and III.
Journal of bacteriology 02/2011; 193(3):715-22. · 3.94 Impact Factor
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ABSTRACT: Mycoplasma penetrans has the ability to change its surface lipoprotein profiles frequently. The P35 family lipoproteins encoded by the mpl genes are key players in this profile variation. The M. penetrans HF-2 genome has 38 mpl genes that form three gene clusters. Most of these mpl genes have an invertible promoter sequence that is responsible for the ON/OFF switching of individual mpl gene expression. Here, we identified the recombinase that catalyses inversions of the mpl gene promoters. We focused on two open reading frames of the M. penetrans HF-2 genome, namely MYPE2900 and MYPE8180, which show significant homology to the tyrosine site-specific recombinase (Tsr) family proteins. Since genetic tools for M. penetrans are still not developed, we cloned the MYPE2900 and MYPE8180 genes and expressed them in Mycoplasma pneumoniae and Escherichia coli. The promoter regions of the mpl genes [p35 (MYPE6810) or p42 (MYPE6630) genes] were also introduced into M. pneumoniae and E. coli cells expressing MYPE2900 or MYPE8180. Inversion of these promoters occurred in the presence of the MYPE2900 gene but not in the presence of the MYPE8180 gene, indicating that the MYPE2900 gene product is the recombinase that catalyses mpl gene promoter inversions. We used a PCR-based method to detect mpl promoter inversion. This method also enabled us to detect inversions of 10 mpl gene promoters in M. penetrans HF-2 cells. All these promoter inversions occurred at the 12 bp inverted repeat (IR) sequences flanking the promoter sequence. The consensus sequence of these IRs was proposed as TAAYNNNDATTA (Y=C or T; D=A, G or T).
Microbiology 05/2009; 155(Pt 4):1241-9. · 3.06 Impact Factor
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ABSTRACT: Despite frequent colonization with Mycoplasma hominis, the invasive disease is rare in neonates. This study describes a neonatal case with meningitis in which M. hominis was isolated from a cerebrospinal fluid sample by culture and detected by PCR. The M. hominis infection was confirmed by elevated metabolic inhibition titers against the isolated M. hominis strain and anti-M. hominis antibodies in serum samples. Minocycline and moxifloxacin were effective against M. hominis, which caused meningitis in the patient. However, the patient exhibited left hemiplegia because of massive brain infarction. Based on data of the previously reported 28 cases in addition to our case, the high morbidity and mortality of the M. hominis central nervous system infection were confirmed; it was assumed to result from delayed diagnosis and ineffective initial therapy. Early diagnosis and prompt initiation of appropriate antimicrobial treatment are necessary for a favorable prognosis. Fourth-generation fluoroquinolones, especially moxifloxacin, deserve wider use in such cases.
The Journal of infection 10/2008; 57(4):338-43. · 4.13 Impact Factor
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ABSTRACT: Mycoplasma pneumoniae clinical isolates obtained between 1995 and 2005 were examined to determine the prevalent genotype. One hundred and twenty-seven strains isolated from bronchitis and pneumonia patients were genotyped by a PCR-RFLP method based on nucleotide sequence polymorphisms of the p1 gene, which encodes the major adhesin protein. The typing results established that 66 of the isolates were group I strains, 45 were group II strains and 16 were group II variants. Analysis of the annual occurrence of these isolates showed a predominance of group II strains between 1995 and 2001 (n=37). No group I strain was found during this period. However, group I strains appeared in the isolates from 2002 (2/5 isolates, 40 %) and increased in specimens taken after 2003, thereby constituting a large proportion of the isolates. In 2004 and 2005, no group II strains were found among the isolates (n=49), although there were nine group II variants. Throat swabs and sputum samples obtained from patients with respiratory infections between 1997 and 2005 were also analysed by PCR-RFLP or a new nested PCR to detect the p1 gene DNA. Typing analysis of these p1 gene DNAs also showed that the group I p1 gene was not present in specimens taken before 2000, but was present and dominant in specimens taken after 2001. These results indicate that, in Japan, the prevalent type of M. pneumoniae changed from a group II strain to a group I strain around 2002.
Journal of Medical Microbiology 05/2008; 57(Pt 4):469-75. · 2.50 Impact Factor
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ABSTRACT: Previously, we reported the establishment of cells with persistent SARS-CoV infection after apoptotic events and showed that both JNK and PI3K/Akt signaling pathways are important for persistence by treatment with inhibitors at the early stages of SARS-CoV infection. However, the mechanisms of establishment of persistent infection are still unclear. In this study, we investigated which signaling pathways play important roles in escape from apoptosis in cells infected with SARS-CoV. In persistently infected cells at 50h.p.i., PI3K/Akt, JNK, p38 MAPK and Bcl-2 were phosphorylated and the protein levels of Bcl-2 and Bcl-xL were increased. When surviving cells were treated with the JNK-specific inhibitor, SP600125, at 50h.p.i., all cells died, suggesting that the JNK signaling pathway is necessary for maintenance of persistently infected cells. Among the signaling pathways in persistently infected cells, Akt and JNK were phosphorylated in SARS-CoV-nucleocapsid (N) protein-expressing Vero E6 cells using vaccinia viral vector (DIs), strongly suggesting that N protein-induced phosphorylation of Akt and JNK are necessary to establish persistence. These results indicated that at least four proteins, Akt, JNK, Bcl-2 and Bcl-xL, are necessary for survival of persistently SARS-CoV-infected cells.
Biochemical and Biophysical Research Communications 09/2006; 347(1):261-5. · 2.48 Impact Factor
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ABSTRACT: Passive agglutination (PA) and immunoglobulin M (IgM), IgA, and IgG enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Mycoplasma pneumoniae were compared with PCR testing of sputum samples obtained from children with lower respiratory tract infections. The sensitivity and specificity of PA were 80.3% and 92.3% at a titer of 1:80. ELISA was found to be less sensitive than PA.
Clinical and Vaccine Immunology 07/2006; 13(6):708-10. · 2.55 Impact Factor
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ABSTRACT: Macrolide-resistant Mycoplasma pneumoniae (MR M. pneumoniae) has been isolated from clinical specimens in Japan since 2000. A comparative study was carried out to determine whether or not macrolides are effective in treating patients infected with MR M. pneumoniae. The clinical courses of 11 patients with MR M. pneumoniae infection (MR patients) treated with macrolides were compared with those of 26 patients with macrolide-susceptible M. pneumoniae infection (MS patients). The total febrile days and the number of febrile days during macrolide administration were longer in the MR patients than in the MS patients (median of 8 days versus median of 5 days [P = 0.019] and 3 days versus 1 day [P = 0.002], respectively). In addition, the MR patients were more likely than the MS patients to have had a change of the initially prescribed macrolide to another antimicrobial agent (63.6% versus 3.8%; odds ratio, 43.8; P < 0.001), which might reflect the pediatrician's judgment that the initially prescribed macrolide was not sufficiently effective in these patients. Despite the fact that the febrile period was prolonged in MR patients given macrolides, the fever resolved even when the initial prescription was not changed. These results show that macrolides are certainly less effective in MR patients.
Antimicrobial Agents and Chemotherapy 02/2006; 50(2):709-12. · 4.84 Impact Factor
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ABSTRACT: To examine the participation of P1 adhesin in gliding of Mycoplasma pneumoniae, we examined the effects of an anti-P1 monoclonal antibody on individual gliding mycoplasmas. The antibody reduced the gliding speed and removed the gliding cells from the glass over time in a concentration-dependent manner but had only a slight effect on nongliding cells, suggesting that the conformational changes of P1 adhesin and its displacement are involved in the gliding mechanism.
Journal of Bacteriology 04/2005; 187(5):1875-7. · 3.83 Impact Factor
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Mayumi Matsuoka,
Mitsuo Narita,
Norio Okazaki,
Hitomi Ohya,
Tsutomu Yamazaki,
Kazunobu Ouchi,
Isao Suzuki,
Tomoaki Andoh, Tsuyoshi Kenri,
Yuko Sasaki,
Atsuko Horino,
Miharu Shintani,
Yoshichika Arakawa,
Tsuguo Sasaki
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ABSTRACT: In recent years, Mycoplasma pneumoniae strains that are clinically resistant to macrolide antibiotics have occasionally been encountered in Japan. Of 76 strains of M. pneumoniae isolated in three different areas in Japan during 2000 to 2003, 13 strains were erythromycin (ERY) resistant. Of these 13 strains, 12 were highly ERY resistant (MIC, > or =256 microg/ml) and 1 was weakly resistant (MIC, 8 microg/ml). Nucleotide sequencing of domains II and V of 23S rRNA and ribosomal proteins L4 and L22, which are associated with ERY resistance, showed that 10 strains had an A-to-G transition at position 2063 (corresponding to 2058 in Escherichia coli numbering), 1 strain showed A-to-C transversion at position 2063, 1 strain showed an A-to-G transition at position 2064, and the weakly ERY-resistant strain showed C-to-G transversion at position 2617 (corresponding to 2611 in E. coli numbering) of domain V. Domain II and ribosomal proteins L4 and L22 were not involved in the ERY resistance of these clinical M. pneumoniae strains. In addition, by using our established restriction fragment length polymorphism technique to detect point mutations of PCR products for domain V of the 23S rRNA gene of M. pneumoniae, we found that 23 (24%) of 94 PCR-positive oral samples taken from children with respiratory infections showed A2063G mutation. These results suggest that ERY-resistant M. pneumoniae infection is not unusual in Japan.
Antimicrobial Agents and Chemotherapy 12/2004; 48(12):4624-30. · 4.84 Impact Factor
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ABSTRACT: Mycoplasma pneumoniae lacks a cell wall but has internal cytoskeleton-like structures that are assumed to support the attachment organelle and asymmetric cell shape of this bacterium. To explore the fine details of the attachment organelle and the cytoskeleton-like structures, a fluorescent-protein tagging technique was applied to visualize the protein components of these structures. The focus was on the four proteins--P65, HMW2, P41, and P24--that are encoded in the crl operon (for "cytadherence regulatory locus"), which is known to be essential for the adherence of M. pneumoniae to host cells. When the P65 and HMW2 proteins were fused to enhanced yellow fluorescent protein (EYFP), a variant of green fluorescent protein, the fused proteins became localized at the attachment organelle, enabling visualization of the organelles of living cells by fluorescence microscopy. The leading end of gliding M. pneumoniae cells, expressing the EYFP-P65 fusion, was observed as a focus of fluorescence. On the other hand, when the P41 and P24 proteins were labeled with EYFP, the fluorescence signals of these proteins were observed at the proximal end of the attachment organelle. Coexpression of the P65 protein labeled with enhanced cyan fluorescent protein clearly showed that the sites of localization of P41 and P24 did not overlap that of P65. The localization of P41 and P24 suggested that they are also cytoskeletal proteins that function in the formation of unknown structures at the proximal end of the attachment organelle. The fluorescent-protein fusion technique may serve as a powerful tool for identifying components of cytoskeleton-like structures and the attachment organelle. It can also be used to analyze their assembly.
Journal of Bacteriology 11/2004; 186(20):6944-55. · 3.83 Impact Factor
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Tsuyoshi Kenri
Nippon rinsho. Japanese journal of clinical medicine 04/2003; 61 Suppl 3:772-8.
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ABSTRACT: Mycoplasma penetrans is a newly identified species of the genus MYCOPLASMA: It was first isolated from a urine sample from a human immunodeficiency virus (HIV)-infected patient. M. penetrans changes its surface antigen profile with high frequency. The changes originate from ON<==>OFF phase variations of the P35 family of surface membrane lipoproteins. The P35 family lipoproteins are major antigens recognized by the human immune system during M. penetrans infection and are encoded by the mpl genes. Phase variations of P35 family lipoproteins occur at the transcriptional level of mpl genes; however, the precise genetic mechanisms are unknown. In this study, the molecular mechanisms of surface antigen profile change in M. penetrans were investigated. The focus was on the 46-kDa protein that is present in M. penetrans strain HF-2 but not in the type strain, GTU. The 46-kDa protein was the product of a previously reported mpl gene, pepIMP13, with an amino-terminal sequence identical to that of the P35 family lipoproteins. Nucleotide sequencing analysis of the pepIMP13 gene region revealed that the promoter-containing 135-bp DNA of this gene had the structure of an invertible element that functioned as a switch for gene expression. In addition, all of the mpl genes of M. penetrans HF-2 were identified using the whole-genome sequence data that has recently become available for this bacterium. There are at least 38 mpl genes in the M. penetrans HF-2 genome. Interestingly, most of these mpl genes possess invertible promoter-like sequences, similar to those of the pepIMP13 gene promoter. A model for the generation of surface antigenic variation by multiple promoter inversions is proposed.
Journal of Bacteriology 02/2003; 185(1):231-42. · 3.83 Impact Factor
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Yuko Sasaki,
Jun Ishikawa,
Atsushi Yamashita,
Kenshiro Oshima, Tsuyoshi Kenri,
Keiko Furuya,
Chie Yoshino,
Atsuko Horino,
Tadayoshi Shiba,
Tsuguo Sasaki,
Masahira Hattori
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ABSTRACT: The complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF-2 strain, was determined. The HF-2 genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs), one set of rRNA genes and 30 tRNA genes. Among the 1038 CDSs, 264 predicted proteins are common to the Mycoplasmataceae sequenced thus far and 463 are M.penetrans specific. The genome contains the two-component system but lacks the essential cellular gene, uridine kinase. The relatively large genome of M.penetrans HF-2 among mycoplasma species may be accounted for by both its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all CDSs. The largest paralog family is the p35 family, which encodes surface lipoproteins including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them form one large cluster in the chromosome. The genetic tree of p35 paralogs suggests the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M.penetrans HF-2 may have acquired diverse repertoires of antigenic variation-related genes to allow its persistent infection in humans.
Nucleic Acids Research 01/2003; 30(23):5293-300. · 8.03 Impact Factor
