Enhancement of cytotoxicity against Vero E6 cells persistently infected with SARS-CoV by Mycoplasma fermentans.

We previously reported that cells with persistent severe acute respiratory syndrome coronavirus (SARS-CoV) infection were established after apoptotic events. In the present study, we investigated the cytopathic effects of dual infection with SARS-CoV and Mycoplasma fermentans on Vero E6 cells. Dual infection completely killed cells and prevented the establishment of persistent SARS-CoV infection. M. fermentans induced inhibition of cell proliferation, but the cells remained alive. Apoptosis was induced easily in M. fermentans-infected cells, indicating that they were primed for apoptosis. These results indicated that M. fermentans enhances apoptosis in surviving cells that have escaped from SARS-CoV-induced apoptosis.

Characteristics of macrolide-resistant Mycoplasma pneumoniae strains isolated from patients and induced with erythromycin in vitro.

Some patients with Mycoplasma pneumoniae infection are clinically resistant to antibiotics such as erythromycin, clarithromycin, or clindamycin. We isolated M. pneumoniae from such patients and found that one of three isolates showed a point mutation in the 23S rRNA gene. Furthermore, 141 EM-sensitive clinical isolates of M. pneumoniae were cultured in broth medium containing 100 microg/ml of erythromycin (EM). Among 11 EM-resistant strains that grew in the medium, point mutations in the 23S rRNA were found in 3 strains at A2063G, 5 strains at A2064G and 3 strains at A2064C. The relationship between the point mutation pattern of these EM-resistant strains and their resistance phenotypes to several macrolide antibiotics was investigated.

Visualization of the attachment organelle and cytadherence proteins of Mycoplasma pneumoniae by immunofluorescence microscopy.

A method was developed for protein localization in Mycoplasma pneumoniae by immunofluorescence microscopy. The P1 adhesin protein was revealed to be located at least at one cell pole in all adhesive cells, as has been observed by immunoelectron microscopy. Cell images were classified according to P1 localization and assigned by DNA content. Cells with a single P1 focus at one cell pole had a lower DNA content than cells with two foci, at least one of which was positioned at a cell pole. Those with one focus at each cell pole had the highest DNA content, suggesting that the nascent attachment organelle is formed next to the old one and migrates to the opposite cell pole before cell division. Double staining revealed that the accessory proteins for cytadherence-HMW1, HMW3, P30, P90, P40, and P65-colocalized with the P1 adhesin in all cells. The localization of cytadherence proteins was also examined in cytadherence-deficient mutant cells with a branched morphology. In M5 mutant cells, which lack the P90 and P40 proteins, HMW1, HMW3, P1, and P30 were focused at the cell poles of short branches, and P65 showed no signal. In M7 mutant cells, which produce a truncated P30 protein, HMW1, HMW3, P1, P90, and P40 were focused, and P65 showed no signal. In M6 mutant cells, which express no HMW1 and a truncated P30 protein, the P1 adhesin was distributed throughout the entire cell body, and no signal was detected for the other proteins. These results suggest that the cytadherence proteins are sequentially assembled to the attachment organelle with HMW1 first, HMW3, P1, P30, P90, and P40 next, and P65 last.

[Clinicobacteriological studies on the nine cases with upper genital tract Mycoplasma hominis infection].

Clinicobacteriological characteristics of nine cases isolated Mycoplasma hominis from the genital tract were studied, and the following results were obtained: elevation of IgG antibodies to M. hominis was measured by ELISA in all cases, but in the MI method only one case showed an elevation of metabolic inhibitory antibody. Convalescent sera from seven patients showed additional and high density bands which were not recognized by acute phase sera in immunoblotting. It was thought that in two patients M. hominis was a causal bacteria for pelvic inflammatory disease (PID). In three cases, it was suggested that M. hominis was related to a premature delivery and idiopathic labor. As infectious symptoms, two patients had body temperatures of more than 38 degrees C but other cases showed 37-37.8 degrees C. Though all cases showed an elevation of CRP, six elevations were slight. As a medication beta-lactam agents were administrated, but their efficacy was not recognized. Furthermore, two patients showed spontaneous recovery in spite of improper antimicrobial agents administration or drainage combined with antimicrobial agents. From the above results. It was thought that M. hominis played a causative role in upper genital tract infection.

Identification of a new variable sequence in the P1 cytadhesin gene of Mycoplasma pneumoniae: evidence for the generation of antigenic variation by DNA recombination between repetitive sequences.

A Mycoplasma pneumoniae cytadhesin P1 gene with novel nucleotide sequence variation has been identified. Four clinical strains of M. pneumoniae were found to carry this type of P1 gene. This new P1 gene is similar to the known group II P1 genes but possesses novel sequence variation of approximately 300 bp in the RepMP2/3 region. The position of the new variable region is distant from the previously reported variable regions known to differ between group I and II P1 genes. Two sequences closely homologous to this new variable region were found within the repetitive sequences outside the P1 gene of the M. pneumoniae M129 genome. This suggests that the new P1 gene was generated by DNA recombination between repetitive sequences and the P1 gene locus. The finding of this new type of P1 gene supports the hypothesis that the repetitive sequences of the M. pneumoniae genome serve as a reservoir to generate antigenic variation of the cytadhesin P1 gene.

Direct evidence of autosomal recessive inheritance of Arg24 to termination codon in purine nucleoside phosphorylase gene in a family with a severe combined immunodeficiency patient.

Purine nucleoside phosphorylase (PNP) deficiency is a rare immunodeficiency disease involving a T-lymphocyte-dysfunction that is fatal unless bone marrow transplantation is successful. In this study we undertook genetic analysis of a patient with PNP deficiency. Sequencing of the PNP gene, which is located on chromosome 14ql3, of the patient led to the identification of three point mutations in exon 2 at amino acid positions 20 (His, silent mutation), 24 (Arg-->termination codon) and 51 (Ser-->Gly). Intrafamilial sequence analysis of exon 2 revealed that both parents were heterozygous for the Arg24 and termination codon 24 alleles. Two of their three children had inherited different homozygous alleles, termination codon 24 for the patient, and Arg24 for his healthy sibling. Transcriptional termination was suggested as the mechanism giving rise to the disorder in this case. A lack of PNP protein was also confirmed by immunoblot analysis of the patient's hemolysate. This could be the first report providing evidence of autosomal recessive inheritance in PNP deficiency by sequence-based analysis.

Identification and characterization of HU protein from Mycoplasma gallisepticum.

A hypothetical ORF of Mycoplasma gallisepticum with a putative 99-amino-acid product (ORF99) was noted previously in the upstream region from the type II topoisomerase gene. The amino acid sequence shows weak homology with the Escherichia coli histone-like protein HU. To identify and characterize the protein product of ORF99, we prepared mouse antiserum against recombinant GST-ORF99 fusion protein. The antiserum reacted with an 11-kDa peptide in the crude cell extract of M. gallisepticum, indicating that this protein is an ORF99 product. ORF99 protein binds to DNA, although its binding affinity is weaker than that of E. coli HU. When ORF99 was cloned in a plasmid and expressed in E. coli cells depleted of HU, Mu phage growth was strongly promoted in the cells, showing the presence of HU activity. The effect of IHF mutation was suppressed when a high level of ORF99 protein was expressed in an E. coli mutant deficient in IHF.

Two patients with premature labour associated with Mycoplasma hominis infection.

Because several reports have suggested that bacterial vaginosis causes premature labour and early rupture of the fetal membranes, the presence of a bacterial flora that causes bacterial vaginosis is thought to be a risk factor for premature labour. The present study investigated two patients with premature delivery and intra-uterine Mycoplasma hominis infection. In microbiological studies, Gram's staining of amniotic fluids revealed numerous neutrophils and epithelial cells but no micro-organisms. Culture of amniotic fluid before antibiotic therapy yielded only M. hominis under anaerobic conditions; aerobic culture was negative. Vaginal discharge taken on the day of delivery yielded no growth in case 1 and M. hominis and Enterococcus faecalis in case 2. Maternal sera showed specific antibodies to M. hominis by ELISA and immunoblotting. As no possible cause of premature labour other than M. hominis infection was detected, it is concluded that the intra-uterine M. hominis infection was associated with premature labour in these patients.

Evaluation of a new method for identification of bacteria based on sequence homology of 16S rRNA gene.

A new identification method for bacteria based on partial sequences of divergent regions of the 16S rRNA gene was evaluated. The method involves PCR-based amplification of 16S rRNA gene fragments, followed by sequencing and comparison of sequences of about 300 nucleotides with those in the database of NCBI (National Center for Biotechnology Information) via the Internet. Most of the bacteria tested could be identified at the species level even if some unread nucleotides were present in the sequence. Although this method still requires improvement, it has the potential to be a highly reliable and practical identification method for bacteria.

Application of PCR for detection of mycoplasma DNA and pestivirus RNA in human live viral vaccines.

PCR techniques were applied for the detection of mycoplasma DNA and pestivirus RNA to 43 lots of live viral vaccines (measles, mumps, rubella, and oral poliomyelitis) produced by six manufacturers in Japan. Although mycoplasma DNA was not detected in any of the vaccines tested, pestivirus RNA was detected in 12 lots (28%). The incidence of contamination among the four viral vaccines was in the range of 20 to 37%, and the incidence among the six manufacturers varied from 0 to 56%.

Evaluation of current sterility tests for human live viral vaccines.

Current sterility tests for human viral vaccines were evaluated. A total of 43 lots of bulk suspension of live viral vaccines (measles, mumps, rubella and oral poliomyelitis) produced by six manufacturers in Japan were evaluated for bacteriostatic and mycoplasmastatic activities. Some of them showed fairly high bacteriostatic and mycoplasmastatic activities, due to antibiotics added during vaccine production. It was concluded that the current sterility test for mycoplasmas is not reliable to detect viable mycoplasmas in live viral vaccines.

Epidemiological study of Mycoplasma pneumoniae infections in japan based on PCR-restriction fragment length polymorphism of the P1 cytadhesin gene.

Two hundred fifty strains of Mycoplasma pneumoniae isolated during the past 20 years in Japan were classified into two groups (I and II) based upon different PCR-restriction fragment length polymorphism patterns of their P1 cytadhesin genes. Clear shifts between the M. pneumoniae groups were observed but did not appear to be correlated with M. pneumoniae epidemic cycles. Patients' sera showed relatively higher levels of antiadhesin antibodies to M. pneumoniae strains homologous with the infecting strain.

Three tandemly repeated structural genes encoding tRNA(f1Met) in the metZ operon of Escherichia coli K-12.

The metZ operon of Escherichia coli K-12 consists of three tandemly repeated structural genes encoding tRNA(f1Met) separated by two well-conserved spacer sequences. A multicopy plasmid carrying an intact metZ operon is unstable, deleting tRNA(f1Met)-coding regions by RecA-dependent recombination during cell growth.

Construction and characterization of an Escherichia coli mutant deficient in the metY gene encoding tRNA(f2Met): either tRNA(f1Met) or tRNA(f2Met) is required for cell growth.

The Escherichia coli metY gene, encoding tRNA(f2Met), was split by the kanamycin-resistance-encoding gene. The resulting mutant exhibited the same growth rate as the wild type, indicating that tRNA(f2Met) is not indispensable as is the case with the metZ gene encoding tRNA(f1Met) [Kenri et al., Gene 103 (191) 31-36]. beta-Galactosidase was produced efficiently from the start codon AUG of the intact lacZ gene or a trpA'::lac'Z fusion gene, in the metY mutant. The lac repressor from the lacI gene and the chimeric protein from a hupB'::lac'Z fusion gene, whose start codons are GUG, were also synthesized efficiently in the insertion mutant. These results provide evidence that tRNA(f2Met) is not essential for growth of E. coli and that the start codons, AUG and GUG, are both recognized by tRNA(f1Met), a major N-formyl methionine-specific tRNA, in the tRNA(f2Met)-depleted cells. We were unable to construct mutants deficient in both tRNA(f1Met) and tRNA(f2Met) by P1 phage-mediated transduction with the metY and metZ mutations. Moreover, the ampicillin-resistance marker of the pUC9 plasmid carrying metZ+ was not cured at 42 degrees C in host cells with the polAts and metY-metZ double mutations. These results indicate that either tRNA(f1Met) or tRNA(f2Met) is required for the growth of E. coli.

Construction and characterization of an Escherichia coli mutant with a deletion of the metZ gene encoding tRNA (f1Met).

The Escherichia coli metZ gene encoding tRNA (f1Met) was replaced by the chloramphenicol-resistance-encoding gene. The resulting mutant exhibited slightly lower growth rates as compared to the wild type at 37 degrees C or 42 degrees C, but grew apparently slower than the latter at 30 degrees C, indicating a slight cold sensitivity of growth. beta-Galactosidase was produced efficiently from the start codon AUG of the intact lacZ gene or trpA'::lac' Z fusion gene, in the metZ deletion mutant. The lac repressor from the lacI gene and the chimeric protein from a hupB' ::lac'Z fusion gene, whose start codons are GUG, were also synthesised in the deletion mutant. These results provide evidence that tRNA (f1Met) is not essential for growth of E. coli and that the start codons, AUG and GUG, are both recognized by tRNA (f1Met), a minor N-formyl methionine-specific tRNA, in the tRNA (f1Met)-depleted cells.