Genome sequence of the repetitive-sequence-rich Mycoplasma fermentans strain M64.

Mycoplasma fermentans is a microorganism commonly found in the genitourinary and respiratory tracts of healthy individuals and AIDS patients. The complete genome of the repetitive-sequence-rich M. fermentans strain M64 is reported here. Comparative genomics analysis revealed dramatic differences in genome size between this strain and the recently completely sequenced JER strain.

Molecular cloning and expression of a novel cholinephosphotransferase involved in glycoglycerophospholipid biosynthesis of Mycoplasma fermentans.

A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using alpha-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5'-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.

The importance of B-cells and ecto-5'nucleotidase in Mycoplasma fermentans infection and the relevance to rheumatoid arthritis.

The aim of this work was to discover if Mycoplasma fermentans, which is known to infect B cells, could be the cause of the raised ecto-5'-nucleotidase observed in the synovial fluid of rheumatoid arthritis patients. The ecto-5'-nucleotidase activity in the patients' serum has been shown to correlate with the erythrocyte sedimentation rate and DNA from the mycoplasma has been found in the synovial fluid. B lymphoblastoid cell lines were exposed to 16 strains of Mycoplasma fermentans and their ecto-5'-nucleotidase, CD73, was measured both biochemically and by mouse antibodies to human ecto 5'-nucleotidase using the fluorescence activated cell sorter. The type strain, PG 18, did not grow with the B cells. Some of the mycoplasma strains (9/15) increased the cellular ecto-5'-nucleotidase activity from twice to 17 fold, and usually showed 5'-nucleotidase activity themselves. At least one strain, M106, induced human 5'-nucleotidase on the normally 5'-nucleotidase negative Daudi and Raji Burkitt's lymphoma cell lines, and increased sevenfold the 5'-nucleotidase on the monocyte/macrophage cell line THP-1. Growing the cells in aged medium increased the level of mycoplasma infection. This mycoplasma-induced enzyme showed a conformational change and an increase in activity with a glycosylation change involving mannose groups. The other group of strains, mostly of respiratory or cell culture origin, usually did not have any 5'-nucleotidase of their own and decreased the B-cell enzyme activity by about half. Electron microscopy and flow cytometry showed that the strain M106 was filamentous and could be found inside the B-cells. The 5'-nucleotidase-inducing strains of M. fermentans may be important in the aetiology of rheumatoid arthritis.

An evaluation of PCR methods to detect strains of Mycoplasma fermentans.

A panel of 30 putative Mycoplasma fermentans strains, isolated from various sources including human, ovine and cell lines, were tested by a previously described polymerase chain reaction (PCR) to confirm their identity by amplification of a conserved 206 bp region of the insertion sequence IS1550. In addition, the application of another PCR based on the major part of the IS1550 element showed one or two products of different length (1144 and 1341 bp) enabling M. fermentans strains to be divided into two types designated as Type A and Type B. A PCR, which amplifies the macrophage activating lipopeptide gene (malp), supported the identification of all the strains as M. fermentans. Thirteen other species of Mycoplasma from human sources gave negative results in these tests, with the exception of Mycoplasma orale, which was detected by both IS1550-PCRs based on the major part and the conserved 206 bp region of the IS1550 element. This study suggests that all M. fermentans isolates possess both the IS1550 element and the malp gene. In contrast to the IS1550, the malp gene is shown to be species-specific and the use of a malp PCR described here could prove to be a useful adjunct to IS1550 detection as confirmation of the presence of M. fermentans in clinical material.

Genotypic characterization of seven strains of Mycoplasma fermentans isolated from synovial fluids of patients with arthritis.

We performed a genotypic characterization of seven strains of Mycoplasma fermentans which have been isolated from the synovial fluid of patients with rheumatoid arthritis (n = 2), spondyloarthropathy (n = 1), and unclassified arthritis (n = 4). We compared them to three reference strains (strains PG18 and K7 and incognitus strain) and to a clinical isolate from the urethra of a patient with nongonococcal urethritis. The characterization methods included electrophoresis of native DNA, arbitrarily primed PCR, and restriction fragment length polymorphism analysis following conventional and pulsed-field gel electrophoresis. Southern blot analysis with a probe internal to an insertion sequence was performed with the restriction products produced by the last two techniques. No extrachromosomal DNA sequences were detected. The M. fermentans strains identified by these methods did not present a unique profile, but they could be separated into two main categories: four articular isolates were genetically related to PG18 and the three other isolates, the urethral isolate, and the incognitus strain were related to K7. We also looked for the presence of the bacteriophage MAV1 (associated with the arthritogenic property of Mycoplasma arthritidis in rodents) in the M. fermentans strains. MAV1 DNA was not detected in either the clinical isolates or the reference strains of M. fermentans.

Genotypic and phenotypic analysis of Mycoplasma fermentans strains isolated from different host tissues.

A correlation was found between the expression of a specific Mycoplasma fermentans surface antigen (Pra, proteinase-resistant antigen) and the site of isolation of the organism from the infected host. Strains which expressed Pra were most frequently associated with cells of bone marrow origin, and strains which lacked expression of Pra were most commonly isolated from the respiratory tract, genital tract, and arthritic joints, i.e., epithelial cell surfaces. Pra was previously shown to be resistant to degradation by proteinases and was hypothesized to play a protective role at the organism surface and perhaps to influence which host tissue site was colonized by the organism. The methods used for this phenotyping scheme required isolation and growth of the mycoplasma in quantities sufficient for immunoblot analysis using monoclonal antibodies. We wanted to determine a more rapid and less cumbersome technique to supplement this method for determining the Pra phenotype directly in clinical specimens. Here we describe PCR studies to investigate the movement of a previously identified M. fermentans insertion sequence (IS)-like element. These data showed a correlation between a specific IS genotype and the Pra+ phenotype. Production of a 160-bp product using a single set of IS-based primers was associated with expression of Pra. The genomic IS location resulting in the 160-bp product was determined by using Southern blot analysis and was found to be a stable insertion site characteristic of genotype I strains. Additional analyses of sequences within and flanking the IS insertion sites revealed another pair of PCR primer sites which resulted in the consistent production of a 450-bp amplicon. The stability of this site was dependent on the absence of the IS-like element between the primer sites. The production of this 450-bp amplicon correlated with the Pra mutant phenotype and was characteristic of genotype II strains. The data showed that the sequence within the IS may be unstable and that reliable genotyping sequences are more easily found in the stable genomic sites which flank the IS element.

Differentiation of strains of Mycoplasma fermentans from various sources by pyrolysis mass spectrometry.

Mycoplasma fermentans has attracted much interest both as a cofactor for the progression of AIDS and as a pathogenic agent in non-AIDS related diseases. Previous studies with serological and genetic techniques suggest that M. fermentans represents a homogeneous group of organisms, with no significant differences identified among the strains examined. In this study, 25 cultures of M. fermentans, including isolates from human sources and tissue culture cells, were compared by pyrolysis mass spectrometry (PMS). It was possible to distinguish the 'type' strain PG-18 from an AIDS-associated M. fermentans strain 'incognitus' by this technique. PMS was also able to differentiate laboratory-induced aminoglycoside-resistant variants from their fully susceptible parents. Four AIDS-associated isolates were distinguished from each other, whilst five European cell culture isolates were shown to be closely related, as were six M. fermentans isolates from an outbreak of acute respiratory infection in Canada. PMS has proved useful in distinguishing isolates of M. fermentans, providing epidemiological data. In addition, PMS may help in determining the likely origin of a given isolate, and in the future may be of use in assessing the role of this micro-organism in human disease.

Detection of serum antibodies against phosphocholine-containing aminoglycoglycerolipid specific to Mycoplasma fermentans in HIV-1 infected individuals.

Mycoplasma fermentans has been associated with acquired immunodeficiency syndrome (AIDS). The lipids extracted from five strains of M. fermentans and eight other species of mycoplasmas were investigated. By using high-performance thin-layer chromatography (HPTLC) and immunostaining on HPTLC-plates with polyclonal and monoclonal antibodies against lipids of M. fermentans, a glycophospholipid GGPL-III was proved to be a specific lipid and important antigen of the species of M. fermentans. Previously we reported that M. fermentans had phosphocholine-containing glycoglycerolipid (GGPL-I and GGPL-III). TLC immunostaining showed that anti-GGPL-III specific antibodies were detected in HIV-1 infected individuals. By employing purified GGPL-III, an enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of specific antibodies to GGPL-III in human serum. This ELISA showed high sensitivity, specificity and good reproducibility. Frequency of antibodies to GGPL-III was significantly higher in sera from HIV-1 infected people (44.6%, 29/65) than from HIV-1 negative healthy donors (1.7%, 2/117) or patients with three kinds of other diseases we investigated (0-8.7%, 0/20-2/23). Thus M. fermentans infection seemed to be correlated with HIV-1 infection although further investigation is essential for clarification of the role of M. fermentans during the long latency period in HIV infected individuals.

Evidence that Lo's mycoplasma (Mycoplasma fermentans incognitus) is not a unique strain among Mycoplasma fermentans strains.

Mycoplasma fermentans incognitus has attracted much interest either as a cofactor for the progression of AIDS or as a pathogenic agent in non-AIDS-related diseases (S.-C. Lo, M. S. Dawson, P. B. Newton III, M. A. Sonoda, J. W.-K. Shih, W. F. Engler, R. Y.-H. Wang, and D. J. Wear, Am. J. Trop. Med. Hyg. 41:364-376, 1989; S.-C. Lo, M. S. Dawson, M. Wong, P. B. Newton III, M. A. Sonoda, W. F. Engler, R.Y.-H Wang, J. W.-K. Shih, H. J. Alter, and D. J. Wear, Am. J. Trop. Med. Hyg. 41:601-616, 1989; S.-C. Lo, J.W.-K. Shih, N.-Y. Yang, C.-Y. Ou, and R. Y.-H. Wang, Am. J. Trop. Med. Hyg. 40:213-226, 1989). In the present study, the genetic and serologic properties of the incognitus strain and other M. fermentans strains were compared. Furthermore, the replication of human immunodeficiency virus type 1 (HIV-1), determined by reverse transcriptase activity and HIV-1 p24 antigen level, in peripheral blood mononuclear cells was evaluated after stimulation with mycoplasma cell lysates. The psb-2.2 viruslike infectious agent DNA probe, used to identify the incognitus strain in the tissues of AIDS and non-AIDS patients by Lo et al. (Am. J. Trop. Med. Hyg. 41:364-376 and 40:213-226, 1989), showed reaction patterns similar to those of two M. fermentans strains isolated from cell cultures but not to that of the type strain PG18. Restriction enzyme patterns of the incognitus strain with EcoRI and HindIII were also similar to those of M. fermentans strains isolated from cell cultures. There were no remarkable differences in the immunoblot profiles between the incognitus strain and the other M. fermentans strains. These results suggest that the incognitus strain is not a unique strain among M. fermentans strains. Further, cell lysates of each M. fermentans strain could also enhance the replication of HIV-1 to a level similar to that of the incognitus strain as determined by the reverse transcriptase activity and the amount of the p24 antigen.