The evolution of Mycoplasma genitalium

Mycoplasma genitalium is the smallest microorganism capable of self-replication. With its small genome, M. genitalium is the best representative of a minimal cell. The comparison of genome evolution among the three urogenital mycoplasmas, M. genitalium, M. hominis, and Ureaplasma parvum, not only indicated that they share a core genome of ~250 protein-encoding genes that correspond to their basic cell metabolism, but also showed a striking difference in their energy-generating pathways. M. genitalium is a sexually transmitted organism associated with nongonococcal urethritis in men and several inflammatory reproductive tract syndromes in women, such as cervicitis, pelvic inflammatory disease, and infertility. The treatment of M. genitalium infections has not yet been standardized. Macrolides are recommended, especially single-dose azithromycin; tetracyclines are responsible for a large number of therapeutic failures without any acquired resistance demonstrated. Acquired resistance to macrolides and fluoroquinolones leading to therapeutic failure has been described.     

Genome size of Mycoplasma genitalium.

The genome size of Mycoplasma genitalium was determined by using restriction enzymes that infrequently cut its DNA. The calculated value of 577 to 590 kilobases is one-fourth smaller than the genome of Mycoplasma pneumoniae, which is considered among the smallest genomes of self-replicating organisms.     

Adherence epitopes of Mycoplasma genitalium adhesin.

The adherence-mediating sites of the 153 kDa adhesin of Mycoplasma genitalium (MgPa-protein) were characterized at the amino acid sequence level using six monoclonal anti-MgPa antibodies which showed adherence-inhibiting activity. For characterization of the regions to which antibody bound, three segments of the adhesin (N-terminal region, a D1-domain located approximately in the middle of the molecule and a D2-domain located near to the C-terminus) were synthesized as overlapping octapeptides. These regions were chosen in analogy to the three domains of Mycoplasma pneumoniae that are involved in the adhesion process. Whereas two monoclonal antibodies (mAb 5B11 and mAb 6F3) bound exclusively to an epitope in the N-region, mAb 3B7 and mAb 6A2 reacted with two distinct epitopes of the D2-domain only. Binding to short synthetic peptides of different regions was analysed for mAb 3A12 (N-region and D1-region) and mAb 2B6 (N-region and D2-region). Close proximity of the N-region and the D2-region in the native MgPa-protein of M. genitalium was indicated in a competitive ELISA test, using freshly harvested M. genitalium cells. Epitope mapping and competition experiments with monoclonal anti-MgPa antibodies revealed interesting differences in the adherence-mediating sites of MgPa and the adhesin (P1-protein) of M. pneumoniae. Whereas a three-dimensional arrangement of protein loops is suggested for both native adhesins, the MgPa-protein and the P1-protein adherence-mediating epitopes are located in non-homologous regions of these two related proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics

The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).     

Persistence of Mycoplasma genitalium following azithromycin therapy

Background

To determine clinical outcomes and cure rates for M.genitalium genital infection in men and women following azithromycin 1 g.

Methodology

Patients attending Melbourne Sexual Health Centre between March 2005 and November 2007 with urethritis/epididymitis, cervicitis/pelvic inflammatory disease and sexual contacts of M.genitalium were tested for M.genitalium by polymerase chain reaction (PCR). M.genitalium-infection was treated with 1 g of azithromycin and a test-of-cure (toc) was performed one month post-azithromycin. Response to azithromycin, and response to moxifloxacin (400 mg daily for 10 days) in individuals with persistent infection post-azithromycin, was determined.

Principal Findings

Of 1538 males and 313 females tested, 161 males (11%) and 30 females (10%) were infected with M.genitalium. A toc was available on 131 (69%) infected individuals (median = 36 days [range 12-373]). Of 120 individuals prescribed azithromycin only pre-toc, M.genitalium was eradicated in 101 (84%, 95% confidence intervals [CI]: 77–90%) and persisted in 19 (16%, 95% CI: 10–23%). Eleven individuals with persistent infection (9%, 95% CI: 5–15%) had no risk of reinfection from untreated-partners, while eight (7%, 95% CI: 3–12%) may have been at risk of reinfection from doxycycline-treated or untreated-partners. Moxifloxacin was effective in eradicating persistent infection in all cases not responding to azithromycin. Patients with persistent-M.genitalium were more likely to experience persistent symptoms (91%), compared to patients in whom M.genitalium was eradicated (17%), p<0.0001.

Conclusion

Use of azithromycin 1 g in M.genitalium-infected patients was associated with unacceptable rates of persistent infection, which was eradicated with moxifloxacin. These findings highlight the importance of follow-up in M.genitalium-infected patients prescribed azithromycin, and the need to monitor for the development of resistance. Research to determine optimal first and second-line therapeutic agents for M.genitalium is needed.     

A minimized motile machinery for Mycoplasma genitalium

The cell wall‐less bacterium Mycoplasma genitalium uses specialized adhesins located at the terminal organelle to adhere to host cells and surfaces. The terminal organelle is a polar structure protruding from the cell body that is internally supported by a cytoskeleton and also has an important role in cell motility. We have engineered a M. genitalium null mutant for MG491 protein showing a massive downstream destabilization of proteins involved in the terminal organelle organization. This mutant strain exhibited striking similarities with the previously isolated MG_218 null mutant strain. Upon introduction of an extra copy of MG_318 gene in both strains, the amount of main adhesins P140 and P110 dramatically increased. These strains were characterized by microcinematography, epifluorescence microscopy and cryo‐electron microcopy, revealing the presence of motile cells and filaments in the absence of many proteins considered essential for cell adhesion and motility. These results indicate that adhesin complexes play a major role in the motile machinery of M. genitalium and demonstrate that the rod element of the cytoskeleton core is not the molecular motor propelling mycoplasma cells. These strains containing a minimized motile machinery also provide a valuable cell model to investigate the adhesion and gliding properties of this human pathogen.     

Treatment of Mycoplasma genitalium. Observations from a Swedish STD Clinic

Objectives

To evaluate therapy for Mycoplasma genitalium infection with doxycycline or azithromycin 1 g compared to five days of azithromycin (total dose 1.5 g).

Methods

A retrospective case study was performed among patients attending the STD-clinic in Falun, Sweden 1998–2005. All patients with a positive PCR test for M. genitalium were routinely offered a test of cure (toc). Response to doxycycline for 9 days, azithromycin 1 g single dose and extended azithromycin (500 mg on day 1 followed by 250 mg o.d. for 4 days) was determined. In patients with treatment failure after azithromycin, macrolide resistance was monitored before and after treatment. Furthermore, the rate of macrolide resistance was monitored for positive specimens available from 2006–2011.

Results

The eradication rate after doxycycline was 43% (48% for women and 38% for men), for azithromycin 1 g 91% (96% for women and 88% for men) and for extended azithromycin 99% (100% for women and 93% for men). Macrolide resistance developed in 7/7 examined (100%) of those testing positive after azithromycin 1 g, but in none of those treated with extended azithromycin. Macrolide resistance before treatment increased from 0% in 2006 and 2007 to 18% in 2011.

Conclusions

These findings confirm the results from other studies showing that doxycycline is inefficient in eradicating M. genitalium. Although azithromycin 1 g was not significantly less efficient than extended dosage, it was associated with selection of macrolide resistant M. genitalium strains and should not be used as first line therapy for M. genitalium. Monitoring of M. genitalium macrolide resistance should be encouraged.     

Detection and discrimination of Mycoplasma pneumoniae and Mycoplasma genitalium by the in vitro DNA amplification.

By using the primers designed on the bases of the sequences of the 16S rRNA genes of Mycoplasma pneumoniae and Mycoplasma genitalium, respectively, specific and sensitive in vitro DNA amplification assay system for the detection and discrimination of these two mycoplasmas was established. The detection limit of the assay was 100 cells for M. pneumoniae and 1,000 cells for M. genitalium. Neither other human mycoplasmas nor oral bacteria existing in human saliva showed any cross-reactions with these primers.     

Functional characterization of the RuvB homologs from Mycoplasma pneumoniae and Mycoplasma genitalium

Homologous recombination between repeated DNA elements in the genomes of Mycoplasma species has been hypothesized to be a crucial causal factor in sequence variation of antigenic proteins at the bacterial surface. To investigate this notion, studies were initiated to identify and characterize the proteins that form part of the homologous DNA recombination machinery in Mycoplasma pneumoniae as well as Mycoplasma genitalium. Among the most likely participants of this machinery are homologs of the Holliday junction migration motor protein RuvB. In both M. pneumoniae and M. genitalium, genes have been identified that have the capacity to encode RuvB homologs (MPN536 and MG359, respectively). Here, the characteristics of the MPN536- and MG359-encoded proteins (the RuvB proteins from M. pneumoniae strain FH [RuvB(FH)] and M. genitalium [RuvB(Mge)], respectively) are described. Both RuvB(FH) and RuvB(Mge) were found to have ATPase activity and to bind DNA. In addition, both proteins displayed divalent cation- and ATP-dependent DNA helicase activity on partially double-stranded DNA substrates. The helicase activity of RuvB(Mge), however, was significantly lower than that of RuvB(FH). Interestingly, we found RuvB(FH) to be expressed exclusively by subtype 2 strains of M. pneumoniae. In strains belonging to the other major subtype (subtype 1), a version of the protein is expressed (the RuvB protein from M. pneumoniae strain M129 [RuvB(M129)]) that differs from RuvB(FH) in a single amino acid residue (at position 140). In contrast to RuvB(FH), RuvB(M129) displayed only marginal levels of DNA-unwinding activity. These results demonstrate that M. pneumoniae strains (as well as closely related Mycoplasma spp.) can differ significantly in the function of components of their DNA recombination and repair machinery.     

A physical map of the Mycoplasma genitalium genome

We report the construction of a physical map of the genome of the human pathogen Mycoplasma genitalium through the use of pulse-field gel electrophoresis. The small size and relative simplicity of this genome permit the arrangement of restriction fragments without having to construct linking clones. The size of the genome has been calculated to be approximately 600 kb and several important genetic determinants have been assigned specific loci on the map.     

Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins

The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.     

Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N⁶-methyladenine (6 mA) and N⁴-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5′-CTAT-3′), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5′-GAN₇TAY-3′/3′-CTN₇ ATR-5′). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.     

A survey of the Mycoplasma genitalium genome by using random sequencing.

A total of 508 random clones from five Mycoplasma genitalium genomic libraries were partially sequenced and analyzed. This resulted in the identification of 291 unique contigs. Sequence information from these clones (100,993 nucleotides), representing approximately 17% of this pathogen's genome, was analyzed by comparison to the DNA and protein sequence data bases. The frequency with which clones could be identified, by virtue of possessing homology to another data base entry, was 46%. Sequence analysis indicated the following. (i) The M. genitalium genome contains many genes involved in various metabolic processes. (ii) Repetitive DNA may comprise as much as 4% of this genome. (iii) The MgPa adhesin gene may be the result of horizontal transfer from an unknown origin. (iv) Not all dinucleotide pairs are present in this genome at the expected frequency. (v) This genome potentially encodes approximately 390 proteins and makes very efficient use of its limited amount of DNA. In addition, this study allowed us to estimate the number of genes involved with various cellular functions.