Mycoplasma pneumoniae Community Acquired Respiratory Distress Syndrome toxin expression reveals growth phase and infection-dependent regulation

Mycoplasma pneumoniae causes acute and chronic respiratory infections, including tracheobronchitis and community acquired pneumonia, and is linked to asthma and an array of extra-pulmonary disorders. Recently, we identified an ADP-ribosylating and vacuolating toxin of M. pneumoniae, designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we analysed CARDS toxin gene (annotated mpn372) transcription and identified its promoter. We also compared CARDS toxin mRNA and protein profiles in M. pneumoniae during distinct in vitro growth phases. CARDS toxin mRNA expression was maximal, but at low levels, during early exponential growth and declined sharply during mid-to-late log growth phases, which was in direct contrast to other mycoplasma genes examined. Between 7% and 10% of CARDS toxin was localized to the mycoplasma membrane at mid-exponential growth, which was reinforced by immunogold electron microscopy. No CARDS toxin was released into the medium. Upon M. pneumoniae infection of mammalian cells, increased expression of CARDS toxin mRNA was observed when compared with SP-4 broth-grown cultures. Further, confocal immunofluorescence microscopy revealed that M. pneumoniae readily expressed CARDS toxin during infection of differentiated normal human bronchial epithelial cells. Analysis of M. pneumoniae-infected mouse lung tissue revealed high expression of CARDS toxin per mycoplasma cell when compared with M. pneumoniae cells grown in SP-4 medium alone. Taken together, these studies indicate that CARDS toxin expression is carefully controlled by environmental cues that influence its transcription and translation. Further, the acceleration of CARDS toxin synthesis and accumulation in vivo is consistent with its role as a bona fide virulence determinant

Mycoplasma hyopneumoniae Transcription Unit Organization: Genome Survey and Prediction

Mycoplasma hyopneumoniae is associated with swine respiratory diseases. Although gene organization and regulation are well known in many prokaryotic organisms, knowledge on mycoplasma is limited. This study performed a comparative analysis of three strains of M. hyopneumoniae (7448, J and 232), with a focus on genome organization and gene comparison for open read frame (ORF) cluster (OC) identification. An in silico analysis of gene organization demonstrated 117 OCs and 34 single ORFs in M. hyopneumoniae 7448 and J, while 116 OCs and 36 single ORFs were identified in M. hyopneumoniae 232. Genomic comparison revealed high synteny and conservation of gene order between the OCs defined for 7448 and J strains as well as for 7448 and 232 strains. Twenty-one OCs were chosen and experimentally confirmed by reverse transcription–PCR from M. hyopneumoniae 7448 genome, validating our prediction. A subset of the ORFs within an OC could be independently transcribed due to the presence of internal promoters. Our results suggest that transcription occurs in ‘run-on’ from an upstream promoter in M. hyopneumoniae, thus forming large ORF clusters (from 2 to 29 ORFs in the same orientation) and indicating a complex transcriptional organization

Genetic Variation in the Complete MgPa Operon and Its Repetitive Chromosomal Elements in Clinical Strains of Mycoplasma genitalium

Mycoplasma genitalium has been increasingly recognized as an important microbe not only because of its significant association with human genital tract diseases but also because of its utility as a model for studying the minimum set of genes necessary to sustain life. Despite its small genome, 4.7% of the total genome sequence is devoted to making the MgPa adhesin operon and its nine chromosomal repetitive elements (termed MgPars). The MgPa operon, along with 9 MgPars, is believed to play an important role in pathogenesis of M. genitalium infection and has also served as the main target for development of diagnostic tools. However, genetic variation in the complete MgPa operon and MgPars among clinical strains of M. genitalium has not been addressed. In this study we examined the genetic variation in the complete MgPa operon (approximately 8.5 kb) and full or partial MgPar sequences (0.4–2.6 kb) in 15 geographically diverse strains of M. genitalium. Extensive variation was present in four repeat regions of the MgPa operon (with homology to MgPars) among and within strains while the non-repeat regions (without homology to MgPars) showed low-level variation among strains and no variation within strains. MgPars showed significant variation among strains but were highly homogeneous within strains, supporting gene conversion as the likely recombination mechanism. When applying our sequence data to evaluate published MgPa operon-based diagnostic PCR assays and genotyping systems, we found that 11 of 19 primers contain up to 19 variable nucleotides and that the target for one of two typing systems is located in a hypervariable repeat region, suggesting the likelihood of false results with some of these assays. This study not only provides new insights into the role of the MgPa operon in the pathogenesis of M. genitalium infection but has important implications for the development of diagnostic tools

Mycoplasma genitalium: An Emerging Cause of Sexually Transmitted Disease in Women

Mycoplasma genitalium is an emerging sexually transmitted pathogen implicated in urethritis in men and several inflammatory reproductive tract syndromes in women including cervicitis, pelvic inflammatory disease (PID), and infertility. This comprehensive review critically examines epidemiologic studies of M. genitalium infections in women with the goal of assessing the associations with reproductive tract disease and enhancing awareness of this emerging pathogen. Over 27,000 women from 48 published reports have been screened for M. genitalium urogenital infection in high- or low-risk populations worldwide with an overall prevalence of 7.3% and 2.0%, respectively. M. genitalium was present in the general population at rates between those of Chlamydia trachomatis and Neisseria gonorrhoeae. Considering more than 20 studies of lower tract inflammation, M. genitalium has been positively associated with urethritis, vaginal discharge, and microscopic signs of cervicitis and/or mucopurulent cervical discharge in seven of 14 studies. A consistent case definition of cervicitis is lacking and will be required for comprehensive understanding of these associations. Importantly, evidence for M. genitalium PID and infertility are quite convincing and indicate that a significant proportion of upper tract inflammation may be attributed to this elusive pathogen. Collectively, M. genitalium is highly prevalent in high- and low-risk populations, and should be considered an etiologic agent of select reproductive tract disease syndromes in women

Analysis Identifying Common and Distinct Sequences among Texas Clinical Strains of Mycoplasma genitalium▿

Mycoplasma genitalium is a human bacterial pathogen linked to urethritis and other sexually transmitted diseases. Here, we assessed the incidence of M. genitalium infection in patients attending a sexually transmitted disease clinic in San Antonio, TX, by use of diagnostic real-time PCR. Overall, 16.8% of women and 15.1% of men were found M. genitalium positive. Regions of the mgpB gene, which encodes the MgPa adhesin, were amplified from positive clinical specimens and evaluated for sequence variability, which demonstrated transmission of the pathogen between sexual partners. Follow-up analysis of a subset of patient specimens revealed reinfection by a different strain of M. genitalium, indicating the absence of protective immunity. Eighteen DNA sequence variants were obtained and compared with all other available clinical sequences. Detailed analysis revealed silent mutations of six amino acid residues within the encoded region of the MgPa adhesin in numerous clinical strains. In addition, missense mutations of limited numbers of amino acids were observed. Alignment of putative amino acid sequences revealed the simultaneous occurrence of several mutations and the existence of identical or similar protein variants in strains from different locations

Determination of Infectious Load of Mycoplasma genitalium in Clinical Samples of Human Vaginal Cells

Mycoplasma genitalium is a leading cause of chlamydia-negative, nongonoccocal urethritis and has been directly implicated in numerous other genitourinary as well as extragenitourinary tract pathologies. Detection of M. genitalium has relied almost entirely on PCR amplification of clinical specimens and evidence of seroconversion since these mycoplasmas are highly fastidious and culture isolation by microbiological techniques is very rare. We have established a combinatorial strategy using confocal immunoanalysis (CIA) and real-time PCR to qualitatively and quantitatively assess patterns of M. genitalium infection in women attending a sexually transmitted disease-related health clinic in San Antonio, Tex. CIA allows spatial examination of mycoplasmas on surfaces and inside human target cells, plus the ability to evaluate cell-to-cell patterns and variances within samples. Real-time PCR permits determination of genome copy numbers of mycoplasmas and human cells by multiplex amplification using mycoplasma gyrA and human RNase P gene sequences, which indicates overall levels of mycoplasma infection and degree of parasitism. These assays are strongly correlated and, in combination, permit detection and elucidation of heretofore-unrecognized patterns of M. genitalium infections in clinical and experimental samples

Genomic Analysis Reveals Mycoplasma pneumoniae Repetitive Element 1-Mediated Recombination in a Clinical Isolate▿

Mycoplasmas are cell wall-less bacteria that evolved by drastic reduction of the genome size. Complete genome analysis of Mycoplasma pneumoniae revealed the presence of numerous copies of four distinct large M. pneumoniae repetitive elements (RepMPs). One copy each of RepMP2/3, RepMP4, and RepMP5 are localized within the P1 operon (MPN140 to MPN142 loci), and their involvement in sequence variation in adhesin P1 and adherence-related protein B/C has been documented. Here we analyzed a clinical strain of M. pneumoniae designated S1 isolated from a 1993 outbreak of respiratory infections in San Antonio, TX. Based on the type of RepMPs within the P1 operon, we classified clinical isolate S1 as type 2 with unique minor sequence variations. Hybridization with oligonucleotide arrays revealed sequence divergence in two previously unsuspected hypothetical genes (MPN137 and MPN138 loci). Closer inspection of this region revealed that the MPN137 and MPN138 loci harbored previously unrecognized unique RepMP1 sequences found only in M. pneumoniae. PCR and sequence analyses revealed a recombination event involving three RepMP1-containing genes that resulted in fusion of MPN137 and MPN138 reading frames and loss of all but a short fragment of another RepMP1-containing locus, MPN130. The multiple copies of unique RepMP1 elements spread throughout the chromosome could allow vast numbers of sequence variations in clinical strains. Comparisons of amino acid sequences showed the presence of leucine zipper motifs in MPN130 and MPN138 proteins in reference strain M129 and the absence of these motifs in the fused protein of S1. The presence of tandem leucine and other repeats points to possible regulatory functions of proteins encoded by RepMP1-containing genes