Oxana Musatovova

Cloning and molecular characterization of two genes encoding adhesion proteins involved in Trichomonas vaginalis cytoadherence

Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagellate Trichomonas vaginalis. Four trichomonad surface proteins (AP65, AP51, AP33 and AP23) mediate cytoadherence. The cDNA encoding the AP65 adhesin was isolated from a phagemid cDNA expression library by screening with antiserum and monoclonal antibody (mAb) raised against the purified trichomonad AP65 protein. Two clones, F11.2 and F11.5, coded for immuno‐crossreactive recombinant proteins that possessed functional properties equal to the T. vaginalis AP65 adhesin. Analysis of full‐length sequences corresponding to the F11.2 and F11.5 cDNAs revealed that both contained 1701‐base open reading frames (ORFs) that encoded proteins of 63 281 daltons and 63087 daltons, respectively. Comparison of the full‐length sequences showed 87% identity at the nucleotide level and 91% identity at the protein level. Restriction‐enzyme mapping and Southern analysis reaffirmed the distinctness of the F11.2 and F11.5 cDNAs, indicating that two different AP65 genes (now called ap65−1 and ap65−2) are present in the T. vaginalis genome in at least two copies each. Northern analysis detected high levels of transcript of ∼1.8 kb for both ap65−1 and ap65‐2 genes in trichomonads grown only in high‐iron medium, confirming the transcriptional regulation of adhesin synthesis by iron. Homology searches revealed significant similarity (38% amino acid identity and 54% nucleotide identity) to malic enzymes. However, purified malic enzyme and mAb to AP65 crossreactive with malic enzyme neither inhibited cytoadherence of T. vaginalis to host cells nor prevented binding of the trichomonad AP65 to HeLa cells in a ligand assay.

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

ABSTRACT 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.

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 genitalium symptoms, concordance and treatment in high-risk sexual dyads.

The objective of this study was to determine the prevalence and concordance of Mycoplasma genitalium (MG) among Mexican American and African American women and their male sexual partners. Secondary objectives were to determine symptoms of MG infection and persistence of MG after antibiotic therapy. Heterosexual couples were tested for MG and interviewed separately regarding symptoms and behavioural/epidemiologic variables at baseline, six and 12 months. The overall prevalence of MG among women and men was 9.5% and 10.6%, respectively. Subjects were five times more likely to be infected with MG if their sexual partner was MG positive. Among men and women, MG prevalence and mean bacterial loads were similar after receiving single-dose azithromycin, doxycycline or no antibiotics. MG was associated with current urethral discharge in men. No clinical symptoms were specifically diagnostic of MG infection in women.

Transcriptional heat shock response in the smallest known self-replicating cell, Mycoplasma genitalium.

Mycoplasma genitalium is a human bacterial pathogen linked to urethritis and other sexually transmitted diseases as well as respiratory and joint pathologies. Though its complete genome sequence is available, little is understood about the regulation of gene expression in this smallest known, self-replicating cell, as its genome lacks orthologues for most of the conventional bacterial regulators. Still, the transcriptional repressor HrcA (heat regulation at CIRCE [controlling inverted repeat of chaperone expression]) is predicted in the M. genitalium genome as well as three copies of its corresponding regulatory sequence CIRCE. We investigated the transcriptional response of M. genitalium to elevated temperatures and detected the differential induction of four hsp genes. Three of the up-regulated genes, which encode DnaK, ClpB, and Lon, possess CIRCE within their promoter regions, suggesting that the HrcA-CIRCE regulatory mechanism is functional. Additionally, one of three DnaJ-encoding genes was up-regulated, even though no known regulatory sequences were found in the promoter region. Transcript levels returned to control values after 1 h of incubation at 37 degrees C, reinforcing the transient nature of the heat shock transcriptional response. Interestingly, neither of the groESL operon genes, which encode the GroEL chaperone and its cochaperone GroES, responded to heat shock. These data suggest that M. genitalium selectively regulates a limited number of genes in response to heat shock.

Characterization of a unique ClpB protein of Mycoplasma pneumoniae and its impact on growth.

ABSTRACT Mycoplasma pneumoniae accounts for 20 to 30% of all community-acquired pneumonia and has been associated with other airway pathologies, including asthma, and a range of extrapulmonary manifestations. Although the entire genomic sequence of M. pneumoniae has been completed, the functions of many of these genes in mycoplasma physiology are unknown. In this study, we focused on clpB, a well-known heat shock gene in other bacteria, to examine its role in mycoplasma growth. Transcriptional and translational analyses of heat shock in M. pneumoniae indicated that clpB is significantly upregulated, reinforcing its status as a critical responder to heat stress. Interestingly, M. pneumoniae ClpB does not use dual translational start points for ClpB synthesis, like other ClpB-characterized bacteria. Biochemical characterization of purified M. pneumoniae recombinant ClpB revealed casein- and lysine-independent ATPase activity and DnaK-DnaJ-GrpE-dependent chaperone activity. An M. pneumoniae mini-Tn4001-integrated, clpB-null mutant was impaired in its ability to replicate under permissive growth conditions, demonstrating the growth-promoting status of ClpB.

Transcriptional starts for cytadherence-related operons of Mycoplasma genitalium

One mechanism of mycoplasma cytadherence possessed by several mycoplasmas, including Mycoplasma genitalium, necessitates coordination of multiple adhesins and adherence-associated proteins. The genes encoding these adherence-related proteins are located in three different regions of the M. genitalium genome and exhibit an operon-like organization with surrounding genes. To understand whether genes encoding adherence-related proteins in M. genitalium are regulated as operons, we performed transcriptional and reverse transcription-polymerase chain reaction (RT-PCR) analyses on the loci mg191 (encoding major cytadhesin P140 localized at the specialized tip organelle) and mg218 (encoding high molecular mass cytadherence-related protein MG218 required for tip-mediated adherence). Primer extension suggested that transcription of mg191 was under the control of two transcriptional starts, one immediately upstream of mg191 (Prm(MG191)) and the other upstream of mg190 (Prm(MG190)). In contrast, mg218 appeared to be transcribed by a single transcriptional start, located upstream of mg217. RT-PCR indicated that transcription was continuous from mg190 to mg192 and mg217 to mg219, suggesting that these loci constitute true operons. Additional data revealed heretofore undetected similarities between adherence-related operons of M. genitalium and Mycoplasma pneumoniae.

Fatal Outcomes in Family Transmission of Mycoplasma pneumoniae

BACKGROUND Mycoplasma pneumoniae continues to be a significant cause of community-acquired pneumonia and, on rare occasions, manifests as fulminant disease that leads to mortality, even in healthy individuals. METHODS We conducted a retrospective study on members of a family who were quarantined by the Centers for Disease Control and Prevention in 2002 for respiratory failure and death of a 15-year-old brother (sibling 1) and a 13-year-old sister (sibling 2). Collected airway, cerebrospinal fluid (CSF), and serum samples from both deceased siblings and serum samples from both parents and the remaining 3 ill siblings (sibling 3-5) were tested using a range of diagnostic assays. Autopsy lung tissue samples from sibling 2 were also assessed using immunohistochemical and immunoelectron microscopic methods. RESULTS Autopsy evaluation of sibling 1 revealed cerebral edema consistent with hypoxic ischemic encepatholopathy and pulmonary findings of bronchiolitis obliterans with organizing pneumonia (BOOP). Postmortem lung examination of sibling 2 revealed lymphoplasmacytic bronchiolitis with intraluminal purulent exudate, BOOP, and pulmonary edema. Results of diagnostic assays implicated the household transmission of M. pneumoniae among all 5 siblings and both parents. Further analysis of lung tissue from sibling 2 demonstrated the presence of M. pneumoniae organisms and community-acquired respiratory distress syndrome toxin. M. pneumoniae was cultured directly from sibling 2 autopsy lung tissue. CONCLUSION Evidence is provided that M. pneumoniae was readily transmitted to all members of the household and that the resulting infections led to a spectrum of individual responses with variation in disease progression, including lymphoplasmacytic bronchiolitis, BOOP, and death.

Synthesis and Distribution of CARDS Toxin During Mycoplasma pneumoniae Infection in a Murine Model

Mice were infected with Mycoplasma pneumoniae and monitored for the synthesis and distribution of the unique adenosine diphosphate-ribosylating and vacuolating Community Acquired Respiratory Distress Syndrome (CARDS) toxin in bronchiolar lavage fluid (BALF) and lung. We noted direct relationships between the concentration of CARDS toxin and numbers of mycoplasma genomes in BALF and the degree of histologic pulmonary inflammation. Immunostaining of lungs revealed extensive colonization by mycoplasmas, including the detection of CARDS toxin in the corresponding inflamed airways. Lung lesion scores were higher during the early stages of infection, decreased gradually by day 14 postinfection, and reached substantially lower values at day 35. Infected mouse immunoglobulin (Ig) M and IgG titers were positive for CARDS toxin as well as for the major adhesin P1 of M. pneumoniae. These data reinforce the proposed pathogenic role of CARDS toxin in M. pneumoniae-mediated pathologies.

Proximal Region of the Gene Encoding Cytadherence-Related Protein Permits Molecular Typing of Mycoplasma genitalium Clinical Strains by PCR-Restriction Fragment Length Polymorphism

ABSTRACT Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified proximal region of the gene encoding cytadherence accessory protein P110 (MG192) revealed DNA sequence divergences among 54 Mycoplasma genitalium clinical strains isolated from the genitourinary tracts of women attending a sexually transmitted disease-related health clinic, plus one from the respiratory tract and one from synovial fluid. Seven of 56 (12.5%) strains exhibited RFLPs following digestion of the proximal region with restriction endonuclease MboI or RsaI, or both. No sequence variability was detected in the distal portion of the gene.