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 83 087 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 approximately 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.
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.
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.
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.
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°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.
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