To enhance the sensitivity of the available real-time PCR systems for the detection of Mycoplasma pneumoniae, we established a method to amplify copies of the repetitive element repMp1. In a study of respiratory tract samples, we found that, compared to the use of the conserved part of the P1 adhesin gene as a monocopy target, the use of the repMp1-PCR showed an increase in the detected genome equivalents by a factor of 22.Mycoplasma pneumoniae is one of the most common causes of upper and lower respiratory tract infections in children and adults. Between 10 and 20% (up to 50%) of all cases of community-acquired pneumonia can be attributed to this cell wallless pathogen. In addition to the frequent occurrence of timeindependent endemic outbreaks in susceptible populations, worldwide epidemics showing 3-to 7-year intervals have been recorded (6,18). While the culture of M. pneumoniae is unreliable and time-consuming, serological tests require the availability of paired sera showing the problem of an age-and time-dependent occurrence of immunoglobulin A and M antibodies (1). PCR approaches have been found to be useful for the rapid, sensitive, and specific detection of M. pneumoniae in respiratory tract specimens. Because of speed and reduced risk of laboratory contaminations, real-time PCR systems increase the significance of the results in the diagnostic procedure to confirm M. pneumoniae infections. Different PCR protocols have been developed targeting in most cases the ATPase gene and the gene of the main P1 adhesin (11). In the latter gene, two copies of repetitive elements could be found occurring all over the genome of the completely sequenced strain M129 (7). The four different repetitive sequences-repMp1, repMp2/3, repMp4, and repMp5-differ in size (147 to 2,701 bp), number of copies (8 to 14), and sequence (15). Investigation of the rep sequences not only is the basis for the differentiation of clinical isolates of M. pneumoniae into subtypes and variants (3,8,16) but also may increase the sensitivity of a PCR approach by amplifying a multicopy target. We sought here to evaluate the sensitivity of a real-time PCR system based on amplification of a part of the repetitive element repMp1 of M. pneumoniae and compare the results with a P1 gene-derived approach targeting a conserved monocopy sequence.M. pneumoniae reference strains M129 (subtype 1, ATCC 29342), FH (subtype 2, ATCC 15531), and the patient isolates 4817 (variant 1; kindly provided by S. A. J. Zaat, Academic Medical Center, Amsterdam, The Netherlands) and ST (characterized as variant 2) (4) were cultivated as described previously (4). To determine the detection limit of the real-time PCR, freshly grown cells of strain M129 were homogenized by using a 27G syringe and tenfold diluted in phosphate-buffered saline. Aliquots (200 l) of the dilutions were used to determine the CFU on PPLO agar (Becton Dickinson, Sparks, MD) and in parallel to prepare the genomic DNAs. Respiratory tract specimens (bronchoalveolar lavage fluids and nasopharyngeal or pha...
The cell wall-less bacterium Mycoplasma pneumoniae is one of the most common agents of respiratory tract diseases in humans. Adhesin-mediated binding of the bacteria to host cells is a crucial step in colonization and subsequent pathogenesis. For the first time, we expressed 16 recombinant proteins covering almost the whole major adhesin P1 and the adherence-associated protein P30 to characterize these proteins immunologically and functionally. We describe a new in vitro assay using several human cell lines in combination with fluorescence-activated cell sorting analysis to screen antisera raised against the recombinant proteins quantitatively for adherence inhibition activity. The protein derived from the nearly C-terminal part of the P1 adhesin (amino acids [aa] 1288 to 1518) and the protein P30 (aa 17 to 274) especially showed prominent immunoreactivity with sera from M. pneumoniae-immunized guinea pigs as well as with M. pneumoniae-positive patient sera. We demonstrate that the same protein regions are involved in mediating cytadherence since antibodies against these adhesin regions decrease mycoplasma adhesion to human cells significantly. For further vaccine studies, we optimized the immunogenic and adherence-mediating properties of the antigen by combining both the P1 and the P30 regions in a novel chimeric protein. Antibodies against this protein show an increased reduction of M. pneumoniae adherence to human bronchial epithelial cells by 95%, which is comparable to results with polyspecific anti-M. pneumoniae animal serum. Our strategy results in a promising defined antigen candidate for reducing or even preventing M. pneumoniae colonization of the respiratory tract in future vaccination studies.Epidemiological studies confirm that between 5 and 10% of all community-acquired pneumonia cases, especially in children and young adults but also in elderly patients, are attributed to the cell wall-less bacterium Mycoplasma pneumoniae (49). Epidemic outbreaks in geographically close populations and the occurrence of extrapulmonary complications of the primary respiratory infections emphasize the significant impact of the agent on public health.Adhesion of M. pneumoniae to the host respiratory epithelium (cytadherence) is an essential first stage of infection and a precondition for successful colonization (19). Even though M. pneumoniae is one of the smallest and simplest microorganisms, M. pneumoniae cells exhibit a complex differentiated cell extension, the attachment organelle, that functions in different processes including cytadherence, cell division, and gliding (1, 32, 39). The analysis of mutants has resulted in the identification of an increasing number of proteins associated with cytadherence of M. pneumoniae (reviewed in reference 33), such as the transmembrane proteins P1 and P30, both densely clustered at the attachment organelle (31). The results of binding experiments and of the characterization of P1-deficient (avirulent) mutants established the role of the protein P1 as the main adhesin of the ...
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