Strategy To Create Chimeric Proteins Derived from Functional Adhesin Regions of Mycoplasma pneumoniae for Vaccine Development

Abstract

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 ...

Role of Mycoplasma pneumoniae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mediating interactions with the human extracellular matrix

“…Western blot analysis revealed a concentration-dependent pattern of the three proteins tested (data not shown). The surface-located part of the membrane-spanning adhesin P1, near the C terminus (Schurwanz et al, 2009), was cleaved after treatment with 10 and 40 mg trypsin ml 21 and no signal was obtained using 100 mg trypsin ml 21 . The cytosolic elongation factor G showed the first cleavage fragment in the presence of 100 mg trypsin ml 21 , indicating a loss of the bacterium's membrane integrity.…”

“…The recombinant GAPDH was diluted to a concentration of 100 mg in 200 ml PBS and mixed with 100 ml complete Freund's adjuvant (Sigma). Primary subcutaneous immunization of a male guinea pig (Charles River), booster immunizations and the collection of serum were carried out as described by Schurwanz et al (2009). Serum was aliquoted and stored at 220 uC until use.…”

“…HeLa cells (human cervical carcinoma cell line, ATCC CCL-2) were cultured as described by Schurwanz et al (2009) in tissue culture flasks containing DMEM medium (Gibco) supplemented with 10 % fetal calf serum (FCS; Invitrogen). Primary human bronchial epithelial cells (Hbe; PromoCell) were grown in Airway Epithelium Medium (PromoCell) according to the manufacturer's recommendations.…”

“…To test the influence of the anti-GAPDH serum on the adherence of M. pneumoniae cells to HeLa cells, an in vitro adhesion inhibition assay was used as reported (Schurwanz et al, 2009). Briefly, a freshly grown M. pneumoniae culture was harvested as described, resuspended in 1 ml PBS and sheared by passing through a 27G needle.…”

“…The PCR product of the MMR was cloned into the pET-30/LIC vector with the ligation independent cloning kit (Novagen) as recommended by the manufacturer. Transformation of Escherichia coli NovaBlue and BL21(DE3), sequencing of the insert, expression, purification and concentration of the N-terminal 66His-tagged recombinant protein were performed as described in a recent study (Schurwanz et al, 2009). The protein concentration was determined by BCA protein assay (Pierce).…”