Cloning and sequence analysis of cytadhesin P1 gene from Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae cytadhesin P1 was purified by monoclonal antibody affinity chromatography followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal 18-aminoacid sequence of P1 was determined and used to design two synthetic oligonucleotides, a 14-mer corresponding to amino acids 1 to 5 and an 18-mer corresponding to amino acids 7 to 12. These oligonucleotides served as hybridization probes for the identification of the P1 gene by Southern blot analysis ofM. pneumoniae DNA. The P1 gene was cloned into plasmid pUC19 and mapped by using appropriate restriction endonucleases. The DNA sequence of the entire P1 gene was determined by subcloning appropriate DNA fragments into bacteriophage M13 and sequencing the DNA by the dideoxy-chain-termination method. The P1 gene contains an open reading frame of 4,881 nucleotides coding for a protein of 1,627 amino acids with a calculated molecular weight of 176,288. Properties of the amino-terminal sequence suggest that protein P1 may be synthesized as a precursor with subsequent processing to a mature protein of a calculated molecular weight of 169,758. Potential antigenic sites were determined by hydrophilicity plots. A computer search revealed that part of the predicted P1 sequence is homologous to cytoskeletal keratin of mammalian species and human fibrinogen alpha chain precursor. These results demonstrate the uniqueness of P1 as a cytadhesin and virulence determinant.

Sialic Acids in Molecular and Cellular Interactions

“…A protein described as P1 adhesin has been suggested to function in Sia-dependent binding. The mature protein has a molecular weight of 169 kDa as determined from its primary amino acid sequence (Su et al, 1987). However, P1 is expressed on both adhesive and nonadhesive M. pneumoniae cells and multiple forms have been found, possibly caused by homologous but not identical copies of the gene on the 182 S0RGE KELM AND ROLAND SCHAUER bacterial genome (Su et al, 1988(Su et al, , 1990(Su et al, , 1991(Su et al, , 1993.…”

Structure, function, and assembly of the terminal organelle of Mycoplasma pneumoniae

“…Synthesized in precursor form, a processing event is required to yield mature P1. The leader peptide [15] consists of a typical, positively charged N-terminus followed by a hydrophobic core that ends with alanine residues in what would ordinarily constitute the 33 and 31 positions of a signal peptidase I cleavage site ( Fig. 3).…”

“…The authors regret that many ¢ne studies were not cited spe-ci¢cally in this review due to the space restrictions. More complete reference listings can be found elsewhere [1,2,13,15].…”

Human antibody response to the major adhesin of Mycoplasma pneumoniae: Increase in titers against synthetic peptides in patients with pneumonia

“…Peptides corresponding to parts of a predicted amino acid sequence of the P1 protein (20) were produced at the Department of Virology, Karolinska Institute, using the method described by Houghten et al (1 1, 12). On the basis of antigenicity predicted according to Jameson & Wo(f (1 5), seven sequences containing a number of amino acids with an antigenicity index 2 1.3 were produced (Table 1).…”

“…The amino acid sequence of the major adhesin of Mycoplasma pneumoniae (cytadhesin P 1) has been determined by cloning the gene and sequencing the DNA (20). For serodiagnostic purposes it is of great interest to define the antigenic sites of the P1 protein responsible for eliciting the antibody response to this strongly antigenic protein ( 1 7).…”