In vivo expression analysis of the P97 and P102 paralog families of Mycoplasma hyopneumoniae

Abstract

The P97 adhesin and P102 genes of Mycoplasma hyopneumoniae each have six paralogs in the genome. We tested whether these genes were expressed during infection. P102 is associated with the mycoplasma and with swine cilia. Further, most of the paralogs were transcribed in vivo in two gene transcriptional units.

FIG. 1.

Paralog gene structure and genome location. The gray bars indicate P102 gene paralogs, and the black bars are P97 gene paralogs. mhp182 is the P102 gene while mhp183 is the gene for P97. The arrows indicate direction of transcription. Not shown are mhp280 and mhp493.

FIG. 2.

Immunogold electron microscopy of mycoplasmas attached to swine cilia during infection. Infected trachea from pigs challenged with strain 232 were excised and epithelial regions prepared for electron microscopy as described. Regions containing ciliated epithelium were sectioned and stained with gold-labeled antibodies. (A) Control section stained with sera from a naïve, specific pathogen-free mouse and immunogold conjugate. (B to E) Sections stained with mouse anti-P102 antibodies followed by conjugate. Note the large numbers of gold particles (arrows). Bar, 0.5 μm.

FIG. 3.

RT-PCR analysis of P97 and P102 paralog gene transcription. A. Open reading frame numbers are shown for each open reading frame examined. Left lanes indicate RNA templates from BAL samples (R); right lanes, genomic DNA template controls (D). P97, intergenic, and P102 RT-PCRs are indicated at the top of the panel. The distance between the open reading frames in base pairs (bp) of each transcriptional unit are also shown. B. Controls. (Upper panel) Lane 1, no reverse transcriptase; lane 2, no RNA; lane 3, no primers; lane 4, P97 primers with cDNA; lane 5, P97 primers with genomic DNA. (Lower panel) These products are from RT-PCRs using RNA isolated from uninfected porcine lung tissue. Lanes 1 to 11 represent RT-PCRs with specific primer pairs for open reading frames mhp275, mhp272, mhp108, mhp107, mhp271, mhp280, mhp684, mhp683, mhp493, mhp385, and mhp384, respectively. Lane 12, P97-P102 primers. Lane 13 represents the pig cyclophilin positive control reaction.