Synthesis, stability, and function of cytadhesin P1 and accessory protein B/C complex of Mycoplasma pneumoniae

Abstract

The genes MPN141 and MPN142 encode the major adhesin P1 and the cytadherence-related B/C proteins (P90/P40), respectively, in Mycoplasma pneumoniae. Using reverse transcriptase PCR we found open reading frames MPN140 to MPN142 constitute a polycistronic transcriptional unit. Cytadherence mutant IV-22 has a frameshift mutation in MPN141 and lacks the P1, B, or C proteins. Recombinant MPN141 and/or MPN142 were introduced into mutant IV-22 by transposon delivery in several configurations, and the levels of the P1, B, and C proteins were assessed by immunoblotting. MPN142 in mutant IV-22 has a wild-type nucleotide sequence, yet the introduction of recombinant MPN141 alone to mutant IV-22, although it restored P1 levels, failed to restore levels of B or C. In contrast, recombinant MPN141 and MPN142 delivered in cis or in trans were sufficient to restore all three proteins. Taken together, our data indicated that some but not all synthesis of B or C is dependent on coupling to the translation of P1 immediately upstream of MPN142 and demonstrated that proteins B and C are not stable in the absence of P1. The linkage of MPN141 and MPN142 at the levels of transcription, translation, and protein stability, in addition to their previously demonstrated colocalization and the requirement of B and/or C for P1 function, reinforces the conclusion that these proteins constitute a multiprotein complex that functions in receptor binding.

FIG. 1.

RT-PCR analysis of the MPN140 to MPN142 locus of M. pneumoniae. (A) Map of the locus in M. pneumoniae. The arrow indicates transcriptional promoter upstream of MPN140. The intergenic sequences are shown above the map, with 12 and 5 nt (in lowercase) separating MPN140 and MPN141, and MPN141 and MPN142, respectively. A predicted stem-loop terminator (not indicated) begins 17 nt downstream of MPN142. Locations corresponding to annealing sites of oligonucleotide primer sets A-G (Table 2) used for RT-PCR are shown below the map. Restriction enzyme sites for PmeI (P) and SphI (Sp) referenced in Fig. 2 are shown. Scale bar is 1 kb. (B) Agarose gel electrophoresis of RT-PCR products. RT-PCRs for primer pairs A-G with wild-type M. pneumoniae RNA template are shown. RT was included (+) or omitted (−). Locations of DNA markers and their sizes in base pairs are indicated.

FIG. 2.

Map of the DNA constructs cloned into IS256L of Tn4001mod of pKV74 or Tn4001cat of pKV104 to create pKV258, pKV264, pKV265, and pKV299. Except for the IS elements, all open reading frames are shown with the 5′ end on the left. The region encoding the 3′ end of MPN140 is not indicated. B, BamHI; Bs, BsaBI; E, EcoRI; S, StuI; Sm/P, SmaI/PmeI junction; M/E/Sp, former SphI site of the P1 operon replaced by MfeI linker and present as an MfeI/EcoRI junction; E or Sm, EcoRI in pKV74 or SmaI in pKV104. The barred symbol indicates a silent change to destroy the site. Gentamicin resistance (Gm^r) is found in pKV74, and chloramphenicol resistance (Cm^r) is found in pKV104. The arrow from Tn4001mod/cat shows the location and direction of the P_OUT promoter. Note that the orientation of the inserts is inverted when cloned into Tn4001mod/cat.

FIG. 3.

Western blot analysis of lysates of wild-type (WT) or mutant (IV-22 or III-4) M. pneumoniae untransformed or transformed with pKV258 or pKV264. Two independent transformants are shown for each plasmid-background combination. A total of 60 μg of protein was used per lane. Blots were probed with antiserum to P1, B, or C as indicated.

FIG. 4.

Western blot analysis of lysates of wild-type (WT) or mutant (IV-22) M. pneumoniae untransformed or transformed with pKV264, pKV299, or pKV264 and pKV299. Three independent transformants are shown for each construct in the mutant IV-22 background except IV-22+pKV264 (see Fig. 3). A total of 60 μg of protein was used per lane. Blots were probed with antiserum to P1, B, or C as indicated.

FIG. 5.

Western blot analysis of lysates of wild-type (WT) or mutant (IV-22 or III-4) M. pneumoniae untransformed or transformed with pKV265. Two independent transformants are shown for each construct-background combination. A total of 60 μg of protein was used per lane. Blots were probed with antiserum to P1, B, or C as indicated.