Molecular characterization of Mycoplasma arthritidis variable surface protein MAA2

Abstract

Earlier studies implied a role for Mycoplasma arthritidis surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability. Here we report the further analysis of MAA2 and the cloning and sequencing of the maa2 gene from two M. arthritidis strains, 158p10p9 and H606, expressing two size variants of MAA2. Triton X-114 partitioning and metabolic labeling with [3H]palmitic acid suggested lipid modification of MAA2. Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y. The maa2 genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats. The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide. The maa2 gene was expressed in Escherichia coli from the lacZ promoter of vector pGEM-T. The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in E. coli. The maa2 gene and upstream DNA sequences were cloned from M. arthritidis clonal variants differing in MAA2 expression state. Expression state correlated with the length of a poly(T) tract just upstream of a putative -10 box. Full-sized recombinant MAA2 was expressed in E. coli from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region.

FIG. 1

Western immunoblot of electrophoretically separated M. arthritidis proteins after treatment of intact cells with carboxypeptidase Y concentrations of 0, 0.075, 0.25, 0.5, 1, and 1.52 mg/ml for 1 h, stained with pooled MAA1- and MAA2-specific MAbs A9a and 7a, respectively. Proteins were electrophoresed on SDS–7.5% (wt/vol) polyacrylamide gels.

FIG. 2

Map of ∼5-kb M. arthritidis 158p10p9 DNA insert in plasmid pM8-2 (derived from pBlueScript) containing sequences hybridizing with the oligonucleotide probe derived from the MAA2 N-terminal amino acid sequence. Base pairs are numbered beginning with the terminal EcoRI site at the 5′ end of the insert. The ORF 1 tandem direct repeat units are indicated as shaded boxes and numbered R1 to R6. A putative Rho-independent transcription terminator between ORFs 1 and 2 is indicated as a hairpin loop. Restriction sites are indicated by arrows. The ∼0.7-kb HP fragment and ∼2.2-kb, ∼0.7-kb, and ∼0.25-kb (HL, HM, and HS, respectively) fragments from this insert that were subcloned to pUC19 for DNA sequencing are also shown.

FIG. 3

Complete nucleotide and deduced amino acid sequences of ORF 1 (maa2) from M. arthritidis 158p10p9. Nucleotides are numbered sequentially from the 5′-terminal EcoRI site. Sites at which DNA sequences differ from those of the maa2 gene from strain H606 are shown as outlined letters. Restriction sites for EcoRI, HindIII, and DraI are printed in lowercase letters. DNA sequences used for preparation of oligonucleotide primers for sequencing and PCR and for use as probes in Southern blotting are underlined. A putative −10 box, ribosome binding site (RBS), and translation terminator are shown in italicized boldface lettering. Repeat units are set apart in brackets and numbered R1 to R6; the first codon of each is printed in boldface. Nucleotides in R1 and R5 that differ from the consensus sequence are circled; amino acids in R1 and R5 that differ from the consensus sequence are underlined. The predicted lipoprotein signal peptide amino acid sequence is boxed. The poly(T) tract is printed in italicized lowercase letters. The inverted repeat downstream from the TAA stop codons is italicized in uppercase letters.

FIG. 4

PCR products (10 μl) amplified from recombinant plasmid pM8-2 (lane 1) and from chromosomal DNA from M. arthritidis H606 (lane 2) and M. arthritidis PG6 (lane 3) by using primers NT and HMPR were electrophoresed on a 1.5% (wt/vol) agarose gel and stained with ethidium bromide. PCR products differed in size in approximately 260-bp increments. Size markers are shown in lane M.

FIG. 5

Southern hybridization of M. arthritidis chromosomal DNA from 20 strains with probes prepared from DNA sequences in two different regions of ORF 1. Nine-microgram samples of chromosomal DNA were digested with EcoRI, electrophoresed on a 0.8% (wt/vol) agarose gel, transferred to nylon membrane, and hybridized with ³²P-labeled oligonucleotide probe NT, from near the 5′ end of the coding sequence (A), and a ³²P-labeled 264-bp HS fragment from within the ORF 1 repeat region (B). All 20 strains contained at least one copy of sequences homologous to NT, while only seven strains contained copies of the sequence from within the repeat region. These seven strains were shown in an earlier study to express the epitope recognized by MAA2-specific MAb 7a, while the others did not (54).

FIG. 6

Expression of recombinant MAA2 in E. coli. (A) E. coli cells containing the maa2 gene ligated into vector pGEM-T in both orientations with respect to the lacZ promoter were grown in the presence and absence of IPTG, lysed, electrophoresed by SDS-PAGE on 7.5% (wt/vol) gels, transferred to nitrocellulose, and stained with MAA2-specific MAb 7a. Lanes contain M. arthritidis 158p10p9 whole-cell lysate (lane 1), E. coli containing the cloned insert in the forward orientation with respect to the lacZ promoter, in the absence (lane 2) and presence (lane 3) of IPTG, E. coli containing the cloned insert in the reverse orientation with respect to the lacZ promoter, in the absence (lane 4) and presence (lane 5) of IPTG, and untransformed E. coli in the absence (lane 6) and presence (lane 7) of IPTG. (B) E. coli cells containing the maa2 gene from M. arthritidis 158p10p9 clonal variants in which expression of MAA2 was switched ON or OFF were grown in the presence or absence of IPTG, lysed, electrophoresed and immunoblotted as described above. Lanes contain E. coli in which maa2 from an OFF variant was ligated into vector pGEM-T in the reverse orientation with respect to the lacZ promoter, in the absence (lane 1) and presence (lane 2) of IPTG, E. coli in which maa2 from an OFF variant was ligated into vector pGEM-T in the forward orientation with respect to the lacZ promoter, in the absence (lane 3) and presence (lane 4) of IPTG, E. coli in which maa2 from an ON variant was ligated into vector pGEM-T in the forward orientation with respect to the lacZ promoter, in the absence (lane 5) and presence (lane 6) of IPTG, and a whole-cell lysate of M. arthritidis strain 158p10p9 (lane 7).