Identification and characterization of the Treponema pallidum tpn50 gene, an ompA homolog

Abstract

Treponema paUlidum is a pathogenic spirochete that has no known genetic exchange mechanisms. In order to identify treponemal genes encoding surface and secreted proteins, we carried out TnphoA mutagenesis of a T. paUlidum genomic DNA library in Escherichia coli. Several of the resulting clones expressed enzymatically active T. pallidum-alkaline phosphatase fusion proteins. The DNA sequence of the 5' portion of a number of the treponemal genes was obtained and analyzed. A recombinant clone harboring plasmid p4A2 that encoded a treponemal protein with an approximate molecular mass of 50,000 Da was identified. Plasmid p4A2 contained an open reading frame of 1,251 nucleotides that resulted in a predicted protein of 417 amino acids with a calculated molecular mass of 47,582 Da. We have named this gene tpnSO in accordance with the current nomenclature for T. pallidum genes. A 1.9-kb HincII-ClaI fragment from p4A2 that contained the tpn5O gene was subcloned to produce p4A2HC2. Comparison of the predicted amino acid sequence of TpN50 with protein sequences in the National Center for Biotechnology Information data base indicated statistically significant homology to the Pseudomonas sp. OprF, E. coli OmpA, Bordetella avium OmpA, Neisseria meningitidis RmpM, Neisseria gonorrhoeae PIII, Haemophilus influenzae P6, E. coli PAL, and LegioneUla pneumophila PAL proteins. These proteins are all members of a family of outer membrane proteins that are present in gram-negative bacteria. The tpn5O gene complemented E. coli ompA mutations on the basis of two separate criteria. First, morphometry and electron microscopy data showed that E. coli C386 (ompA lpp) cells harboring plasmid vector pEBH21 were rounded while cells of the same strain harboring p4A2HC2 (TpN50+), pWW2200 (OprF+), or pRD87 (OmpA+) were rod shaped. Second, E. coli BRE51 (MC4100 AsuL4-ompA) cells harboring pEBH21 grew poorly at 42°C in minimal medium, while the growth of BRE51 cells harboring p4A2HC2 was similar to that of the parental MC4100 cells. These results demonstrate that the TpN50 protein is functionally equivalent to the E. coli OmpA protein. If TpN50 functions in a similar fashion in T. paUlidum, then it may be localized to the treponemal outer membrane. * Corresponding author. t This work is dedicated to the memory of Philip J. Bassford, Jr. His knowledge, friendship, and enthusiasm are deeply missed.

Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture

“…The complete sequence lor tpn50 was obtained from two genomic clones wifh overlapping inserts. Except tor minor discrepancies (absence of nucleotides 409-411, substitution of G for C at nucleotide 1014, and substitution ot A for C at nucleotide 1082 (data not shown), the nucleotide and amino acid sequences were identical to those pLiblished by Hardham and Stamm (1994). De Mot and Vanderieyden {1994) recently identified a group of 28 peptidoglycan-associated membrane proteins from diverse bacterial species with extended homology to the C-terminus of Pseudomonas fluorescens OprF; thts group inciudes all of (he polypeptides reported to be homologous to TpNSO (Hardham and Stamm, 1994).…”

“…Except tor minor discrepancies (absence of nucleotides 409-411, substitution of G for C at nucleotide 1014, and substitution ot A for C at nucleotide 1082 (data not shown), the nucleotide and amino acid sequences were identical to those pLiblished by Hardham and Stamm (1994). De Mot and Vanderieyden {1994) recently identified a group of 28 peptidoglycan-associated membrane proteins from diverse bacterial species with extended homology to the C-terminus of Pseudomonas fluorescens OprF; thts group inciudes all of (he polypeptides reported to be homologous to TpNSO (Hardham and Stamm, 1994). Computer-based sequence alignments revealed that the C-terminal third of TpN50 has homology with three psptidoglycan-associated proteins of Gram-positive bacteria (i.e.. Bacillus subtilis MofB, B. subtilis OrfB, and BaciHus megaterium OrfB) as well as those of Gram-negative bacteria {data not shown).…”

“…TpGMDs also provided a convenient method for localizing TpN50, a recently characterized candidate T", paHidum outer membrane protein with sequence homologies fo a number of bacterial membrane proteins {Hardham and Stamm, 1994). In their initial report, Hardham and Stamm (1994) did not provide information regarding the native form of TpN50. Our experiments not only failed to identify this protein on the 7, pallidutn surface, they indicated that its cellular location was simiiar to that of TpN60, a cytoplasmic chaperonin.…”

“…Total magniiication in all photos is --1250. Hardham and Stamm (1994) recently identified a treponemal antigen {designated TpN50) with sequence homology to a number of bacterial outer membrane proteins, including outer membrane protein A {OmpA) of E. coll and the major outer membrane protein (OprF) of Pseudomonas aeruginosa. We next investigated whether TpGMDs could be used to determine the cellular location of this candidate T. pallidum outer membrane protein.…”

“…We next investigated whether TpGMDs could be used to determine the cellular location of this candidate T. pallidum outer membrane protein. To generate the requisite antiserufn for these studies, the tpn50 gene was amplified as a 1.2 kb polymerase chain reaction {PCR) product from r. paHidum chromosomal DNA using primers derived from the reported nudeoticSe sequence of tpnSO {Hardham and Stamm. 1994) and cloned in frame as a fusion with GST, Transfonnants expressed an IPTG-inducibie 74 kDa protein which reacted with rat anti-GST antiserum and rabbit and human syphilitic sera exlensively adsorbed with E. coii whole cell lysates (data not shown).…”