Genetic characterization and partial sequence determination of a Treponema pallidum operon expressing two immunogenic membrane proteins in Escherichia coli

Abstract

A detailed physical and genetic map of a previously cloned 5.5-kilobase segment of Treponema pallidum DNA is described. This segment expressed two proteins that are cell membrane associated in Escherichia coli. The structural genes of these treponemal membrane proteins, tmpA and tmpB, are coordinately expressed, and transcription in E. coli can start ftom at least two different treponemal promnoters. The tmpA and tmpB proteins are the products of in vivo proteolytic cleavage from precursor proteins which are 2 and 4 kilodaltons larger, respectively, than the mature proteins. Because the sizes of the corresponding proteins produced in T. pallidum were identical to those of the mature membrane proteins in E. coil, we concluded that a similar proteolytic processing takes place in both E. coli and T.paflidum6 Although tmpA and tmpB were controlled by the same transcription signals, tmpB was expressed to a higher extent thlah tmpA, and only the tmpB product could be overproduced by placing the left lambda promoter in front bf the structural genes. The nucleotide sequence of the T. pallidum tmpA geite was established. This is the first T. paUidim gene sequenced. Codon usage and the nature of transcriptional and translational signals ate discussed. The deduced amino acid sequence indicated the presence of a sequence that was characteristic for a s;gnal peptide. This sequence information allowed the construction of hybrid genes coding for proteins having I-galactosidase enzyme activity as well as TmpA epitopes. The enzymelinked antigen was expressed at a high level in E. coli when transcriptional and translational signals from coliphage A were used. In this case the protein produced was a sandwich protein consisting of 21 aminio acids of the A cro protein, 204 amnino acids of the T. pallidam TmpA protein, and 1,020 amino lcids of the E. coli X-galictosidase. The potedtial use of this enzyme-linked antigen for the serodiagnosis of syphilis is discussed. Treponema pallidum is the causative agent of syphilis. Because the microorganism cannot be grown in vitro, little is known about the factors that contribute to its virulence and the immunologic response duting a syphilitic infection. For this reason various groups have cloned genes of T. pallidum that encode for immunogenic proteins (17, 24, 27, 30). These recent studies have shown that T. pallidum DNA can be expressed well in Escherichia coli, and treponemal transcriptional and translational signals appear to be recognized by the E. coli machinery for protein synthesis. Recently, we screened a cosmid library with T. pallidum DNA inserts for the production of T. pallidum antigens (27). Among the 800 clones scteened, one particular phenotype was found most frequently. Of 16 antigen-producing clones,

Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae

“…resulting in a frameshift. This particular arrangement has not been reported before for trxA and trxB genes, but it has been described for other genes, for example for tmpA and tmpB genes of Treponema paltidum (Hansen e^ at.. 1985). In other bacteria, such as S. clavuligerus.…”

Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane‐spanning sequences using a novel alkaline phosphatase expression vector

“…To determine T. pallidum gene representation. 250 randomly chosen APexpressing clones were probed with oligonucleotides made to the amino-terminal sequences of the T. pallidum genes encoding the impA (Hansen et al. 1985;Schouls et al.…”

“…and that the lipid moiety of these fusion proteins results in their partitioning into the hydrophobic Triton X-114 detergent phase. One such gene encoding an acylated AP fusion was initially identified by use of an oligonucleotide probe corresponding to the published sequence of the T. pailidum gene, tmpA (Hansen et a/., 1985). This fusion protein, identified as Tp92, partitioned into the Triton X-114 detergent phase and was shown to incorporate tritiated palmitate.…”

Reactivity of recombinant treponema pallidum (r‐Tp) antigens with anti‐Tp antibodies in human syphilitic sera evaluated by ELISA

“…We constructed Tp genes expressing three forms of recombinant proteins: mature antigens, antigens containing signal sequences, and antigens fused with GST at the NH 2 terminal of the mature antigen. The amino acid sequences of the antigens containing signal sequences were as follows (12)(13)(14)(15)(16)(17):…”

“…The major immunoreactive antigens of these membrane proteins have been reported to have a MW of 47, 42, 17, and 15KDa based on WB analy-sis (8)(9)(10). The putative amino acid sequences of 47, 42, 17, and 15KDa have been demonstrated (11)(12)(13)(14)(15)(16)(17). Here, we describe several forms of expressed r-Tp antigens related to the membrane proteins mentioned above and the detection of anti-Tp antibodies by means of indirect ELISA, and compare the ELISA results with those of the conventional TPHA method.…”