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Treponema pallidum major sheath protein homologue Tpr K is a target of opsonic antibody and the protective immune response.

Centurion-Lara A, Castro C, Barrett L, Cameron C, Mostowfi M, Van Voorhis WC, Lukehart SA - J. Exp. Med. (1999)

Bottom Line: One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium.Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum.This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Washington, Seattle, Washington 98195, USA. acentur@u.washington.edu

ABSTRACT
We have identified a family of genes that code for targets for opsonic antibody and protective immunity in T. pallidum subspecies pallidum using two different approaches, subtraction hybridization and differential immunologic screening of a T. pallidum genomic library. Both approaches led to the identification of a polymorphic multicopy gene family with predicted amino acid homology to the major sheath protein of Treponema denticola. One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium. Reverse transcription polymerase chain reaction analysis of T. pallidum reveals that Tpr K is preferentially transcribed in the Nichols strain of T. pallidum. Antibodies directed to purified recombinant variable domain of Tpr K can opsonize T. pallidum, Nichols strain, for phagocytosis, supporting the hypothesis that this portion of the protein is exposed at the surface of the treponeme. Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum. This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.

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Ethidium bromide– stained gel showing PCR amplification of Nichols strain cDNA  using primers for unique sequences of the variable domains  of tpr A–L. The lane labeled “+” shows the result of amplification using  control primers (demonstrates sufficient amounts of target cDNA in the  sample) and lanes 1–12 show the results when amplification was performed using primers specific for each tpr gene. Total RNA was extracted  from a known number of freshly harvested Nichols strain treponemes,  followed by treatment of the RNA sample with RNAse free DNAse A.  First-strand cDNA was made by random priming, and PCR amplification  was performed using primers specific for each tpr gene (Table I). DNAse  A treatment of the RNA samples, lack of amplification of RNA without  cDNA synthesis, and determination of primer efficiency ruled out the  possibility that these results represent either DNA contamination or PCR  efficiency artifacts (data not shown).
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Figure 3: Ethidium bromide– stained gel showing PCR amplification of Nichols strain cDNA using primers for unique sequences of the variable domains of tpr A–L. The lane labeled “+” shows the result of amplification using control primers (demonstrates sufficient amounts of target cDNA in the sample) and lanes 1–12 show the results when amplification was performed using primers specific for each tpr gene. Total RNA was extracted from a known number of freshly harvested Nichols strain treponemes, followed by treatment of the RNA sample with RNAse free DNAse A. First-strand cDNA was made by random priming, and PCR amplification was performed using primers specific for each tpr gene (Table I). DNAse A treatment of the RNA samples, lack of amplification of RNA without cDNA synthesis, and determination of primer efficiency ruled out the possibility that these results represent either DNA contamination or PCR efficiency artifacts (data not shown).

Mentions: To examine transcription of the tpr genes in T. pallidum, a group of oligonucleotide primers specific for each of the tpr was prepared (Table I). Fig. 3 shows the RT-PCR analysis of tpr transcription from T. pallidum subspecies pallidum, Nichols strain. A strong signal was detectable after 35 cycles for tpr K, whereas weak signal was observed for tpr E and H. After five more PCR cycles, signal was discernible for all the tpr genes (data not shown). The preponderance of tpr K product was not due to an increased efficiency of tpr K amplification, because the PCR assay for tpr K was less efficient with limiting dilutions of genomic DNA than with other tpr genes (see Materials and Methods). The PCR products represent amplification of mRNA, and not contaminating genomic DNA because (a) the RNA was pretreated with DNAse, and (b) performing the PCR with the RNA preparation before the cDNA synthesis step yielded no product. RT-PCR on T. pallidum, Nichols strain, isolated from rabbit testicles on days 5, 10, and 15 after infection all demonstrated a preponderance of tpr K mRNA; smaller amounts of tpr B, E, H, or I mRNA were detected weakly and variably (data not shown). We conclude that tpr K is transcribed to a higher degree than the other tpr genes by T. pallidum, Nichols strain. The preferential expression of Tpr K in the Nichols strain is supported by the selective development of Tpr K–specific antibody and reactive T cells in Nichols- infected rabbits (our unpublished results). Because it is not possible to produce clonal populations of T. pallidum, we are uncertain whether the variable transcription of the other tpr genes described above occurs in subpopulations of treponemes within our Nichols strain or in all bacterial cells of the Nichols strain.


Treponema pallidum major sheath protein homologue Tpr K is a target of opsonic antibody and the protective immune response.

Centurion-Lara A, Castro C, Barrett L, Cameron C, Mostowfi M, Van Voorhis WC, Lukehart SA - J. Exp. Med. (1999)

Ethidium bromide– stained gel showing PCR amplification of Nichols strain cDNA  using primers for unique sequences of the variable domains  of tpr A–L. The lane labeled “+” shows the result of amplification using  control primers (demonstrates sufficient amounts of target cDNA in the  sample) and lanes 1–12 show the results when amplification was performed using primers specific for each tpr gene. Total RNA was extracted  from a known number of freshly harvested Nichols strain treponemes,  followed by treatment of the RNA sample with RNAse free DNAse A.  First-strand cDNA was made by random priming, and PCR amplification  was performed using primers specific for each tpr gene (Table I). DNAse  A treatment of the RNA samples, lack of amplification of RNA without  cDNA synthesis, and determination of primer efficiency ruled out the  possibility that these results represent either DNA contamination or PCR  efficiency artifacts (data not shown).
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Figure 3: Ethidium bromide– stained gel showing PCR amplification of Nichols strain cDNA using primers for unique sequences of the variable domains of tpr A–L. The lane labeled “+” shows the result of amplification using control primers (demonstrates sufficient amounts of target cDNA in the sample) and lanes 1–12 show the results when amplification was performed using primers specific for each tpr gene. Total RNA was extracted from a known number of freshly harvested Nichols strain treponemes, followed by treatment of the RNA sample with RNAse free DNAse A. First-strand cDNA was made by random priming, and PCR amplification was performed using primers specific for each tpr gene (Table I). DNAse A treatment of the RNA samples, lack of amplification of RNA without cDNA synthesis, and determination of primer efficiency ruled out the possibility that these results represent either DNA contamination or PCR efficiency artifacts (data not shown).
Mentions: To examine transcription of the tpr genes in T. pallidum, a group of oligonucleotide primers specific for each of the tpr was prepared (Table I). Fig. 3 shows the RT-PCR analysis of tpr transcription from T. pallidum subspecies pallidum, Nichols strain. A strong signal was detectable after 35 cycles for tpr K, whereas weak signal was observed for tpr E and H. After five more PCR cycles, signal was discernible for all the tpr genes (data not shown). The preponderance of tpr K product was not due to an increased efficiency of tpr K amplification, because the PCR assay for tpr K was less efficient with limiting dilutions of genomic DNA than with other tpr genes (see Materials and Methods). The PCR products represent amplification of mRNA, and not contaminating genomic DNA because (a) the RNA was pretreated with DNAse, and (b) performing the PCR with the RNA preparation before the cDNA synthesis step yielded no product. RT-PCR on T. pallidum, Nichols strain, isolated from rabbit testicles on days 5, 10, and 15 after infection all demonstrated a preponderance of tpr K mRNA; smaller amounts of tpr B, E, H, or I mRNA were detected weakly and variably (data not shown). We conclude that tpr K is transcribed to a higher degree than the other tpr genes by T. pallidum, Nichols strain. The preferential expression of Tpr K in the Nichols strain is supported by the selective development of Tpr K–specific antibody and reactive T cells in Nichols- infected rabbits (our unpublished results). Because it is not possible to produce clonal populations of T. pallidum, we are uncertain whether the variable transcription of the other tpr genes described above occurs in subpopulations of treponemes within our Nichols strain or in all bacterial cells of the Nichols strain.

Bottom Line: One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium.Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum.This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Washington, Seattle, Washington 98195, USA. acentur@u.washington.edu

ABSTRACT
We have identified a family of genes that code for targets for opsonic antibody and protective immunity in T. pallidum subspecies pallidum using two different approaches, subtraction hybridization and differential immunologic screening of a T. pallidum genomic library. Both approaches led to the identification of a polymorphic multicopy gene family with predicted amino acid homology to the major sheath protein of Treponema denticola. One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium. Reverse transcription polymerase chain reaction analysis of T. pallidum reveals that Tpr K is preferentially transcribed in the Nichols strain of T. pallidum. Antibodies directed to purified recombinant variable domain of Tpr K can opsonize T. pallidum, Nichols strain, for phagocytosis, supporting the hypothesis that this portion of the protein is exposed at the surface of the treponeme. Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum. This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.

Show MeSH
Related in: MedlinePlus