Quantitative Analysis oftprGene Expression inTreponema pallidumIsolates: Differences among Isolates and Correlation with T-Cell Responsiveness in Experimental Syphilis

Abstract

Transcriptional analysis of the tpr genes in Treponema pallidum subsp. pallidum (referred to here as simply T. pallidum) has been limited to date, and yet the expression of members of this gene family is likely relevant to the pathogenesis of syphilis. Recently, immunological studies and semiquantitative mRNA analysis led to the hypothesis of the modulation of tpr gene transcription during infection and suggested that various strains of T. pallidum might differentially express these genes. In this study we developed a real-time amplification assay to quantify the tpr mRNAs with respect to the 47-kDa lipoprotein message and to compare transcript levels among four different strains of T. pallidum. In addition, we analyzed the lymphocyte responsiveness pattern toward the Tpr antigens in late experimental syphilis to identify tpr genes that had been expressed during the course of infection. The T-cell response has been implicated in clearance of treponemes from early lesions, and some of the Tprs were identified as strong targets of the cellular immune response. We show that message for many of the tpr genes can be detected in treponemes harvested at the peak of early infection. Interestingly, tprK seems to be preferentially expressed in almost every strain, and it is uniformly the target of the strongest cellular immune response. These studies demonstrate the differential expression of certain tpr genes among strains of T. pallidum, and further studies are needed to explore the relationship between tpr gene expression and the clinical course of syphilis in infected individuals.The Treponema pallidum repeat (tpr) genes constitute a promising branch of research on venereal syphilis and treponemal diseases in general (6,11,21). This paralogous gene family was identified in part by using a combination of experimental approaches (3), but all 12 members were extensively described by the genomic sequence of T. pallidum subsp. pallidum (referred to here as simply T. pallidum), Nichols strain (8). Subsequently, interest in the tpr genes has continued to rise in parallel to the corpus of data relative to these potential virulence factors (4,5,13,(17)(18)(19)22). The predicted Tpr antigens can be divided into three subfamilies, based upon predicted amino acid sequence homology: subfamily I (TprC, -D, -F, and -I), subfamily II (TprE, -G, and -J), and subfamily III (TprA, -B, -H, -K, and -L). Subfamily I and II members show, within subfamilies, common amino and carboxyl termini, separated by unique central domains that differ in sequence and length. In the Nichols strain, TprC and TprD are identical, and TprF is characterized by a truncated central domain and absence of the conserved COOH-terminal region due to a ϳ1-kb deletion and a frameshift mutation (3). Comparatively, less homology can be found among subfamily III Tprs (3). In the Nichols strain, tprA also contains a frameshift at nucleotide 712, which generates two open reading frames (ORFs), A1 and A2, for this gene (3). A reanalysis of the predicted cellular location of...

Quantitation of rabbit cytokine mRNA by real-time RT-PCR

“…Single use aliquots of the standard dilutions were placed in siliconized tubes and stored in −80°C to ensure DNA stability. Real time RT-PCR quantification of T. pallidum levels in lesion biopsies during experimental syphilis was accomplished using the method for measuring the expression of the gene for the treponemal 47-kDa lipoprotein (TpN47) described extensively elsewhere (Giacani et al, 2007).…”

“…We assessed bacterial burden during lesion progression by examining the mRNA levels of a treponemal gene, tpN47, that encodes for a highly-expressed lipoprotein unique to T. pallidum (Giacani et al, 2007). Lesions became clinically apparent on day 4 following intradermal infection and reached maximum size by day 13, averaging 11-15 mm in diameter.…”

Identification of the Treponema pallidum subsp. pallidum TP0092 (RpoE) Regulon and Its Implications for Pathogen Persistence in the Host and Syphilis Pathogenesis

“…For this work, the TP0574 gene (encoding the 47-kDa lipoprotein) was used as a reference for normalization. The rationale for choosing TP0574 as a reference gene for T. pallidum has been previously discussed in detail (51). To obtain standards for quantification of T. pallidum factor mRNA, fragments of each T. pallidum factor gene and TP0574 amplified from Nichols DNA were cloned into the pCRII-TOPO vector (Life Technologies).…”

“…RNA purification was performed, following the Ultraspec manufacturer's instructions, as already described (50). After RNA extraction, DNase I (Life Technologies, Grand Island, NY) treatment was performed, and samples were checked for residual DNA contamination by PCR amplification as already reported (51). Reverse transcription (RT) of DNA-free RNA was performed using the Superscript II first-strand synthesis kit (Life Technologies) with random hexamers according to the manufacturer's protocol.…”

“…The final plasmid construct was then purified using the Qiagen plasmid minikit and linearized using the EcoRV restriction enzyme (New England BioLabs). Plasmid linearization, copy number calculation, and generation of standard curves for message quantification were performed as previously described (51). Amplification conditions for individual genes are shown in Table 2.…”