Comparative genomic analysis of Chlamydia trachomatis oculotropic and genitotropic strains

Abstract

Chlamydia trachomatis infection is an important cause of preventable blindness and sexually transmitted disease (STD) in humans. C. trachomatis exists as multiple serovariants that exhibit distinct organotropism for the eye or urogenital tract. We previously reported tissue-tropic correlations with the presence or absence of a functional tryptophan synthase and a putative GTPase-inactivating domain of the chlamydial toxin gene. This suggested that these genes may be the primary factors responsible for chlamydial disease organotropism. To test this hypothesis, the genome of an oculotropic trachoma isolate (A/HAR-13) was sequenced and compared to the genome of a genitotropic (D/UW-3) isolate. Remarkably, the genomes share 99.6% identity, supporting the conclusion that a functional tryptophan synthase enzyme and toxin might be the principal virulence factors underlying disease organotropism. Tarp (translocated actin-recruiting phosphoprotein) was identified to have variable numbers of repeat units within the N and C portions of the protein. A correlation exists between lymphogranuloma venereum serovars and the number of N-terminal repeats. Single-nucleotide polymorphism (SNP) analysis between the two genomes highlighted the minimal genetic variation. A disproportionate number of SNPs were observed within some members of the polymorphic membrane protein (pmp) autotransporter gene family that corresponded to predicted T-cell epitopes that bind HLA class I and II alleles. These results implicate Pmps as novel immune targets, which could advance future chlamydial vaccine strategies. Lastly, a novel target for PCR diagnostics was discovered that can discriminate between ocular and genital strains. This discovery will enhance epidemiological investigations in nations where both trachoma and chlamydial STD are endemic.

FIG. 1.

Circular representation of the A/Har-13 chromosome. First (outer) circle, alignment of each ORF identified in A/HAR-13 with ORFs in the D/UW-3 chromosomal sequence. Those ORFs that share homology (E value cutoff of ≥1e-10⁻¹⁰) in D/UW-3 are indicated in orange. Those ORFs lacking a D/UW-3 homolog are indicated in blue. Second circle, location of RNA encoding regions; tRNAs in dark green, rRNAs in yellow. Third circle, light green and red represent GC skew calculated as C/G, 20-kb sliding window, with 500-bp incremental steps. Red and light-green profiles signify regional prevalence of C over G compared to the average CG value for the entire chromosome. Fourth circle, regional GC% (over a 20-kb sliding window) compared to the total 41.27% GC% for the chromosome. Red indicates regions with a GC% above the total average, while light green indicates regions with a GC% below the average. The relative locations of the origin of replication (ORI) and terminus of replication (TER) are also indicated.

FIG. 2.

Alignment of the Tarp orthologs from A/HAR-13, D/UW-3, and L2/LGV-434; ClustalV (18) protein alignment of A/HAR-13 Tarp (this study), D/UW-3 Tarp (39), and L2/LGV-434 Tarp (10). The first 8 aa of the ∼50-aa amino-terminal repeat motifs are shown with underlying brackets, and the first 8 aa of the ∼120-aa carboxy-terminal repeat motifs are shown with overlying brackets.

FIG. 3.

PCR amplification of the CT456/Tarp 5′ and 3′ deletions in the 15 C. trachomatis reference strains. (A) Schematic representation of the Tarp-encoding genes for serovars A (A/HAR-13), D (D/UW-3), and L2 (L2/LGV-434). The open boxes indicate the coding regions present in the strain of interest (in relation to the strain containing the unaltered sequence), while the lines delineate the deleted region(s). The arrows indicate the relative locations of the PCR primer binding sites. (B) Agarose gel of the PCR amplification results for the 5′ deletion region. Predicted product sizes from the known sequences are 565 bp (A/HAR-13), 607 bp (D/UW-3), and 972 bp (L2/LGV-434). A 1-μg 50-bp DNA ladder is loaded in lane 1, and 1-μg 1-kb ladder is loaded in lane 17 (Invitrogen). (C) PCR amplification results for the 3′ deletion region. Predicted product sizes for the known sequences are 908 bp (A/HAR-13), 463 bp (D/UW-3), and 197 bp (L2/LGV-434). The size markers and lanes are the same as in panel B.

FIG. 4.

Distribution of SNPs per ORF in A/HAR-13 versus D/UW-3. The number of SNPs located within a given ORF is indicated on the x axis. The total number of ORFs containing the indicated number of SNPs is indicated on the y axis.

FIG. 5.

MHC class I and II haplotype epitope mapping of PmpFs from both A/HAR-13 and D/UW-3. PmpF is indicated as an open box (top) and is 1,034 aa in size in both strains. The protein is predicted to be a member of a family of autotransporter proteins (15). As such, the predicted passenger domain, translocation unit, and site of signal sequence cleavage (SS↓) are indicated above the open box. The PmpF sequences from both strains were aligned, and the location of each amino acid substitution is indicated with a vertical line within the open box. Two regions were identified via this alignment as containing a disproportionate number of the amino acid substitutions. Region I (87 aa) is located within the vertical dashed lines, while region II (378 aa) is located within the shaded region. The sequence of each protein was analyzed with the algorithm SYFPEITHI (33) to predict the locations of the MHC class I and II epitopes. The haplotypes included in this analysis are indicated on the right, with predicted epitopes identified for each haplotype located in the same row. Only epitopes giving a predictive score of 25 or greater are shown. Class I epitopes ranged in size from 8 to 10 aa, while class II epitopes were 15 aa in size. Epitopes that were found within conserved regions of the protein are indicated in blue. Epitopes found in regions shown to contain amino acid substitutions are indicated in red.

FIG. 6.

Diagnostic PCR amplification identifying oculotropic versus genitotropic strains. (A) Comparison of the ORF arrangement for the genitotropic strain D/UW-3 versus that of the oculotropic strain A/Har-13. The arrows indicate the relative primer binding sites for the diagnostic PCR amplification with the predicted base pair product size noted for each. The location of the 125-bp ocular-specific deletion is also indicated relative to D/UW-3. In addition to the 125-bp deletion, there is a single-nucleotide deletion located in CTA0934, accounting for the 126-bp difference in PCR product size between the two strains. (B) PCR amplification of the 15 C. trachomatis reference serovars. The sizes of the 1-kb DNA ladder (Invitrogen) bands shown are 1,018, 517/506, 396, 344, and 298 bp (from top to bottom).