Lenka Mikalová

Whole Genome Sequences of Three Treponema pallidum ssp. pertenue Strains: Yaws and Syphilis Treponemes Differ in Less than 0.2% of the Genome Sequence

Background The yaws treponemes, Treponema pallidum ssp. pertenue (TPE) strains, are closely related to syphilis causing strains of Treponema pallidum ssp. pallidum (TPA). Both yaws and syphilis are distinguished on the basis of epidemiological characteristics, clinical symptoms, and several genetic signatures of the corresponding causative agents. Methodology/Principal Findings To precisely define genetic differences between TPA and TPE, high-quality whole genome sequences of three TPE strains (Samoa D, CDC-2, Gauthier) were determined using next-generation sequencing techniques. TPE genome sequences were compared to four genomes of TPA strains (Nichols, DAL-1, SS14, Chicago). The genome structure was identical in all three TPE strains with similar length ranging between 1,139,330 bp and 1,139,744 bp. No major genome rearrangements were found when compared to the four TPA genomes. The whole genome nucleotide divergence (dA) between TPA and TPE subspecies was 4.7 and 4.8 times higher than the observed nucleotide diversity (π) among TPA and TPE strains, respectively, corresponding to 99.8% identity between TPA and TPE genomes. A set of 97 (9.9%) TPE genes encoded proteins containing two or more amino acid replacements or other major sequence changes. The TPE divergent genes were mostly from the group encoding potential virulence factors and genes encoding proteins with unknown function. Conclusions/Significance Hypothetical genes, with genetic differences, consistently found between TPE and TPA strains are candidates for syphilitic treponemes virulence factors. Seventeen TPE genes were predicted under positive selection, and eleven of them coded either for predicted exported proteins or membrane proteins suggesting their possible association with the cell surface. Sequence changes between TPE and TPA strains and changes specific to individual strains represent suitable targets for subspecies- and strain-specific molecular diagnostics.

Sequencing-based molecular typing of treponema pallidum strains in the Czech Republic: all identified genotypes are related to the sequence of the SS14 strain.

A set of 415 clinical samples isolated from 294 patients suspected of having syphilis collected in the Czech Republic between 2004 and 2010 was tested for the presence of treponemal DNA. Standard serological tests showed that 197 patients were syphilis-seropositive and 97 patients were syphilis-seronegative. In each sample, PCR tests for polA (TP0105), tmpC (TP0319), TP0136, TP0548 and 23S rRNA genes were performed. Samples taken from 91 patients were PCR-positive. Molecular typing of treponemal DNA was based on the sequencing of TP0136, TP0548 and 23S rRNA genes. Treponemal DNA was typeable in samples taken from 64 PCR-positive patients and 9 different genotypes were found. The proportion of treponemal strains resistant to macrolide antibiotics was 37.3%. In the DNA samples taken from 39 patients, a parallel treponemal typing approved by Centers for Disease Control and Prevention was performed. The variants of arp and tpr genes appear to combine independently with sequence variants of TP0136, TP0548 and 23S rRNA genes.

Genome Analysis of Treponema pallidum subsp. pallidum and subsp. pertenue Strains: Most of the Genetic Differences Are Localized in Six Regions

The genomes of eight treponemes including T. p. pallidum strains (Nichols, SS14, DAL-1 and Mexico A), T. p. pertenue strains (Samoa D, CDC-2 and Gauthier), and the Fribourg-Blanc isolate, were amplified in 133 overlapping amplicons, and the restriction patterns of these fragments were compared. The approximate sizes of the genomes investigated based on this whole genome fingerprinting (WGF) analysis ranged from 1139.3–1140.4 kb, with the estimated genome sequence identity of 99.57–99.98% in the homologous genome regions. Restriction target site analysis, detecting the presence of 1773 individual restriction sites found in the reference Nichols genome, revealed a high genome structure similarity of all strains. The unclassified simian Fribourg-Blanc isolate was more closely related to T. p. pertenue than to T. p. pallidum strains. Most of the genetic differences between T. p. pallidum and T. p. pertenue strains were accumulated in six genomic regions. These genome differences likely contribute to the observed differences in pathogenicity between T. p. pallidum and T. p. pertenue strains. These regions of sequence divergence could be used for the molecular detection and discrimination of syphilis and yaws strains.

Resequencing of Treponema pallidum ssp. pallidum Strains Nichols and SS14: Correction of Sequencing Errors Resulted in Increased Separation of Syphilis Treponeme Subclusters

Background Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, is a highly clonal bacterium showing minimal genetic variability in the genome sequence of individual strains. Nevertheless, genetically characterized syphilis strains can be clearly divided into two groups, Nichols-like strains and SS14-like strains. TPA Nichols and SS14 strains were completely sequenced in 1998 and 2008, respectively. Since publication of their complete genome sequences, a number of sequencing errors in each genome have been reported. Therefore, we have resequenced TPA Nichols and SS14 strains using next-generation sequencing techniques. Methodology/Principal Findings The genomes of TPA strains Nichols and SS14 were resequenced using the 454 and Illumina sequencing methods that have a combined average coverage higher than 90x. In the TPA strain Nichols genome, 134 errors were identified (25 substitutions and 109 indels), and 102 of them affected protein sequences. In the TPA SS14 genome, a total of 191 errors were identified (85 substitutions and 106 indels) and 136 of them affected protein sequences. A set of new intrastrain heterogenic regions in the TPA SS14 genome were identified including the tprD gene, where both tprD and tprD2 alleles were found. The resequenced genomes of both TPA Nichols and SS14 strains clustered more closely with related strains (i.e. strains belonging to same syphilis treponeme subcluster). At the same time, groups of Nichols-like and SS14-like strains were found to be more distantly related. Conclusion/Significance We identified errors in 11.5% of all annotated genes and, after correction, we found a significant impact on the predicted proteomes of both Nichols and SS14 strains. Corrections of these errors resulted in protein elongations, truncations, fusions and indels in more than 11% of all annotated proteins. Moreover, it became more evident that syphilis is caused by treponemes belonging to two separate genetic subclusters.

Whole Genome Sequence of Treponema pallidum ssp. pallidum, Strain Mexico A, Suggests Recombination between Yaws and Syphilis Strains

Background Treponema pallidum ssp. pallidum (TPA), the causative agent of syphilis, and Treponema pallidum ssp. pertenue (TPE), the causative agent of yaws, are closely related spirochetes causing diseases with distinct clinical manifestations. The TPA Mexico A strain was isolated in 1953 from male, with primary syphilis, living in Mexico. Attempts to cultivate TPA Mexico A strain under in vitro conditions have revealed lower growth potential compared to other tested TPA strains. Methodology/Principal Findings The complete genome sequence of the TPA Mexico A strain was determined using the Illumina sequencing technique. The genome sequence assembly was verified using the whole genome fingerprinting technique and the final sequence was annotated. The genome size of the Mexico A strain was determined to be 1,140,038 bp with 1,035 predicted ORFs. The Mexico A genome sequence was compared to the whole genome sequences of three TPA (Nichols, SS14 and Chicago) and three TPE (CDC-2, Samoa D and Gauthier) strains. No large rearrangements in the Mexico A genome were found and the identified nucleotide changes occurred most frequently in genes encoding putative virulence factors. Nevertheless, the genome of the Mexico A strain, revealed two genes (TPAMA_0326 (tp92) and TPAMA_0488 (mcp2-1)) which combine TPA- and TPE- specific nucleotide sequences. Both genes were found to be under positive selection within TPA strains and also between TPA and TPE strains. Conclusions/Significance The observed mosaic character of the TPAMA_0326 and TPAMA_0488 loci is likely a result of inter-strain recombination between TPA and TPE strains during simultaneous infection of a single host suggesting horizontal gene transfer between treponemal subspecies.

Molecular typing of Treponema pallidum in the Czech Republic during 2011 to 2013: increased prevalence of identified genotypes and of isolates with macrolide resistance.

ABSTRACT From January 2011 to December 2013, a total of 262 samples, from 188 patients suspected of having syphilis were tested for the presence of treponemal DNA by PCR amplification of five chromosomal loci, including the polA (TP0105), tmpC (TP0319), TP0136, TP0548, and 23S rRNA genes. Altogether, 146 samples from 103 patients were PCR positive for treponemal DNA. A set of 81 samples from 62 PCR-positive patients were typeable, and among them, nine different genotypes were identified. Compared to a previous study in the Czech Republic during 2004 to 2010, the number of genotypes detected among syphilis patients in a particular year increased to six in both 2012 and 2013, although they were not the same six. The proportion of macrolide-resistant clinical isolates in this 3-year study was 66.7%.

Whole genome sequence of the Treponema Fribourg-Blanc: unspecified simian isolate is highly similar to the yaws subspecies.

Background Unclassified simian strain Treponema Fribourg-Blanc was isolated in 1966 from baboons (Papio cynocephalus) in West Africa. This strain was morphologically indistinguishable from T. pallidum ssp. pallidum or ssp. pertenue strains, and it was shown to cause human infections. Methodology/Principal Findings To precisely define genetic differences between Treponema Fribourg-Blanc (unclassified simian isolate, FB) and T. pallidum ssp. pertenue strains (TPE), a high quality sequence of the whole Fribourg-Blanc genome was determined with 454-pyrosequencing and Illumina sequencing platforms. Combined average coverage of both methods was greater than 500×. Restriction target sites (n = 1,773), identified in silico, of selected restriction enzymes within the Fribourg-Blanc genome were verified experimentally and no discrepancies were found. When compared to the other three sequenced TPE genomes (Samoa D, CDC-2, Gauthier), no major genome rearrangements were found. The Fribourg-Blanc genome clustered with other TPE strains (especially with the TPE CDC-2 strain), while T. pallidum ssp. pallidum strains clustered separately as well as the genome of T. paraluiscuniculi strain Cuniculi A. Within coding regions, 6 deletions, 5 insertions and 117 substitutions differentiated Fribourg-Blanc from other TPE genomes. Conclusions/Significance The Fribourg-Blanc genome showed similar genetic characteristics as other TPE strains. Therefore, we propose to rename the unclassified simian isolate to Treponema pallidum ssp. pertenue strain Fribourg-Blanc. Since the Fribourg-Blanc strain was shown to cause experimental infection in human hosts, non-human primates could serve as possible reservoirs of TPE strains. This could considerably complicate recent efforts to eradicate yaws. Genetic differences specific for Fribourg-Blanc could then contribute for identification of cases of animal-derived yaws infections.

Origin of modern syphilis and emergence of a pandemic Treponema pallidum cluster

The abrupt onslaught of the syphilis pandemic that started in the late fifteenth century established this devastating infectious disease as one of the most feared in human history1. Surprisingly, despite the availability of effective antibiotic treatment since the mid-twentieth century, this bacterial infection, which is caused by Treponema pallidum subsp. pallidum (TPA), has been re-emerging globally in the last few decades with an estimated 10.6 million cases in 2008 (ref. 2). Although resistance to penicillin has not yet been identified, an increasing number of strains fail to respond to the second-line antibiotic azithromycin3. Little is known about the genetic patterns in current infections or the evolutionary origins of the disease due to the low quantities of treponemal DNA in clinical samples and difficulties in cultivating the pathogen4. Here, we used DNA capture and whole-genome sequencing to successfully interrogate genome-wide variation from syphilis patient specimens, combined with laboratory samples of TPA and two other subspecies. Phylogenetic comparisons based on the sequenced genomes indicate that the TPA strains examined share a common ancestor after the fifteenth century, within the early modern era. Moreover, most contemporary strains are azithromycin-resistant and are members of a globally dominant cluster, named here as SS14-Ω. The cluster diversified from a common ancestor in the mid-twentieth century subsequent to the discovery of antibiotics. Its recent phylogenetic divergence and global presence point to the emergence of a pandemic strain cluster.

Structure of rrn operons in pathogenic non- cultivable treponemes: sequence but not genomic position of intergenic spacers correlates with classification of Treponema pallidum and Treponema paraluiscuniculi strains

This study examined the sequences of the two rRNA (rrn) operons of pathogenic non-cultivable treponemes, comprising 11 strains of T. pallidum ssp. pallidum (TPA), five strains of T. pallidum ssp. pertenue (TPE), two strains of T. pallidum ssp. endemicum (TEN), a simian Fribourg-Blanc strain and a rabbit T. paraluiscuniculi (TPc) strain. PCR was used to determine the type of 16S–23S ribosomal intergenic spacers in the rrn operons from 30 clinical samples belonging to five different genotypes. When compared with the TPA strains, TPc Cuniculi A strain had a 17 bp deletion, and the TPE, TEN and Fribourg-Blanc isolates had a deletion of 33 bp. Other than these deletions, only 17 heterogeneous sites were found within the entire region (excluding the 16S–23S intergenic spacer region encoding tRNA-Ile or tRNA-Ala). The pattern of nucleotide changes in the rrn operons corresponded to the classification of treponemal strains, whilst two different rrn spacer patterns (Ile/Ala and Ala/Ile) appeared to be distributed randomly across species/subspecies classification, time and geographical source of the treponemal strains. It is suggested that the random distribution of tRNA genes is caused by reciprocal translocation between repetitive sequences mediated by a recBCD-like system.

Comparison of CDC and sequence-based molecular typing of syphilis treponemes: tpr and arp loci are variable in multiple samples from the same patient

BackgroundMolecular typing of syphilis-causing strains provides important epidemiologic data. We tested whether identified molecular subtypes were identical in PCR-positive parallel samples taken from the same patient at a same time. We also tested whether subtype prevalence differs in skin and blood samples.ResultsEighteen syphilis positive patients (showing both positive serology and PCR), with two PCR-typeable parallel samples taken at the same time, were tested with both CDC (Centers for Disease Control and Prevention) and sequence-based typing. Samples taken from 9 of 18 patients were completely typed for TP0136, TP0548, 23S rDNA, arp, and tpr loci. The CDC typing revealed 11 distinct genotypes while the sequence-based typing identified 6 genotypes. When results from molecular typing of TP0136, TP0548, and 23S rDNA were analyzed in samples taken from the same patient, no discrepancies in the identified genotypes were found; however, there were discrepancies in 11 of 18 patients (61.1%) samples relative to the arp and tpr loci. In addition to the above described typing, 127 PCR-positive swabs and whole blood samples were tested for individual genotype frequencies. The repetition number for the arp gene was lower in whole blood (WB) samples compared to swab samples. Similarly, the most common tpr RFLP type “d” was found to have lower occurrence rates in WB samples while type “e” had an increased occurrence in these samples.ConclusionsDifferences in the CDC subtypes identified in parallel samples indicated genetic instability of the arp and tpr loci and suggested limited applicability of the CDC typing system in epidemiological studies. Differences in treponemal genotypes detected in whole blood and swab samples suggested important differences between both compartments and/or differences in adherence of treponeme variants to human cells.