Michal Strouhal

Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays

BackgroundSyphilis spirochete Treponema pallidum ssp. pallidum remains the enigmatic pathogen, since no virulence factors have been identified and the pathogenesis of the disease is poorly understood. Increasing rates of new syphilis cases per year have been observed recently.ResultsThe genome of the SS14 strain was sequenced to high accuracy by an oligonucleotide array strategy requiring hybridization to only three arrays (Comparative Genome Sequencing, CGS). Gaps in the resulting sequence were filled with targeted dideoxy-terminators (DDT) sequencing and the sequence was confirmed by whole genome fingerprinting (WGF). When compared to the Nichols strain, 327 single nucleotide substitutions (224 transitions, 103 transversions), 14 deletions, and 18 insertions were found. On the proteome level, the highest frequency of amino acid-altering substitution polymorphisms was in novel genes, while the lowest was in housekeeping genes, as expected by their evolutionary conservation. Evidence was also found for hypervariable regions and multiple regions showing intrastrain heterogeneity in the T. pallidum chromosome.ConclusionThe observed genetic changes do not have influence on the ability of Treponema pallidum to cause syphilitic infection, since both SS14 and Nichols are virulent in rabbit. However, this is the first assessment of the degree of variation between the two syphilis pathogens and paves the way for phylogenetic studies of this fascinating organism.

Complete genome sequence of Treponema paraluiscuniculi, strain Cuniculi A: The loss of infectivity to humans is associated with genome decay

Treponema paraluiscuniculi is the causative agent of rabbit venereal spirochetosis. It is not infectious to humans, although its genome structure is very closely related to other pathogenic Treponema species including Treponema pallidum subspecies pallidum, the etiological agent of syphilis. In this study, the genome sequence of Treponema paraluiscuniculi, strain Cuniculi A, was determined by a combination of several high-throughput sequencing strategies. Whereas the overall size (1,133,390 bp), arrangement, and gene content of the Cuniculi A genome closely resembled those of the T. pallidum genome, the T. paraluiscuniculi genome contained a markedly higher number of pseudogenes and gene fragments (51). In addition to pseudogenes, 33 divergent genes were also found in the T. paraluiscuniculi genome. A set of 32 (out of 84) affected genes encoded proteins of known or predicted function in the Nichols genome. These proteins included virulence factors, gene regulators and components of DNA repair and recombination. The majority (52 or 61.9%) of the Cuniculi A pseudogenes and divergent genes were of unknown function. Our results indicate that T. paraluiscuniculi has evolved from a T. pallidum-like ancestor and adapted to a specialized host-associated niche (rabbits) during loss of infectivity to humans. The genes that are inactivated or altered in T. paraluiscuniculi are candidates for virulence factors important in the infectivity and pathogenesis of T. pallidum subspecies.

Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases.

ABSTRACT Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45°C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47°C and DmbB is 57°C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.

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.

Genome Differences between Treponema pallidum subsp. pallidum Strain Nichols and T. paraluiscuniculi Strain Cuniculi A

ABSTRACT The genome of Treponema paraluiscuniculi strain Cuniculi A was compared to the genome of the syphilis spirochete Treponema pallidum subsp. pallidum strain Nichols using DNA microarray hybridization, whole-genome fingerprinting, and DNA sequencing. A DNA microarray of T. pallidum subsp. pallidum Nichols containing all 1,039 predicted open reading frame PCR products was used to identify deletions and major sequence changes in the Cuniculi A genome. Using these approaches, deletions, insertions, and prominent sequence changes were found in 38 gene homologs and six intergenic regions of the Cuniculi A genome when it was compared to the genome of T. pallidum subsp. pallidum Nichols. Most of the observed differences were localized in tpr loci and the vicinity of these loci. In addition, 14 other genes were found to contain frameshift mutations resulting in major changes in protein sequences. Analysis of restriction target sites representing 0.34% of the total genome length and DNA sequencing of three PCR products (0.46% of the total genome length) amplified from Cuniculi A chromosomal regions and comparison to the Nichols genome revealed a sequence similarity of 98.6 to 99.3%. These results are consistent with a close genetic relationship among the T. pallidum strains and subspecies and a strong, but relatively divergent connection between the human and rabbit pathogens.

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.

A retrospective study on genetic heterogeneity within Treponema strains: Subpopulations are genetically distinct in a limited number of positions

Background Pathogenic uncultivable treponemes comprise human and animal pathogens including agents of syphilis, yaws, bejel, pinta, and venereal spirochetosis in rabbits and hares. A set of 10 treponemal genome sequences including those of 4 Treponema pallidum ssp. pallidum (TPA) strains (Nichols, DAL-1, Mexico A, SS14), 4 T. p. ssp. pertenue (TPE) strains (CDC-2, Gauthier, Samoa D, Fribourg-Blanc), 1 T. p. ssp. endemicum (TEN) strain (Bosnia A) and one strain (Cuniculi A) of Treponema paraluisleporidarum ecovar Cuniculus (TPLC) were examined with respect to the presence of nucleotide intrastrain heterogeneous sites. Methodology/Principal Findings The number of identified intrastrain heterogeneous sites in individual genomes ranged between 0 and 7. Altogether, 23 intrastrain heterogeneous sites (in 17 genes) were found in 5 out of 10 investigated treponemal genomes including TPA strains Nichols (n = 5), DAL-1 (n = 4), and SS14 (n = 7), TPE strain Samoa D (n = 1), and TEN strain Bosnia A (n = 5). Although only one heterogeneous site was identified among 4 tested TPE strains, 16 such sites were identified among 4 TPA strains. Heterogeneous sites were mostly strain-specific and were identified in four tpr genes (tprC, GI, I, K), in genes involved in bacterial motility and chemotaxis (fliI, cheC-fliY), in genes involved in cell structure (murC), translation (prfA), general and DNA metabolism (putative SAM dependent methyltransferase, topA), and in seven hypothetical genes. Conclusions/Significance Heterogeneous sites likely represent both the selection of adaptive changes during infection of the host as well as an ongoing diversifying evolutionary process.

Molecular Typing of Syphilis-causing Strains Among Human Immunodeficiency Virus-positive Patients in Antwerp, Belgium

Centers for Disease Control and Prevention and sequencing-based treponeme typing was used to analyze 72 blood samples, collected from human immunodeficiency virus and syphilis co-infected patients during 2014 to 2015 in Antwerp, Belgium. Twenty-nine (40.3%) isolates were polymerase chain reaction positive for Treponema pallidum, and all tested were macrolide-resistant. Four genotypes were identified by sequencing-based typing including two new genotypes, U4NR8 and SU9R8, whereas enhanced Centers for Disease Control and Prevention typing revealed 7 subtypes.

Human Treponema pallidum 11q/j isolate belongs to subsp. endemicum but contains two loci with a sequence in TP0548 and TP0488 similar to subsp. pertenue and subsp. pallidum, respectively

Background Treponema pallidum subsp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). An unusual human TEN 11q/j isolate was obtained from a syphilis-like primary genital lesion from a patient that returned to France from Pakistan. Methodology/Principal findings The TEN 11q/j isolate was characterized using nested PCR followed by Sanger sequencing and/or direct Illumina sequencing. Altogether, 44 chromosomal regions were analyzed. Overall, the 11q/j isolate clustered with TEN strains Bosnia A and Iraq B as expected from previous TEN classification of the 11q/j isolate. However, the 11q/j sequence in a 505 bp-long region at the TP0488 locus was similar to Treponema pallidum subsp. pallidum (TPA) strains, but not to TEN Bosnia A and Iraq B sequences, suggesting a recombination event at this locus. Similarly, the 11q/j sequence in a 613 bp-long region at the TP0548 locus was similar to Treponema pallidum subsp. pertenue (TPE) strains, but not to TEN sequences. Conclusions/Significance A detailed analysis of two recombinant loci found in the 11q/j clinical isolate revealed that the recombination event occurred just once, in the TP0488, with the donor sequence originating from a TPA strain. Since TEN Bosnia A and Iraq B were found to contain TPA-like sequences at the TP0548 locus, the recombination at TP0548 took place in a treponeme that was an ancestor to both TEN Bosnia A and Iraq B. The sequence of 11q/j isolate in TP0548 represents an ancestral TEN sequence that is similar to yaws-causing treponemes. In addition to the importance of the 11q/j isolate for reconstruction of the TEN phylogeny, this case emphasizes the possible role of TEN strains in development of syphilis-like lesions.

Treponema pallidum 11qj Subtype May Correspond to a Treponema pallidum Subsp. Endemicum Strain.

Unusual 11qj subtype from 1 isolate was identified in a population of syphilis patients in Paris.1 Later on, Mikalova et al.2 suggested that this 11qj subtype may belong to a Treponema pallidum subsp. pertenue (TPE) rather than a T. pallidum subsp. pallidum (TPA) strain and was further commented by Lukehart and Giacani.3 Pathogenic treponemal strains (TPA, TPE, T. pallidum subsp. endemicum [TEN]) are morphologicaly similar, undistinguishable by serology and exhibit important gene identity.