COMPLETE GENOME SEQUENCE OF TREPONEMA PALLIDUM THE SYPHILIS SPIROCHETE PDF

Abstract Background: Syphilis spirochete Treponema pallidum ssp. Increasing rates of new syphilis cases per year have been observed recently. Results: The 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, single nucleotide substitutions transitions, 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.

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Abstract Background: Syphilis spirochete Treponema pallidum ssp. Increasing rates of new syphilis cases per year have been observed recently. Results: The 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, single nucleotide substitutions transitions, 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. Conclusion: The 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.

After a period of decline in the s, the other repeats. DDT sequencing of 12 such regions number of reported cases of primary and secondary syph- revealed 5 real SNPs, 6 false positives, and one position ilis has been raising annually since in the United with 2 alleles within the SS14 population intrastrain het- States [2].

Sequencing of the 1. Therefore these questionable SNPs were not Nichols strain of TPA in [3] greatly stimulated study included in the final sequence, unless they were verified of this unculturable pathogen. One important direction by DDT sequencing data not shown. An additional 62 not yet developed is use of the Nichols sequence for com- positions out of the SNPs identified by CGS were parative studies to determine variation between different DDT sequenced.

To sample strains on a sufficient scale, rapid, SNPs in the second sequencing stage but there were 57 inexpensive, and highly accurate sequencing methods are regions encompassing oligonucleotide targets where needed.

Traditional whole genome shotgun sequencing sequence changes could not be determined. These repre- methods using dideoxy-terminators WGS-DDT are rela- sented possible hypervariable regions with multiple dif- tively slow and costly to be applied to numerous samples.

In 21 of the 38 cases, mostly closely spaced genomes. DDT sequencing of []. In vitro test- sequenced regions of at least bp in both directions. Nichols strain sequencing of regions identified by WGF Table 3. Alto- was isolated in in Washington, D. Weinstock, unpublished results were found in these DDT sequenced regions 42, bp. To compare these closely related, yet pheno- positive results. Weinstock, In the first mapping stage of CGS, no regions with signifi- unpublished results using a microarray of TPA coding cantly stronger labeled SS14 DNA signals were observed, sequences [18] and thus was not completely random.

These results indi- indicated candidate regions of variation encompass- cate an error frequency comparable to or lower than that ing 1 or more overlapping oligonucleotide targets. The of high quality finished DDT sequence.

Assessment of reproducibility of CGS experiments Physical mapping of treponemal chromosome To test the reproducibility of the method, the genome of To verify the complete sequence of SS14 strain, to screen TPA SS14 was sequenced twice with the CGS approach, for possible discrepancies in cross-reacting repeat regions using 2 independent DNA isolations from two subse- tpr genes and insertions of unique sequences, WGF was quent inoculations of rabbit testes i.

This physical mapping approach showed the respectively. When most of the variable genomic regions order of the ORFs along the chromosome is identical to were excluded from the analysis CGS cannot identify Nichols genome and 4 large indel regions were identified. A dele- shown , three loci showed intrastrain heterogeneity in tion in TP and an insertion in TP—TP com- one of the two SS14 DNA isolations, with one allele iden- prised tandem repeats of 24 and 60 bp, respectively.

The reproducibility of the CGS previously [19]. Moreover, intrastrain heterogeneity in the method is thus likely to be limited by the presence of SNP Nichols strain was observed in regions comprising clusters and influenced by genetically different subpopu- TP—TP and upstream of TP with one allele lations in the test sample.

Sequence changes of variable regions V1—V7 of strain. A similar region was previously described in TP, tprK, were not included, because sequences of another syphilitic strain Chicago, [GenBank:AY] these regions were found to differ greatly in both length and was found to contain a sequence similar to tprK and and sequence within the SS14 population, in agreement is believed to be recipient site of the tprK conversion [21].

Altogether, three large indels were not detected by CGS. Obtained data have been used to compile the sequence of We suggest probable reasons for this fact are 1 the length the SS14 genome [GenBank:CP]. Frameshift mutations and other changes affecting protein Hypervariable regions are listed in Table 5 and include length are presented in Table 4. TP was predicted to be a virulence factor [26] and Sequence changes in four cases led to fusion of ORFs was experimentally verified to be an antigen [27].

Three of these genes TP, TP, TP and human infections [27,28] and was found to serve as were predicted to code for possible surface protein viru- fibronectin and laminin binding protein [29].

The distribution of SNPs in coding and non-coding and TP hypothetical protein and in the intergenic sequences of SS14 was not significantly different. ORFs region between tprI and tprJ.

Consensus sequences were represent Altogether, intrastrain genetic heterogeneity com- 17 SNPs 5. The frequency of SNPs was different polymorphism in a homopolymeric stretch Table 7. The highest fre- quency of SNPs was in hypothetical genes, lowest in Discussion housekeeping genes.

In addition, housekeeping genes had Obtaining the complete genome sequence of a second the lowest number of SNPs altering amino acid sequences syphilis spirochete SS14 shows the utility of the CGS indicating conservation of these gene products.

This is the first application of this approach to sequence an entire Identification of intrastrain variability in TPA population genome. This approach can be used when highly similar Because DDT sequencing of some PCR products did not genomes are investigated and one genome sequence of result in an unambiguous sequence, WGS-DDT sequenc- closely related organism is known.

The CGS strategy rep- ing of small insert libraries was performed. Analysis of resents a rapid days to weeks and scalable methodology libraries and PCR products revealed multiple intrastrain to sequence multiple syphilitic strains and clinical iso- sequence variants in TP tprC , TP coding for lates.

The Tpr protein family includes 12 Treponema There are some of the TPA-specific limitations of this pallidum repeat proteins, found uniquely in this bacterium approach to whole genome sequencing.

Because the CGS and showing sequence similarity to major sheath protein strategy uses genomic DNA as a probe, accuracy is affected of Treponema denticola. Repeat regions found to be affected by sequence changes representing a hybridize to more than one oligonucleotide on a tiling higher proportion than the whole genome rate Altogether 53 SNPs the variants detected.

Precautions have to be taken when and 38 intrastrain variable nucleotide positions, with at inspecting tpr regions and others arp gene, TP least one allele identical to the sequence of the Nichols which cross-react based on sequence similarity. Such genome, were found in tpr genes V1—V7 regions of tprK regions, together with highly variable regions, need to be were excluded from this analysis. Based on the fact that analyzed by WGF and sequenced by DDT to reveal true tpr genes share a high degree of similarity on the DNA nucleotide changes and numbers of repeated regions.

Multiple repeated sequences. Multiple alleles of tpr genes were sequence variants in the Nichols strain population were described among and within TPA strains [,32,33] both described previously and identified in this work, and and some TPA repeated regions tpr genes, arp gene were hybridization based sequence changes discovery in these used as loci for typing of clinical isolates []. Finally, the accu- didate sequences to screen clinical isolates and have racy of the genome sequence produced by CGS is potential to be used as typing markers of strains and iso- dependent on the accuracy of the reference genome lates.

In addition, different strains of TPA have already sequence. As suggested by two newly revealed frameshifts been tested for association with higher risk for neuroinva- in Nichols strain sequence, discovered sequence changes sion in rabbits [39] and identification of underlying have to be verified in Nichols sequence to describe real sequence changes will enable prediction of such risks.

The sequence changes compared to Nichols genome. Both Nichols and SS14 antigenicity data, these could represent pseudogenes. The examples of interstrain and simply scalable method to assess genome-wide heterogeneity and multiple alleles in a population of hap- genetic variability within TPA strains and subspecies, loid organisms are candidates for antigenic variation, con- which share a very unusual degree of sequence similarity tingency genes and other types of SSR short sequence and lack genome rearrangements as shown in this study.

Sequence var- washed in non-stringent wash buffer followed by two 30 iants could be readily used for molecular typing and iden- sec washes in 0. Moreover, the ability to now sequence array design and procedure were described previously [4]. This is a significant development for an position 4 for each strand and 48, bases were organism of important public health impact, but for sequenced in total. Because mutations are sequenced in which standard bacterial genetic methods are untenable.

Methods DNA isolation Dideoxy-terminator sequencing of heterologous SS14 TPA strains Nichols and SS14 were maintained by rabbit genome regions inoculation and purified by Hypaque gradient centrifuga- After the second sequencing stage of the array analysis, tion as described previously [40]. These regions were sequenced by DDT sequencing.

These fragments were cloned into the Mutation mapping microarrays were designed to map pUC18 vector resulting in small insert libraries and mutations by selecting a 29 mer oligonucleotide every 7 recombinant plasmids isolated from at least 48 colonies bases for both strands of the complete TPA Nichols were DDT sequenced to multiple coverage using pUC18 genome sequence [3], [GenBank:AE].

All , primers. Whole genome fingerprinting Arrays were hybridized to digested, labeled genomic DNA Whole genome fingerprinting was performed as described of Nichols and SS14 strains separately and processed as previously [44].

The chromosomal DNA was amplified in described in [4] with an additional step after second wash Treponema pallidum interval TPI regions with in stringent buffer consisting of staining with a solution median length of 12, The primer pairs for these stringent wash buffer. The Cy3 signal was amplified by amplifications are listed in Table S2 See additional file 1: secondary labeling of the DNA with biotinylated goat Supplemental data.

The resulting fingerprints of TPA 5. SARS coronavirus using high-throughput, high-density rese- quencing arrays. Genome Res , 14 3 Nucleotide sequence accession number 6.

Antimicrob Agents Chemother , 50 6 GMW designed the study. PM performed genome Nature Genet , 38 12 JFP contributed to 9. All authors read overproduction in Streptomyces coelicolor A3 2. J Bacteriol and approved the final manuscript. Infect Immun , 75 7 Additional file 1 Genome Res , 16 6 J Bacteriol , 5 Miura K, Rikihisa Y: Virulence potential of Ehrlichia chaffeensis Acknowledgements strains of distinct genome sequences.

Infect Immun , This work was supported by grants from the U. Public Health Service to 75 7 R01 AI and T. Antimicrob Agents Chemother , 32 2 World Health Organization: Global prevalence and incidence of Bacteriol , 5

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Complete genome sequence of Treponema pallidum, the syphilis spirochete.

Abstract Background: Syphilis spirochete Treponema pallidum ssp. Increasing rates of new syphilis cases per year have been observed recently. Results: The 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, single nucleotide substitutions transitions, 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.

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Complete genome sequence of Treponema pallidum, the syphilis spirochete

You can help by adding to it. December Three subspecies of T. Treponema carateum, the cause of pinta, remains a separate species because no isolate is available for DNA analysis. They are composed of the intermediate filament -like protein CfpA cytoplasmic filament protein A. Although the filaments may be involved in chromosome structure and segregation or cell division, their precise function is unknown. Their DNA sequences are more than It revealed that T.

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