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Molecular Studies in Treponema pallidum Evolution: Toward Clarity?

Posted by plosntds_team on 14 Jan 2008 at 23:04 GMT

Following is an excerpt from an upcoming Expert Commentary by Mulligan et al, to be published in PLoS Neglected Tropical Diseases next week.

Molecular Studies in Treponema pallidum Evolution: Toward Clarity?

Connie J. Mulligan 1, Steven J. Norris 2, Sheila A. Lukehart 3

1 Department of Anthropology, University of Florida, Gainesville, Florida, United States of America
2 Department of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, Houston, Texas, United States of America,
3 Department of Medicine, University of Washington, Seattle, Washington, United States of America

A New Study: On the Origin of the Treponematoses—A Phylogenetic Approach

Harper and colleagues [18] have examined a number of chromosomal regions in a total of 23 strains/samples representing the three T. pallidum subspecies and have identified regions of mutation (single nucleotide polymorphisms [SNP] or indels). Some of these polymorphisms have been previously described and others are novel. Importantly, this analysis included two new yaws samples from Guyana, representing the only South American yaws samples evaluated to date. Based upon 17 regions of mutation in the established strains, the authors constructed a phylogenetic tree of the concatenated sequences, and propose that this tree identifies the yaws subspecies as being the oldest, with the bejel and syphilis subspecies evolving subsequently. Unfortunately the DNA from the two new yaws samples from Guyana was too degraded to conduct extensive sequencing, so these samples were not included in the phylogenetic analysis. From these samples, the authors selected a subset of seven genetic regions for analysis. Based upon the homology of four SNPs in the Guyana samples with the group of pallidum strains, the authors conclude that the syphilis subspecies evolved from New World yaws strains.

Strengths and Limitations of the Study

The basis of the claim for a New World origin of venereal syphilis is sequence similarity between the Guyana yaws samples and the pallidum strains. However, the sequence similarity is based on only four SNPs. Furthermore, three of the SNPs cause non-synonymous changes and occur in a very short region (~15 amino acids) of the tprI protein. This is an extraordinarily high rate of evolutionary change in a genus that has been characterized by very little change. Two of the SNPs (tprI 137 and 151) are shown to have evolved two independent times according to the authors’ network analysis (Fig. 3 of Harper et al. [18]), a result that again makes little sense in a genus characterized by very little variation. One of those SNPs (tprI 151) also differs between two pertenue strains CDC-1 and CDC-2, which are strains that might be predicted to be identical given the very close geographical and chronological proximity of isolation. Finally, tprI is thought to be involved in pathogenesis [19] and thus is subject to the effects of natural selection, which violates the assumptions of phylogenetic analysis. Clearly the tprI locus is atypical of the treponemal genome and, thus, is not the best choice when trying to resolve the decades-old debate concerning the origin of venereal syphilis.

Additionally, the phylogenetic and network analyses presented by Harper et al. are contradictory in that the phylogeny supposedly supports the evolution of pallidum from endemicum (Fig 2 of Harper et al. [18]) but the network (Fig 3 of Harper et al. [18]) is used to infer the origin of pallidum from New World pertenue strains. Part of the problem may be the fact that the phylogeny does not show significant structure, contrary to the authors’ claims. When the tree is redrawn to show only branches with minimal 50% bootstrap support, the pertenue cluster disappears and all three subspecies, plus the simian isolate, branch off the most basal branch simultaneously (see Figure 1). This means that no evolutionary order can be inferred. Furthermore, since all strains were collected contemporaneously (at least on an evolutionary time scale), the branch lengths should all be approximately equivalent since a phylogeny reflects only mutational evolution (i.e. all treponemal strains should be equidistant from their common ancestor). The fact that the pallidum strains have longer branch lengths does not mean they evolved more recently, but instead is consistent with an argument for increased recombination or selection along the pallidum branch, i.e. essentially any phenomenon that violates the evolution-by-mutation-only assumption of a phylogenetic analysis. It is also perhaps noteworthy that the pallidum strains are all from the New World except for two strains (South Africa and Madras), whereas the endemicum and pertenue strains are all from the Old World (with the exception of the new Guyana strains). Thus, the reported sequence homology between the Guyana and pallidum strains may simply reflect geographic clustering of New World vs. Old World strains. It would be interesting, though perhaps not possible, to examine older European or Asian pallidum strains to see whether the phylogeny is altered.

Caution, therefore, must be used in drawing conclusions about the evolution of “subspecies” that may represent a biological continuum, rather than discrete agents. Certainly, firm conclusions should not be based upon a few SNPs in two samples taken from a single location.

Next Steps

Despite the limitations of the Harper et al. analysis (as acknowledged by the authors in the discussion), the results reinforce another long-term question: How could the limited divergence between Treponema species and subspecies give rise to the observed differences in pathogenesis? Examination of ~7 kb of sequence resulted in the identification of only 26 nucleotide substitutions among T. pallidum strains, or one difference for every 275 bp (99.6% identity). As indicated in the article, this figure most likely represents a gross overestimate of the overall degree of heterogeneity, because some of the DNA segments were selected because of their high variability. A recent report by Strouhal et al. [20] compared the sequences of T. pallidum subsp. pallidum (Nichols) and Treponema paraluiscuniculi (Strain Cuniculi A), the closely related spirochete that causes venereal spirochaetosis in rabbits but is not pathogenic to humans. A genome-wide analysis using microarray and whole genome restriction mapping indicated that the overall sequence similarity is in the range of 98.6 to 99.3%. Most of the differences identified are within tpr genes or neighboring genes. Based on these two studies and prior evidence, it is possible that genes within the tpr loci are primarily responsible for the differences in disease manifestations and host susceptibility.

T. pallidum is one of the few human bacterial pathogens that have not been cultivated in vitro, obviating experimental approaches such as mutational analysis and complementation to definitively identify virulence determinants. How then can our knowledge of genes related to treponemal evolution and pathogenesis be further refined? One possible approach is whole genome sequencing of multiple strains and comparison of the resulting sequences. The ongoing goal of inexpensive, ‘personalized’ human genome sequences has resulted in the development of multiple novel sequencing approaches; some of these methods can yield a high redundancy bacterial genome sequence for ~US$400 in reagent costs [21]. The new methodologies tend to yield shorter sequences per template (25 to 100 bp) and to have a higher error rate than Sanger sequencing. These shortcomings make the newer technologies more applicable to genome re-sequencing (e.g. the analysis of the closely related pathogenic Treponema strains) and can be ameliorated in part by combining the results of two or more sequencing technologies.

Because of the paucity of available endemicum strains and New World pertenue isolates, new approaches may be needed to analyze archival DNA specimens. Kolman et al. [22] were able to identify T. pallidum sequences by PCR amplification of DNA extracted from deformed bones (saber shins) in 200-year-old skeletal remains from Easter Island. Processes have been developed for isolation, whole genome amplification, and sequencing of DNA from individual bacterial cells [23,24]. These methods, perhaps coupled with laser capture microscopy or techniques for dissecting out organisms, could be utilized to recover and obtain sequence information from ancient bones or preserved tissue specimens with treponemal infections.


18. Harper KN, Ocampo PS, Steiner BM, George RW, Silverman MS, et al. (2007) On the Origin of the Treponematoses: A Phylogenetic Approach. PLoS Negl Trop Dis 2(1) e148. doi:10.1371/journal.pntd.0000148

19. Giacani L, Sambri V, Marangoni A, Cavrini F, Storni E, et al. (2005) Immunological evaluation and cellular location analysis of the TprI antigen of Treponema pallidum subsp. pallidum. Infect Immun 73: 3817-3822.

20. Strouhal M, Smajs D, Matejkova P, Sodergren E, Amin AG, et al. (2007) Genome differences between Treponema pallidum subsp. pallidum strain Nichols and T. paraluiscuniculi strain Cuniculi A. Infect Immun 75: 5859-5866.

21. Bentley DR (2006) Whole-genome re-sequencing. Curr Opin Genet Dev 16: 545-552.

22. Kolman CJ, Centurion-Lara A, Lukehart SA, Owsley DW, Tuross N (1999) Identification of Treponema pallidum subspecies pallidum in a 200-year-old skeletal specimen. J Infect Dis 180: 2060-2063.

23. Marcy Y, Ouverney C, Bik EM, Losekann T, Ivanova N, et al. (2007) Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proc Natl Acad Sci U S A 104: 11889-11894.

24. Zhang K, Martiny AC, Reppas NB, Barry KW, Malek J, et al. (2006) Sequencing genomes from single cells by polymerase cloning. Nat Biotechnol 24: 680-686.

RE: Molecular Studies in Treponema pallidum Evolution: Toward Clarity?

kharper replied to plosntds_team on 24 Jan 2008 at 01:00 GMT

This is our response towards this partial commentary by Mulligan et al. A complete point-by-point response to their commentary can be found in the discussion section for that article.

Molecular Studies in Treponema pallidum Evolution: Toward Reality
Kristin N. Harper1, Bret M. Steiner2, Michael Silverman3 and George J. Armelagos4

1 Population Biology, Ecology, and Evolution Department, Emory University, Atlanta, GA
2 Laboratory Research and Reference Branch, Division of Sexually Transmitted Diseases, U.S. Centers for Disease Control and Prevention, Atlanta, GA
3 University of Toronto, ON, Canada
4 Anthropology Department, Emory University, Atlanta, GA

Our claim for a New World origin of venereal syphilis is based on sequence similarity between the Guyana subsp. pertenue samples and the subsp. pallidum strains. Our sequence comparison consisted of 17 SNPs that differentiated subsp. pallidum from subsp. pertenue and endemicum strains. The Guyana subsp. pertenue samples resembled syphilis-causing strains at four of these SNPs; at most, other non-venereal strains were identical at only one. Mulligan et al object that three of these identical SNPs come from a single gene, tprI. They neglect to mention that the fourth SNP, a synonymous substitution in the gpd gene, is on the other side of the genome and tells the same story. They argue that, "the tprI locus is atypical of the treponemal genome and, thus, not the best choice when trying to resolve the decades-old debate concerning the origin of venereal syphilis." While we clearly explained that the tpr genes may be atypical of the genome in our article, and thus emphasized the importance of the supporting SNP from the gpd gene, in their 2006 paper Drs. Mulligan and Lukehart created phylogenies entirely from the tpr genes and used them to make inferences about the treponemes’ evolutionary past [4]. Much of their discussion in that article centered on the tprI gene in particular, in which they asserted that recombination did not play an important role and in which substitutions “were assumed to evolve in a clocklike manner.” In the commentary, Mulligan et al state that the CDC-1 and CDC-2 strains of subsp. pertenue should have identical genetic signatures at the tprI gene, since they were collected in West Africa at similar times. For this reason, they seem to have changed their mind regarding the suitability of this gene for evolutionary inference. However, if yaws did indeed originate in Africa, then we would expect to find a greater amount of variation in African strains than in those from other locations.
Mulligan and colleagues make the point that the two New World subsp. pertenue samples were taken from one location. In fact, although these two samples were both taken from Guyana, the villages involved are at a significant distance from each other. Travel between them requires a combination of bush plane and boat. The communities therefore do not mix, and, therefore, these are truly two separate samplings.
Mulligan and colleagues’ discussion of our phylogeny is riddled with errors in terminology and misunderstanding of basic evolutionary concepts. This impairs their interpretation of our study and makes it difficult to understand their criticism. They frequently refer to the study of genes that are subject to natural selection or recombination as violating the “assumptions of phylogenetic analysis.” There are no universal assumptions of phylogenetic analysis. The assumptions of each phylogeny depend on the methods used and the problem addressed. Drs. Mulligan and Lukehart must be aware of this fact, having published a paper full of phylogenies based on the members of the recombining tpr gene family [4]. We assume that what they refer to is that in genes subject to these processes, the molecular clock cannot be used. Similarly, Mulligan et al state that because the “strains were collected contemporaneously, the branch lengths should all be approximately equivalent, since a phylogeny reflects only mutational evolution.” They also write that selection violates the “evolution-by-mutation-only assumption of a phylogenetic analysis.” Again, they confuse phylogenetic analysis and the molecular clock, while also asserting that only neutral substitutions result from mutation. We presume that what they meant to say is that the use of the molecular clock assumes that neutral substitutions accumulate in a clockwork manner, but substitutions subject to positive selection may not.
It is our understanding, then, that Mulligan and coworkers’ main concern is that recombination and positive selection are present in our data and complicate the interpretation of the phylogeny’s branch lengths in terms of time elapsed. However, we performed rigorous tests for recombination (p. 5) and for positive selection (using maximum likelihood methods to estimate the ratio of non-synonymous to synonymous substitutions along each branch of the phylogeny), and found evidence of neither. If Mulligan et al assert that there is rampant recombination and selection present in these data, they are obligated to provide evidence to support their assertion. Although they cite the long length of the branch leading to syphilis-causing strains as evidence of one or both of these processes at work, this branch could also be explained by the recent divergence of this subspecies and the time dependence of molecular rate estimates [13], which is consistent with our conclusions.
Mulligan and colleagues’ other main contention is that our phylogeny lacks significant structure. They state that the phylogeny was created from 17 SNPs/INDELS. This is incorrect. Our phylogeny was created from a total of 70 SNPs and 12 INDELS, found in 20 different genetic regions; 26 of these SNPs and 5 of these INDELS occurred between T. pallidum strains. In their reanalysis of our data, they fail to provide their methodology, and thus it is unclear which 17 substitutions were used and which were not to redraw the phylogeny. However, having only analyzed a subset of our data, it is to be expected that the authors did not identify some of the clades that we found in our analysis. They also fail to mention that the relative order of divergence of the subspecies found in our phylogeny was further supported by the average nucleotide difference of each subspecies from T. paraluiscuniculi, the outgroup (p. 9).
Mulligan et al express concern that, in our phylogeny syphilis-causing strains appear to be most closely related to subsp. endemicum strains, while in our network analysis syphilis-causing strains appear most closely related to New World subsp. pertenue strains. But, as they note earlier in their commentary, the New World subsp. pertenue strains were not included in the phylogeny because extensive sequencing could not be performed upon them. Therefore, the phylogeny could not show that syphilis-causing strains were most closely related to New World subsp. pertenue strains. In the network analysis, in which all strains were included, syphilis-causing strains were most closely related to New World subsp. pertenue strains, with subsp. endemicum strains following. Figures three and four in our paper are consistent.
In one of their most surprising objections, Mulligan et al suggest that the resemblance between syphilis-causing strains and New World subsp. pertenue strains may be due to geographic clustering. There is no evidence in any peer-reviewed publication that suggests geographic clustering between subspecies occurs. Are they again proposing the Unitarian hypothesis, which posits no genetic differences between the subspecies, is viable? While their idea is an interesting one, there is absolutely no evidence to support it.
Mulligan et al conclude by asking a fascination question: “How could the limited divergence between Treponema species and subspecies give rise to the observed differences in pathogenesis?” This is the question that drives the ongoing work in our laboratories. However, having argued earlier that there is no biological basis for transmission route in the T. pallidum subspecies, we do not understand how Mulligan et al can now assert that variation in the tpr genes most likely underlies biological differences between the subspecies. The authors call for additional sequencing of strains, which we wholeheartedly support. They also propose that more effort be put into sequencing archival strains, in particular into sequencing ancient treponemal DNA. Sadly, the recent literature questions our ability to do this. Mulligan and Lukehart’s previous paper , in which DNA was amplified from a 200-year-old bone sample from Easter Island, is cited. However, amplification of ancient treponemal DNA has never been replicated, and, in their study, Mulligan and colleagues did not provide data that could be used to assess its quality, such as the sequence of individual clones reflecting damage consistent with an age of 200 years. Attempts to amplify ancient treponemal DNA have met with no success, even when a variety of medically diagnosed samples from different geographies and different temporal periods, including the twentieth century, were assessed [14-16]. It has long been suspected that difficulty in identifying treponemes inside diagnostic bone lesions is attributable to the relative absence of the bacteria in tertiary-stage infection [17]. Last year, a rabbit model was used to obtain empirical evidence that, after initial infection, it is difficult to amplify treponemal DNA from bone [16]. Because of the invasiveness of attempting to amplify treponemal aDNA, as well as the method’s poor track record, the scientific community is increasingly skeptical of the utility of this approach.
Mulligan and coworkers misrepresent the most basic facts in our paper, such as the number of regions that we included in our phylogeny. Their failure to accurately present the most basic evolutionary concepts, such as the molecular clock and the uses of phylogenetics, introduces unnecessary confusion. In reality, recent molecular studies have clarified the evolutionary history of the treponemes while providing new questions for future studies [1,5].


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