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Retraction vindicated?

Two new studies seek to validate the results of a retracted 2004 paper on parasite-to-host gene transfer, but skepticism lingers

By | March 28, 2011

The microparasite that causes Chagas disease really can integrate bits of its genetic material into its host's genome, where it can then be inherited by the host's offspring, according to two studies published in linkurl:PLoS ONE;http://f1000.com/5406956?key=7dy1rrt6b35c8v1 and PLoS Neglected Tropical Diseases (PLoS NTD).
Trypanosoma cruzi forms in blood smear from patient with African trypanosomiasis.
Image: Wikimedia commons, CDC/Dr. Myron G. Schultz
The findings, which may represent the first documentation of lateral genetic transfer from parasite to host and subsequent vertical transfer to the host's progeny, appear to confirm the results of a retracted 2004 Cell paper, but lingering uncertainties in the data leave some scientists unconvinced. "I think it's very exciting, and could explain a lot of things about Chagas disease," said genome biologist linkurl:Cédric Feschotte;http://www.uta.edu/biology/feschotte/ at the University of Texas in Arlington, who was not involved in the research. "The implications are potentially huge, but extraordinary claims require extraordinary lines of evidence...and it's still missing some crucial pieces of evidence," such as confirmation of the integration sites and conclusive evidence of DNA integration in the offspring. "I'm not completely convinced," agreed linkurl:Julie Dunning Hotopp;http://medschool.umaryland.edu/facultyresearchprofile/viewprofile.aspx?id=20308 University of Maryland School of Medicine, who did not participate in the research. "But I'm excited by the possibility. Viruses are known to integrate into the human genome, but most people think bacterial DNA and parasite DNA don't integrate." Chagas is a tropical parasitic disease that can lead to life-threatening heart damage. It's transmitted by the flagellate protozoan Trypanosoma cruzi, but the details by which T. cruzi causes Chagas have long baffled researchers. In 2004, Antônio Teixeira of the Chagas Disease Multidisciplinary Research Laboratory at the University of Brasilia and colleagues found that T. cruzi could transfer genetic material to its rabbit, chicken, and human hosts. The researchers suggested that such lateral transfer, specifically of the parasite's mitochondrial kinetoplast DNA (kDNA), may contribute to the disease by disrupting host gene function and causing an autoimmune response. They published their findings in a July issue of Cell, also reporting evidence of parasite DNA in the progeny of infected individuals. But the following year, Cell linkurl:retracted the paper;http://www.cell.com/abstract/S0092-8674%2804%2900630-0 without the authors' consent and linkurl:without much explanation,;http://www.the-scientist.com/article/display/22782/ stating that there were "concerns" regarding the specific sites of DNA integration, and that the data did "not provide strong enough evidence for the central hypothesis," according to independent reviewers.
Trypanosoma cruzi in monkey heart.
Image: Wikimedia commons, CDC/ Dr. L.L. Moore, Jr.
linkurl:Nancy Sturm,;http://www.mimg.ucla.edu/faculty/campbell/ a University of California, Los Angeles, researcher who helped with all three manuscripts, said she was "absolutely shocked" to learn about the retraction. "It seemed like it was all kind of done behind closed doors." Sturm added that her impression was that there were problems Teixeira's identification and analysis of the specific sites of DNA integration, which she said is one important but relatively small part. Those who find fault with the paper cite the techniques used that are highly-prone to methodological artifacts, and additional tests that would have been needed to confirm that integrated DNA got passed on to offspring. "[The retraction was] an extremely dramatic course of action because it basically stigmatizes the authors," said linkurl:Etienne Joly;http://f1000.com/thefaculty/member/1613334334127718 of the CNRS in France, who was not involved in the research. "They will have ten times more hurdles to jump because everyone suspects that there was misconduct. Retracting it without proving there was fraud, misconduct or gross misinterpretation -- that's totally unacceptable as far as I'm concerned." Lynne Herndon, the president and CEO of Cell at the time of the retraction, declined to provide comment to The Scientist in 2005, and Emilie Marcus, the current executive editor of Cell Press, declined to comment when contacted late last year. Marcus did not respond to further requests for comment as this article went to press. Teixeira said he responded to many requests from Cell for clarification of the data prior to the retraction -- to which Cell simply responded it was forced to retract the paper, according to Teixeira -- and has since appealed for the paper to be returned to the literature, to no avail. Confident in his data, Teixeira's strategy has been simply to move on. "My way of fighting back is producing more information," he said. He has now published two studies that he claims validate his previous results. In the first paper, published in linkurl:PLoS ONE;http://f1000.com/5406956?key=7dy1rrt6b35c8v1 in February 2010, Teixeira and his colleagues once again claim they've shown evidence of lateral DNA transfer from T. cruzi kDNA to various retrotransposable LINE elements of its human hosts, with progenies of five families demonstrating the vertical transmission of such integration. The other study, published online today (March 29) in PLoS NTD, extends their findings to a chicken model, where the parasite itself cannot be passed on through the egg. As such, Teixeira claims, any evidence of T. cruzi DNA in the offspring of infected individuals must be a result of DNA integration and transmission through the germline. Once again, the team provides evidence of lateral and vertical DNA transfer from parasite to host, with affected chickens showing Chagas-like symptoms. "The findings in PLoS ONE paper and in the PLoS NTD paper data confirm and extend all our previous data and information in the Cell 2004 unilaterally retracted paper," Teixeira told The Scientist. "The data and the information in the Cell paper are correct, and the results in it are consistent with the conclusions drawn." "In the chicken model you definitely see the integration and the passing on of only the DNA sequence, and not the parasite, to the progeny," agreed Sturm -- who is not listed as an author on any of the papers, but is acknowledged for her comments on the manuscript. But not everyone is convinced. Much of the evidence presented in all three papers is based on PCR, which revealed bits of hybrid DNA containing genetic sequences from both the trypanosome and its host. But PCR is highly prone to artifacts, said Dunning Hotopp. "Any time you do a PCR and you have a sequence that's common between two pieces of DNA, you have the potential to create a chimera," which is then amplified during the subsequent rounds of PCR, she explained. "The results of the method could be chimeras that look like lateral gene transfer. I don't think that's what happening," she added, "but I think that's still a black mark against the paper." According to some researchers, Teixeira and his team could easily quell the controversy surrounding the three papers. To validate the PCR results, Feschotte noted, it's important to use specific primers located in the regions flanking the suspected insertion and verify the sequence. "That would really be the nail in the coffin," he said, but "they don't do that." Another potential validation is to visualize the suspected integration sites using a technique known as a southern blot, something Teixeira and his team did. Unfortunately, some researchers did not find the results of these tests conclusive. There is a high burden of proof for lateral DNA transfer that is not always met, Dunning Hotopp said. "This is often a problem with lateral gene transfer papers. When you read the paper it looks fine but when you go into the databases and look at the sequences, it's not as convincing." Still, most scientists who have seen Teixeira's results think that there is some truth to the findings; that T. cruzi-infected individuals likely integrate the parasite's kDNA into their genomes, and even passing it on to their offspring, if not at the frequencies reported by Teixeira and his colleagues. "I still think he's onto something really exciting. I think that what they've uncovered about Chagas is really important," Joly said. "I have the feeling they don't do the right experiments to ask the right questions. Even if you have an interesting observation in the first place, if you document it badly then you give a bad name to the observation." M.M. Hecht et al., "Inheritance of DNA transferred from American trypanosomes to human hosts," linkurl:PLoS ONE,;http://f1000.com/5406956?key=7dy1rrt6b35c8v1 5: e918, 2010. A.R.L. Teixeira et al., "Trypanosoma cruzi in the chicken model: Chagas-like heart disease in the absence of parasitism," PLoS Negl Trop Dism, 5: e1000, 2011.
**__Related stories:__*** linkurl:Bacterial genes jump to host;http://www.the-scientist.com/news/display/53552/
[30th August 2007]*linkurl:Scientists protest Cell retraction;http://www.the-scientist.com/article/display/22782/
[29th September 2005]

**__Related F1000 Evaluations:__***linkurl:Inheritance of DNA transferred from American trypanosomes to human hosts;http://f1000.com/5406956?key=7dy1rrt6b35c8v1
M.M. Hecht et al., PLoS ONE5: e918, 2010. Evaluated by Etienne Joly, CNRS.*linkurl:Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon;http://f1000.com/1060778?key=4jyp6mm26q065d4
A. Simões-Barbosa et al., Mem Inst Oswaldo Cruz, 101:833-43, 2006. Evaluated by Paul J Brindley, George Washington Univ.
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Comments

Avatar of: Fred Schaufele

Fred Schaufele

Posts: 52

March 31, 2011

I have read none of the papers. But really, this has to be the most straightforward study of anything that any of us would ever do in a lab: Are there virus sequences inserted in the host genome that were not there prior to infection? Are those now in the progeny? End of story.\n\nI'm confused. Why the controversy? This is not technically challenging to complete(subclone genome DNA, pull out insertions, sequence to identify insertion site, check prior to infection, possibly for chimerism in the infected subject and for genome-wide insertion in progeny). If the authors provided that, then it's pretty much a done deal. If the authors did not provide that, then it shouldn't be published. Have we collectively somehow lost the essence of experimental design, execution and conclusions? It's embarassing that we'd be even having this discussion in an online forum.
Avatar of: Mike Waldrep

Mike Waldrep

Posts: 155

March 31, 2011

Interesting! I hope that everyone has a great April Fool's Day! That goes for last year and all the other years that I've missed.
Avatar of: DON MERLO

DON MERLO

Posts: 1

April 4, 2011

It was proven in the mid 1970's that the plant pathogen Agrobacterium tumefaciens transfers over 20 kbp of plasmid DNA to plant cells, wherein it is stably integrated into the chromosomes and transmitted to progeny cells in a Mendelian fashion. Since then, several species of fungi and even cultured mammalian cells have also been transfomed in the lab by Agrobacterium.
Avatar of: Eugene Semon

Eugene Semon

Posts: 16

April 6, 2011

When wondering about something straightforward that suddenly becomes "mysterious", as the previous post states, the question to ask is cui bono?
Avatar of: Eugene Semon

Eugene Semon

Posts: 16

April 6, 2011

I meant the comment by Fred Schaufele.

April 13, 2011

For decades I have been skeptical that random mutation, alone, could drive evolutionary adaptation and, as a consequence, I welcome each new increment of evidence leading toward the discovery of ANY MECHANISM or mechanisms that might be more intellectually satisfying to me than that unlikely one. Do two viruses invade a single eukaryote cell simultaneously, and a new or chimeric virus emerge sometimes? Does a virus ever enter a cell and the cell survive and maintain an ability to survive and reproduce? The odds may be astronomically averse to it, but the number of invasions are, after all, astronomical in number. (Obviously I am not a molecular biologist, or I would know of research results relating to these possibilities. But it takes only one example to rule in a possibility, on the one hand, and an infinite number of examples to rule out a possibility... isn't that correct?) Exchanges of chromosomes, or fragments of broken chromosomes, whenever and however they might occur, could participate on evolutionary change (heritable adaptation), could it not? Attempts at gene therapy, by use of vectors, are in their infancy. And progress in our understanding of heritable RNA/DNA anomalies will surely come. Who knows for a CERTAINTY that no mechanisms remain undiscovered, whereby RNA/DNA exchanges do not occur in rare, but evolutionarily significant ways, to provide ways outside of mere random mutation, in coming up with serendipitous adaptation challenge appropriate innovations within species, or across species? My intuition tells me there just have to be other mechanisms in play, in providing adaptive changes. Non-deleterious random mutations that actually produce some immediate advantage are remarkably rare, and random mutations that would, at each increment, provide advantage and yet contribute to a chain of such traits resolving into a complex organ or system, such as the eye or the human visual system, would seem next to impossible, if we had not the hard evidence to know seeing does occur. And exchanges of RNA/DNA sequences may be rarer even than such fortunate serendipitous results. But any entrenched assumption that no exchange of RNA/DNA across host and pathogen, or host and symbiotic occupant, to evolutionary advantage in a complex eukaryote... wouildn't we be hard pressed to rule out such possibilities? And if the notion that certain kinds might occur is deemed so unlikely as to deserve no research money or time, then maybe we will get lucky and stumble over one now and then until we discover we've been missing a boat. It's just a thought...

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