Pasteur Institute

Bacterial Gene Transfer Gets Sexier.

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Mycobacterium smegmatis can donate larger portions of its genome to other bacteria than previously thought, approaching the level of gene shuffling seen in sexual reproduction.

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n what appears to be a novel form of bacterial gene transfer, or conjugation, the microbeMycobacterium smegmatis can share multiple segments of DNA at once to fellow members of its species, according to a study published today (July 9) in PLOS Biology. The result: the generation of genetic diversity at a pace once believed to be reserved for sexual organisms.

“It is a very nice study providing clear evidence that, in Mycobacterium smegmatis at least, conjugation underlies much of species diversity,” said Richard Meyer, who studies conjugation at The University of Texas at Austin, in an email toThe Scientist.

Traditionally, transfer of genetic material through conjugation has been considered an incremental process. Plasmids mediate the transfer of short segments of DNA, one at a time, between pairs of touching bacterial cells, often conferring such traits as antibiotic resistance.

But M. smegmatis, a harmless bacterium related to the pathogen M. tuberculosis, appears to use a more extensive method of gene shuffling, endowing each recipient cell with a different combination of new genes. The researchers dubbed this form of conjugation “distributive conjugal transfer.” “We can generate a million [hybrid bacteria] overnight, and each of those million will be different than each other,” said coauthor Todd Gray, a geneticist at the New York State Department of Health’s Wadsworth Center.

Coauthor Keith Derbyshire, also a geneticist at the Wadsworth Center, and colleagues had previously published data indicating that M. smegmatis used a novel form of conjugation, but the new study confirms and expands on their suspicions using genetic data. The researchers compared the whole genome sequences of donor and recipient bacteria before and after the massive gene transfers.

The researchers found that, after the transfers, up to a quarter of the recipient bacteria’s genomes were made up of donated DNA, scattered through the chromosomes in segments of varying lengths.

According to the authors, the diversity resulting from distributive conjugal transfer approaches that achieved by meiosis, the process of cell division that underlies sexual reproduction. “The progeny were like meiotic blends,” said Derbyshire. “The genomes are totally mosaic.”

The genes and machinery behind distributive conjugal transfer remain largely unknown, but Gray, Derbyshire, and colleagues have zeroed in on a region of the genome that may determine whether a bacterium becomes a DNA donor or recipient. The region encodes the ESX-1 family of proteins, which are also involved in secreting molecules from M. tuberculosis that give the bacterium its pathogenicity.

The researchers suspect distributive conjugal transfer is important in multiple species of Mycobacteria. Earlier this year, Roland Brosch, a tuberculosis researcher at the Pasteur Institute in France, and colleagues sequenced various strains of the pathogenic M. canettii, which is closely related to M. tuberculosis, and found they were genetically variable—possible evidence of distributive conjugal transfer, according to Gray and Derbyshire. Brosch said he had not yet been able to demonstrate distributive conjugal transfer in M. canettii, however, and he noted that such large-scale gene transfer is unlikely to be occurring in M. tuberculosis, which is a highly genetically homogenous species that shows little sign of recent horizontal gene transfer.

Brosch agreed with Derbyshire and Gray that distributive conjugal transfer could have been important in the evolutionary history of the Mycobacteria genus as a whole, however.  Gray pointed out that understanding the prevalence of distributive conjugal transfer could change views on the time scale of mycobacterial evolution. “I think it’s really going to open some eyes about how quickly things can change,” he said.

Asked whether distributive conjugal transfer could be happening in bacteria outside of theMycobacterium genus, Derbyshire said it remained a mystery, but added: “It’s likely to be more prevalent than currently is known.”

T.A. Gray, “Distributive conjugal transfer in Mycobacteria generates progeny with meiotic-like genome-wide mosaicism, allowing mapping of a mating identity locus,” PLOS Biology, 11: e1001602, 2013.

Source: the-scientist.com

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sexual reproduction , mycobacteria, DNA sequencing, diversity, conjugation, bacterial evolution andbacteria

Cells of the future: making living tissue from dead bodies.

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At the Pasteur Institute in Paris, a scientist opens a standard kitchen refrigerator and pulls out a clear plastic vial filled with cherry-colored liquid. A small, soft, fleshy lump sits on the bottom. It is a piece of muscle, taken from a deceased 44-year-old Frenchman. The laboratory will use its stem cells to grow a brand new strip of living muscle in the hopes that — one day — post-mortem stem cells can provide sick or injured people with a whole new source of body parts.

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The near-miraculous properties of stem cells have intrigued medical researchers for years. With their ability to divide repeatedly and fabricate other cells, they are ideal for reconstructing or repairing tissue. Embryonic stem cells can give rise to any organ, while most adult stem cells are limited to their own origin — neural stem cells make neurons, those from the skin form skin, and so on. Adult stem cells are constantly regenerating our blood and skin, and they also mend tissue that has been damaged by injury or disease.

Now a group of French researchers from the Pasteur Institute have discovered another awe-inspiring property: stem cells can survive without oxygen. This means that even when the body is dead, the stem cells continue living, in a state of reduced metabolism.

The team was led by Dr. Fabrice Chrétien, a histologist and neuropathologist who made a curious observation about five years ago while performing autopsies. Even after a corpse’s muscle tissue had started to atrophy, he saw healthy-looking cells that resembled stem cells, with a normal nucleus and intact DNA. One day he ran a test on the corpse of a young adult, taking a muscle tissue biopsy and putting it into a container with culture. His suspicions were confirmed when the stem cells started to multiply. “Honestly, I was a little shocked,” he recalled. “Shocked because I had done a biopsy on an individual who had been dead for four days, and in the box of culture the cells proliferated, becoming more numerous every day. They were alive — and yet the person was undeniably dead. It makes you think twice about the definition of death.”

In a living person, certain stem cells spend long periods of time in a quiescent state, not doing much of anything until they are activated due to stress, disease or injury. This quiescence permits them to survive and maintain their potency even under hostile conditions, whether radiation treatment for cancer or a workout at the gym. Once the onslaught has passed, they reawaken and multiply to repair the injured body part.

What Chrétien’s team discovered is that they can resist anoxia, or total oxygen deprivation. He explained that inside all our cells we have little organs called mitochondria that convert oxygen into energy. When there is no oxygen, the mitochondria produce toxins that destroy the cells. “We were stupefied to see that when we removed oxygen from the environment, stem cells got rid of their mitochondria,” he said. “As a result, their DNA was not damaged.” The stem cells stopped breathing and went into a dormant state.

The Pasteur team has tested human muscle from the arm, leg and abdomen, as well as bone marrow from mice. They only work with adult stem cells. (Aside from the controversy of sacrificing embryos for research or medical purposes, Chrétien said that fetal cells that proliferate endlessly can lead to cancer.) They’ve procured viable stem cells from human bodies up to 17 days after death — the oldest corpses they have access to — and from mice up to 14 days post-mortem.

On a recent spring day, Chrétien’s colleague, Dr. Pierre Rocheteau, walked me around the lab in a new building on the campus of the venerable Pasteur Institute. He explained that the piece of muscle tissue I saw would be “digested” by enzymes, then put through a machine that discards everything but the dormant stem cells. These would go into a plastic box with culture (glucose, serum and the like) at 37 degrees Celsius, and after three weeks, a thin, whitish layer of muscle would cover the bottom of the box.

I peered into a microscope at a sample after two weeks in culture and saw a number of little spots, all different shapes and sizes. Each stem cell had a black dot of DNA in the center. Rocheteau said they were moving a lot, but the motion was not visible to the naked eye. Then he showed me a time-lapse video of post-mortem stem cells zipping around erratically, stretching out until they split in two, multiplying exponentially, colliding and fusing. Another video displayed the final result, a strip of gently throbbing muscle.

Pasteur Institute policy forbids journalists from seeing the lab mice, but Chrétien told me the team has performed several transplants, injecting bone marrow from a 4-day-old mouse corpse into living specimens that had been irradiated to destroy their own marrow. “It worked magnificently,” he said. “All the mice survived.” This augurs well for his belief that one day human corpses can provide an additional source of stem cells for medical purposes, such as repairing muscle withered by muscular dystrophy or transplanting bone marrow for leukemia patients. He said that corpses can also be a useful source of stem cells for molecular screening in the pharmacological industry.

Is corpse harvesting necessary?

After meeting with Chrétien, I spoke with Dr. Vijay Gorantla, an associate professor of surgery at the University of Pittsburgh, who has been studying the possibilities of using cadaveric bone marrow to improve hand and face transplants. There is an important linguistic difference here — Gorantla procures his marrow from brain-dead “cadavers” whose hearts are still beating, as opposed to “corpses” who are dead in every sense of the term. His team’s research involves retrieving vertebral bone marrow at the same time as a donated body part and injecting it into a transplant recipient. The idea is to trick the body into accepting the hand and its foreign DNA without needing a lifetime of immunosuppressive drugs.

In the course of these experiments, he, too, was struck by the resiliency of stem cells. A colleague, Dr. Albert Donnenberg, developed a protocol for sterilizing pieces of vertebral bone with bleach or hydrogen peroxide. Despite the harshness of this chemical treatment, the stem cells maintained their counts and viability. Not only that, he found he could hold the vertebral bodies on ice for up to 72 hours before extracting the stem cells, and they were still just fine. “There’s something in them that prevents them from dying or offers them this capacity to survive,” Gorantla said. He was intrigued by Chrétien’s findings, and imagined that one day, after further screening for infection, there could be a worldwide registry connecting patients with deceased bone marrow donors.

Other people I spoke with were more skeptical about the utility of corpses for bone marrow transplants. Dr. Willis Navarro, medical director of transplant services for the National Marrow Donor Program in Minneapolis, said that source is not a major issue. The chance of an American patient finding a living match who is willing and able to donate bone marrow is 66 to 93 percent, and umbilical cords from newborn babies can also be harvested for embryonic-like stem cells.

Chrétien believes this still isn’t enough. He said an adult patient generally needs more than one umbilical cord. And in many parts of the world — including the United States but not France, where it’s illegal –parents can privately bank their own offspring’s cord blood in case the child needs it later, making it unavailable to the general public.

In any case, much research remains to be done regarding sterility before any human receives injections of post-mortem cells. The slightest risk of infection from bacteria in a corpse would prove fatal for a leukemia patient with a destroyed immune system. Chrétien estimates it will take at least five more years of study before corpses can be viable sources. “And you shouldn’t really wait 17 days post-mortem. We did that to prove it could be done, but it’s but not ideal. I think within 48 hours after death you can have a good quantity of very effective stem cells without any problems of sterility.”

Maintaining cells’ “stem-ness”

In the meantime, his team’s discovery also offers better ways to isolate and store stem cells. Storage can be problematic because as soon as stem cells are dissociated from tissue they start to proliferate like mad, eventually exhausting their capacity to multiply, or their “stem-ness.” But when they are deprived of oxygen and kept at 4 degrees Celsius, they hibernate for up to a month. This dormancy is reversible: the cells awaken and resume their normal activity after being put in culture or transplanted into a living body.

Currently, Chrétien’s team is studying the possible repercussions of their discovery on cancer treatments that consist of cutting off a tumor’s blood supply and starving it of oxygen. He said it would be catastrophic if cancer stem cells didn’t die with the rest of the tumor but instead went to sleep, only to wake up later and make new tumors. “We don’t want an upsurge of metastasis in a few years,” he explained. Though the research is in its early stages, he has found that cancer stem cells are in fact sensitive to oxygen deprivation in vitro. However, he cannot say if that is the case inside an actual person.

Indeed, it seems that not all of the body’s stem cells react the same way to different aggressors. Researchers at the McKnight Brain Institute in Gainesville, Fla., led a collaboration with investigators at the Kennedy Space Center, looking at the effects of cosmic rays on the brain. Surprisingly, they found that quiescent stem cells in the brain are extremely sensitive to cosmic radiation — a simulated mission to Mars showed up to 65 percent of them at risk of dying. According to Dr. Dennis Steindler, who directed the McKnight Brain Institute (and the study), this result contradicts the assumption that cancer tumors return after chemotherapy or radiotherapy because quiescence protects their stem cells.

Steindler said that understanding the metabolic requirements of different kinds of stem cells and how they behave under stress will provide scientists with valuable insight “extremely relevant to cancer research.” This knowledge can shine a light on other diseases, too. As Chrétien noted, “We are starting to see that the quantity of oxygen varies widely in different tissues of the body, and it’s not just chance — it plays a very particular role in cell fate.”

Stem cell research heralds a revolution in medical care. Cellular therapy can turn doctors into engineers of the human body, reconstructing tissue or building new organs without surgery. The fact that some stem cells have superhuman qualities makes the range of possibilities even larger. It is true that stem cells play a small role in practical medicine today. But, Chrétien predicted, “they will be enormously important tomorrow.”

Source: Smart Planet

 

 

A cure for HIV: where we’ve been, and where we’re headed.

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2013 marks the 30th anniversary of the discovery of HIV.130 Years of HIV Science: Imagine the Future, a meeting at the Pasteur Institute in Paris, France, in May, 2013, sought to celebrate successes in countering the HIV/AIDS epidemic and to map out the challenges ahead.

The successes have been spectacular. Antiretroviral therapy (ART) has transformed what was once a death sentence into a chronic manageable disease. ART not only prolongs life, but dramatically reduces HIV transmission. ART is now available to 8 million people living with HIV in low-income and middle-income countries.2 In 2011, the numbers of new infections declined by 50% in 25 countries—many in Africa, which has the largest burden of disease.2 These advances are a result of transformative science, advocacy, political commitment, and effective partnerships with affected communities.

However, substantial challenges exist to maintain access to and funding for lifelong ART to the more than 34 million people with HIV. The costs of delivering ART are overwhelming many organisations and public health systems; we must continue to search for alternatives to lifelong treatment to benefit patients at manageable costs to health systems. With that aim, the International AIDS Society (IAS) global scientific strategy,3 Towards An HIV Cure, was launched in 2012.

Reports of both sterilising cure (elimination of all HIV-infected cells) and functional cure (long-term control of HIV replication after ART) have raised hope that a cure for HIV can be achieved—at least in a subset of individuals. The first and only reported case of sterilising cure was Timothy Brown, the Berlin patient, an HIV-infected man given a bone marrow transplant for acute myeloid leukaemia. The donor was naturally resistant to HIV because of a mutation in the CCR5 gene—a critical protein required by HIV to enter and infect cells.4 Brown stopped ART very soon after transplantation and he remains free of HIV after 6 years.

The Mississippi baby seems to be the first case of functional cure of an infant due to ART given 30 h after birth.5 After 18 months, ART was stopped and the infant continues to have undetectable HIV in blood or tissue. Deborah Persaud and colleagues, who studied the baby, don’t yet fully understand what cured the infant. Very early treatment might prevent formation of latent reservoirs for HIV, at least in an infant with an immature immune system. Careful follow up and further studies will be needed to see if this approach can be replicated in more infants, and then on a larger scale.

In the VISCONTI cohort,6 14 patients in France have maintained control of their HIV infection for a median of 7·5 years after ART interruption.6 These so-called post-treatment controllers were diagnosed and treated with ART during primary HIV infection (on average within 10 weeks after infection), for a median of 3 years before discontinuation. Patients in this cohort do not have the same distinct immunological profile seen in elite controllers, who naturally control HIV in the absence of ART.6 The VISCONTI study potentially shows the benefits of early ART on the size of the reservoir. Further studies of reservoir size in patients who initiate ART in chronic infection but with high CD4 counts are to be presented at IAS 2013, Kuala Lumpur, Malaysia (Hocqueloux, WEAB0102; Chéret, WEAB0101).

Bone marrow transplantation, from a donor without a mutation in CCR5, might substantially reduce or even eliminate the HIV reservoir. Two patients with lymphoma from Boston (MA, USA) were given chemotherapy, radiotherapy, and stem cell transplantation while on continuous ART. Several years after transplantation, HIV DNA had disappeared from both patients’ blood and tissues.7 An update on the Boston patients is anticipated at IAS 2013 (Henrich, WELBA05).

The other approach to tackle HIV persistence in patients taking ART is to lure HIV out of its hiding place in resting T cells. Activating latent virus might lead to death of the cell or make the virus ready for immune-mediated clearance. A range of licensed drugs that modify gene expression, including viral gene expression, are in clinical trials in HIV-infected patients on ART. Two studies8, 9 have reported that HIV latency can be activated with the histone deacetylase inhibitor vorinostat.

There are now 15 HIV-cure-related trials being done worldwide.3 Clinical trials include investigations of increasingly potent histone deacetylase inhibitors, and of gene therapy to eliminate the CCR5 receptor from patient-derived cells.

HIV-cure-related trials raise many complex issues. Giving potentially toxic interventions to patients doing very well on ART needs careful assessment. At this early phase of research, participants will be unlikely to derive any direct benefits. Understanding risk—benefit, ethical issues, and the expectations and perspectives of the community will all be discussed and debated at IAS 2013 and the preceding IAS workshop, Towards an HIV Cure.

Developments towards a cure for HIV are exciting—for scientists, for clinicians, and most importantly, for patients. But we need to be realistic. Finding a cure will be a long and tough road, and will take many more years to achieve. We are at the very beginning, although many now believe that it might be possible to find a cure, at least for a small proportion of infected people.

We need to take inspiration from the many people who have delivered so much in the past 30 years, and continue to imagine, continue to innovate, and continue to work together towards an HIV cure—for everyone.

Source: Lancet

 

Outbreak of Illness and Death Among Children in Cambodia.

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The outbreak appears to have been caused by enterovirus 71.

In early July 2012, an outbreak of severe illness with high mortality was reported by the Ministry of Health in Cambodia. According to a WHO report dated July 13, 78 cases in 14 provinces had been identified since April, mostly in children aged ❤ years.

Investigation focused on the 61 children who met the case definition, of whom 54 had died. Illness manifestations included respiratory symptoms, fever, and generalized neurological abnormalities; children who died usually did so within 24 hours after hospital admission. Samples from 31 patients were tested for a variety of pathogens by Institut Pasteur du Cambodge, and “most” tested positive for enterovirus 71 (EV-71); a few also tested positive for dengue virus or Streptococcus suis. On July 15, 2012, authorities announced that no additional cases had been noted in Cambodia. Investigators believed that the use of steroids, which can suppress the immune system, worsened the illness in many of the patients.

Comment: EV 71 — a member of the picornavirus family — was first isolated in the late 1960s. It has been associated with outbreaks worldwide, most recently in Asia. Infection with EV 71, like that with other enteroviruses, ranges from asymptomatic to lethal and can manifest as rashes, diarrhea, respiratory symptoms, meningitis, hand-foot-mouth disease (HFMD), or myocarditis. Less commonly, it has been associated with acute flaccid paralysis, encephalitis, Guillain-Barré syndrome, and pulmonary edema and hemorrhage.

HFMD is most often caused by coxsackievirus A16 (another enterovirus) but is also caused by EV 71. According to the WHO, HFMD usually begins with fever, poor appetite, malaise, and sore throat. One or 2 days after fever onset, painful sores develop on the tongue, gums, and inside of the cheeks, beginning as small red blistering spots and then often becoming ulcers. A nonitchy skin rash develops over 1 or 2 days, with flat or raised red spots that may blister. Usually located on the palms of the hands and soles of the feet, the rash can also appear on the buttocks or genitals. Generally, HFMD is spread from person to person by direct contact with nose or throat discharges, saliva, fluid from blisters, or stool of infected persons. Transmissibility is greatest during the first week of the illness but can last for several weeks. No vaccine or antiviral agent has proven effective in preventing or treating EV 71 infection.

Source: Journal Watch Infectious Diseases