Completion of the entire hepatitis C virus life cycle in genetically humanized mice

More than 130 million people worldwide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease. Antiviral treatments are only partially effective against HCV infection, and a vaccine is not available. Development of more efficient therapies has been hampered by the lack of a small animal model. Building on the observation that CD81 and occludin (OCLN) comprise the minimal set of human factors required to render mouse cells permissive to HCV entry, we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice. Here we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo. Blunting antiviral immunity in genetically humanized mice infected with HCV results in measurable viraemia over several weeks. In mice lacking the essential cellular co-factor cyclophilin A (CypA), HCV RNA replication is markedly diminished, providing genetic evidence that this process is faithfully recapitulated. Using a cell-based fluorescent reporter activated by the NS3-4A protease we visualize HCV infection in single hepatocytes in vivo. Persistently infected mice produce de novo infectious particles, which can be inhibited with directly acting antiviral drug treatment, thereby providing evidence for the completion of the entire HCV life cycle in inbred mice. This genetically humanized mouse model opens new opportunities to dissect genetically HCV infection in vivo and provides an important preclinical platform for testing and prioritizing drug candidates and may also have utility for evaluating vaccine efficacy.

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A respiratory nitrate reductase active exclusively in resting spores of the obligate aerobe Streptomyces coelicolor A3(2)

Summary

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From Parasites to Protectors

Socially parasitic ants can serve as protective symbionts for their fungus-growing hosts in the face of attacks by predatory raiding species.

From The Scientist

A Megalomyrmex symmetochus ant (top) confronts a Gnamptogenys hartmani raider ant (bottom)ANDERS ILLUMSocially parasitic Megalomyrmex ants establish colonies in the gardens of fungus-growing ants, where they restrict the growth and reproduction of their hosts by feeding on resident broods and clipping the wings of virgin queens. But according to a study out today (September 9) in the Proceedings of the National Academy of Sciences, the presence of these parasitic guests can be beneficial when the host colony comes under attack from another species of ant—the guests use their potent alkaloid venom to fight the raiders and defend the fungal gardens.

“This is really intriguing,” said evolutionary biologist Joan Herbers of Ohio State University, who was not involved in the study. “It’s a novel demonstration that in a certain context, when this nasty predatory ant is around, the parasite can protect the host.” In other words, the parasite-host relationship has shifted to a context-dependent mutualism in which the cost to the host is compensated for by protection against a shared enemy. “That hadn’t been appreciated before for social insects,” Herbers added.

“The findings also highlight how ‘parasitic’ or ‘mutualistic’ relationships are not so clear-cut, and [instead] belong on a continuum of interaction between the two,” Natasha Mehdiabadi, an entomologist at the Smithsonian Institution in Washington D.C., who was not involved in the study, told The Scientist in an e-mail.

Working in the field in Panama, the Smithsonian Institution’s Rachelle Adams saw that one species of venom-alkaloid-producing ants, Megalomyrmex symmetochus, was infiltrating host Sericomyrmex amabilis colonies in greater numbers than other socially parasitic species typically would. She also noticed that the guest colonies boasted far more worker ants than usual. “It wasn’t just the queen and a few workers, as you typically find,” she said, “there were hundreds of workers patrolling the cavities in the host colonies.”

One explanation might be that the guests were at risk of attack by host workers. But the guests’ chemical weapons easily overpower host workers’ biting defenses, so Adams and colleagues wondered whether the hordes of guest workers might be essential to the survival of their own queen in another way. They hypothesized that M. symmetochus might act as defenders when host colonies are attacked by the destructive agro-predator Gnamptogenys hartmani, which Adams had observed invading the same colonies on occasion. To test the idea, she and her colleagues observed interactions among the three species in the lab.

In a series of encounters staged in petri dishes, M. symmetochus workers—which attack their adversaries with toxic venom—were much more effective than S. amabilis workers—which use their mandibles to cut off foes’ extremities—at killing G. hartmani raiders. The guest ants were also significantly less likely to be killed by the raiders. What’s more, some raiders daubed in venom by guest ants were later attacked by members of their own raiding party, suggesting that M. symmetochus venom is both toxic and somehow confuses the G. hartmani ants.

When the ants interacted on fungus garden fragments in the lab, the guest ants remained more effective at killing raiders than their hosts, who needed much larger numbers to mount any counter-attack against even just one or two raiders. Because G. hartmani usually attack with a force of 100 or more, the researchers concluded that hosting a guest ant colony would likely have substantial fitness payoffs for M. symmetochus if the risk of raids was high.

Adams and colleagues also showed that even when the raiders could not directly interact with guest ants, they preferred to initiate attacks on colonies without M. symmetochus compared to those with the guests. This suggested that the very presence of guest ants, communicated by the volatile chemicals they emit, deters G. hartmani.
“They’re still parasites because they’re extracting resources,” said Adams. “But in the context of this scenario, [the parasites] use the same chemical weaponry that helps them to invade the host colony against this other raiding species.” Adams compared M. symmetochus workers to the Medieval mercenaries that protected cities for pay.
To Herber’s mind, the work provides a neat illustration of how coevolved relationships can be transformed from purely parasitical to partially mutualistic. “Most—if not all—mutualistic interactions evolved from a parasitic relationship,” she said. “So how do we get from parasitic to mutualistic? Well, this [study] shows us one way.”
Mehdiabadi added that the study also emphasizes the importance of considering all the players influencing such coevolutionary arms races. “A mutualism doesn't involve two mutualists in the absence of other organisms, and a host-parasite interaction doesn't involve only the two and no others. All players need to be studied, and their interactions need to be examined.”
Adams said her team now plans to investigate whether the same results can be found in the field. The researchers also want to see if another socially parasitic ant species from the same genus, Megalomyrmex adamsae—named after Adams, who discovered the species—also protects its fungus-farming hosts.
R.M.M. Adams et al., “Chemically armed mercenary ants protect fungus-farming societies,” PNAS, doi: 10.1073/pnas.1311654110, 2013.

Obesity via Microbe Transplants

Physical traits like obesity and leanness can be “transmitted” to mice, by inoculating the rodents with human gut microbes. A team of scientists led by Jeffrey Gordon from the Washington University School of Medicine in St. Louis found that germ-free mice put on weight when they were transplanted with gut microbes from an obese person, but not those from a lean person.

The team also showed that a “lean” microbial community could infiltrate and displace an “obese” one, preventing mice from gaining weight so long as they were on a healthy diet. The results were published today (September 5) in Science.

Gordon emphasized that there are many causes of obesity beyond microbes. Still, he said that studies like these “provide a proof-of-principle for ameliorating diseases.” By understanding how microbes and food interact to influence human health, researchers may be able to design effective probiotics that can prevent obesity by manipulating the microbiome.

The human gut is home to tens of trillions of microbes, which play crucial roles in breaking down food and influencing health. Gordon’s group and others have now shown that obese and lean people differ in their microbial communities. Just last week, the MetaHIT consortium showed that a quarter of Danish people studied had a very low number of bacterial genes in their gut—an impoverished state that correlated with higher risks of both obesity and metabolic diseases.

However, descriptive studies like these cannot tell scientists whether such microbial differences are the cause of obesity or a consequence of it. “A lot of correlations are being made between microbe community configurations and disease states, but we don’t know if these are casual or causal,” said Gordon. By using germ-free mice as living laboratories, Gordon and his colleagues aim to start moving “beyond careful description to direct tests of function,” he added.

“It’s extremely exciting and powerful to go from descriptive studies in humans to mechanistic studies in mice,” said Oluf Pedersen, an endocrinologist who was involved in the MetaHIT studies. “That’s beautifully illustrated in this paper.”

Gordon lab graduate student Vanessa Ridaura inoculated the germ-free mice with gut microbes from four pairs of female twins, each in which one person was obese and the other had a healthy weight. Mice that received the obese humans’ microbes gained more body fat, put on more weight, and showed stronger molecular signs of metabolic problems.

Once the transplanted microbes had taken hold in their guts, but before their bodies had started to change, Ridaura housed the two groups of mice together. Mice regularly eat one another’s feces, so these cage-mates inadvertently introduced their neighbors’ microbes to their own gut communities. Gordon called this the “Battle of the Microbiota.”

These co-housing experiments prevented the mice with “obese” microbes from putting on weight or developing metabolic problems, while those with the “lean” microbes remained at a healthy weight.

Gordon explains that the obese microbe communities, being less diverse than the lean ones, leave many “job openings” within the gut—niches that can be filled by the diverse lean microbes when they invade. “And obviously, those job openings aren’t there in the richer, lean gut community,” he said. “That’s why the invasion is one-directional.” 

“But if invasion is so robust, why then isn’t there an epidemic of leanness?” asked Gordon. “The answer appears to be, in part, diet.”

In her initial experiments, Ridaura fed the mice standard chow, which is high in fiber and plant matter. She also blended up two new recipes, designed to reflect extremes of saturated fat versus fruit and vegetable consumption associated with Western diets.

If the mice were fed food low in fat and high in fruit and vegetables, Ridaura found the same results as before—the lean microbes could cancel out the effect of the obese ones. But when the mice were fed food low in fruit and vegetables and high in saturated fat, those with obese gut microbes still gained weight, no matter who their neighbors were.

This may be because the best colonizers among the lean communities were the Bacteroidetes—a group of bacteria that are excellent at breaking down the complex carbohydrates found in plant foods. When the mice ate plant-rich diets, the Bacteroidetes could fulfill a metabolic role that was vacant in the obese gut communities. When the mice ate unhealthy, plant-poor diets, “these vacancies weren’t there and the organisms couldn’t establish themselves,” said Gordon.

“We’re now trying to identify particular sets of organisms that can do what the complete community does,” Gordon added. The ultimate goal is to create a set of specific bacteria that could be safely administered as a probiotic that, along with a defined diet, could help these beneficial microbes to establish themselves and might effectively prevent weight gain.

“This study is an inspiration for us at MetaHIT,” said Pedersen. “It would be very interesting to take stools or cultures from extreme cases within our samples—people who have very rich or very poor gut microbiomes—and inoculate them into germ-free mice. . . . Now that we have a proof-of-concept, it’s obvious for us to follow up our findings through these studies.”

V.K. Ridaura et al., “Gut microbiota from twins discordant for obesity modulate metabolism in mice,” Science, doi: 10.1126/science.1241214, 2013.

Microbial Fuel Factories

Microbial Fuel Factories

The paper
M.W. Keller et al., “Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide,” PNAS, 110:5840-45, 2013.

The finding
Genetically engineered microorganisms could be used to produce fuels and industrial products, but some microbes’ metabolisms get in the way, degrading or blocking synthesis of the products scientists want. By keeping an extreme heat-loving microorganism below its accustomed temperature, University of Georgia biochemist Michael Adams and colleagues deactivated many of the hyperthermophile’s own metabolic processes and engineered a pathway that could ultimately be manipulated to produce liquid fuel.

The methods
Adams and colleagues took five genes from one archaebacterium (Metallosphaera sedula) that uses hydrogen gas as an energy source to incorporate carbon dioxide into organic compounds, and inserted those genes into the hyperthermophile Pyrococcus furiosus, another archaebacterium. The modified microbe produced 3-hydroxypropionic acid, a valued industrial compound used in acrylic plastics production.

The innovation
The hyperthermophile P. furiosus grows best at 100 °C, but the scientists cultured it near 70 °C, a temperature at which “it’s sort of a close-to-inactive bowl of cytoplasm, whereas the genes we engineered into it are maximally active at 70 degrees,” says Adams. By shifting the culturing temperature, the team enabled the engineered microbe to efficiently perform new tasks without interference from its usual metabolic processes.

The model
“This is exciting work that extends frontiers in several important ways,” demonstrating both a new way to make useful organic chemicals and a strategy for genetically engineering a heat-loving microbe, says Lee Lynd, a metabolic engineer at Dartmouth College.

Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens

Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens

The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections. Conversely, disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens^{1, 2, 3, 4}. How pathogens capitalize upon the failure of microbiota-afforded protection is largely unknown. Here we show that two antibiotic-associated pathogens, Salmonella enterica serovar Typhimurium (S. typhimurium) and Clostridium difficile, use a common strategy of catabolizing microbiota-liberated mucosal carbohydrates during their expansion within the gut. S. typhimurium accesses fucose and sialic acid within the lumen of the gut in a microbiota-dependent manner, and genetic ablation of the respective catabolic pathways reduces its competitiveness in vivo. Similarly, C. difficile expansion is aided by microbiota-induced elevation of sialic acid levels in vivo. Colonization of gnotobiotic mice with a sialidase-deficient mutant of Bacteroides thetaiotaomicron, a model gut symbiont, reduces free sialic acid levels resulting in C. difficile downregulating its sialic acid catabolic pathway and exhibiting impaired expansion. These effects are reversed by exogenous dietary administration of free sialic acid. Furthermore, antibiotic treatment of conventional mice induces a spike in free sialic acid and mutants of both Salmonella and C. difficile that are unable to catabolize sialic acid exhibit impaired expansion. These data show that antibiotic-induced disruption of the resident microbiota and subsequent alteration in mucosal carbohydrate availability are exploited by these two distantly related enteric pathogens in a similar manner. This insight suggests new therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.

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A bacterial symbiont is converted from an inedible producer of beneficial molecules into food by a single mutation in the gacA gene

Abstract

Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria’s host is a “farmer” clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer’s spore production and depresses a nonfarmer’s spore production. Genome sequence and phylogenetic analyses identify a derived point mutation in the food strain that generates a premature stop codon in a global activator (gacA), encoding the response regulator of a two-component regulatory system. Generation of a knockout mutant of this regulatory gene in the nonfood bacterial strain altered its secondary metabolite profile to match that of the food strain, and also, independently, converted it into a food source. These results suggest that a single mutation in an inedible ancestral strain that served a protective role converted it to a “domesticated” food source.

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