Tag Archives: Nature

All hail the new hepatitis C mouse model

With existing treatments only partially effective with major adverse effects and no vaccine currently available, hepatitis C virus (HCV) infection is a major health problem worldwide. An estimated 120 million people are chronically infected around the world and, therefore, at increased risk of liver damage (fibrosis and cirrhosis) and liver cancer. Research into potential new vaccines and therapies for HCV has been severely hampered by the lack of a small animal mouse model, often a crucial research tool to investigate disease progression and to test new drugs. Now, US scientists have for the first time made a genetically humanized mouse model for hepatitis C, which could prove vital in HCV infection research.
HCV is spread via blood-to-blood contact; anything from blood transfusions, sharing contaminated needles in injection drug use, and, as Pamela Anderson found out, contaminated tattoo needles. Diagnosis can be problematic and disease progression can be unpredictable, infected individuals range from being asymptomatic, to clearing the virus naturally or suffering progressive liver damage that, ultimately, leads to liver failure and need for transplantation.
Mice are normally resistant to HCV infection, only humans and chimpanzees are naturally permissive to HCV, and at least four human factors are critical for HCV entry, claudin 1, occuldin, CD81 and scavenger receptor type B class I (SCARBI). In their paper published this week in Nature, Marcus Dorner and colleagues built on existing knowledge that in vitro rodent cells only need to express occludin and CD81 to enable HCV entry. They reasoned that expressing these key human genes (CD81 and occludin) in mice could make living animals susceptible to HCV infection.
The scientists made mice that expressed human SCARB1, claudin 1, occludin and CD81 using an adenovirus as a vector to deliver the human genes into the mouse liver. Although mouse liver cells expressed these human genes (5% of cells expressed all four genes, whilst 18–25% expressed both CD81 and occludin), infecting these mice with HCV and proving they were infected was the major stumbling block as HCV infection in murine cells in vitro and in vivo is inefficient. Even though mice were infected with bioluminescent HCV (tagged with firefly luciferase), which can be easily detected if they replicated (the cells would ‘glow’), bioluminescent signals were not above background levels making it difficult to detect the virus. As an alternative approach, the mice were engineered to express the luciferase reporter whilst the HCV genome was engineered to express a protein that activates the bioluminesce reporter gene, such that delivery and replication of HCV in the liver leads to a bioluminescent signal. In this way, the researchers showed that all mice expressing at least human occludin and CD81 could indeed be infected with HCV. They then went on to validate their new model and demonstrated the in vivo role of SCARB1 in viral entry and uptake into host cells. Furthermore, the study authors managed to block HCV entry using passive immunisation (transfer of readymade anti-HCV antibodies) in the humanized mice. A promising HCV vaccine candidate (a recombinant vaccine virus vector expressing HCV proteins that has been shown to work in chimps) was also tested in the model mice and was shown to induce immunity and partial protection against HCV infection.
“To our knowledge, this is the first time that any step in the viral life cycle has been recapitulated in a rodent simply by the expression of human genes,” write the study authors. This new mouse model should enable scientists to closely study hepatitis C disease progression in a small animal model that is more amenable to lab research. Hopefully, new improved strategies (both drugs and vaccines) against HCV can be developed and used to guide any future clinical trials.


ResearchBlogging.orgDorner, M., Horwitz, J., Robbins, J., Barry, W., Feng, Q., Mu, K., Jones, C., Schoggins, J., Catanese, M., Burton, D., Law, M., Rice, C., & Ploss, A. (2011). A genetically humanized mouse model for hepatitis C virus infection Nature, 474 (7350), 208-211 DOI: 10.1038/nature10168



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Altruistic bacterial charity workers help protect their vulnerable stressed out kin

US scientists have found that a small minority of highly antibiotic-resistant bacteria will produce and share a molecule, indole, that can activate survival mechanisms in less-resistant cells to enable the whole bacterial population to survive stressful environments despite the fact that production of this signalling molecule weakens the fitness of bacteria.

The increasing incidence of antibiotic resistance and the emergence of so-called ‘superbugs’ are of huge importance to medicine and society as a whole with the ever-increasing likelihood of a return to a world without antibiotics. This potentially disastrous public health crisis led the Infectious Diseases Society of America to launch the “10x’20” initiative in which they call for a global commitment to research and develop 10 new, effective antibiotic drugs by 2020. As a complement to this drug development, research into how bacteria develop this resistance could provide crucial clues for the rational design of new antimicrobial agents.

Henry Lee and colleagues investigated the population dynamics of antibiotic resistance. They grew a vat of E. coli with increasing amounts of the antibiotic norfloxacin and then took samples of the bacteria and monitored the percentage of bacteria that became resistant to the antibiotic. The scientists found an individual isolate that was highly resistant to norfloxacin (even higher than the greatest norfloxacin levels tested in their bioreactor). These bacteria produced indole, which is known to aid tolerance to stress in E. coliindole induces anti-stress mechanisms such as drug efflux pumps that help drive out toxic substances from the bacterial cell—although its production can reduce the overall fitness of the bacteria. Moreover, indole boosts the antibiotic resistance of the whole bacterial population and not just the select few that produce it. This population-based resistance was not drug specific and was even observed when the scientists challenged E. coli with gentamicin, which is a different type of antibiotic (with a different mode of action) to the quinolone norfloxacin.

The researchers conclude that under antibiotic stress, a few drug-resistant mutants will endure a fitness cost to produce and share the benefits of the metabolite indole to “shield the less-resistant bacteria from antibiotic insult” and enable these ‘weak’ bacteria to survive.

ResearchBlogging.orgLee HH, Molla MN, Cantor CR, & Collins JJ (2010). Bacterial charity work leads to population-wide resistance. Nature, 467 (7311), 82-5 PMID: 20811456

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Sleeping sickness—a new cure for a neglected disease?

Scientists have validated a new drug target, the Trypanosoma brucei enzyme N-myristoyltransferase, in the fight against sleeping sickness, and have already identified and tested an inhibitor against this enzyme that successfully cures T. brucei infection in mice.

The study, published in the journal Nature, provides a much-needed boost to research into neglected tropical diseases, which are often associated with poverty.

Image provided by Wikipedia

Sleeping sickness—also known as African trypanosomiasis—is a disease caused by by the parasite T. brucei, which itself is transmitted to humans via the tsetse fly. African trypanosomiasis is endemic in regions of Sub-Saharan Africa and WHO estimates suggest that 50,000-70,000 people are currently infected with the parasite and 30,000 people die from the infection every year. The disease can cause significant morbidity and mortality and consists of two stages; stage 1, where parasites are present in the blood, lymph and interstitial fluid, and the more serious stage 2, with parasites present in the central nervous system (CNS). Without treatment, the severe neurological symptoms of the disease—confusion, extreme fatigue and sleep cycle disturbances—can ultimately lead to an irreversible and progressive mental decline ending in coma and death.

The few treatments available in our arsenal against African trypanosomiasis are out-dated and can have poor efficacy and serious side effects. Previous work has already proposed that N-myristoyltransferase is a potential target for the treatment of parasitics diseases, including African trypanosomiasis. This enzyme adds myristate (a common saturated fatty acid) to many eukaryotic and microbial proteins, a process which is required for their biological activity. Julie Frearson and colleagues, have now made a breakthrough in the discovery and development of effective, low toxicity drugs to treat sleeping sickness by investigating compounds that affect T. brucei N-myristoyltransferase.

The researchers first screened a library of 62,000 lead-based compounds to test their effectiveness at inhibiting N-myristoyltrasnferease and in preventing proliferation of the blood stage form of T. brucei. They found one compound—DDD85646—was very potent at inhibiting myristoylation and trypanosome growth during in vitro tests. The scientists then tested the efficacy of this compound in animal models of trypanosomiasis. They found that DDD85646 was well-tolerated and effectively cured acute trypanosomiasis in mice. Furthermore, DDD85646 is trypanocidal—it rapidly killed trypanosomes in both in vitro and in vivo assays. Finally, the investigators confirmed that DDD85646 truly acts “on target” against the T. brucei N-myristoyltransferase, and they also characterised the peptide pocket in which the inhibitor binds the target enzyme.

A possible drawback in using an inhibitor against N-myristoyltransferase as a trypanocidal drug is that humans also produce this enzyme. More research is needed into the inhibitor DDD85646 to improve its selectivity (ensuring that it is specific only for ­N-myristoyltransferase produced by trypanosomes) and to determine whether it can also penetrate the CNS and effectively kill parasites during the late-stage of trypanosomiasis. Crucially, clinical trials will be needed to ensure the compound is safe to use in humans. Only then will any future drugs for sleeping sickness, based on this research by Frearson et al., be seriously considered for production by big pharmaceutical companies.

ResearchBlogging.orgFrearson, J., Brand, S., McElroy, S., Cleghorn, L., Smid, O., Stojanovski, L., Price, H., Guther, M., Torrie, L., Robinson, D., Hallyburton, I., Mpamhanga, C., Brannigan, J., Wilkinson, A., Hodgkinson, M., Hui, R., Qiu, W., Raimi, O., van Aalten, D., Brenk, R., Gilbert, I., Read, K., Fairlamb, A., Ferguson, M., Smith, D., & Wyatt, P. (2010). N-myristoyltransferase inhibitors as new leads to treat sleeping sickness Nature, 464 (7289), 728-732 DOI: 10.1038/nature08893

*see also African trypanosomes just love social networking

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The MetaHIT catalogue 2010— your gut microbiome directory

An international team of scientists have produced a catalogue of genes from the micro-organisms that live in our gut (the gut microbiome), and it is the first published work from the MetaHIT (Metagenomics of the Human Intestinal Tract) project. “This gene catalogue contains virtually all of the prevalent gut microbial genes in our cohort, provides a broad view of the functions important for bacterial life in the gut and indicates that many bacterial species are shared by different individuals,” write Junjie Qin and colleagues.

The research, published last week in the journal Nature, pieces together a staggering 576.6 gigabases of gene sequence to assemble and characterise 3.3 million non-redundant microbial genes from faecal samples from124 European individuals. The results provide a vital clue to the microbial species which are prevalent, and surprisingly common between different individuals, in the human gut.

The human body hosts trillions of micro-organism, most of which live in our gut. These gut bacteria are hugely important for human life, not only do they help us to get vital energy from the food we eat but changes in the types of micro-organisms in the gut are thought to contribute to bowel disease and obesity.

The researchers used an Illumina Genome Analyser to deep sequence DNA from faecal samples from Danish and Spanish adults who were healthy, overweight and obese, or had inflammatory bowel disease. This approach is called metagenomics and directly analyses genetic material from environmental samples, which means that organisms can be studied in their natural habitat and allows otherwise difficult-to-culture micro-organisms to be studied.

Qin et al. generated almost 200 times more metagenomic sequence data from the gut than had been produced in previous studies. The scientists found that their gene set was 150 times bigger than the human gene complement and included most of the known human intestinal microbial genes. Furthermore, their analysis revealed that 99% of the genes they identified were bacterial and that a common core of bacterial species existed in each person— including members of the Bacteriodetes and the Firmicutes, which have already been shown to be abundant in the gut environment. Finally, they used their gene catalogue to uncover the bacterial functions which are important for life in this habitat, such as synthesis of short-chain fatty acids, vital amino acids and vitamins, and the breakdown of complex polysaccharides.

“We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively,” conclude the investigators who hope that their extensive catalogue of the human gut microbiome will enable future studies of the association between microbial genes and human phenotypes, disease and living habits from birth to old age.

ResearchBlogging.orgQin, J., Li, R., Raes, J., Arumugam, M., Burgdorf, K., Manichanh, C., Nielsen, T., Pons, N., Levenez, F., Yamada, T., Mende, D., Li, J., Xu, J., Li, S., Li, D., Cao, J., Wang, B., Liang, H., Zheng, H., Xie, Y., Tap, J., Lepage, P., Bertalan, M., Batto, J., Hansen, T., Le Paslier, D., Linneberg, A., Nielsen, H., Pelletier, E., Renault, P., Sicheritz-Ponten, T., Turner, K., Zhu, H., Yu, C., Li, S., Jian, M., Zhou, Y., Li, Y., Zhang, X., Li, S., Qin, N., Yang, H., Wang, J., Brunak, S., Doré, J., Guarner, F., Kristiansen, K., Pedersen, O., Parkhill, J., Weissenbach, J., Antolin, M., Artiguenave, F., Blottiere, H., Borruel, N., Bruls, T., Casellas, F., Chervaux, C., Cultrone, A., Delorme, C., Denariaz, G., Dervyn, R., Forte, M., Friss, C., van de Guchte, M., Guedon, E., Haimet, F., Jamet, A., Juste, C., Kaci, G., Kleerebezem, M., Knol, J., Kristensen, M., Layec, S., Le Roux, K., Leclerc, M., Maguin, E., Melo Minardi, R., Oozeer, R., Rescigno, M., Sanchez, N., Tims, S., Torrejon, T., Varela, E., de Vos, W., Winogradsky, Y., Zoetendal, E., Bork, P., Ehrlich, S., & Wang, J. (2010). A human gut microbial gene catalogue established by metagenomic sequencing Nature, 464 (7285), 59-65 DOI: 10.1038/nature08821


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Host factors help influenza virus replication

German researchers have identified hundreds of host cell genes that affect influenza A virus replication. The work by Alexander Karlas and colleagues and published online in the journal Nature could help identify new drug targets which could be useful against a broad range of influenza viruses.

Influenza A viruses are a global public health threat that cause seasonal flu epidemics and periodic pandemics, the most recent major outbreak being the infamous swine flu pandemic that originated in Mexico in 2009. The influenza virus has a high mutation rate, which means that drugs and vaccines can become ineffective rapidly during a flu outbreak. The influenza virus can only replicate inside living cells and so novel drugs that target host cell functions required for virus replication are an attractive research area.

Karlas et al. conducted a genome-wide RNA interference screen in conjunction with a luciferase reporter assay to identify host cells factor that are important for influenza virus replication in human cells. First, they transfected human epithelial lung cells with 62,000 siRNAs against ~17,000 annotated and ~6,000 predicted human genes and 48 h later they infected the same cells with influenza A H1N1 virus and used immunofluorescence microscopy to check the virus infection rates. Secondly, they transferred the virus supernatants from the lung cells onto human embryonic kidney cells which contained an influenza-virus-specific luciferase reporter that bioluminesces in the presence of the virus to further quantify the virus infection and replication rates.

The scientists identified 287 human genes which appeared to be important in influenza virus replication, including the nuclear export factor genes NXF1 and XPO1, which have already been shown to be important for flu virus replication, and several genes which are connected with pre-mRNA splicing. They confirmed that siRNAs against ~59% (168 out of 287) of these genes decreased the number of endemic H1N1 and the 2009 pandemic H1N1 influenza viruses in human cells by at least five times. Interestingly, a subset of the same siRNAs also decreased replication of the highly pathogenic avian H5N1 influenza A strain. Furthermore, they found that a small molecule inhibitor of CDC-like kinase 1 reduced influenza virus replication by two orders of magnitude, because of impaired splicing of viral M2 messenger RNA. Finally, in vivo mouse studies confirmed the importance of the cell cycle regulator p27 in virus replication, p27-/- knockout mice were infected with H1N1 virus and 2 days later the researchers observed that the viral load within the lungs of the mice was significantly reduced.

This study “provided new and comprehensive information on host cell determinants of replication, and uncovered potential targets for novel antiviral strategies…against a broad spectrum of influenza viruses” write the authors and presents more information on the interaction between viruses and the human host.

ResearchBlogging.orgKarlas, A., Machuy, N., Shin, Y., Pleissner, K., Artarini, A., Heuer, D., Becker, D., Khalil, H., Ogilvie, L., Hess, S., Mäurer, A., Müller, E., Wolff, T., Rudel, T., & Meyer, T. (2010). Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication Nature DOI: 10.1038/nature08760

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M cells, gatekeepers or gateway to the gut

Glycoprotein 2 is the M cell receptor for type I pili on bacteria and is important for the immune response to these bacteria, according to research by Hase and colleagues published last week in the journal Nature.

The mucosal immune system is one of the largest components of our immune system and is hugely important for protecting mucosal surfaces (like our gastrointestinal tract) from harmful pathogens. Our guts are home to trillions of commensal bacteria which live quite happily there causing us no harm whatsoever. The gastrointestinal tract is protected from these bacteria (or other damaging substances) by a layer of tightly packed epithelial cells which form a barrier against any bacteria or molecules penetrating the gut. However, Microfold (M) cells are specialised intestinal cells located over mucosal lymphoid tissue called Peyer’s patches which are potential entry points into the host. M cells sample microorganisms or molecules in our gut and help transport them across the epithelial cell barrier (a process called antigen transcytosis) to deliver to professional immune cells (like macrophages, T cells or dendritic cells) to stimulate a protective immune response. In essence, M cells act like CCTV cameras to survey the gut area for anything that is out of the ordinary, or potentially harmful, and then present them to our immune system (essentially the police and the law courts) to sort those bad ‘uns out.

Previous work had shown that antigen transcytosis by M cells is important for mucosal immune responses but little was known about the mechanism involved. The researchers used microarray to scan the entire genome for specific molecules associated with M cells and found that glycoprotein 2 (GP2) was expressed in M cells in both human and mouse Peyer’s patches. Using immunoelectron microscopy they showed that GP2 was localised to the apical surface of M cells (the surface exposed to the commensal bacteria in the gut). Furthermore, they found that GP2 bound a variety of commensal and pathogenic enterobacteria (Escherichia coli, Salmonella enterica serovar Enteritidis and Salmonella Typhimurium), and more specifically bound to FimH expressed in the bacterial type I pilus (filamentous projections on the bacterial surface which are important for adhesion). Three-dimensional imaging revealed that GP2 accumulates around E. coli and S. Typhimurium as they are internalised in M cells and deletion of GP2 in mice reduced the uptake of type-I-piliated bacteria. After bacteria are translocated through M cells in a GP2-dependent manner they were captured by dendritic cells. Furthermore, GP2 was important for induction of mucosal immune responses against specific bacterial antigens (proteins that stimulate an immune response). Bacteria deficient in FimH lost the ability to bind GP2, had reduced entry into Peyer’s patches and induced a weak helper T cell and antibody immune response. Similarly, mice lacking GP2 had reduced helper T cell and antibody immune responses after challenge with bacteria expressing FimH.

This paper highlights the biological importance of GP2-dependent M cell antigen transcytosis as part of immunosurveillance in the intestine for bacteria expressing FimH. More work is needed to fully understand exactly what happens to the bacteria after they are delivered to immune cells and tissues by the M cells. Finally, M cells are thought to be a promising target for oral vaccinations to induce a protective immune response and this work shows that GP2 may be a possible vaccine target.

Although M cells act as a great surveillance system for the gut there are always a few bacteria that abuse the system. Shigella is one particular deviant bacterium that cannot normally invade the apical surface of intestinal epithelial cells and so uses the M cells to breach the epithelial cell barrier and gain access to their basolateral surface. Here, they can successfully invade, replicate in the intestinal epithelium, and wreak havoc on the gut by causing shigellosis or bacillary dysentery.

Hase, K., Kawano, K., Nochi, T., Pontes, G., Fukuda, S., Ebisawa, M., Kadokura, K., Tobe, T., Fujimura, Y., Kawano, S., Yabashi, A., Waguri, S., Nakato, G., Kimura, S., Murakami, T., Iimura, M., Hamura, K., Fukuoka, S., Lowe, A., Itoh, K., Kiyono, H., & Ohno, H. (2009). Uptake through glycoprotein 2 of FimH+ bacteria by M cells initiates mucosal immune response Nature, 462 (7270), 226-230 DOI: 10.1038/nature08529

Further reading

Jang, M.H. et al., (2004) Intestinal villous M cells: An antigen entry site in the mucosal epithelium. Proceedings of the National Academy of Sciences, 101, p.6110-6115.

Schroeder, G.N. and Hilbi, H. (2008) Molecular pathogenesis of Shigella spp.: Controlling host cell signalling, invasion and death by Type III secretion. Clinical Reviews Microbiology, 21, p.134-156.

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Be noble, cheats don’t always prosper

Social amoebae have evolved resistance to cheaters, which makes sure that the amoebae work together for the good of the group, say scientists published in Nature last week.

Social amoebae, such as Dictyostelium, co-operate together to form multicellular, fruiting bodies when they reproduce. However, as always some try to cheat the system and reap the benefits without any of the costs, in this case getting more of their fair share of spores into the fruiting bodies than the other Dictyostelium. Cheats must pay though, with lower fitness and can be discriminated against by the other amoebae.

The scientists randomly mutated a group of Dictyostelium and then mixed them with a strain of cheating amoebae (called cheater C). Most of the mutant amoebae got cheated on by the cheater C’s, and died out. However, during this process cheater-resistant strains evolved (called resistant to cheater C), and started to grow. The resistant strains turned out to be noble amoebae, they didn’t take advantage of the weaker mutant strains that had already been bullied by the cheater C’s. The evolution of cheater-resistant strains helps preserve co-operative behaviour in the social amoebae and reduces the number of cheaters in a population. So the moral of this story is…..cheats don’t always prosper and sometimes it is better to work together for the good of society.

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