Tag Archives: PNAS

Microbial forensics: the science behind the Amerithrax investigation

Nearly a decade after the postal anthrax attacks in the USA that killed 5 individuals and infected more than 20 people, scientists have revealed the measures used to trace the Bacillus anthracis strain used in the bioterror attack in a new paper available online for free from Proceedings of the National Academy of Sciences. A groundbreaking mix of genomics and microbiology were used as part of the criminal investigation  into the 2001 anthrax attacks (called Amerithrax); microbial forensics proved key to identifying the exact flask from which the anthrax spores were taken.

Rasko and colleagues used highly accurate whole-genome sequencing and comparative genomics (against the B. anthracis Ames ancestor, believed to be the progenitor of all Ames lab samples and used as a gold standard reference strain in the USA) to determine the source strain of B. anthracis used in the letter attacks. First, the scientists took spore samples from some of the letters and grew them in the lab. A number of morphological variants were observed in these letter-isolated bacterial samples (yellow or yellow–grey coloured rather than the usual grey–white of wild-type anthrax colonies) and all had diminished abilities to sporulate. These variants were then sequenced and compared with genomes sequences of the gold standard Ames ancestor to identify four distinct loci with genetic mutations (three of which were in B. anthracis sporulation pathways, specifically regulation of a key protein, Spo0F) in the morpholigical variants—features unique to the isolated anthrax variants. None of these variants were found to be prevalent in the environment (even in the areas associated with the Amerithrax investigation).

Ultimately, using comparisons with genomes of repository anthrax sources, the anthrax spores used to lace the letters were found to have a unique genetic fingerprint; anthrax batches were eventually traced back to a source flask (RMR-1029) in the lab of Dr Bruce Ivins (a key suspect in the subsequent criminal investigation who later committed suicide before a criminal case could be brought to trial).

The study authors conclude that the B. anthracis bioterror attack investigations “taught us important lessons about the integration of whole-genome sequencing for forensic applications”, although they do concede that their methods might not applicable to other bioterror agents.

ResearchBlogging.orgRasko, D., Worsham, P., Abshire, T., Stanley, S., Bannan, J., Wilson, M., Langham, R., Decker, R., Jiang, L., Read, T., Phillippy, A., Salzberg, S., Pop, M., Van Ert, M., Kenefic, L., Keim, P., Fraser-Liggett, C., & Ravel, J. (2011). Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1016657108

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CFTR aids Listeria escape into cell cytosol

Courtesy of CDC/ Dr. Balasubr Swaminathan; Peggy Hayes

The intracellular pathogen Listeria monocytogenes must escape the vacuole formed during entry into the host cell to replicate in its preferred environment—the cell cytosol—and continues its life cycle. Although the pore-forming bacterial toxin listeriolysin O is vital for Listeria escape and virulence, new research by Radtke and colleagues published online in PNAS shows that a host cell protein, CFTR (cystic fibrosis transmembrane conductance regulator, which forms a chloride ion channel that, incidentally, when dysfunctional results in cystic fibrosis), promotes escape of L. monocyotgenes from intracellular vacuoles.

Radtke et al. reasoned that, as the intravacuolar environment is dynamic and likely modulated by a variety of proteins, regulation of ion flux whilst Listeria is inside a vacuole could affect its subsequent escape from this membrane-bound organelle. The researchers confirmed that CFTR was endogenously expressed by mouse macrophages and addition of a CFTR inhibitor did not affect uptake of Listeria into host cells but did reduce the number of intracellular bacteria, indicating that the bacteria might be trapped within the vacuole. Using macrophages isolated from either wild-type mice or mice carrying the CFTR mutation associated with human cystic fibrosis, they found that defects in CFTR led to delayed intracellular replication (indicative of a defect in vacuole escape). Finally, the researchers conclude that CFTR potentially promotes escape of Listeria by controlling the flux of chlorides into the vacuole—a high chloride concentration seems to increase both the oligomerisation and haemolytic activity of listeriolysin O, the key bacterial toxin needed for escape.


Little is known about the role of ion transport in the context of bacterial infection and it would be interesting to see whether other ion channels and transporters also contribute to the virulence of Listeria and other intracellular bacteria.


 ResearchBlogging.orgRadtke, A., Anderson, K., Davis, M., DiMagno, M., Swanson, J., & O’Riordan, M. (2011). Listeria monocytogenes exploits cystic fibrosis transmembrane conductance regulator (CFTR) to escape the phagosome Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1013262108

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Spreading Salmonella—hyper-replicating bacteria act as a reservoir for dissemination

New research reveals how Salmonella enterica spread in the gut and gallbladder—a subpopulation of Salmonella primed for invasion rapidly replicate in the host cell cytosol such that bacteria-laden cells are extruded out of the epithelial-cell layer releasing invasive Salmonella into the gastrointestinal and biliary lumen. Leigh Knodler and colleagues write that other mucosal-dwelling pathogens could use this “host cell process as an exit strategy”.

Salmonella species can cause a range of infections from typhoid fever to food poisoning. Ordinarily, the intracellular bacteria Salmonella enterica resides and replicates within a membrane-bound vacuole in epithelial cells. During its life cycle, the bacteria are adapted to survive within a wide range of environmental niches within the human host (including cells such as enterocytes and macrophages and organs such as the spleen and gastrointestinal tract).

Knodler et al. observed a subpopulation of Salmonella that were ‘hyper-replicating’; these bacteria were doubling in number at almost five times the rate of the overall population of bacteria in the epithelial cell. Not only that, these bacteria were rapidly proliferating not in the Salmonella-containing vacuole, but in the host cell cytosol (which is believed to be nutrient rich) and were ready to invade other cells (they expressed type III secretion system 1 components and flagella, virulence factors that are required for invasion). Moreover, epithelial cells overloaded with these hyper-replicating cytosolic Salmonella were forced out of the apical side of the epithelial-cell layer—just as when dying cells are extruded out of the epithelium during the normal rapid turnover of epithelial cells that occurs to maintain the gut epithelium. Subsequently, invasive bacteria are released into the lumen and are primed and ready to infect new cells. The extruded host cells then die in a caspase-1-dependent manner and trigger the production of the proinflammatory cytokine interleukin 18—a process which could, in part, explain the high levels of mucosal inflammation observed in Salmonella infections of the gut and gallbladder.

ResearchBlogging.orgKnodler, L., Vallance, B., Celli, J., Winfree, S., Hansen, B., Montero, M., & Steele-Mortimer, O. (2010). Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia Proceedings of the National Academy of Sciences, 107 (41), 17733-17738 DOI: 10.1073/pnas.1006098107

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Monkeypox infections on the increase in Africa

The incidence of a smallpox-like disease—caused by the monkeypox virus—has increased 20-fold in the Demoncratic Republic of Congo (DRC) over the past 30 years, according to new research published online in the journal PNAS. The findings suggest that, as smallpox vaccination programmes ceased in the DRC in 1980, people are now immunologically ‘naïve’ to orthopoxviruses (including monkeypox and smallpox viruses) and could be at an increased risk of infection by this family of viruses.

The monkeypox virus can cause a serious zoonotic disease (potentially transmitted to humans from rodent species such as squirrels as well as primates such as monkeys) that is similar to smallpox. Smallpox vaccinations not only helped eradicate the smallpox virus, but also provided cross-protective immunity to monkeypox virus infection.

Anne Rimoin and colleagues assessed the burden of human monkeypox in the DRC by analyzing surveillance data for this disease from 2005–2007 in regions known to be endemic for the virus, a feat which had not been conducted since the early 1980s. In their up-to-date surveillance data across nine health zones in central DRC, 760 human monkeypox cases were confirmed by laboratory tests and the researchers found that the average annual cumulative incidence of monkeypox virus infection was 5.53 cases per 10,000 people across all zones. Men, children under the age of 15 years and people living in forested areas or who had not received a prior smallpox vaccination were at an increased risk of monkeypox infection whilst those who had been vaccinated against smallpox had a 5.2-fold lower risk of infection by the monkeypox virus than unvaccinated individuals. Rimoin et al. then compared the 1980s active surveillance data with their new 2000s findings from the same health zone and found that the incidence of human monkeypox in this zone increased substantially from 0.72 cases per 10,000 people in the 1980s to 14.42 cases per 10,000 people between 2005 and 2007.

The investigators add that “entire households are now mostly or completely [smallpox] vaccine naive”, which could result in increased human-human transmission between different generations within the same house. Furthermore, Rimoin and colleagues argue that because of several limitations (including poor access to remote areas) their study could have under-reported the true incidence of human monkeypox and the observed dramatic increase in disease incidence could in fact be a “conservative estimate”. Improved and continued disease surveillance will be needed to assess the true burden of monkeypox virus infection on public health in African populations, and could aid the development and implementation of strategies to reduce the risk of monkeypox virus infection.

ResearchBlogging.orgAnne W. Rimoin, Prime M. Mulembakani, Sara C. Johnston, James O. Lloyd Smith, Neville K. Kisalu, Timothee L. Kinkela, Seth Blumberg, Henri A. Thomassen, Brian L. Pike, Joseph N. Fair, Nathan D. Wolfe, Robert L. Shongo, Barney S. Graham, Pierre Formenty, E, & Major (2010). Major increase in human monkeypox incidence
30 years after smallpox vaccination campaigns
cease in the Democratic Republic of Congo Proceedings of the National Academy of Sciences USA (Published ahead of print 30th August 2010) DOI: 10.1073/pnas.1005769107

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Silencing human RSV infection

A new therapy based on RNA interference (RNAi) successfully reduces respiratory syncytial virus (RSV) infection in humans. The study, published free online in PNAS, demonstrates that therapeutic RNAi-based drugs are clinically effective, and suggests that similar ‘silencing’ therapies could be useful against other respiratory pathogens.

RSV is an RNA virus that infects the lungs to cause respiratory tract infections—especially in the lower respiratory tract. RSV infection can be severe in immunocompromised patients, the elderly and young children; in the US alone, the virus has a ten times higher mortality rate in young children than the influenza virus and is the most common cause of infant hospitalisation. No vaccine for RSV exists, and the only approved drug treatment, ribavirin, has limited use and effectiveness. Current treatment strategies for RSV infection are only supportive—namely oxygen and fluids until the infection naturally resolves. Previous work has shown that a small interfering RNA (siRNA) drug—called ALN-RSV01—effectively silences a RSV protein that is critical for virus replication, and has a considerable antiviral effect in a mouse model of RSV infection. Evidence for the clinical effectiveness of siRNA drugs to treat disease in humans is, however, lacking.

DeVincenzo and colleagues tested whether ALN-RSV01 was an effective antiviral drug in adult, human volunteers who were infected with wild-type RSV. The investigators enrolled 88 healthy participants into a double-blind, placebo-controlled trial. They randomly assigned the volunteers to receive a nasal spray containing either ALN-RSV01 or saline as a placebo control. This nasal spray was administered 2 days prior to, and 3 days after, inoculation with RSV. They found that treatment with the siRNA nasal spray decreased the number of people infected with RSV by 38%, with the greatest reduction in people’s symptoms between 4–7 days after RSV inoculation. This antiviral effect was not related to the concentration of proinflammatory cytokines (such as tumor necrosis factor and interferon α) in the nose or whether the participants had pre-existing antibodies against RSV. Furthermore, the scientists showed that intranasal ALN-RSV01 was well tolerated and safe to use in humans.

DeVincenzo et al. argue that their findings represent a “significant advance in the development of human therapies…[and] a definitive demonstration in humans of an RNAi effect using a synthetic siRNA.” More clinical trials are needed to determine whether intranasal ALN-RSV01 can reduce RSV infection in children and adults that are naturally infected by the virus, and to determine the optimal dose and frequency for ALN-RSV01 administration that produces the best antiviral effect and clinical outcome. The results from this study also demonstrate a “broader potential” for “locally delivered siRNAs as unique anti-infective drugs against other respiratory pathogens.”

ResearchBlogging.orgDeVincenzo, J., Lambkin-Williams, R., Wilkinson, T., Cehelsky, J., Nochur, S., Walsh, E., Meyers, R., Gollob, J., & Vaishnaw, A. (2010). A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0912186107

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A gene polymorphism helps protect against malaria, but makes you vulnerable to lupus

A polymorphism in the human gene FCGR2B is associated with susceptibility to systemic lupus erythematosus (SLE), but it is also associated with protection against malaria, according to a new study published in PNAS. The polymorphism was most common in people of Southeast Asian and African origin (i.e. populations from areas endemic for malaria), implying that its protective effects against malaria could provide a survival advantage and explain its prevalence in these populations despite the increased risk of SLE.

Systemic lupus erythematosus is a chronic and systemic autoimmune disorder whilst malaria is an infectious disease caused by Plasmodium parasites. These two disparate diseases are in fact linked by their association with the gene FCGR2B, which is involved in immune regulation and encodes the Fc gamma receptor IIb. A single nucleotide change in FCGR2B, termed rs1050501, codes for a threonine instead of isoleucine in the FCGR2B protein, which in turn leads to a non-functional Fc gamma receptor IIb (FcγRIIbT232) that has been shown to be important for resistance against Plasmodium. Furthermore, rs1050501 had already been associated with SLE and the frequency of this allele was found to vary between people of different ethnicities—it is more common in Southeast Asians or East Africans than Caucasians.

Lisa. C. Willcocks and colleagues analysed the genotypes of 819 people with SLE from Hong Kong compared with 1,026 ethnically matched controls, and 326 Caucasian patients with SLE compared with 1,296 controls in the “largest study of this FCGR2B SNP in SLE performed so far.” They found that SLE was strongly associated with FcγRIIbT232 in both ethnic groups. Next, the investigators genotyped rs1050501 in children from an area in Kenya endemic for malaria—they analysed control children as well as children who had suffered from either repeated episodes of mild malaria or a severe form of malaria. They found children homozygous for FcγRIIbT232 (the rs1050501 allele was present on each chromosome pair) were protected against severe malaria. In contrast, the same allele did not protect against bacterial infection.

Willcocks et al. state that “the high mortality from malaria has resulted in the strongest known force for evolutionary selection in the recent history of the human genome.” They argue that the protective effects of FcγRIIbT232 against malaria could explain why the mutant receptor is most common in Africans and Southeast Asians; the frequency of the rs1050501 allele, like sickle-cell disease and thalassemia, being related to malarial endemicity. “Malaria seems to have driven retention of a polymorphism predisposing to a polygenic autoimmune disease, and this may begin to explain the ethnic differences seen in frequency of that disease,” write the authors.

ResearchBlogging.orgWillcocks, L., Carr, E., Niederer, H., Rayner, T., Williams, T., Yang, W., Scott, J., Urban, B., Peshu, N., Vyse, T., Lau, Y., Lyons, P., & Smith, K. (2010). A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0915133107

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Major bacterial pathogen jumped from humans to poultry

It’s not much fun being a broiler chicken. Not only will you eventually end up on someone’s plate but you get tarnishedBroiler, white background2 with the same brush as all of bird-kind and blamed as a harbinger of an infectious microorganism (bird flu) that could cause the next great flu pandemic. However, new research shows that the spread of pandemic pathogens also jumps from humans to poultry, according to a study by Lowder and colleagues published online in the early edition of the journal PNAS.

Staphylococcus aureus is a bacteria commonly found on our skin (it is perhaps more notoriously known for MRSA, the strain of S. aureus which is resistant to the antibiotic methicillin and is a serious nosocomial (acquired in hospital) infection). S. aureus infection is also a major cause of infectious disease in poultry. It causes skeletal infections (or bacterial chondronecrosis osteomyelitis, BCO) that leads to lameness in chickens. BCO emerged in the 70’s and now has a huge economic impact on the global broiler chicken industry (a multibillion dollar industry controlled by a small number of multinational companies who supply a limited number of breeds to the global market).

Scientists from the University of Edinburgh and Robert Koch Institut (Germany) examined the population genetics of S. aureus isolates from the past 54 years, which were taken from healthy and diseased chickens in 8 countries across 4 continents and also different species of reared game and wild birds. The scientists used a type of sequencing called MLST which arranges the isolates into groups with similar sequence identity called clonal complexes. They found that most of the isolates were from a single sequence type, ST5, which is commonly associated with humans and MRSA strains. Further analysis traced the evolution of the poultry ST5 group; the isolates had descended from a single S. aureus that had jumped from a human host to infect poultry ~ 38yrs ago in Poland. The poultry ST5 group has rapidly spread across the world, probably due to distribution by the global poultry industry. However, the related human ST5 group is present in distinct geographical clusters and is rarely spread intercontinentally. Furthermore, the poultry ST5 group evolved to be successful poultry pathogens by acquiring novel mobile genetic elements from other poultry S. aureus strains and losing genes which are important for human disease. The poultry ST5 group are successful at causing disease because they are more resistant to killing by chicken heterophils (an immune cell found in chickens, equivalent to human neutrophils). This makes it harder for the chicken to fight the infection since heterophils are important for the innate immune response which protects against S. aureus infection.

This data highlights the huge influence human activity has on the emergence of animal pathogens and demonstrates just how globalisation helps spread infectious microorganisms across the world. Additionally, it suggests that farm livestock should be regularly screened to identify newly emerging bacterial pathogens.

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