Genetically engineered mosquitos resist dengue fever virus

Aedes aegypti Dengue has represented a significant public health burden for a number of decades. Given the lack of dengue-specific drugs and limited availability of licensed vaccine, new methods for prevention and control are urgently needed. Researchers investigated whether genetic manipulation of the mosquitoes’ native JAK/STAT pathway-mediated anti-DENV defense system could be used to render mosquitoes more resistant to infection. They generated Aedes aegypti mosquitoes overexpressing the JAK/STAT pathway components Dome and Hop under the control of a bloodmeal-inducible, fat body-specific vitellogenin promoter. These genetically modified mosquitoes showed an increased resistance to DENV infection, likely because of higher expression of DENV restriction factors and lower expression of DENV host factors, as indicated by transcriptome analyses. Expression of the transgenes had a minimal impact on mosquito longevity; however, it significantly impaired the mosquitoes’ fecundity. Bloodmeal-inducible fat body-specific overexpression of either Hop or Dome did not affect mosquito permissiveness to either ZIKV or CHIKV infection, suggesting a possible specialization of JAK/STAT pathway antiviral defenses. This is the first to provide a proof-of-concept that genetic engineering of the mosquitoes’ JAK/STAT immune pathway can be used to render this host more resistant to DENV infection.

 

Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus. (2017) PLoS Negl Trop Dis 11(1): e0005187. doi: 10.1371/journal.pntd.0005187
We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway’s antiviral role.

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Siderophores – more than stealing iron

Siderophores - more than stealing iron Siderophores are small molecular iron chelators that are produced by microbes and whose most notable function is to sequester iron from the host and provide this essential metal nutrient to microbes. Recent studies have proposed additional, noncanonical roles for siderophores, including the acquisition of noniron metals and modulation of host functions. Recently, siderophores secreted by Klebsiella pneumoniae during lung infection have been shown to induce stabilization of the transcription factor HIF-1α, increase the expression of proinflammatory cytokines in the lung, and promote dissemination of K. pneumoniae to the spleen. Thus, their study demonstrated novel roles for siderophores in vivo, beyond iron sequestration. The interaction of siderophores with host cells further promotes the pathogenicity of K. pneumoniae and is likely relevant for other pathogens that also secrete siderophores in the host.

Siderophores: More than Stealing Iron. MBio. 2016 Nov 15;7(6). pii: e01906-16. doi: 10.1128/mBio.01906-16

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Drug resistance in hookworms

Hookworms More than 2 billion people around the world are infected with intestinal helminths, parasitic worms that can cause disease, complicate pregnancies, and stunt the growth of children. A number of drugs are currently used to treat hookworms, one of the most common helminths to infect humans, but many worry that prolonged use of the drugs could lead to drug-resistant worms. Now researchers have described a rapid test that can monitor hookworm DNA for drug resistance mutations.

 

Isothermal Diagnostic Assays for Monitoring Single Nucleotide Polymorphisms in Necator americanus Associated with Benzimidazole Drug Resistance. (2016) PLoS Negl Trop Dis 10(12): e0005113. doi: 10.1371/journal.pntd.0005113
Hookworms are amongst the major STHs and the second most prevalent intestinal hel- minth of humans. Large-scale treatment with the benzimidazoles (BZs) albendazole or mebendazole is the major control strategy against STHs in mass drug administration (MDA) programs. Prolonged and repeated treatment with the same anthelmintics has led to the emergence of widespread BZ resistance in veterinary parasites which is caused by a single nucleotide polymorphism at codon 200, 167 or 198 in the β-tubulin gene. There is a considerable concern that prolonged use of the same anthelmintics with suboptimal effi- cacy against hookworms, may select for resistant parasites and favour the development of resistance. We developed a novel genotyping assay to screen for β-tubulin polymorphisms in N. americanus, using the SmartAmp2 method. SmartAmp2 is a unique genotyping technology that detects a mutation under isothermal conditions with high specificity and sensitivity. The N. americanus SNP detection assay is rapid, sensitive and highly specific and has the potential to be used in the field for the detection of SNPs associated with BZ resistance.

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As I look back

Brain Having been involved in microbiology for so long it’s sometimes difficult to see the progress we are making. When I started out as a microbiologist in the 1970s the impact of molecular biology on microbiology was overwhelming, even if it took us a few years to think about what we should do with our new tools beyond expressing foreign proteins in microbes. With the development of PCR in the 1980s the flood of new sequence data and new species increased, and the 1990s brought us into the genomics era with the start of the Human Genome Project. But around the millenium it all got a bit cloudy for me and I wasn’t sure where we were headed any longer.

Many people would argue that CRISPR has been the great leap forward of the last decade, but I’m not so sure. To me it’s just another tool, following on directly from cloning and PCR. When people involved the first human genome CRISPR trials tell you they are “a huge undertaking and not very scalable“, they may have a point. As the NHS crumbles in the UK, what hope will there ever be for rolling out such expensive technologies worldwide? Anyway, back ten years….

As we accummulated more and more metagenomes, I still wasn’t sure were we were going. Was this just stamp collecting? Of course I shouldn’t have worried, because blue skies research is never a waste. The true revalation of the past decade has been the link between gut bacteria and the brain – the real game changer (Gut Microbes and the Brain: Paradigm Shift in Neuroscience). Who could ever have seen that one coming? The news that Parkinson’s Disease may be triggered (in those with a genetic predisposition) by the balance and shifts of gut flora is the real future. Forget expensive genome engineering, the future worldwide looks like faecal transplants (in the short term) and diets, probiotics and pills of freeze dried bacteria in the medium term. This is already happening in clinics to treat C. difficile colitis and other bowel conditions, but extending these treatments to the brain is the early days of a revolution. Forget moonshot-scale expenditure, the future is all about the bugs in your gut. The future’s bright. The future’s brown.

 

Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson’s Disease. Cell 167(6): 1469–1480.e12, 1 December 2016. doi: 10.1016/j.cell.2016.11.018
The intestinal microbiota influence neurodevelopment, modulate behavior, and contribute to neurological disorders. However, a functional link between gut bacteria and neurodegenerative diseases remains unexplored. Synucleinopathies are characterized by aggregation of the protein α-synuclein (αSyn), often resulting in motor dysfunction as exemplified by Parkinson’s disease (PD). Using mice that overexpress αSyn, we report herein that gut microbiota are required for motor deficits, microglia activation, and αSyn pathology. Antibiotic treatment ameliorates, while microbial re-colonization promotes, pathophysiology in adult animals, suggesting that postnatal signaling between the gut and the brain modulates disease. Indeed, oral administration of specific microbial metabolites to germ-free mice promotes neuroinflammation and motor symptoms. Remarkably, colonization of αSyn-overexpressing mice with microbiota from PD-affected patients enhances physical impairments compared to microbiota transplants from healthy human donors. These findings reveal that gut bacteria regulate movement disorders in mice and suggest that alterations in the human microbiome represent a risk factor for PD.

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Dengue vaccine reduces disease burden where dengue infections are common

Dengue virus The first available dengue vaccine, CYD-TDV (Dengvaxia), is estimated to reduce the burden of dengue and be potentially cost effective in settings where infections with dengue are common.

 

The Long-Term Safety, Public Health Impact, and Cost- Effectiveness of Routine Vaccination with a Recombinant, Live-Attenuated Dengue Vaccine (Dengvaxia): A Model Comparison Study. (2016) PLoS Med 13(11): e1002181. doi: 10.1371/journal.pmed.1002181
Large Phase III trials across Asia and Latin America have recently demonstrated the efficacy of a recombinant, live-attenuated dengue vaccine (Dengvaxia) over the first 25 mo following vaccination. Subsequent data collected in the longer-term follow-up phase, however, have raised concerns about a potential increase in hospitalization risk of subsequent dengue infections, in particular among young, dengue-naïve vaccinees. We here report predictions from eight independent modelling groups on the long-term safety, public health impact, and cost-effectiveness of routine vaccination with Dengvaxia in a range of transmission settings, as characterised by seroprevalence levels among 9-y-olds (SP9). These predictions were conducted for the World Health Organization to inform their recommendations on optimal use of this vaccine. Dengvaxia has the potential to reduce the burden of dengue disease in areas of moderate to high dengue endemicity. However, the potential risks of vaccination in areas with limited exposure to dengue as well as the local costs and benefits of routine vaccination are important considerations for the inclusion of Dengvaxia into existing immunisation programmes. These results were important inputs into WHO global policy for use of this licensed dengue vaccine.

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Would you like Salmonella with your salad?

Salad Leaves New research from my colleagues at the University of Leicester shows that “salad juice” from damaged leaved in bagged salads can stimulate the growth of Salmonella, even at refrigerator temperatures. Although this research did not look for evidence of Salmonella in bagged salads, it does show how Salmonella grows on salad leaves when they are damaged. Research published recently by the Food Standards Agency reported that annually there are more than 500,000 cases of food poisoning in the UK. While poultry meat was the most common source of infection, some 48,000 of food poisoning cases were from fresh produce: vegetables, fruit, nuts and sprouting seeds. Salmonella was the pathogen that caused the greatest number of hospital admissions – around 2,500 per year.

This work strongly emphasises the need for salad growers to maintain high food safety standards as even a few Salmonella cells in a salad bag at the time of purchase could be become many thousands by the time a bag of salad leaves reaches its use by date, even if kept refrigerated. Even small traces of juices released from damaged leaves can make the pathogen grow better and become more able to cause disease.

Salad leaf juices enhance Salmonella growth, fresh produce colonisation and virulence. Applied and Environmental Microbiology, 18 November 2016, doi: 10.1128/AEM.02416-16.
We show in this report that traces of juices released from salad leaves as they became damaged can significantly enhance Salmonella enterica salad leaf colonisation. Salad juices in water increased Salmonella growth by 110% over the un-supplemented control, and in host-like serum based media by more than 2400-fold over controls. In serum based media salad juices induced growth of Salmonella via provision of Fe from transferrin, and siderophore production was found to be integral to the growth induction process. Other aspects relevant to salad leaf colonisation and retention were enhanced, such as motility and biofilm formation, which increased over controls by >220% and 250% respectively; direct attachment to salad leaves increased by >350% when a salad leaf juice was present. In terms of growth and biofilm formation the endogenous salad leaf microbiota was largely unresponsive to leaf juice, suggesting that Salmonella gains a marked advantage from fluids released from salad leaf damage. Salad leaf juices also enhanced pathogen attachment to the salad bag plastic. Over 5 days refrigeration (a typical storage time for bagged salad leaves) even traces of juice within the salad bag fluids increased Salmonella growth in water by up to 280-fold over control cultures, as well as enhancing salad bag colonisation, which could be an unappreciated factor in pathogen fresh produce retention. Collectively, this study shows that exposure to salad leaf juice may contribute to the persistence of Salmonella on salad leaves, and strongly emphasizes the importance of ensuring the microbiological safety of fresh produce.

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Drug Resistance Mutations Enhance Growth of Malaria Parasite

Mosquito Some mutations that enable drug resistance in the malaria-causing parasite Plasmodium falciparum may also help it grow.

Plasmodium falciparum is a single-celled parasite that infects the human bloodstream and causes the most severe form of malaria. Some strains of P. falciparum have evolved resistance to antimalarial drugs, including the commonly used drug chloroquine. Often, chloroquine resistance mutations hinder P. falciparum’s ability to infect the bloodstream and grow. However, a previous study discovered that a uniquely mutated version of the P. falciparum gene known as pfcrt provides drug resistance while avoiding the detrimental impact of growth seen with more widely distributed mutated pfcrt variants. In a new study, an allele of the pfcrt gene called Cam734, which has been found in certain regions in Southeast Asia, was shown to increase growth rates in living parasites.

Cam734 helps to maintain an electrochemical gradient that allows the protein encoded by pfcrt to thwart the cellular effects of chloroquine. These new findings broaden understanding of Cam734, the second most common variant of the pfcrt gene in Southeast Asia. The findings identify multiple intracellular processes and multidrug resistance phenotypes impacted by changes in PfCRT and can help inform future malaria treatment efforts.

 

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology. (2016) PLoS Pathog 12(11): e1005976. doi: 10.1371/journal.ppat.1005976
Point mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) earlier thwarted the clinical efficacy of chloroquine, the former gold standard, and consti- tute a major determinant of parasite susceptibility to antimalarial drugs. Recently, we reported that the highly mutated Cambodian PfCRT isoform Cam734 is fitness-neutral in terms of parasite growth, unlike other less fit isoforms such as Dd2 that are outcompeted by wild-type parasites in the absence of CQ pressure. Using pfcrt-specific zinc-finger nucle- ases to genetically dissect the Cam734 allele, we report that its unique constituent mutations directly contribute to CQ resistance and collectively offset fitness costs associated with intermediate mutational steps. We also report that these mutations can contribute to resis- tance or increased sensitivity to multiple first-line partner drugs. Using isogenic parasite lines, we provide evidence of changes in parasite metabolism associated with the Cam734 allele compared to Dd2. We also observe a close correlation between CQ inhibition of hemozoin formation and parasite growth, and provide evidence that Cam734 PfCRT can modulate drug potency depending on its membrane electrochemical gradient. Our data highlight the capacity of PfCRT to evolve new states of antimalarial drug resistance and to offset associated fitness costs through its impact on parasite physiology and hemoglobin catabolism.

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Bugs In Space

Spaceman Bacteria behave differently in space, as indicated by reports of reduced lag phase, higher final cell counts, enhanced biofilm formation, increased virulence, and reduced susceptibility to antibiotics. These phenomena are theorized, at least in part, to result from reduced mass transport in the local extracellular environment, where movement of molecules consumed and excreted by the cell is limited to diffusion in the absence of gravity-dependent convection. However, to date neither empirical nor computational approaches have been able to provide sufficient evidence to confirm this explanation. Molecular genetic analysis findings, conducted as part of a recent spaceflight investigation, support the proposed model. This new paper reposring research conducted aboard the International Space Station indicates an overexpression of genes associated with starvation, the search for alternative energy sources, increased metabolism, enhanced acetate production, and other systematic responses to acidity – all of which can be associated with reduced extracellular mass transport.

A Molecular Genetic Basis Explaining Altered Bacterial Behavior in Space. (2016) PLoS ONE 11(11): e0164359. doi: 10.1371/journal.pone.0164359

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Cells fight back against virus factories

Rabies virus stress granules This week I’ve been talking to first year students about cell biology, discussing how much the environment of the cell varies from one site to another within the cell. Viruses “know” this and much virus replication is localized at particular sites within the cell, not just occurring haphazardly. The first example of this is to be discioved were Negri bodies, the virus factories induced in rabies virus-infected cells. But how does the cell respond?

Exposure of cells to environmental stresses, such as heat shock and viral infection, induces a cellular response leading to the formation of Stress Granules composed of stalled translation initiation complexes (RNA-binding proteins and mRNA). Subsequent inhibition of host translation contibutes to cell survival. Viruses modulate or interfere with Stress Granule formation to control virus replication and antiviral responses, but differences exist in the dynamics and outcome of the stress responses induced by various viruses. A new paper shows that Rabies virus (RABV) induces the formation of Stress Granules in infected cells. Stress Granules are highly dynamic structures that increase in size by fusion events, exhibit transient assembly or persist throughout infection. They localize close to viral factories, cytoplasmic structures characteristic of RABV infection involved in viral replication and transcription. Viral messenger RNAs, but not viral genomic RNA, are transported from the factories to Stress Granules, indicating the communication between both compartments. RABV-induced cellular stress is dependent on double-stranded RNA-activated protein kinase (PKR). PKR also participates in innate immune responses through the induction of the Interferon-B gene. These results give an insight on new and important aspects of RABV infection and host antiviral stress responses.

 

Rabies Virus Infection Induces the Formation of Stress Granules Closely Connected to the Viral Factories. (2016) PLoS Pathog 12(10): e1005942. doi: 10.1371/journal.ppat.1005942
Stress granules (SGs) are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV) induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1), T-cell intracellular antigen 1 (TIA-1) and poly(A)-binding protein (PABP) in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri bodies (NBs). Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection.

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