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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Nanoparticle vaccines against dengue fever?

Dengue virus-infected cells Every year more than 350 million people in over 120 countries contact dengue fever, which can cause symptoms ranging from aching muscles and a skin rash to life-threatening haemorrhagic fever. Researchers have struggled to create effective vaccines against dengue virus, in part because four distinct serotypes of the virus cause dengue fever and a vaccine must immunize against all four individually. Attempts at using live dengue viruses to develop a dengue fever vaccine have often led to an imbalance in immunity to the four dengue serotypes. Previous infection with one serotype of dengue, or protection against just one serotype, can lead to more severe disease if a person contracts other serotypes, so it’s vital that vaccines are available that specifically target all four strains.

To create a new dengue virus vaccine researchers designed nanoparticles of various shapes and sizes. Each nanoparticle was studded with copies of DENV2-E protein, a key protein from serotype 2 of the virus. After immunization with the DENV2-E nanoparticles, mice had a specific antibody response to serotype 2 of the dengue virus, but not the other three serotypes. Compared to mice vaccinated with only the soluble DENV2-E proteins, the nanoparticle formulations led to a stronger immune response.

Clearly this enabling research is still a long way from an effective human vaccine against dengue fever. Future studies will be required to test whether the antibody levels prevent dengue infection as well as whether similar nanoparticles can be develop for all dengue serotypes. At the same time, it is difficult to imagine that the next few years will not bring a host of candidate vaccines based on nanoparticles.


Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses. (2016) PLoS Negl Trop Dis 10(10): e0005071. doi: 10.1371/journal.pntd.0005071
Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simulta- neous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial effi- cacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been pre- cisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of dif- ferent sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT1 nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT© nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.

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The skin is a reservoir for African trypanosomes

Trypanosome Human African trypanosomiasis – sleeping sickness – is a potentially fatal disease, which currently affects ~3,500 people in sub-Saharan Africa. The disease is caused by parasites called African trypanosomes and is spread by tsetse flies. Controlling these biting insects, combined with surveillance and treatment, reduces the impact of outbreaks of the disease and the World Health Organisation (WHO) hopes to eliminate sleeping sickness by 2020. A new paper suggests that this target might be overambitious.

Detection of trypanosomes in the skin is not well documented, although there are descriptions of cutaneous symptoms associated with African trypanosomiasis. This paper reports the investigation of a possible anatomical reservoir in the skin and provides evidence of T.b. brucei, (a causative agent of animal trypanosomiasis) and the human-infective trypanosome, T.b. gambiense, invading the extravascular tissue of the skin (including but not restricted to the adipose tissue) and undergoing onward transmission despite undetected vascular parasitaemia. It also provides evidence of localisation of trypanosomes within the skin of undiagnosed humans. The presence of a significant transmissible population of T. brucei in this anatomical compartment is likely to impact future control and elimination strategies for both animal and human trypanosomiases.

The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes. eLife 2016; 5: e17716 doi: 10.7554/eLife.17716
The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly in the case of Human African Trypanosomiasis caused by Trypanosoma brucei gambiense. We hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively show that substantial quantities of trypanosomes exist within the skin following experimental infection, which can be transmitted to the tsetse vector, even in the absence of detectable parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-evaluation of current diagnostic methods and control policies. More broadly, our results indicate that transmission is a key evolutionary force driving parasite extravasation that could further result in tissue invasion-dependent pathology.

Trypanosomiasis: Skin deep

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Zoonotic Hepatitis E Virus

Zoonotic Hepatitis E Virus During the past ten years, several new hepatitis E viruses (HEVs) have been identified in various animal species. In parallel, the number of reports of indigenous hepatitis E in Western countries has increased as well, raising the question of what role these possible animal reservoirs play in human infections.

This review describes recent discoveries of animal HEVs and their classification within the Hepeviridae family, their zoonotic and species barrier crossing potential, possible use as models to study hepatitis E pathogenesis and transmission pathways identified from animal sources.

Zoonotic Hepatitis E Virus: Classification, Animal Reservoirs and Transmission Routes. (2016) Viruses 8(10): 270. doi:10.3390/v8100270.

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Herpesviruses as Pioneers in Cell Biology

HCMV Intracytoplasmic vesicular transport is well established; nucleo-cytoplasmic transport has so far been thought to be restricted to passage through the nuclear pore either passively, if size permits, or via karyopherin-mediated active transport. This limits transport in and out of the nucleus to particles of a maximum of 39 nm. With a diameter of 120 nm, herpesvirus capsids, which are assembled in the nucleus but mature to infectious virions in the cytosol, are unable to pass through the nuclear pore. It has become clear in the last decade that they leave the nucleus and traverse the nuclear envelope by a vesicle-mediated process that entails budding of nucleocapsids at the inner nuclear membrane, forming a primary enveloped virion in the perinuclear space. The primary envelope then fuses with the outer nuclear membrane.

Long thought to be specific for herpesviruses, this pathway has recently also been suggested to function in the export of large ribonucleoprotein (RNP) complexes during development of Drosophila. Common between the two is the involvement of kinases (viral, cellular, or both) to phosphorylate and soften the nuclear lamina allowing access of the ‘cargo’ (i.e., viral nucleocapsids or cellular RNPs) to the INM as well as morphological similarities [6]. The cellular AAA+ ATPase TorsinA has also been proposed to be involved in both processes. Thus the notion was developed that herpesviruses have actually co-opted a hitherto cryptic cellular transport pathway for their replication.


Vesicular Nucleo-Cytoplasmic Transport – Herpesviruses as Pioneers in Cell Biology. Viruses 2016, 8 (10): 266. doi:10.3390/v8100266
Herpesviruses use a vesicle-mediated transfer of nuclear-assembled nucleocapsids through the nuclear envelope for maturation in the cytoplasm. The molecular basis for this novel vesicular nucleo-cytoplasmic transport is beginning to be elucidated in detail. The heterodimeric viral nuclear egress complex, conserved within the classical herpesviruses, mediates vesicle formation from the inner nuclear membrane by polymerization into a hexagonal lattice followed by fusion of the vesicle membrane with the outer nuclear membrane. Mechanisms of capsid inclusion as well as vesicle-membrane fusion, however, are largely unclear. Interestingly, a similar transport mechanism through the nuclear envelope has been demonstrated in nuclear export of large ribonucleoprotein complexes during Drosophila neuromuscular junction formation, indicating a widespread presence of a novel concept of cellular nucleo-cytoplasmic transport.

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Fungal Super Glue – Biofilms

Candida albicans biofilm Biofilms are arguably the most common state of microbial growth found in nature and in patients infected with pathogenic organisms. A feature of prokaryotic and eukaryotic biofilms is their production of an extracellular matrix. The matrix provides a protective environment for biofilm cells, offering a three-dimensional framework for both surface adhesion and cell cohesion. In addition, this extracellular material controls cell dispersion from the biofilm and provides a nutrient source for the community. The physical barrier formed by the matrix is also clinically relevant, as it shields cells from environmental threats, including immune cells and antimicrobial drugs used for treatment. This defensive characteristic has been demonstrated for biofilms formed by diverse fungal pathogens, including Aspergillus, Candida, Cryptococcus, and Saccharomyces.

Biofilm-associated Candida infections are the fourth cause for nosocomial infections (predominantly infecting medical devices), which may lead to systemic infection associated with high mortality rates. Candida spp. are also the most common cause of mucosal infection of the oral and vaginal sites, where biofilm infection has been increasingly recognized. Despite the ubiquitous nature of the biofilm matrix, we are only beginning to understand the synthesis and composition of this material for a handful of species. This review discusses components of the extracellular matrix of fungal biofilms, including their synthesis, structure, and function.

Fungal Super Glue: The Biofilm Matrix and Its Composition, Assembly, and Functions. (2016) PLoS Pathog 12(9): e1005828. doi: 10.1371/journal.ppat.1005828

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