Climbing the walls : behavioural manipulation of insects by baculoviruses

Parasites often have severe effects on their hosts by causing developmental and physiological alterations in their hosts. These alterations may contribute to parasite growth, reproduction and survival. For example, host development may be inhibited so that more nutrients become available for the parasite. Parasites can also interfere with host behavior as a strategy to increase parasite survival or transmission. This phenomenon is known as ‘parasitic manipulation’ or ‘behavioural manipulation’. Although many examples of parasitic manipulation are known, the genetic basis underlying such manipulations is largely unexplored. A thorough understanding of how parasites manipulate their hosts’ behavior is therefore lacking, but it can be hypothesized that parasites carry specific genes that induce these behavioural alterations. Such ‘behavioural’ parasite genes likely affect one or more host proteins directly or via the expression of specific target genes in the host, which subsequently leads to altered behaviour. Understanding the details of such interactions between parasite and host is important as parasitic manipulation is thought to be wide spread in nature and to be a strong driver of the co-evolutionary arms race between parasite and host. Furthermore, the strategies employed by parasites to alter behavior likely provide important insights in the molecular mechanism of many behavioural processes. Chapter 2 of this thesis reviews our current understanding of the mechanisms of behavioural manipulation in invertebrates. It discusses known examples of behavioural manipulation and the present knowledge on the mechanistic basis of these manipulations. Furthermore, an overview of host genes and proteins that play a conserved role in behavioural traits in different invertebrate species is given. These genes and proteins are worthwhile to be studied in more detail in the context of parasitic manipulation, as they might be suitable targets for parasites to induce behavioural changes. This thesis focuses on behavioural manipulation in insect hosts by baculoviruses. Baculoviruses are DNA viruses that infect the larval stages of mainly lepidopteran insects. These viruses alter host behaviour in multiple ways. They induce hyperactivity in the larvae, which likely contributes to virus dispersal over a large area. In addition, baculoviruses alter host climbing behaviour leading to death at elevated positions, a phenomenon known as ‘tree-top disease’ or ‘Wipfelkrankheit’. This latter manipulation is thought to contribute to optimal virus dispersal on plant foliage. In the research described in this thesis baculoviruses and their lepidopteran insect hosts are used as a model system to study molecular mechanisms of behavioural manipulation. In Chapter 3 of this thesis the involvement of the protein tyrosine phosphatase (ptp) gene from the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in the induction of hyperactivity of the beet armyworm Spodoptera exigua was studied. A homolog of this gene in Bombyx mori nucleopolyhedrovirus (BmNPV) was previously shown to be important in hyperactivity in the silkworm B. mori. The results in Chapter 3 showed that the AcMNPV ptp gene induces hyperactive behaviour in S. exigua larvae and that the phosphatase activity of the encoded PTP enzyme is crucial for this behavioural change. Phylogenetic inference revealed that the baculovirus ptp is presumably transferred from a (ancestral) lepidopteran host to a baculovirus. Within the family Baculoviridae, ptp is only present in group I NPVs, which are a group of phylogenetically related baculoviruses. It is hypothesized that ptp-induced hyperactivity is an evolutionarily conserved strategy of group I NPVs to manipulate host behaviour. To obtain insights into the target proteins of the baculovirus PTP enzyme to achieve hyperactive behaviour in infected caterpillars, a PTP substrate analysis was performed. Chapter 4 describes host and viral proteins that were found to co-purify with AcMNPV PTP. Many of these host proteins are known to be important in signalling pathways and behavioural traits. For one of these proteins, 14-3-3 z, mRNA transcript levels were found to be significantly higher in wild type AcMNPV-infected S. exigua larvae as compared to larvae infected with a mutant virus from which the ptp gene has been deleted (AcMNPV Δptp). The 14-3-3 protein is a known activator of the enzymes tryptophan hydroxylase and tyrosine hydroxylase, which in turn are required for synthesis of the neurotransmitters serotonin and dopamine. These signalling molecules are both important determinants in hyperactive behaviour in various organisms, and are associated with behavioural manipulation in several parasite-host systems. In Chapter 9 a model is proposed of how the putative interaction between baculovirus PTP and host 14-3-3 zmay lead to hyperactive behaviour. Within the baculoviruses two different genes that encode protein tyrosine phosphatases, ptp and ptp2, are found. While the ptp gene induces hyperactivity (described in Chapter 3), no function has yet been assigned to the ptp2 gene. Chapter 5 describes the functional characterization of the baculovirus ptp2 gene. PTP2 protein carries a conserved consensus sequence that is characteristic for mitogen-activated protein kinase (MAPK) phosphatases. SeMNPV ptp2 induced a mild apoptosis and caspase activation in insect cells upon transient expression, which may be related to its putative function as MAPK phosphatase. Several host proteins that co-purified with SeMNPV PTP2 have known functions in apoptosis and/or MAPK signalling, rendering them promising candidate proteins to be involved in SeMNPV PTP2-induced apoptosis and possibly MAPK signalling. Whether PTP2 also has any behavioural effect is unknown, but the data from this chapter indicate that PTP2 likely has a cellular function during virus infection. Baculoviruses are known to alter host climbing behaviour, commonly leading to death at elevated positions (tree-top disease). In Chapter 6 the hypothesis was tested that baculovirus-induced hyperactive behaviour and tree-top disease are induced by a single baculovirus gene. To this aim the effect of the hyperactivity-inducing ptp gene (Chapter 3) on tree-top disease was investigated. The results demonstrated that AcMNPV ptp, known to cause hyperactive behaviour in S. exigua, is not involved in tree-top disease in this host. This indicates that hyperactivity and tree-top disease induced by baculoviruses are governed by independent mechanisms. Furthermore, a moulting-dependent effect on tree-top disease in S. exigua was found, which may relate to physiological and/or ecological differences between moulted and unmoulted larvae. In the next chapter (Chapter 7) the effect of AcMNPV infection on tree-top disease was investigated for two different host species, Trichoplusia ni and S. exigua. Data show that in T. ni larvae AcMNPV induces tree-top disease, causing death at elevated positions. In contrast, in S. exigua a moulting-dependent effect on the height at death was observed, as was also described in Chapter 6. Furthermore, in this chapter the role of the AcMNPV egt gene, encoding ecdysteroid UDP glucosyl transferase, on tree-top disease in T. ni and S. exigua larvae was analysed. A homolog of this gene causes tree-top disease in Lymantria dispar larvae infected with L. dispar (Ld) MNPV. The results (Chapter 7) show that AcMNPV egt does not play a role in the observed death at elevated positions in the two host systems studied. This indicates that the role of egt in tree-top disease may not be conserved among members of the family Baculoviridae. In addition to the mechanisms employed by the generalist baculovirus AcMNPV to alter climbing behaviour, the effect of the specialist baculovirus S. exigua (Se) MNPV on tree-top disease in its only known host S. exigua was studied. In Chapter 8 it is shown that SeMNPV induces tree-top disease by triggering an aberrant response to light, and this positive phototaxis leads to death at elevated positions. A hypothesis is put forward that SeMNPV hijacks a host behavioural pathway that is involved in light perception to induce this positive phototactic response. Overall, the results of this thesis show that hyperactivity and tree-top disease are induced by baculoviruses through independent mechanisms and that distinct baculovirus species presumably use different genes and proximate mechanisms to induce tree-top disease. While the baculovirus ptp gene induces hyperactivity, possibly by targeting host 14-3-3 z, the baculovirus ptp2 gene may function as a pro-apoptotic gene. The baculovirus egt gene does not have a conserved function in tree-top disease, indicating that other viral genes may underlie this host manipulative strategy. This thesis also demonstrates that tree-top disease in SeMNPV-infected caterpillars is the result of a strong attraction to light. Parasitic manipulation is a fascinating biological phenomenon that can provide crucial information on how behavioural traits are controlled at the molecular level. The research described in this thesis provides several new insights in the mechanisms by which parasites manipulate the behaviour of their hosts.</p

Characterization of the pro-apoptotic function of eIF5A in human cancer cell lines

Eukaryotic translation initiation factor 5A (eIF5A) is the only known protein containing the unique amino acid, hypusine. eIF5A is present in all eukaryotic cells and is highly conserved, but its function is not well understood. The present investigation was undertaken to study the regulatory role of eIF5A in the induction of apoptosis in human cancer cell lines. Suppression of eIF5A1 using specific siRNA was shown to have no effect on the growth and proliferation of mammalian cells, although inhibition of the post-translational hypusination of eIF5A1 resulted in G1 phase cell cycle arrest. Treatment of HT-29 human colon adenocarcinoma cells with eIF5A1 siRNA did, however, reduce their sensitivity to pro-apoptotic stimuli including nitric oxide, Actinomycin D, proteaosome inhibition and serum starvation. Furthermore, over-expression of eIF5A1 in HT-29 cells or Hela S3 human cervical carcinoma cells using adenovirus constructs strongly induced apoptosis in a time-dependent manner. The pro-apoptotic effect of eIF5A1 appears to reflect at least in part its ability to activate the mitochondrial pathway of apoptosis in that its up-regulation resulted in dissipation of mitochondrial ∆Ψm, release of cytochrome c, activation of caspase 9 and caspase 3 and translocation of Bax from the cytosol to mitochondria. Similar effects were observed following treatment with eIF5A2, a second isoform of human eIF5A, and in addition eIF5A2 induced up-regulation of cleaved Bcl-2 which is thought to be pro-apoptotic. A mutant of eIF5A1 in which the conserved lysine, lysine50, that is post-translationally modified to hyusine was switched to alanine [eIF5A1(K50A)] also proved capable of inducing apoptosis by activating the mitochondrial pathway. As well, eIF5A1 and eIF5A1(K50A) both induced strong up-regulation of p73, a homolog of the tumor suppressor p53, in Hela S3 cells containing null p53. The finding that up-regulation of eIF5A1 also resulted in activation of caspase 8 indicates that it may be involved in regulation of the death receptor pathway of apoptosis as well. This contention is further supported by confocal microscopy studies indicating that, following its up-regulation, eIF5A1 localizes to the inner surface of the plasma membrane in a time-dependent manner that correlates temporally with the induction of apoptosis. eIF5A1 and its post-translationally modified forms were isolated by 2-dimensional Western blotting and sequenced by mass spectrometry. These analyses indicated that eIF5A1 containing unmodified lysine50 is the form of the protein that accumulates coincident with induction of apoptosis either by up-regulation of eIF5A1 or treatment with NO. These observations, together with the finding that eIF5A1(K50A) is capable of inducing apoptosis, indicate that it is the unhypusinated form of eIF5A1 that is apoptogenic

Drosophila, metabolomics and insecticide action

The growing problem of insecticide resistance is jeopardising current pest control strategies and current insecticide development pipelines are failing to provide new alternatives quickly enough. Metabolomics offers a potential solution to the bottleneck in insecticide target discovery. As a proof of concept, metabolomics data for permethrin exposed Drosophila melanogaster was analysed and interpreted. Changes in the metabolism of amino acids, glycogen, glycolysis, energy, nitrogen, NAD+, purine, pyrimidine, lipids and carnitine were observed along with markers for acidosis, ammonia stress, oxidative stress and detoxification responses. Many of the changed metabolites and pathways had never been linked to permethrin exposure before. A model for the interaction of the observed changes in metabolites was proposed. From the metabolic pathways with the largest changes, candidate genes from tryptophan catabolism were selected to determine if the perturbed pathways had an effect on survival when exposed to permethrin. Using QPCR it was found that all genes in the entire pathway were downregulated by permethrin exposure with the exception of vermilion suggesting an active response to try and limit flux through tryptophan catabolism during permethrin exposure. Knockdown of the tryptophan catabolising genes vermilion, cinnabar and CG6950 in Drosophila using whole fly RNAi resulted in changes in susceptibility to permethrin for both topical and oral routes of exposure. Knockdown of the candidate genes also caused changes in susceptibility when the insecticides fenvalerate, DDT, chlorpyriphos and hydramethylnon were orally administered. These results show that tryptophan catabolism knockdown has an effect on surviving insecticides with a broad range in mode of action. Symptoms that occur in Drosophila during exposure to the different insecticides were also noted. To gain further understanding into the mechanisms affecting survival, tissue specific knockdown was performed revealing tissue and gender specific changes in survival when vermilion, cinnabar and CG6950 are knocked down. Metabolomics was performed on the knockdown strains to determine the efficacy of the knockdowns on tryptophan catabolism and to identify any knock-on effects. The results indicate that tryptophan metabolite induced perturbations to energy metabolism and glycosylation also occur in Drosophila along with apparent changes in the absorption of ectometabolites. As the knockdown of vermilion, cinnabar and CG6950 tended to result in reduced susceptibility to insecticides, they would make poor targets for insecticidal compounds, however, they may be the first examples of genes that are not directly involved in insecticide metabolism or cuticle synthesis that increase insecticide tolerance in Drosophila. As the first metabolomics data set showed evidence for oxidative stress during permethrin exposure, preliminary work was begun for identifying the tissue specificity and timing of oxidative stress in both Dipterans and Lepidopterans using Drosophila and Bombyx mori as models. In Drosophila oxidative stress did not begin immediately suggesting that the insecticide itself is not a cause, however, a rapid increase in oxidative stress occured over a six hour period after a day of oral exposure implicating catabolites of permethrin. Bombyx were highly susceptible to permethrin showing oxidative stress in the Malpighian tubule and silk gland when exposed. This study has shown that metabolomics is highly effective at identifying pathways which modulate survival to insecticide exposure. It has also brought insight into how insecticide induced pathology may cause death. Data has also been generated which could help characterize the putative transaminase CG6950

Tudor staphylococcal nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis

Tudor staphylococcal nuclease (TSN; also known as Tudor-SN, p100, or SND1) is a multifunctional, evolutionarily conserved regulator of gene expression, exhibiting cytoprotective activity in animals and plants and oncogenic activity in mammals. During stress, TSN stably associates with stress granules (SGs), in a poorly understood process. Here, we show that in the model plant Arabidopsis thaliana, TSN is an intrinsically disordered protein (IDP) acting as a scaffold for a large pool of other IDPs, enriched for conserved stress granule components as well as novel or plant-specific SG-localized proteins. While approximately 30% of TSN interactors are recruited to stress granules de novo upon stress perception, 70% form a protein-protein interaction network present before the onset of stress. Finally, we demonstrate that TSN and stress granule formation promote heat-induced activation of the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1), the plant orthologue of mammalian AMP-activated protein kinase (AMPK). Our results establish TSN as a docking platform for stress granule proteins, with an important role in stress signalling

Understanding the Molecular Basis of Pathogenesis of White Spot Syndrome Virus

The aquaculture industry has a substantial role to play in securing future food supply, but its expansion is currently limited by disease. In shrimp aquaculture White Spot Disease (WSD) outbreaks, caused by White Spot Syndrome Virus (WSSV), are the major limiter as they rapidly culminate in 100% mortality and lack prophylactics or therapeutics. Losses associated with WSD are estimated at $1 billion USD annually. All crustaceans are considered to be susceptible to WSSV but the degree of susceptibility varies dramatically between species from the highly susceptible penaeid shrimp to highly resistant European shore crab (Carcinus maenas), which is capable of harbouring the virus for long periods of time without any disease symptoms. This differential susceptibility between crustacean hosts offers the opportunity to investigate how they differ in their molecular responses to WSSV, in an attempt to identify the molecular mechanisms responsible for susceptibility to this pathogen. The thesis therefore aimed to investigate the host-WSSV molecular interactions occurring within two crustaceans with differing susceptibility to WSD in order to construct hypotheses for the likely molecular pathways that could be responsible for tolerance to WSD in crustaceans. Further, the thesis also explored the role of environmental abiotic factors in WSSV susceptibility. To address these aims I first conducted an in-depth literature review of the impacts of the environment on WSD outbreaks. I identified links between rapidly changing environmental conditions and WSD occurrence and substantial gaps in the study of many single and combined factors including dissolved oxygen, CO2, pH and nitrogen concentrations. Secondly, a WSSV infection trial was carried out in (highly susceptible) Penaeusvannamei and (mRNA and miRNA) transcriptomes produced. Transcriptome analysis of susceptible P. vannamei pointed towards increased susceptibility via rapid subversion of processes to enhance WSSV entry, evasion of cellular recycling components and cellular stress with a clear absence of significant immune responses during the early stages of the disease. During early time points, differentially expressed miRNAs were predicted to regulate cytoskeleton polymerisation, phagocytic activity, osmoregulation and metabolism with a hypothesised role in enhancing WSSV infection. The reduced immune responses observed in shrimp may be partially explained by differentially expressed miRNAs such as pPva-miR-H, which reduced immune- priming transcript expression (Dscam) and pPva-miR-K and –N, which regulated apoptosis at 24 and 36 hours post injection (hpi) to favour the virus. The ability of miRNAs to also enhance nucleotide and lipid metabolism from 24 hpi likely further supports virus replication. The P. vannamei transcriptome was then compared to an equivalent dataset in resistant C. maenas, which showed limited transcriptional responses during early time points. Critical alterations to transcripts involved in endocytosis and innate immunity, suggested that crabs may exhibit an enhanced ability to prevent WSSV entry and clear invading virions and that the associated transcripts may be key to resisting WSD. In particular, the significant upregulation of Cma-miR-92b, which may negatively regulate the translation of viral E3 ligase, immediate-early protein in WSSV-recalcitrant crabs should be explored further. For both susceptible and resistant crustaceans many novel miRNAs were identified, contributing to the current lack of crustacean genomic resources. These require further study to determine their significance in the WSSV infection. Based on the comparison of the transcriptome profiles between these two species with contrasting susceptibility to WSD, I propose the hypothesis that a two-pronged mechanism of reduced virion endocytosis (resulting from the inhibition of internal vesicle budding by dynamin-1), downregulation of transcripts that mediate cellular transport and increased viral clearance in crabs not replicating WSSV (by upregulation of apoptosis-associated transcripts) may underpin the mechanism of WSD resistance in crabs. In contrast, I hypothesise that the increased susceptibility of shrimp to WSD arises due to the suppression of both the innate immune responses during early infection and immune-priming molecule Dscam (by pPva-miR-H) during late infection. Further exploration of these key components in the molecular response to WSSV may lead to potential avenues to explore treatments for this devastating disease in aquaculture.Centre for Environment, Fisheries and Aquaculture Scienc

Molecular characterisation and functional analysis of eEF1B subunits in mammals

During the elongation of the polypeptide chain in eukaryotic protein synthesis, GTP-bound eukaryotic translation elongation factor 1A recruits the aminoacyl tRNA to the A-site of the ribosome. The GDP-GTP recycling is catalysed by the elongation factor 1B complex (eEF1B) which in higher eukaryotes consists of three different subunits: alpha, delta and gamma. Previous studies on eEF1B focused mainly on biochemical analysis and reports of overexpression in tumours and correlation to decreased survival rate but not a lot is known about is biology. The aim of this PhD is to characterise the eEF1B subunits at the molecular level in view of their potential involvement in tumourigenesis using a variety of bioinformatic and laboratory techniques. All three subunits were found to be ubiquitously expressed at mRNA and protein levels in all mouse tissues analysed. In addition, eEF1Bβ has several transcript variants in mice derived from alternative splicing and multiple isoforms, including a brain and testis specific heavier isoform and a muscle-specific form in addition to other forms. The characteristics of each eEF1B subunit were catalogued by further bioinformatic analysis. eEF1Bα was not detectable at early mouse developmental stages, eEF1Bβ showed stronger expression at pre-natal and early post-natal stages than adult stage whereas eEF1Bγ is ubiquitously expressed at similar levels throughout mouse development. In adult mice and human tissues, eEF1B subunits appeared to be expressed in different cell types and cell sub-populations. Surprisingly, cytoplasmic and some nuclear expression was observed in vivo. This nuclear expression pattern could not be observed in cell lines and it was not related to the cell cycle stage in vitro. The expression of eEF1B subunits did not change during the cell cycle except eEF1Bγ which was highly expressed in S-phase arrested cells. Knockdown by siRNAs of eEF1B subunits leads to decreased proliferation, increased number of cells in G0/G1 phase and increase in apoptosis in HeLa, HCT116, DLD1 and HepG2 cells. In contrast, overexpression in HeLa cells with a V5-tagged constructs lead to increased proliferation, increased number of cells in the G2/M phase and increased viability. Knockdown of eEF1Bα and eEF1Bβ leads to a reduction in eEF1Bγ levels; it is therefore possible that the phenotype shown by the knockdown of each subunit individually might be due to the reduced levels of eEF1Bγ. However, overexpression of each subunit did not affect the protein levels of the other subunits. The presence of multiple forms, the complex expression pattern and distribution of each eEF1B subunit in mouse and human tissues, and the knockdown and overexpression effect on cells suggests that the eEF1B complex might have different quaternary forms throughout development and in different cell types, possibly a more intricate role in translation, potential non-canonical functions any of which may be implicated in the potential role of eEF1B subunits in tumourgenesis

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

This work was supported by the National Institute of General Medical Sciences [GM131919].In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.PostprintPeer reviewe

Synonymous codons affect polysome spacing, protein production and protein folding stress: studies of bacterial translation using ribosome profiling

The acquisition of protein secondary and tertiary structure depends on the primary sequence of amino acids. However, predicting a protein's folded structure is difficult even with the knowledge of its sequence. It has been suggested that, in addition to encoding the amino acid sequence, genes also encode kinetic information which regulates the ribosome's translation rate. This information might guide nascent protein folding during translation. With the advent of ribosome profiling, a high-throughput sequencing technique which quantifies ribosome density on mRNA, it is now possible to investigate this hypothesis in greater detail. Here, a new way to analyze ribosome profiling data is presented, confirming that ribosome profiling detects ribosome pauses at slow codons. This method is able to precisely determine the locations of the ribosome aminoacyl and peptidyl transfer sites within the ribosome footprint. Next, a simulation tool which models the progression of ribosomes along an mRNA is used to explore the effects of translation initiation and elongation rates on protein expression. This tool can be used to generate testable predictions for how changing the translation rate should affect various experimental observables, including ribosome density. New experimental data, collected from the bacterium Escherichia coli, demonstrate that the sequence of the Firefly (Photinus pyralis) Luciferase mRNA affects its ribosome occupancy. Importantly, ribosome occupancy is differentially influenced by synonymous codons. These data also show that Luc expression is controlled by the 15 codons immediately downstream of the start codon and that greater Luciferase expression levels progressively activate the heat shock response. However, this response appears to saturate, suggesting that the overexpression of foreign proteins in E. coli readily overwhelms the endogenous chaperone system. This result demonstrates that expression level, rather than translation kinetics, determines the yield of folded Luciferase protein in E. coli