Host-Pathogen Interactions as a Resource-Competition Problem

Malnutrition and obesity are two of the most prevalent nutritional conditions affecting humans around the world today. As well as their obvious pathological effects on body condition and general well-being, sufferers may also have a reduced capacity to resist infections. In light of this, there has been a growth in the use of nutritional supplements aimed at boosting immunity, practiced both by the medical establishment (e.g. iron and vitamin supplements) and on the medical fringes. Whilst there is little doubt that certain key nutrients can play an important role in fighting infections, many of the studies to date have been conducted on single nutrients in isolation, without considering how they interact with other components of the diet, and without a robust framework for assessing their efficacy or mechanisms of action. The aim of the current proposal is to combine the theories provided by nutritional biology, ecological immunology and community ecology to develop a new framework that will allow us to test ideas about the importance of nutrition in determining the outcome of infections. Insects and their pathogens can provide useful models for understanding human diseases. This is because the insect's relatively simple immune system shows striking parallels with the human innate immune system. Moreover, insects are often easier to work with than humans, yet exhibit many of the same nutritional behaviours, such as obesity, anorexia and self-medication. We will use the caterpillar, Spodoptera littoralis, and the bacterium, Bacillus subtilis, as a model system for exploring the effects of macronutrients on innate immunity and pathogen resistance. Our previous studies have shown that healthy insects perform best on a diet that is roughly balanced in terms the relative amounts of protein (P) and carbohydrate (C), the two most important macronutrients for insects. If the insect eats too much or too little P or C, then it performs less well, indicating that this is the 'optimal' diet when uninfected. In contrast, insects that have been infected with pathogens, like B. subtilis, have much higher survival rates if they eat a diet extremely rich in protein. Moreover, if they are given a choice, infected insects will feed on a diet with a high protein content to aid their survival. We will use a step-wise experimental approach to determine whether we can predict the outcome of pathogen infections by understanding how the host and pathogen fare when grown in isolation. In particular, we will test the null hypothesis that the outcome of the infection (e.g. whether the insect lives or dies) is determined solely by the nutritional requirements of the host and pathogen. We will use 20 different artificial diets that vary in their P:C composition, so that we force the insects to ingest different amounts of P and C. 1. We will start by establishing how the diet of the insect affects the nutritional composition of its blood, which is where the bacteria will feed. 2. We will then grow the bacteria in a range of broths that have the same nutritional composition as the insect blood on each diet, but without blood cells and other immune defences. This will show us the nutritional requirements of the bacterium in the absence of the host. 3. We will then determine how the insect host's immune defences perform in the absence of the pathogen by injecting the insect with dead bacteria to stimulate the immune response, which we will be quantified using genetic and other methods. These first three experiments will allow us to determine how the performance of the host (insect) and pathogen (bacteria) differs on different diets and to make predictions about winners and losers based on assumptions about who controls nutrient use. 4. Finally, we will inject caterpillars with live bacteria and quantify the outcome in terms of bacterial growth rates and insect growth/survival, allowing us to test these predictions.

The proposed study aims to use the geometric framework for nutritional ecology to determine the combined effects of dietary protein (P) and carbohydrate (C) on the outcome of bacterial infections in a model insect-pathogen system: Spodoptera littoralis larvae infected with the gram-positive bacterium Bacillus subtilis. In so doing, our overarching null hypothesis is that the growth rate of the bacterial pathogen and the expression of the host's immune effector systems (haemocytes, enzymes, immune genes, etc.) are constrained by different nutritional resources (specific macronutrients and their components) and that the outcome of the infection reflects these differences in their nutrient-dependencies. To test this hypothesis, we will conduct 4 key experiments: 1) Larvae will be fed one of 20 chemically-defined diets that differ in their P:C ratios and amounts. We will then determine the relative amounts of amino acids and sugars in the insect haemolymph, which is what the bacteria will feed on during an infection 2) This will allow us to produce 20 different artificial media varying in amino acid/sugar composition to mimic insect blood, but lacking the haemocytes and other immune effectors, allowing us to quantify the resource requirements of B. subtilis in the absence of the host 3) Larvae will again be fed on the 20 different diets and half will receive an inoculation of B. subtilis to stimulate an immune response, which will then be quantified via immune gene expression, enzyme assays, and differential haemocyte counts, so quantifying the resource requirements of the host immune response in the absence of the pathogen 4) In the final experiment, the host and pathogen will come together by infecting S. littoralis with an LD50 dose of live B. subtilis. The outcome of the interaction across the 20 diets will be quantified by measuring bacterial growth rates, host immune responses, nutrient dynamics, and ultimately the survival/death rates of the insect.

In the developing world in particular, protein-energy malnutrition is a major contributor to the high mortality and morbidity rates from infectious diseases, such as TB, malaria and HIV/AIDS. Understanding the mechanisms generating these effects is of pressing importance. However, progress is hampered by the lack of a systematic framework for cataloguing these effects, designing robust experiments and practicing effective nutritional therapies. Quantitative and qualitative models developed by nutritional ecologists, including Simpson (project partner), are beginning to make important contributions to our understanding a range of nutritional pathologies, such as human obesity, anorexia, and healthy ageing. We envisage that the same theoretical frameworks will make similar inroads in understanding the impact of nutritional inequalities on parasite-induced morbidity and mortality. Thus, in the medium to long term, the most direct beneficiaries of our research are likely to be the medical community. Simpson's translational work on the nutritional bases of human obesity and on healthy ageing arose originally from research on insect models, such as locusts and caterpillars. The current proposal takes the same approach by focussing initial studies on a well understood insect-pathogen system, to refine approaches and test proofs of concept. Only once this has been established can our research programme switch to other model systems closer to human infections, such as mice, and ultimately humans. Thus the main impact is likely to be by establishing new theoretical frameworks to inform experimental designs and therapy programmes. Our research may also be of benefit to livestock managers and other areas of commercial benefit. For example, Simpson's nutritional models underpin current research associations with one of the world's largest aquaculture companies, CERMAQ and their research arm, EWOS Innovation, to run feeding trials on Atlantic salmon in Norway and Finland. We envisage that findings from the current study could be used to inform management practices for commercially damaging diseases of farmed fish, such as sea lice, Gyrodactylus and infectious salmon anaemia. Conservation biologists may also benefit from our findings. Simpson is involved in a project to examine the effects of nutrition on the endangered kakapo parrot in New Zealand and with the conservation of bees in the UK as part of a recent BBSRC consortium. Other longer term beneficiaries of this research are likely to be with governments and institutes involved in controlling insect crop pests. Wilson's research with the same model insect (Spodoptera caterpillars) is already heavily engaged with the Department of Agriculture and Food Security in Tanzania, exploring how to develop novel pest control strategies. Insights into how the nutritional requirements of insects and their natural enemies, mediated by their host plants, determines the outcome of the interaction may have some knock-on effects for designing novel inter-cropping strategies. The potential exploitation of our research findings will be enhanced by the co-location of the Enterprise and Business Partnerships centre within the Lancaster Environment Centre, which specializes in the nurturing of spin-out companies and small businesses in the environmental and biological sector.