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Effect of different host genetic backgrounds in the response against oral bacterial infection
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Os organismos vivos interagem naturalmente no seu habitat de formas que se estendem sobre uma vasta panóplia de interações bióticas, e de entre as quais o parasitismo parece ser a mais comum e bem-sucedida. O seu sucesso deve-se maioritariamente a um delicado e dinâmico equilíbrio entre a transmissão do parasita e a sua virulência, propriedades que são usualmente atribuídas exclusivamente ao patogénio. Contudo, estas características podem variar quer natural quer experimentalmente, dependendo de condições ambientais, da genética do hospedeiro e das comunidades microbianas que constituem a microbiota. Naturalmente, para sobreviver a uma infeção parasítica o hospedeiro tem de ser apto para montar uma resposta imune competente e eficaz. Em traços gerais, a resposta imune é constituída por mecanismos de resistência, que visam limitar a infeção bloqueando ou eliminado o patogénio e reduzindo a carga parasitária, e por mecanismos de tolerância à doença, que resultam numa melhoria no vigor ou estado de saúde do hospedeiro sem implicar uma redução da carga parasitária. A mosca da fruta, Drosophila melanogaster, passa grande parte do seu ciclo de vida em matéria orgânica em decomposição contactando intimamente com microrganismos, muitos deles patogénicos. Como tal, este organismo possui um vasto conjunto de respostas imunes que o protegem de bactérias, fungos, parasitas e vírus. Embora não possua sistema imunitário adaptativo, presente em vertebrados, a Drosophila tem sido amplamente usada como organismo modelo para estudar imunidade inata, devido a conservação evolutiva destes mecanismos. As defesas imunes da mosca da fruta consistem em: 1) Imunidade comportamental, que tem como propósito evitar contacto com microrganismos patogénicos para prevenir uma possível infeção; 2) Imunidade epitelial, que consiste em barreiras físicas que protegem contra o estabelecimento de microrganismos no interior do corpo; 3) Imunidade celular que depende da ação de células móveis e sésseis (hemócitos) presentes na cavidade corporal; 4) Imunidade humoral que tem por base a produção e secreção de péptidos antimicrobianos para a hemolinfa. Um dos múltiplos patogénios naturais capazes de infetar Drosophila é Pseudomonas entomophila, cujo genoma se encontra totalmente sequenciado e muitos dos seus fatores de virulência identificados, tornando este microrganismo num modelo ideal para estudos de imunidade em D. melanogaster. P. entomophila causa ativação da imunidade sistémica mesmo quando ingerida, e a sua patogenicidade neste cenário deve-se à capacidade que esta bactéria tem de persistir no sistema digestivo da mosca, excretando múltiplas substâncias tóxicas que causam danos no intestino. Muitos dos seus genes associados à sua entomopatogenicidade foram identificados como, por exemplo, genes que codificam toxinas, explicando o efeito altamente nefasto que esta bactéria tem quando ingerida pela mosca da fruta. Dada a importância do background genético do hospedeiro em face de uma infeção a que responde através de mecanismos de resistência e tolerância à doença, este trabalho tem como ambição compreender qual o contributo relativo de cada um destes mecanismos neste processo. Para este fim, infetámos um conjunto de 75 linhas do Drosophila Genetic Reference Panel (DGRP) com P. entomophila. O DGRP é um painel que consiste de cerca de 200 linhas isogénicas, sequenciadas na sua totalidade, que permite decompor a variabilidade genética presente numa população natural de Drosophila, constituindo uma poderosa ferramenta para análises de associação genética. Usando este sistema previamente estabelecido no laboratório o nosso objetivo é caracterizar dinâmicas de infeção em linhas do DGRP por P. entomophila. Com a recolha de dados relativos à sobrevivência após infeção, caracterizámos diferentes dinâmicas de sobrevivência dependentes no background genético do hospedeiro. Através da análise combinada de sobrevivência e dados referentes a carga parasitária recolhidos, inferimos fenótipos relativos a resistência e tolerância à doença neste conjunto de linhas do DGRP. No futuro, a extensão dos protocolos à totalidade do DGRP, juntamente com alguns ajustes que visam melhorar a qualidade dos dados recolhidos, poderá levar à identificação através de análises de associação genética (GWAS) de genes envolvidos em mecanismos quer de resistência quer de tolerância à doença.
Organisms coexist naturally in their environment, interacting through a vast array of biotic interactions of which parasitism seems to be the most common and successful. This success comes from a balance between parasite transmission and virulence, traits which are often seen as properties of the pathogen. However, these traits can vary both experimentally or spontaneously, depending on environment, host genetics and the microbial communities forming the microbiota. Therefore, to survive infection it is particularly important that hosts are able to mount strong and competent immune responses that comprise resistance and tolerance mechanisms. The first is responsible for limiting infection by blocking or eliminating the pathogen, decreasing the parasitic load, while the second reveals itself in the increase in health status or fitness of the host without decreasing the parasitic load. The fruit fly, Drosophila melanogaster spends its whole life cycle in decaying organic matter in close contact with pathogenic microorganisms. Therefore, it possesses an array of immune mechanisms to protect itself against infecting bacteria, fungi, parasites or viruses. Although lacking the presence of an adaptive immune system, present in vertebrates, Drosophila has been widely used as model system for the study of innate immune defences, due to their evolutionary conservation. Fruit fly immune defences consist of: 1) Behavioural immunity, which involves avoiding the pathogen in order to prevent infection or reduce pathogen exposure; 2) Physical or epithelial immunity, which is composed by physical barriers against the establishment of microorganisms in the body; 3) Cellular component, which consists in the action of free-floating and sessile blood cells (haemocytes), that circulate through the body cavity; 4) Humoral immunity, that relies on the production and secretion of antimicrobial peptides (AMPs) to the haemolymph. One of the several natural Drosophila pathogens is Pseudomonas entomophila which has its genome fully sequenced and its virulence factors identified, making it a prime bacterial model for infection in D. melanogaster. P. entomophila is able to activate a systemic immune response in the fruit fly even through ingestion. Under oral infection, pathogenicity is strictly dependent on its ability to persist in the gut and the excretion of toxic substances that disrupt host physiology. A lot of its genes have also been associated with its entomopathogenicity. For example, genes that encode toxins explain why infection with this bacterium is so deleterious to the gut, causing irreversible damage to flies. Given that the host genetic background influences infection outcome through defence mechanisms of resistance and disease tolerance, this work aimed at shedding light on the contributions of each mechanism upon oral infection with a pathogen. As such, we infected a subset of 75 Drosophila Genetic Reference Panel (DGRP) lines with a natural pathogen of D. melanogaster, P. entomophila. The DGRP is a panel that consists of around 200 fully sequenced isogenic lines that allows for the breakdown and analysis of the genetic variability of a natural population, constituting a powerful tool for Genome Wide Association Studies (GWAS). Using this lab established system our objective was to characterize infection dynamics of oral infection by P. entomophila in DGRP lines. By gathering survival data upon infection, we were able to characterize the differences in infection outcome caused by differences in host genetic background. We also created several categories of survival profile based on mortality dynamics over the course of infection. We also measured bacterial loads such as initial inoculum, bacterial loads upon death and set point bacterial loads, in order to characterize how the host influences pathogen dynamics during infection. By combining all the data, we were able to infer phenotypes of resistance and disease tolerance on a set of DGRP lines. In the future, amplification of the dataset to the full DGRP, together with the identified necessary adjustments to improve data quality, may lead to Genome Wide Association Studies (GWAS) and the identification of genes that are involved in the mechanisms of resistance and disease tolerance.
Organisms coexist naturally in their environment, interacting through a vast array of biotic interactions of which parasitism seems to be the most common and successful. This success comes from a balance between parasite transmission and virulence, traits which are often seen as properties of the pathogen. However, these traits can vary both experimentally or spontaneously, depending on environment, host genetics and the microbial communities forming the microbiota. Therefore, to survive infection it is particularly important that hosts are able to mount strong and competent immune responses that comprise resistance and tolerance mechanisms. The first is responsible for limiting infection by blocking or eliminating the pathogen, decreasing the parasitic load, while the second reveals itself in the increase in health status or fitness of the host without decreasing the parasitic load. The fruit fly, Drosophila melanogaster spends its whole life cycle in decaying organic matter in close contact with pathogenic microorganisms. Therefore, it possesses an array of immune mechanisms to protect itself against infecting bacteria, fungi, parasites or viruses. Although lacking the presence of an adaptive immune system, present in vertebrates, Drosophila has been widely used as model system for the study of innate immune defences, due to their evolutionary conservation. Fruit fly immune defences consist of: 1) Behavioural immunity, which involves avoiding the pathogen in order to prevent infection or reduce pathogen exposure; 2) Physical or epithelial immunity, which is composed by physical barriers against the establishment of microorganisms in the body; 3) Cellular component, which consists in the action of free-floating and sessile blood cells (haemocytes), that circulate through the body cavity; 4) Humoral immunity, that relies on the production and secretion of antimicrobial peptides (AMPs) to the haemolymph. One of the several natural Drosophila pathogens is Pseudomonas entomophila which has its genome fully sequenced and its virulence factors identified, making it a prime bacterial model for infection in D. melanogaster. P. entomophila is able to activate a systemic immune response in the fruit fly even through ingestion. Under oral infection, pathogenicity is strictly dependent on its ability to persist in the gut and the excretion of toxic substances that disrupt host physiology. A lot of its genes have also been associated with its entomopathogenicity. For example, genes that encode toxins explain why infection with this bacterium is so deleterious to the gut, causing irreversible damage to flies. Given that the host genetic background influences infection outcome through defence mechanisms of resistance and disease tolerance, this work aimed at shedding light on the contributions of each mechanism upon oral infection with a pathogen. As such, we infected a subset of 75 Drosophila Genetic Reference Panel (DGRP) lines with a natural pathogen of D. melanogaster, P. entomophila. The DGRP is a panel that consists of around 200 fully sequenced isogenic lines that allows for the breakdown and analysis of the genetic variability of a natural population, constituting a powerful tool for Genome Wide Association Studies (GWAS). Using this lab established system our objective was to characterize infection dynamics of oral infection by P. entomophila in DGRP lines. By gathering survival data upon infection, we were able to characterize the differences in infection outcome caused by differences in host genetic background. We also created several categories of survival profile based on mortality dynamics over the course of infection. We also measured bacterial loads such as initial inoculum, bacterial loads upon death and set point bacterial loads, in order to characterize how the host influences pathogen dynamics during infection. By combining all the data, we were able to infer phenotypes of resistance and disease tolerance on a set of DGRP lines. In the future, amplification of the dataset to the full DGRP, together with the identified necessary adjustments to improve data quality, may lead to Genome Wide Association Studies (GWAS) and the identification of genes that are involved in the mechanisms of resistance and disease tolerance.
Descrição
Tese de mestrado, Biologia Evolutiva e do Desenvolvimento, Universidade de Lisboa, Faculdade de Ciências, 2021
Palavras-chave
Imunidade Drosophila melanogaster Pseudomonas entomophila Resistência Tolerância à doença Teses de mestrado - 2021