Investigating bacterial virulence using an alternative host-pathogen model

毒力被定义为细菌病原体通过产生病原体入侵和在宿主中繁殖所必需的因子来伤害宿主生物的能力。为了了解细菌病原体的行为，必须对这些因素进行识别和功能表征。*我的研究计划的长期目标是确定影响鱼类的致病菌产生的毒力因子的分子基础，以及抑制或调节它们以预防或治疗养鱼场细菌感染的方法。为了达到这个目标，我使用了一种方法，将细菌基因组学与使用另一种宿主模型盘状盘齿龙（Dictyostelium disideum）结合起来，盘状盘齿龙是一种土壤变形虫，是细菌的天然捕食者。盘状芽孢杆菌是研究嗜冷致病菌毒力的一个特别有用的工具，包括那些影响鱼类如鲑鱼的致病菌。*在接下来的五年里，我的研究计划将继续关注沙门氏菌气单胞菌的毒力。这种细菌是一种鱼类病原体，对加拿大的水产养殖产生重大负面影响。利用变形虫宿主模型，我们已经证明这种细菌对应激条件很敏感，应激条件会导致基因组修饰，影响主要的毒力因子，包括3型分泌系统，从而导致毒力丧失。沙门氏菌的基因组可塑性是由于其基因组中存在丰富的可移动遗传元件。利用多个沙门氏菌菌株的下一代测序和基因分型，我们有初步证据表明存在新的未表征的毒力因子。*基于沙门氏菌重要毒力因子受到移动遗传元件活性影响的假设，我们提出该细菌的其他毒力因子也受到移动遗传元件活性的影响，可能成为预防或治疗沙门氏菌感染的靶点。鉴定和鉴定沙门氏菌毒力因子，并评估其对天然产物中分离化合物或通过这些化合物刺激的移动遗传元件的直接作用的敏感性是一种很有前途的方法。*因此，我的研究计划将集中在以下短期目标上：(1)通过以盘状芽孢杆菌为宿主的遗传筛选和比较基因组学，确定新的沙门氏菌毒力因子。(2)利用各种方法，包括对鱼类的挑战，对这些新的毒力因子进行功能表征。这部分研究还将涉及开发新的遗传工具，以有效地操纵沙门氏菌基因。(3)鉴定可直接抑制毒力因子或通过刺激可移动遗传因子破坏其基因的水产养殖相容化合物。这些化合物对沙门氏菌毒力的影响将以盘状芽孢杆菌为宿主进行筛选。最有希望的化合物将在鱼身上进行测试。*我的研究项目将更好地了解沙门氏菌的毒力因素。基于我们正在进行的努力，了解沙门氏菌的致病基础和本提案中设定的目标，我们有信心，我们将能够通过利用其基因组可塑性来调节和抑制这种细菌的毒性。这项研究对于开发针对毒力因子的新型抗菌药物至关重要，这些药物不仅可以在兽医领域替代抗生素，而且可以为人类健康服务。

Virulence is defined as the ability of a bacterial pathogen to injure a host organism by producing factors that are essential for the pathogen to invade and multiply in the host. These factors must be identified and functionally characterized in order to understand the behaviour of bacterial pathogens.*The long-term objective of my research program is to identify the molecular basis of the virulence factors produced by pathogenic bacteria affecting fish as well as ways to inhibit or modulate them to prevent or treat bacterial infections in fish farms. I use an approach that combines bacterial genomics with the use of the alternative host model Dictyostelium discoideum, a soil amoeba that is a natural predator of bacteria, to achieve this goal. D. discoideum is a particularly useful tool for studying the virulence of psychrophilic pathogenic bacteria, including those that affect fish such as salmonids.*Over the next five years, my research program will continue to focus on the virulence of Aeromonas salmonicida. This bacterium is a fish pathogen that has a major negative impact on aquaculture in Canada. Using the amoeba host model, we have shown that this bacterium is sensitive to stressful conditions, which cause genomic modifications that affect major virulence factors, including the type three secretion system, leading to a loss of virulence. The genomic plasticity of A. salmonicida results from the presence of abundant mobile genetic elements in its genome. Using next generation sequencing and genotyping of multiple A. salmonicida strains, we have preliminary evidence of the presence of new uncharacterized virulence factors.*Based on the hypothesis that important virulence factors of A. salmonicida are affected by the activity of mobile genetic elements, we propose that other virulence factors in this bacterium are also affected by the activity of mobile genetic elements and may be targets for the prevention or treatment of A. salmonicida infections. The identification and characterization of A. salmonicida virulence factors and the evaluation of their sensitivity to the direct effect of compounds isolated from natural products or through the action of mobile genetic elements stimulated by these compounds is a promising approach.*My research program will thus focus on the following short-term objectives: (1) Identify new virulence factors of A. salmonicida by genetic screening using D. discoideum as a host and by comparative genomics. (2) Functionally characterize these new virulence factors using various approaches, including challenges in fish. This part of the study will also involve the development of new genetic tools to efficiently manipulate A. salmonicida genes. (3) Identify aquaculture-compatible compounds that directly inhibit virulence factors or disrupt their genes by stimulating mobile genetic elements. The effect of these compounds on A. salmonicida virulence will be screened using D. discoideum as a host. The most promising compounds will then be tested on fish.*My research program will provide a better understanding of the virulence factors of A. salmonicida. Based on our on-going effort to understand the pathogenic basis of A. salmonicida and the objectives set out in this proposal, we are confident that we will be able to modulate and inhibit the virulence of this bacterium by taking advantage of its genomic plasticity. This research is crucial for the development of novel anti-bacterial drugs that target virulence factors and that can be used as alternatives to antibiotics not only in the veterinary field but also for human health.