Differential proteome profiling of bacterial culture supernatants reveals candidates for the induction of oral immune priming in the Red Flour Beetle

Most organisms are hosts to a wide range of symbionts and commensals, but they might also encounter parasites and pathogens during their lifetime. Therefore, immune strategies to recognize and counteract potential threats have evolved across all kingdoms of life. The most elaborate immune defences are found in vertebrate animals, where responses are classically divided into innate and adaptive. Insects lack the cellular machinery needed to provide adaptive immunity. However, over the past decades, work on immune priming has shown that to a certain degree, insects are indeed capable of specifically recognizing, memorizing, and counteracting certain pathogens. An important piece of the puzzle are the pathogen-associated molecules that induce these immune priming responses. Here, we make use of the model system Tribolium castaneum (T. castaneum) and its pathogen Bacillus thuringiensis (B. thuringiensis), to compare the proteomes of two closely related B. thuringiensis strains that either induce priming via the oral route, or not. We identify proteins that might be immunostimulatory in T. castaneum. The Cry3Aa toxin, an important plasmid-encoded virulence factor of Bacillus thuringiensis bv. tenebrionis (hereafter called Btt) was found to be one of the most promising candidates. We made use of additional B. thuringiensis strains varying in the possession of the Cry-carrying plasmid to experimentally test for its relevance for priming. Our findings will help future studies to focus on specific candidates to perform experiments on the mechanisms and evolution of immune priming.

绝大多数生物均为多种共生菌与共栖生物的宿主，且在生命周期中亦可能遭遇寄生虫与病原体。因此，识别并抵御潜在威胁的免疫策略，已在生命的所有界别中演化形成。脊椎动物拥有结构最为复杂的免疫防御系统，其免疫应答传统上分为固有免疫与适应性免疫两类。昆虫缺乏介导适应性免疫所需的细胞机制。然而在过去数十年间，针对免疫预激（immune priming）的研究表明，昆虫在一定程度上确实能够特异性识别、记忆并抵御特定病原体。可诱导此类免疫预激应答的病原体相关分子，正是破解该谜题的重要一环。本研究以模式生物赤拟谷盗（Tribolium castaneum, T. castaneum）及其病原体苏云金芽孢杆菌（Bacillus thuringiensis, B. thuringiensis）为研究系统，对比两株亲缘关系相近的苏云金芽孢杆菌菌株的蛋白质组——二者分别可经口途径诱导免疫预激，或无法诱导该应答。本研究鉴定出了赤拟谷盗体内可能具有免疫刺激活性的蛋白质。苏云金芽孢杆菌暗黑生物变种（Bacillus thuringiensis bv. tenebrionis，下称Btt）的重要质粒编码毒力因子Cry3Aa毒素，被鉴定为最具潜力的候选蛋白之一。本研究进一步利用携带或不携带Cry质粒的不同苏云金芽孢杆菌菌株，通过实验验证该毒素在免疫预激中的作用相关性。本研究结果可为未来聚焦特定候选蛋白开展免疫预激机制与演化相关实验的研究提供参考。