Phage-encoded Shiga Toxin as a Bacterial Defense Against Predation: Shiga Toxin Induction, Uptake and Toxicity in Tetrahymena Thermophila

细菌的外毒素是已知的最致命的物质之一。这些外毒素通过灭活因子和/或在真核生物中普遍保守的途径杀死真核细胞。当对哺乳动物进行研究时，这些毒素会引起从霍乱到白喉到肠出血性腹泻等疾病。编码这些毒素的基因通常是由细菌病毒（噬菌体）编码的。环境DNA测序（宏基因组分析）表明，约15%的噬菌体（包括游离的和并入细菌的）编码外毒素基因。每毫升海水中约有106个噬菌体，每克沉积物中约有108个噬菌体，噬菌体中编码任何特定外毒素基因的基因出现的频率远远超过潜在动物宿主的数量。此外，这些噬菌体编码的外毒素基因在从相应的人类疾病不流行的环境中分离出来的噬菌体中被发现的频率很高。这些观察结果表明，人类和其他易感哺乳动物既不是这些毒素的最初目标，也不是主要目标。因此，对这些毒素进化的任何人为影响可能不如人类和其他多细胞生物出现之前发生的影响那么重要。如果不是哺乳动物，那么“谁”才是无处不在的外毒素的真正目标。细菌死亡的一个主要原因是被单细胞真核捕食者消耗，如纤毛虫和其他原生动物。细菌已经进化出许多不同的方法来抵御这些捕食者，包括变大和高速逃跑。通过溶原性噬菌体的溶解诱导，在摄入前或摄入后协调释放噬菌体编码的外毒素，是另一种可能的抗捕食者防御策略。原生动物是一种吞噬细胞，它们与哺乳动物的吞噬细胞，尤其是巨噬细胞有许多共同的特征。噬菌体编码的外毒素，如经过充分研究的志贺毒素（Stx），通过攻击所有真核细胞（单细胞和多细胞）共有的特征和途径来杀死真核细胞。由于真核捕食者（如纤毛虫和其他原生动物）的捕食是细菌死亡的主要来源，这些观察结果表明，外毒素可能是作为反捕食者（抗原生动物）防御策略的一部分而出现的。因此，这些毒素的进化可能发生在多细胞生物出现之前。因此，在原生动物和它们的细菌猎物之间的进化战争中，人类可能是无辜的旁观者。利用自然发生的捕食者-猎物相互作用，本项目的研究探索了噬菌体编码的外毒素（如志贺毒素）如何被用作细菌抗捕食者武器库的一部分。这些研究将提供细菌对捕食反应的分子细节，通过确定1)嗜热t细胞如何向编码毒素的噬菌体的溶原细菌发出信号以产生志贺毒素，以及2)这种外毒素如何进入并引起这些纤毛虫的细胞毒性。其中一些机制显然是纤毛虫所独有的，而另一些可能在高等真核生物中保守。拟议的研究将为布法罗大学的研究生和本科生提供假设驱动的研究经验，并通过合作，为默西赫斯特学院的本科生提供假设驱动的研究经验，默西赫斯特学院是位于宾夕法尼亚州伊利的一所小型文理学院。在Koudelka实验室，学生们学会了如何提出重要的生物学问题，并利用他们的观察对科学理解做出重要贡献。在过去的15年里，库德尔卡博士指导了22名本科生和高中生，其中50%是少数族裔或女性。所有PI的学员都在研究或医学领域取得了成功。这些研究的结果将有助于确定捕食和细菌对它的反应，如何影响微生物群落的进化和结构。

Intellectual meritExotoxins of bacteria are among the most deadly substances known. These exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. When studied in the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. The genes encoding these toxins are usually encoded by viruses of bacteria (bacteriophages). Environmental DNA sequencing (metagenomic analyses) indicates that ~15% of all bacteriophage (both free and incorporated into bacteria) encode an exotoxin gene. With approximately 106 bacteriophages /mL of seawater and 108 bacteriophages/gram of sediment, the frequency of occurrence of the genes encoding any particular exotoxin gene in bacteriophage far exceeds the number of potential animal hosts. Moreover, these bacteriophage-encoded exotoxin genes are found at high frequency in phages isolated from environments where the corresponding human diseases are not prevalent. These observations suggest the hypothesis that humans and other susceptible mammals are neither the original nor primary targets of these toxins. Hence any anthropogenic effects on the evolution of these toxins may not be as significant as those that occurred before the dawn of man and other multicellular organisms. If not mammals, then "who" are the true targets of the ubiquitous exotoxins. A major source of bacterial mortality is consumption by single-celled eukaryotic predators, such as ciliates and other protozoa. Bacteria have evolved many different methods to fend off these predators, including becoming oversized and fleeing at high speeds. Coordinated release of phage-encoded exotoxins, either at the pre- or post-ingestional states via lytic induction of lysogenic bacteriophage comprises another possible antipredator defense strategy. Protozoa are phagocytotic cells and they share many features with mammalian phagocytes, especially macrophages. The phage-encoded exotoxins like the well-studied Shiga toxin (Stx), kill eukaryotic cells by attacking features and pathways that are common to all eukaryotes both, uni- and multi-cellular. Since predation by eukaryotic predators (e.g., ciliates and other protozoa), is a major source of bacterial mortality, these observations suggest that exotoxins may have arisen as part of an antipredator, (antiprotozoan) defense strategy. Thus the evolution of these toxins may have occurred before the appearance of multicellular organisms. Hence humans may be innocent bystanders in the evolutionary battle between protozoans and their bacterial prey.Using a naturally occurring predator-prey interaction, the studies in this project explore aspects of how bacteriophage-encoded exotoxins like Shiga toxin are used as part of the bacterial anti-predator arsenal. These studies will provide the molecular details of the bacterial response to predation by determining 1) how T. thermophila signals bacteria that are lysogenic for toxin-encoding phages to produce Shiga toxin and 2) how this exotoxin enters and causes cytotoxicity in these ciliates. Some of these mechanisms are apparently unique to the ciliates and others may be conserved in higher eukaryotes. Broader Impacts The proposed research will provide a hypothesis-driven research experience to both graduate and undergraduate students at the University at Buffalo institution and through collaborations, undergraduate students at Mercyhurst College, a small, liberal arts college located in Erie, PA. In the Koudelka lab, students have learned how to ask important biological questions and used their observations to make important contributions to scientific understanding. Dr. Koudelka has supervised 22 undergraduates and high school students in the last 15 years, 50% of which were members of underrepresented minority groups or women. All of the PI's trainees have gone on to successful careers in research or medicine. The results of these studies will help define how predation and bacterial responses to it, influence the evolution and structure of microbial communities.