Grogan Christina, Bennett Marissa, Moore Shannon, Lampe David
Department of Biological Sciences, Bayer School of Natural and Environmental Sciences, Duquesne University, Pittsburgh, PA, United States.
Front Microbiol. 2021 Feb 16;12:633667. doi: 10.3389/fmicb.2021.633667. eCollection 2021.
Mosquitoes vector many pathogens that cause human disease, such as malaria that is caused by parasites in the genus . Current strategies to control vector-transmitted diseases are hindered by mosquito and pathogen resistance, so research has turned to altering the microbiota of the vectors. In this strategy, called , symbiotic bacteria are genetically modified to affect the mosquito's phenotype by engineering them to deliver antiplasmodial effector molecules into the midgut to kill parasites. One paratransgenesis candidate is , a Gram-negative, rod-shaped bacterium colonizing the midgut, ovaries, and salivary glands of sp. mosquitoes. However, common secretion signals from and closely related species do not function in . Here, we report evaluation of 20 native N-terminal signal sequences predicted from bioinformatics for their ability to mediate increased levels of antiplasmodial effector molecules directed to the periplasm and ultimately outside the cell. We tested the hypothesis that by increasing the amount of antiplasmodials released from the cell we would also increase parasite killing power. We scanned the SF2.1 genome to identify signal sequences from extra-cytoplasmic proteins and fused these to the reporter protein alkaline phosphatase. Six signals resulted in significant levels of protein released from the bacterium. Three signals were successfully used to drive the release of the antimicrobial peptide, scorpine. Further testing in mosquitoes demonstrated that these three strains were able to suppress the number of oocysts formed after a blood meal containing to a significantly greater degree than wild-type , although prevalence was not decreased beyond levels obtained with a previously isolated siderophore receptor signal sequence. We interpret these results to indicate that there is a maximum level of suppression that can be achieved when the effectors are constitutively driven due to stress on the symbionts. This suggests that simply increasing the amount of antiplasmodial effector molecules in the midgut is insufficient to create superior paratransgenic bacterial strains and that symbiont fitness must be considered as well.
蚊子是许多导致人类疾病的病原体的传播媒介，比如由疟原虫属寄生虫引起的疟疾。当前控制媒介传播疾病的策略受到蚊子和病原体抗性的阻碍，因此研究转向改变媒介的微生物群。在这种被称为“共生菌转基因策略”的方法中，共生细菌经过基因改造，通过工程手段使其将抗疟效应分子输送到中肠以杀死寄生虫，从而影响蚊子的表型。一种用于共生菌转基因策略的候选细菌是嗜沫栖热菌，它是一种革兰氏阴性、杆状细菌，定殖于冈比亚按蚊的中肠、卵巢和唾液腺。然而，来自嗜沫栖热菌及其近缘物种的常见分泌信号在冈比亚按蚊中不起作用。在此，我们报告了对通过生物信息学预测的20个嗜沫栖热菌天然N端信号序列介导抗疟效应分子向周质并最终向细胞外增加水平的能力的评估。我们测试了这样一个假设，即通过增加从细胞释放的抗疟物质的量，我们也会增强杀灭寄生虫的能力。我们扫描了嗜沫栖热菌SF2.1基因组以识别来自胞外蛋白的信号序列，并将这些序列与报告蛋白碱性磷酸酶融合。六个信号导致嗜沫栖热菌释放出显著水平的蛋白质。三个信号成功用于驱动抗菌肽蝎毒素的释放。在蚊子中的进一步测试表明，这三种嗜沫栖热菌菌株能够比野生型嗜沫栖热菌更显著地抑制在含有疟原虫的血餐后形成的卵囊数量，尽管感染率降低的程度没有超过用先前分离的铁载体受体信号序列所达到的水平。我们将这些结果解释为表明当效应分子因共生菌受到压力而组成性驱动时，存在一个可以达到的最大抑制水平。这表明仅仅增加中肠中抗疟效应分子的量不足以产生更优的共生菌转基因菌株，还必须考虑共生菌的适应性。
