Centro de Genómica y Bioinformática, Facultad de Ciencias, Universidad Mayor, Santiago, Chile.
Consorcio Tecnológico de Sanidad Acuícola, Ictio Biotechnologies S.A., Santiago, Chile.
Front Immunol. 2020 Nov 12;11:544718. doi: 10.3389/fimmu.2020.544718. eCollection 2020.
, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by and contribute to the development of new effective therapeutic strategies against SRS.
，一种侵袭性的细胞内病原体，是鲑鱼立克次氏体败血病（SRS）的病原体。这是一种慢性多系统疾病，在智利鲑鱼养殖中产生高死亡率和巨大损失，威胁着鲑鱼产业的可持续性。先前的报告表明，能够在鲑鱼巨噬细胞中存活和复制，诱导抗炎环境和有限的溶酶体反应，这可能与宿主免疫逃避机制有关，有利于细菌的存活。目前针对 （基于抗生素和疫苗的使用）的控制和预防策略并没有在对抗感染方面取得预期的成功。这使得揭示宿主-病原体相互作用以开发更有效的治疗策略变得迫在眉睫。在这项研究中，我们通过共聚焦显微镜分析溶酶体 pH 值和蛋白水解能力，评估了 IgM 珠治疗对感染 Atlantic 鲑鱼巨噬细胞富集细胞培养物中溶酶体活性的影响。通过测量乳酸脱氢酶（LDH）活性释放来量化细胞溶解，评估了 IgM 珠对感染细胞中 诱导的细胞毒性的影响。通过 qPCR 定量 rDNA 拷贝数和计数胞外和胞内环境中的集落形成单位（CFU）来确定细菌负荷。我们的结果表明，抗体刺激通过降低溶酶体 pH 值并增加该细胞器内的蛋白水解活性来促进溶酶体活性。此外，在感染的 Atlantic 鲑鱼巨噬细胞中孵育 IgM 珠会降低细菌诱导的细胞毒性并减少细菌负荷。总的来说，我们的结果表明，用 IgM 珠刺激感染的细胞可以逆转由细菌感染诱导的溶酶体活性的调节，促进巨噬细胞的存活和细菌的消除。这项工作代表了理解 建立的细菌逃避机制的新的重要证据，并有助于开发针对 SRS 的新的有效治疗策略。
