Influence of Streptococcus suis SLY and DltA on the crosstalk between innate immune cells in different host compartments

猪链球菌是猪上呼吸道常见的定植菌，但也可引起严重的全身性疾病，如脑膜炎和败血症。然而，S.猪链球菌感染及其毒力相关因子，即孔形成毒素猪溶血素（SLY）和D-丙氨酸-D-丙氨酰载体连接酶（DltA）的作用仍然没有完全了解。到目前为止，已知SLY通过溶解孔形成损害不同的宿主细胞，并诱导炎症反应，导致细胞因子的释放，特别是单核细胞/巨噬细胞的释放，以及嗜中性粒细胞的募集。已知DltA对细胞壁中脂磷壁酸的D-丙氨酰化增加了对抗菌肽（AMP）的抗性和S.嗜中性粒细胞。我们的目的是阐明SLY和DltA在S.猪呼吸道定植、通过血流全身传播和侵入中枢神经系统。因此，我们将重点关注它们对先天免疫细胞的影响以及单核细胞/巨噬细胞和中性粒细胞之间的串扰。我们假设宿主隔室差异地影响SLY和DltA的表达，反之亦然，SLY和DltA通过调节中性粒细胞和单核细胞/巨噬细胞之间的串扰来影响宿主的先天免疫应答，从而有助于抵抗。S. suis对immune免疫defense防御mechanism机制.为了研究这一点，我们将使用复杂的体外细胞培养系统，密切模仿在体内的情况下，三个主要的主机隔室：共培养系统的原代呼吸道上皮细胞分化下的气液界面条件和肺泡巨噬细胞，猪精密切割肺切片，重建的全血模型，和血液脑脊液屏障模型。将用S. suis血清2型野生型菌株（wt）、其同基因突变体Δsly、ΔdltA和ΔdltAΔsly以及相应的互补突变菌株。我们将首先通过定量实时PCR和Western印迹分析sly和dltA在三个隔室中的表达，然后研究中性粒细胞和单核细胞/巨噬细胞对S.猪野生型和sly-和dltA-缺陷型突变体。我们将集中于中性粒细胞胞外陷阱的形成，活性氧的产生，吞噬活性，以及某些细胞因子和AMP的释放。最后，我们将研究S.猪感染影响中性粒细胞迁移、炎症反应和吞噬能力，反之亦然。本文详细研究了猪对沙门氏菌的天然免疫反应。猪链球菌在三个不同区室中的感染有助于更好地了解S.猪作为入侵病原体的定殖者，以及SLY和DltA如何参与这一过程。

Streptococcus suis is a frequent colonizer of the upper respiratory tract of pigs, but can also cause severe systemic diseases like meningitis and septicemia. However, pathogenesis of S. suis infection and the role of its virulence-associated factors, namely the pore-forming toxin suilysin (SLY) and the D-alanine-D-alanyl carrier ligase (DltA), is still not fully understood. So far, it is known that SLY damages different host cells by lytic pore formation and induces an inflammatory response leading to the release of cytokines, especially by monocytes/macrophages, and the recruitment of neutrophils. D-alanylation of lipoteichoic acids in the cell wall by DltA is known to increase resistance against antimicrobial peptides (AMPs) and phagocytosis of S. suis by neutrophils. Our aim is to clarify the role of SLY and DltA in S. suis colonization of the porcine respiratory tract, systemic dissemination via the bloodstream, and invasion of the central nervous system. Thereby we will focus on their influence on innate immunity cells and the crosstalk between monocytes/macrophages and neutrophils. We hypothesize that the host compartments influence the expression of sly and dltA differentially and that vice versa SLY and DltA influence the host’s innate immune response by modulating the crosstalk between neutrophils and monocytes/macrophages contributing to the resistance of. S. suis towards immune defense mechanisms. To investigate this, we will use complex in vitro cell culture systems that closely mimic the in vivo situation of the three main host compartments: a co-culture system of primary respiratory epithelial cells differentiated under air-liquid interface conditions and alveolar macrophages, porcine precision-cut lung slices, a reconstituted whole blood model, and the blood cerebrospinal fluid barrier model. Infection experiments will be performed with S. suis serotype 2 wild-type strain (wt), its isogenic mutants Δsly, ΔdltA, and ΔdltAΔsly as well as the respective complemented mutant strains. We will start with the analysis of sly and dltA expression in the three compartments by quantitative real-time PCR and Western blotting, followed by the investigation of the neutrophil and the monocyte/macrophage response towards S. suis wt and sly- and dltA-deficient mutants. We will focus on the formation of neutrophil extracellular traps, reactive oxygen species production, phagocytic activity, and the release of certain cytokines and AMPs. Finally, we will investigate how secretions from monocytes/macrophages induced by S. suis infection influence neutrophils regarding their transmigration, inflammatory response, and phagocytic capacity, and vice versa. The detailed investigation of the innate immune response towards S. suis infection in the three different compartments will help to better understand the switch of S. suis as a colonizer to an invasive pathogen, and how SLY and DltA are involved in this process.