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的常见菌，但也可引起严重的全身性疾病，如脑膜炎和败血症。然而，猪链球菌感染的发病机制及其毒力相关因子，即成孔毒素suilysin （SLY）和d -丙氨酸- d -丙氨酰载体连接酶（DltA）的作用仍不完全清楚。到目前为止，已知SLY通过裂解孔形成破坏不同的宿主细胞，并诱导炎症反应，导致细胞因子的释放，特别是单核/巨噬细胞的释放和中性粒细胞的募集。已知DltA对细胞壁中脂质胆酸的d -丙烯酰化会增加对抗菌肽（amp）的抗性和中性粒细胞对猪链球菌的吞噬作用。我们的目的是阐明SLY和DltA在猪链球菌在猪呼吸道的定植、通过血液的全身传播和入侵中枢神经系统中的作用。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/巨噬细胞和中性粒细胞之间的串扰。我们假设宿主细胞室对sly和dltA的表达有不同的影响，反之亦然，sly和dltA通过调节中性粒细胞和单核/巨噬细胞之间的串扰来影响宿主的先天免疫反应，从而促进免疫耐受。对免疫防御机制的研究。为了研究这一点，我们将使用复杂的体外细胞培养系统来模拟三种主要宿主室的体内情况：在气液界面条件下分化的原代呼吸上皮细胞和肺泡巨噬细胞的共培养系统、猪精确切割的肺切片、重建的全血模型和血脑脊液屏障模型。感染实验将用猪链球菌血清2型野生型菌株（wt）、其等基因突变株Δsly、ΔdltA和ΔdltAΔsly以及各自的补充突变株进行。我们将首先通过定量实时PCR和Western blotting分析三个区室中sly和dltA的表达，然后研究中性粒细胞和单核/巨噬细胞对猪链球菌wt和sly和dltA缺陷突变体的反应。我们将重点关注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，我们将研究猪链球菌感染诱导的单核/巨噬细胞分泌如何影响中性粒细胞的转运、炎症反应和吞噬能力，反之亦然。对猪链球菌感染的先天免疫反应的详细研究将有助于更好地理解猪链球菌从定植体到侵袭性病原体的转变，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.