Experimental research on pathogenesis of Streptococcus suis infection

Abstract

The dissertation presents an experimental research on the pathogenic mechanism of meningitis of the streptococcal infection of pigs based on study of the pathogenetic processes of pyroptosis through the expression of mRAN of pyroptosis-related genes and proteins and changes in morphologyof endothelial cells of brain microvessels of white mice infected with Streptococcus suis serotype 2. Streptococcus suis (S. suis) is an important zoonotic pathogen that can cause many diseases in pigs, such as sepsis, arthritis, endocarditis and meningitis, of which meningitis is the most serious. There are 35 serotypes of, and serotype 2 is the most virulent. At the same time, Streptococcus suis serotype 2 (S. suis 2) can also infect humans, causing serious public health problems. Although S. suis 2 has attracted great attention worldwide, the research on its pathogenesis is still limited. The adhesion of pathogenic bacteria to the surface of host cells or tissues and its subsequent invasion and diffusion are the key steps of pathogenic bacteria. And the interaction between pathogen and host is involved in all of these processes. Therefore, to study the pathogenic mechanism of pathogenic bacteria is to study the interaction between pathogenic bacteria and host. This paper described several common virulence factors, such as CPS, SLY, MRP, EF, SAO, Srt, FBPS, SadP and Eno. Under the actions of virulence factors, S. suis 2 adheres and colonize to the mucosal and epithelial surface of host cells. Then S. suis 2 invades into deeper tissues and bloodstream. If S. suis 2 in the blood doesn't cause fatal sepsis, іt can go to the third stage. The third stage is to cross the Blood brain barrier (BBB) and to get access to the central nervous system (CNS), and ultimately causes meningitis. During pathogenesis, S. suis 2 interacts with multiple cells of the host, such as neutrophils, macrophages, epithelial cells, and microvascular endothelial cells, to evade the innate or adaptive immunity of the host. Cells are the basic unit of life, and cell death plays an important role in the body's metabolism, the occurrence and development of diseases. Pyroptosis is a form of programmed cell death. Pyroptosis is significantly different from other cell death methods (such as apoptosis, necrosis, etc.) in morphological characteristics, occurrence mechanism, and mechanism of action. When a cell undergoes pyroptosis, the nucleus condenses to form a pyroptotic body, numerous pores appear in the cell membrane, the cell swells and ruptures, releasing its contents. Caspase family is a homologous and structurally similar proteolytic enzyme in cytoplasm, which selectively recognizes and cleaves peptide bonds behind downstream target aspartic acid residues. Caspase-1,4,5,11 can induce pyroptosis through different pathways. Besides caspases, gasdermin also plays an important role in pyroptosis. Gasdermins (GSDMs) are a family of functionally diverse proteins expressed in a variety of cell types and tissues. The Gasdermin family includes 6 members, of which gasdermin D is the executor of pyroptosis. Upon cleavage by activated capsese, gasdermen D can be divided into N and C segments. Among them, the N fragment can form pores in the cell membrane, leading to cell swelling, rupture, outflow of cytokines and other contents, triggering the body's immune response, and leading to pyroptosis. The occurrence of pyroptosis can be divided into the classical pathway and the non-classical pathway. The classical pathway mainly depends on caspase-1, while the non-canonical pathway depends on the activation of caspase-4/5/11. In addition, there are uncommon caspase-3/8-mediated pathway and granzyme-mediated pathway. As a way of cell death, pyroptosis is inextricably linked to disease. Inflammasomes and cytokines produced in the process of pyroptosis can trigger an inflammatory response in the body, and an excessive inflammatory response can lead to diseases, such as infectious diseases, neurological diseases, and tumors. In infectious diseases, pyroptosis is closely related to the infection of a variety of bacteria, fungi and viruses, and Pathogen-associated molecular patterns (PAMPs) and Lipopolysaccharide (LPS) can be recognized by corresponding inflammasomes and caspases, respectively, and activate the downstream pyroptotic pathways. Pathogen infection is the main way to induce pyroptosis. In cardiovascular diseases, a high-fat environment can induce an increase in reactive oxygen species (ROS), trigger endothelial cell pyroptosis, and exacerbate the development of atherosclerosis (AS). In the nervous system, cell death is involved in the pathogenesis of the progression of degenerative diseases of the central nervous system, such as Alzheimer's disease (AD), Parkinson's disease (PD), and stroke. In terms of tumors, pyroptosis can inhibit the occurrence and development of tumors, and at the same time, as a pro-inflammatory death, pyroptosis can form a microenvironment suitable for tumor cell growth, thereby promoting tumor growth. In this study, S. suis 2 was used to infect mouse brain microvascular endothelial cell (bEnd.3). First, the infection conditions were screened. The results showed that the optimal infection number was 100:1 and the optimal infection time was 12 hours. Brain microvascular endothelial cells were divided into 4 groups: control group, LPS+ Adenosine triphosphate (ATP) infection group, S. suis 2 infection group and S. suis 2+ Ac-YVAD-CMK (CMK, caspase-1 inhibitor) infection group. The following three groups were infected with Streptococcus suis type 2 according to the above conditions, and multiple replicates were performed simultaneously. After 12 hours, the cells and cell supernatants were collected for different tests. Total RNA was extracted, RNA concentration was measured, and cDNA was obtained by reverse transcription. cDNA was detected by qPCR for mRNA expression of cytokines. Compared with the control group, the mRNA expression levels of caspase-1, il-18 and il-1beta in LPS+ATP group and S. suis 2 group were higher than those in the control group, indicating that a large number of cytokines related to pyroptosis were secreted. At the same time, mRNA expression levels of caspase-1, il-18 and il-1beta in S. suis 2 +CMK group were significantly lower than those in S. suis 2 +CMK group, indicating that CMK inhibitor played an inhibitory role. At the same time, protein was extracted from the collected cells, the protein concentration was measured, and then WB test was carried out to detect the protein expression level of related genes. Compared with the control group, the protein expressions of IL-18, IL-1β,caspase-1,GSDMD and GSDME in LPS+ATP group were significantly up-regulated, increasing by 0.477, 0.088, 0.378, 1.118 and 3.05 times, respectively. The protein expressions of IL-18, IL-1β,caspase-1,GSDMD and GSDME in S. SUIS 2 group were significantly up-regulated, increasing by 1.024, 0.066, 0.376, 0.453 and 1.654 times, respectively. Compared with S. SUIS 2 group, protein expression in S. suis 2+CMK group was significantly decreased by 0.6, 0.396, 0.298, 0.743 and 0.586 times, respectively. The results showed that S. suis 2 infection of brain microvascular endothelial cells caused intense inflammatory reaction and even pyroptosis of cells. At the same time, the inhibitor CMK played a good inhibitory effect. In addition, the protein content of IL-6, IL-10, IL-18, IL-1beta, caspase-1 in the cell culture supernatant was also detected. Compared with control group, the relative expression of TNF-α, IL-6, IL-10, IL-18, IL-1β in cell supernatant of SS group and LPS+ATP group was significantly increased, and the difference was statistically significant (P < 0.01). Compared with S. suis 2 group and LPS+ATP group, the relative expression level of TNF-α, IL-6, IL-10, IL-18, IL-1βin S. suis 2 + CMK group was significantly decreased, and the difference was statistically significant (P < 0.01), indicating that coke death caused by S. suis 2 and LPS+ATP could be inhibited by CMK. At the same time, At the same time, the release rate of lactate dehydrogenase in the cell supernatant was also measured, and the results showed that the release rate of lactate dehydrogenase in LPS+ATP group and S. suis 2 group was much higher than that in the control group, and the difference was extremely significant. In S. suis 2 + CMK group, the release rate of lactate dehydrogenase decreased sharply under the action of the inhibitor. In order to observe the morphology of infected cells, the collected cells were also observed by transmission electron microscopy. The results of electron microscopy showed that the cell membrane of LPS+ATP group and S. suis 2 group was broken and the cell contents were leaked out. Combined with the above other results, it can be concluded that under the infection of S. suis 2, brain microvascular endothelial cells produced an inflammatory response and pyroptosis occurred. In the dissertation work on the basis of research are substantiated the pathogenic mechanism of Streptococcus suis, inhibitor protection to develop target drugs and vaccine for meningitis, reduce losses of pig industry and promote the healthy development of animal husbandry.