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Granulomatous diseases caused by Nocardia seriously endanger the health of cultured fish. These bacteria are widely distributed, but prevention and treatment methods are very limited. Chronic granulomatous inflammation is an important pathological feature of Nocardia infection. However, the molecular mechanisms of granuloma formation and chronic inflammation are still unclear. Constructing a granuloma infection model of Nocardia is the key to exploring the pathogenesis of the disease. In this study, we established a granuloma model in the liver of largemouth bass (Micropterus salmoides) and assessed the infection process of Nocardia seriolae at different concentrations by analysing relevant pathological features. By measuring the expression of pro‐inflammatory cytokines, transcription factors and a pyroptosis‐related protein, we revealed the close relationship between pyroptosis and chronic inflammation of granulomas. We further analysed the immunofluorescence results and the expression of pyroptosis‐related protein of macrophage infected by N. seriolae and found that N. seriolae infection induced macrophage pyroptosis in vitro. These results were proved by flow cytometry analysis of infection experiment in vivo. Our results indicated that the pyroptosis effect may be the key to inducing chronic inflammation in the fish liver and further mediating granuloma formation. In this study, we explored the molecular mechanism underlying chronic inflammation of granulomas and developed research ideas for understanding the occurrence and development of granulomatous diseases in fish. [ABSTRACT FROM AUTHOR]

Nocardia seriolae can lead to the development of granulomatous lesions in infected fish, but the exact mechanism responsible for this effect is not fully understood. Here, we assessed the impact of autophagy on granuloma formation through pathological observations and analysis of infection characteristics in N. seriolae -infected largemouth bass (Micropterus salmoides). In our model of chronic inflammatory granulomas, we observed that the expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in infected fish may be regulated by autophagy during granuloma formation. Through the infection experiments in vivo , we measured the expression of autophagy-related genes and autophagic processes. The results showed that N. seriolae induced autophagy and possibly enhanced the fusion of autophagosomes with lysosomes. Examination of immunofluorescence and ultrastructure of infected head kidney primary macrophages and measurement of the expression of pro-inflammatory cytokines revealed that autophagy in macrophages promoted the fusion of autophagosomes and lysosomes while inhibiting the expression of IL-1β and TNF-α. Structural biology analysis of microtubule-associated protein 1 A/1B-light chain 3 (LC3) demonstrated that structure basis and function of autophagy may highly conserved from largemouth bass to humans. In conclusion, our findings suggest that autophagy may play a key role in regulating the formation of chronic inflammatory granulomas, which provides a novel scientific foundation for understanding the pathogenic mechanism of N. seriolae. • Infection of Micropterus salmoides with Nocardia seriolae induces autophagy. • Autophagy inhibits the expression of pro-inflammatory cytokines in primary head kidney macrophages of Micropterus salmoides. • Autophagy may be one of the key factors regulating chronic inflammatory granuloma in the liver of Micropterus salmoides. [ABSTRACT FROM AUTHOR]

Yersinia ruckeri , as a facultative intracellular pathogen, can cause two different infection phenotypes of fish. An important phenotype is diffuse acute inflammation (DAI), which is mainly characterized by systemic hemorrhage and multiple tissues damage. Another phenotype is enteric redmouth symptom (EM) with predominantly digestive system damage, while slight damage to other tissues and organs. At present, the causes and mechanisms of heterogeneity of Y. ruckeri infection are not clear. According to our previous research and related bioinformatics analysis, it is suggested that the heterogeneity of infection of Y. ruckeri may be related to the activation of intracellular pattern recognition receptor (NLRP3). Furthermore, combined with the transmission electron microscope observation and ultrastructural pathological analysis of DAI and EM phenotypes, it is hypothesized that Y. ruckeri infection may mediate the occurrence of DAI by inducing mitochondrial damage and activation of NLRP3. In this study, we established DAI and EM phenotypes of channel catfish (Ictalurus punctatus) through the artificial infection experiment. Subsequently, we observed the pathological changes and characteristics of different tissues in DAI and EM phenotypes, such as liver, spleen, kidney, intestine and gill. In addition, the mitochondrial damage of head kidney mediated by Y. ruckeri infection was confirmed by transmission electron microscope observation. Furthermore, by detecting the expression of proinflammatory factors and related pattern recognition receptors in various tissues, we preliminarily clarified the molecular characteristics of immune response in DAI and EM phenotype fish. Moreover, the head kidney macrophages isolated from DAI and EM were observed through transmission electron microscope, and the expression levels of NLRP3, Caspase-1 and IL-1β were detected. Finally, the correlation between mitochondrial damage induced by Y. ruckeri infection and NLRP3 signal transduction was preliminary revealed. Taken together, our results showed that the inflammatory DAI phenotype of fish mediated by Y. ruckeri infection is closely related to mitochondrial damage and NLRP3 activation, which will provide theoretical basis for further study on the pathogenic mechanism of Y. ruckeri infection and clinical diagnosis of the disease, and also provide some insights into the role of NLRP3 in the natural immunity of aquatic animals. [Display omitted] • Yersinia ruckeri infection is capable of inducing varying degrees of inflammation. • Yersinia ruckeri infection mediated serious damage to mitochondria in DAI phenotype. • Yersinia ruckeri infection mediated severe activation of NLRP3 in DAI phenotype. [ABSTRACT FROM AUTHOR]

Granulomatous chronic inflammation is a typical pathological characteristic of largemouth bass (Micropterus salmoides) infected by Nocardia seriolae , and it is also an important breakthrough to solve the problem of pathogen prevention and treatment. Spleen, the target organ of N. seriolae infection, is significant for studying pathogenic mechanism. However, histopathological characteristics and immunological molecular mechanisms of the development of splenic granulomas in fish have not been extensively studied. In the initial process of exploring the molecular mechanism of N. seriolae infectious splenic granuloma formation, we accidentally found positive signals of necroptosis-related gene by immunohistochemical staining, suggesting that necroptosis may be involved in the formation of granulomas. Nevertheless, the role of necroptosis in the formation of splenic granuloma is still unclear. In this study, we established a granuloma model in the spleen of largemouth bass infected by N. seriolae. Through multiple tissue staining, we compared splenic granulomas with hepatic and renal granulomas, and preliminarily clarified the histopathological characteristics of splenic granulomas and the formation pattern of fibrosis, calcium salt deposition and other lesions. Notably, we found that the expression of inflammatory factors and necroptosis-related molecules in spleen were significantly upregulated during N. seriolae infection. Further immunohistochemical staining showed that the splenic granulomas were accompanied by extensive and intense p-MLKL-positive signals in the peripheral tissue. In addition, western blot and immunofluorescence results showed that N. seriolae could induce necroptosis in primary spleen cells of largemouth bass in vitro. Our data indicated that necroptosis is closely related to the formation of splenic granulomas in largemouth bass, and provide some ideas for the study of the formation mechanism of fish granuloma. [Display omitted] • Construction of Nocardia seriolae granuloma model of largemouth bass. • Necroptosis of spleen epithelial cells was induced by Nocardia seriolae infection. • Nocardia seriolae mediated chronic inflammation through necroptosis. [ABSTRACT FROM AUTHOR]

In the process of continuous combat between pathogens and fish, bacteria have evolved a series of unique strategies to evade the defenses of the natural immune system. The bacterial type IV secretion system (T4SS), as an important multi-protein complex, plays an important role in the process of bacterial infection of the host. However, the T4SS effector protein and its function during bacterial infection remain to be elucidated. A typical T4SS is present in the integrative and conjugative element ICE (r2) of Yersinia ruckeri SC09. In this study, we found that SC09-T4SS mediated the virulence of bacteria and improved the viability of bacteria in eukaryotic cells. SC09-T4SS also mediated the inhibition of pro-inflammatory signals, thereby assisting in the immune escape of bacteria during infection. These effects of SC09-T4SS were due to the secretion of molecules such as STIR-1, STIR-2, and STIR-3. We believe that SC09-T4SS secretion systems are common in bacteria, and our research may provide a general rule of bacterial pathogenicity. • The SC09-T4SS assists Yersinia ruckeri immune escape. • STIR-1, STIR-2, STIR-3 are secreted by SC09-T4SS. • VirB3–4 is an important component of SC09-T4SS. [ABSTRACT FROM AUTHOR]