Protease SfpB plays an important role in cell membrane stability and immune system evasion in Streptococcus agalactiae.

Bacteria possess the ability to develop diverse and ingenious strategies to outwit the host immune system, and proteases are one of the many weapons employed by bacteria. This study sought to identify S. agalactiae additional serine protease and determine its role in virulence. The S. agalactiae THN0901 genome features one S8 family serine peptidase B (SfpB), acting as a secreted and externally exposed entity. A S8 family serine peptidase mutant strain (ΔsfpB) and complement strain (CΔsfpB) were generated through homologous recombination. Compared to the wild-type strain THN0901, the absorption of EtBr dyes was significantly reduced (P < 0.01) in ΔsfpB, implying an altered cell membrane permeability. In addition, the ΔsfpB strain had a significantly lower survival rate in macrophages (P < 0.01) and a 61.85 % lower adhesion ability to the EPC cells (P < 0.01) compared to THN0901. In the in vivo colonization experiment using tilapia as a model, 210 fish were selected and injected with different bacterial strains at a concentration of 3 × 106 CFU/tail. At 6, 12, 24, 48, 72 and 96 h post-injection, three fish were randomly selected from each group and their brain, liver, spleen, and kidney tissues were isolated. Subsequently, it was demonstrated that the ΔsfpB strain exhibited a markedly diminished capacity for colonization in tilapia. Additionally, the cumulative mortality of ΔsfpB in fish after intraperitoneal injection was reduced by 19.92-23.85 %. In conclusion, the findings in this study have demonstrated that the SfpB plays a significant role in S. agalactiae cell membrane stability and immune evasion. The immune evasion is fundamental for the development and transmission of invasive diseases, the serine protease SfpB may be a promising candidate for the development of antimicrobial agents to reduce the transmission of S. agalactiae.

Gill lesions are the main cause of death in yellowfin seabream (Acanthopagrus latus) following infection with Amyloodinium ocellatum.

Amyloodiniosis, caused by the ectoparasite Amyloodinium ocellatum, affects the healthy development of mariculture. This study used a local infection method to identify the pathogenic target organ responsible for the death of infected fish. Comparing the relationship between the abundance of trophonts in gills and skin with the mortality of infected fish using local infection showed that severe gill infections cause the mortality of infected fish. At the 40 % survival rate of infected fish, the parasite abundance in the gill was 14,167 ± 4371. The gill filaments of the infected fish were structurally disordered, with pronounced lesions associated with the presence of trophonts, such as epithelial cell degeneration and massive lymphocytic infiltration. However, the skin showed no obvious pathological changes. The TUNEL assay showed a significant presence of apoptotic cells concentrated in the area of A. ocellatum infection. The trophonts on the gills developed faster than those parasitising the skin and fins. Microbiome analysis revealed that at the phylum level, Proteobacteria, Bacteroidota, and Firmicutes are abundant in the skin, while Verrucomicrobiota, Bacteroidota, and Proteobacteria are abundant in the gills of A. latus. Furthermore, A. ocellatum infection significantly reduced (p < 0.05) the richness and diversity of the gill microbial community of A. latus. Infection by A. ocellatum increased the relative abundance of several putative pathogenic bacteria (Flavobacterium and Nocardia) in the gill and skin of A. latus, possibly increasing the likelihood of disease in the host. In conclusion, these results evidenced that severe gill infections by A. ocellatum cause mortality in infected fish, which clarifies the direction for exploring the pathogenesis of amyloodiniosis.

Establishment of a gill cell line from yellowfin seabream (Acanthopagrus latus) for studying Amyloodinium ocellatum infection of fish.

Amyloodinium ocellatum is among the most devastating protozoan parasites, causing huge economic losses in the mariculture industry. However, the pathogenesis of amyloodiniosis remains unknown, hindering the development of targeted anti-parasitic drugs. The A. ocellatum in vitro model is an indispensable tool for investigating the pathogenic mechanism of amyloodiniosis at the cellular and molecular levels. The present work developed a new cell line, ALG, from the gill of yellowfin seabream (Acanthopagrus latus). The cell line was routinely cultured at 28°C in Dulbecco's modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS). ALG cells were adherent and exhibited an epithelioid morphology; the cells were stably passed over 30 generations and successfully cryopreserved. The cell line derived from A. latus was identified based on partial sequence amplification and sequencing of cytochrome B (Cyt b). The ALG was seeded onto transwell inserts and found to be a platform for in vitro infection of A. ocellatum, with a 37.23 ± 5.75% infection rate. Furthermore, scanning electron microscopy (SEM) revealed that A. ocellatum parasitizes cell monolayers via rhizoids. A. ocellatum infection increased the expression of apoptosis and inflammation-related genes, including caspase 3 (Casp 3), interleukin 1 (IL-1), interleukin 10 (IL-10), tumour necrosis factor-alpha (TNF-α), in vivo or in vitro. These results demonstrated that the in vitro gill cell monolayer successfully recapitulated in vivo A. latus host responses to A. ocellatum infection. The ALG cell line holds great promise as a valuable tool for investigating parasite-host interactions in vitro.

Cordyceps militaris polysaccharides modulate gut microbiota and improve metabolic disorders in mice with diet-induced obesity.

BACKGROUND: Cordyceps militaris is an edible and medicinal fungus, and its polysaccharides are among its main pharmacological components. They can display immunomodulation, anti-oxidation, anti-inflammation, anti-hypolipidemic, and other functions. The anti-obesity effect of C. militaris polysaccharides (CMP) is not yet fully understood, however. RESULTS: In this study, a CMP diet intervention was applied over a 4 week period to mice with obesity induced by a high-fat diet (HFD), followed by profiling of obesity-induced dyslipidemia, low-grade inflammation, and gut dysbiosis. The results suggested that CMP could significantly reduce HFD-induced obesity, alleviate obesity-induced hyperlipidemia and insulin resistance, and ameliorate systemic inflammation, showing a promising ability to protect mice from obesity. Further analyses revealed that CMP could regulate obesity-induced gut dysbiosis by restoring the phylogenetic diversity of gut microbiota. It could also increase the relative abundance of short-chain fatty acid (SCFA)-producing bacteria, while down-regulating the level of bacteria that were positively related to the development of obesity. A correlation analysis showed that Helicobacter, Allobaculum, Clostridium XVIII, Parabacteroides, Ligilactobacillus, Faecalibaculum, Adlercreutzia, and Mediterraneibacter were positively related to obese phenotypes. CONCLUSION: This study highlights the potential of CMP as a prebiotic agent to protect obese individuals from metabolic disorders and gut dysbiosis. © 2022 Society of Chemical Industry.

In vitro bioactivity and degradability of ß-tricalcium phosphate porous scaffold fabricated via selective laser sintering.

Porous scaffolds consisting of ß-tricalcium phosphate (ß-TCP) were successfully fabricated via selective laser sintering. The scaffolds had a controlled microstructure and totally interconnected porous structure. The microstructure and mechanical properties were studied. The bioactivity and degradability of scaffolds were evaluated through the simulated body fluid (SBF) cultivation experiment. The formation of a biologically active carbonate apatite layer on the surface after immersion in SBF was demonstrated using scanning electron microscope, energy dispersive X-ray, and Fourier transform infrared spectroscopy. Fast nucleation and growth of the carbonate apatite crystals were observed to occur all through the specimen surfaces. The phenomenon was explained in terms of the distribution and dispersion of inorganic phases in the scaffolds and the ionic activity products of the apatite in the SBF. The calculation results of weight loss and Ca/P molar ratio also suggest the good bioactivity and degradability of the scaffolds. These indicate that the ß-TCP porous ceramic scaffold is a potential candidate scaffold for bone tissue engineering.

Inhibiting thioredoxin glutathione reductase is a promising approach to controlling Cryptocaryon irritans infection in fish.

Marine cultured fish often suffer from Cryptocaryon irritans infection, which causes enormous mortality. C. irritans is resistant to oxidative damage induced by zinc. To develop an effective drug to control the parasite, a putative thioredoxin glutathione reductase (CiTGR) from C. irritans was cloned and characterized. CiTGR was designed as a target to screen for inhibitors by molecular docking. The selected inhibitors were tested both in vitro and in vivo. The results showed that CiTGR is located in the nucleus of the parasite, possesses a common pyridine-oxidoreductases redox active center, and lacks a glutaredoxin active site. Recombinant CiTGR exhibited high TrxR activity but low glutathione reductase activity. Shogaol was found to significantly suppress TrxR activity and enhance toxicity of zinc on C. irritans (P < 0.05). The abundance of C. irritans on the fish body decreased significantly after oral administration of shogaol (P < 0.05). These results implied that CiTGR could be used to screen for drugs that weaken the resistance of C. irritans to oxidative stress, which is critical for controlling the parasite in fish. This paper deepens the understanding of the interaction between ciliated parasites and oxidative stress.

Mechanical properties' improvement of a tricalcium phosphate scaffold with poly-l-lactic acid in selective laser sintering.

To improve the mechanical properties of a scaffold fabricated via selective laser sintering (SLS), a small amount (0.5-3 wt%) of poly-l-lactic acid (PLLA) is added to the ß-tricalcium phosphate (ß-TCP) powder. The fracture toughness of the scaffold prepared with the mixture powder containing 1 wt% PLLA increases by 18.18% and the compressive strength increases by 4.45% compared to the scaffold prepared from the ß-TCP powder. The strengthening and toughening is related to the enhancement of ß-TCP sintering characteristics via introducing a transient liquid phase in SLS. Moreover, the microcracks caused by the volume expansion due to the ß-α phase transformation of TCP are reduced because of the PLLA inhibition function on the phase transformation. However, PLLA additive above 1 wt% would lead to a PLLA residue which will decrease the mechanical properties. The experimental results show that PLLA is an effective sintering aid to improve the mechanical properties of a TCP scaffold.