Identification and application of an endophytic fungus Arcopilus aureus from Panax notoginseng against crop fungal disease.

Endophytic fungi are important microbial resources for developing novel antibacterial and antifungal drugs to prevent and control crop diseases. Panax notoginseng has been used as a Chinese medicinal herb for a long time, as it has various bioactivities. However, information on endophytic fungi isolated from Panax notoginseng is rare. In this study, an endophytic fungus known as SQGX-6, which was later identified as the golden hair fungus Arcopilus aureus, was isolated from Panax notoginseng. SQGX-6 was extracted using ethyl acetate, and the active components of the fungus were identified using ultra-performance liquid chromatography-mass spectrometry (UHPLC-MS). The antifungal and antioxidant activities of the extract were determined and evaluated in vitro and in vivo. SQGX-6 and its extract inhibited the growth of Corn stalk rot (Fusarium graminearum), Corn southern leaf blight (Helminthosporium maydis), and Tomato gray mold (Botrytis cinerea) in vitro. The free radical scavenging rates for 2,2-Diphenyl-1-pyridinyl hydrazide (DPPH) radical scavenging activity, 3-Ethylbenzothiazoline-6-Sulfonic Acid Radical scavenging (ABTS) activity were also downregulated by the SQGX-6 extract. In vivo, the SQGX-6 extract inhibited the mycelial growth rates of the three aforementioned fungi and downregulated malondialdehyde (MDA) content and upregulated peroxidase (POD) and phenylalanine ammonia-lyase (PAL) content in fruits, leading to significant reduction in damage to cherry tomatoes caused by Botrytis cinerea. UHPLC-MS was performed to identify various active substances, including Alkaloids, Azoles, Benzofurans, Coumarins, Flavonoids, Organic acids, Phenols, and plant growth regulators contained in the extract. These results suggested that the endophytic fungus SQGX-6 of Panax notoginseng and its extract have excellent antifungal and antioxidant activities, and thus, it is an important microbial resource for the developing novel drugs against plant fungal infections.

The two-component system CpxA/CpxR is critical for full virulence in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, a major bacterial porcine respiratory tract pathogen causing pig pleuropneumonia, has resulted in high economic losses worldwide. The mutation of the two-component system CpxAR strongly impacted the virulence of A. pleuropneumoniae, but the underlying regulatory mechanism remained unclear. Here, we found that CpxAR positively regulated the cpxDCBA gene cluster involved in polysaccharide capsule export. A capsular layer was confirmed in wild-type cells by transmission electron microscopy, whereas cpxAR and cpxD mutants were non-capsulated. The mutants for polysaccharide capsule export gene cpxD exhibited non-capsulated and were strongly impaired in virulence for mice, indicating a major role of CPS export system in virulence. We then demonstrated that CpxR directly regulated the transcription of the CPS export gene cluster cpxDCBA. Taken together, our data suggested that CpxAR is a key modulator of capsule export that facilitates A. pleuropneumoniae survival in the host.

Molecular Characterization of Outer Capsid Proteins VP5 and VP7 of Grass Carp Reovirus.

Aquareovirus, which is a member of the Reoviridae family, was isolated from aquatic animals. A close molecular evolutionary relationship between aquareoviruses and mammalian orthoreoviruses was revealed. However, the functions of the aquareovirus genome-encoded proteins are poorly understood. We investigated the molecular characteristics of the outer capsid proteins, namely, VP5 and VP7, of grass carp reovirus (GCRV). The peptides VP5 and VP7 were determined using in-gel tryptic digestion and mass spectrometry. Recovered peptides represented 76% and 66% of the full-length VP5 and VP7 sequences, respectively. Significantly, two-lysine acetylation, as well as two-serine and two-threonine phosphorylation modifications, were first revealed in VP5. We found that the initial amino acid in VP5 was Pro43, suggesting that a lower amount of VP5 remained uncleaved in virions at the autocleavage site (Asn42-Pro43). Further biochemical evidence showed that the cleaved VP5N/VP5C conformation was the major constituent of the particles. Moreover, early cleavage fragments of VP7 and enhanced infectivity were detected after limited tryptic digestion of GCRV, indicating that stepwise VP7 cleavage is essential for VP5 conformational rearrangement. Our results provide insights into the roles of posttranslational modifications in VP5 and its association with VP7 in the viral life cycle.

The CpxA/CpxR two-component system mediates regulation of Actinobacillus pleuropneumoniae cold growth.

Introduction: To survive in various hostile environments, two-component system is an adaptive mechanism for diverse bacteria. Activity of the CpxA/CpxR two-component system contributes to coping with different stimuli, such as pH, osmotic and heat stress. Methods: However, the role of the CpxA/CpxR system in cold resistance is little-known. In this study, we showed that CpxA/CpxRwas critical for A. pleuropneumoniae growth under cold stress. Results: ß-Galactosidaseanalysis showed that CpxA/CpxR positively regulated the predicted cold stress gene cspC. The mutant for cold stress gene cspC was impaired in the optimal growth of A. pleuropneumoniae under cold stress. Furthermore, electrophoretic mobility shift assays demonstrated that CpxR-P could directly regulate the transcription of the cold stress gene cspC. Discussion: These results presented in this study illustrated that the CpxA/CpxR system plays an important role in cold resistance by upregulating expression of CspC. The data give new insights into how A. pleuropneumoniae survives in cold stress.

Transcriptomic analysis of porcine PBMCs in response to Actinobacillus pleuropneumoniae reveals the dynamic changes of differentially expressed genes related to immuno-inflammatory responses.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, for which the mortality rate is high. Host peripheral blood is a body site for the immune clearance of pathogens mediated by release of inflammatory factors. However, "out of control" inflammatory factor release can contribute to host death. To further understand the changes in the transcription level of immune-related effectors, samples of peripheral blood mononuclear cells (PBMCs) collected from piglets at different stages of infection (0, 24 and 120 h) were sequenced on an Illumina HiSeq™ 4000 platform. We found 3818 differentially expressed genes (DEGs) in the 24 h-infection group compared to the 0 h-infection group (Pb24-Vs-Pb0). DEGs mainly involved in the Gene ontology and KEGG pathways that included nucleic acid metabolism regulation, cell growth, cell differentiation, and organ morphological maintenance were not significantly enriched (P > 0.05). However, DEGs associated with protein kinase activity, receptor activation, metabolism, local adhesion and immune inflammatory responses were significantly enriched in Pb120-Vs-Pb24 (P < 0.05), as were those related to the T cell receptor signalling pathway, with most being down-regulated compared to the preceding stage (Pb24-Vs-Pb0). In PBMCs there were some changes in glucose metabolism, local adhesion and the immune inflammatory response (Pb120-Vs-Pb0). In addition, up-regulated DEGs, such as IL8, IL1ß, and CCL2, and were significantly enriched in immune-inflammatory related pathways compared to the uninfected stage, although they began to decline after 24 h.

A guard-killer phage cocktail effectively lyses the host and inhibits the development of phage-resistant strains of Escherichia coli.

In recent years, after the emergence of a large number of multidrug-resistant bacteria, phages and phage-associated products for the prevention and control of bacterial disease have revealed prominent advantages as compared with antibiotics. However, bacteria are susceptible to becoming phage-resistant, thus severely limiting the application of phage therapy. In this study, Escherichia coli cells were incubated with lytic bacteriophages to obtain mutants that were resistant to the lytic phages. Then, bacteriophages against the phage-resistant variants were isolated and subsequently mixed with the original lytic phage to prepare a novel phage cocktail for bactericidal use. The data showed that our phage cocktail not only had notable bactericidal effects, including a widened host range and rapid lysis, but also decreased the generation and mutation frequency of phage-resistant strains in vitro. In addition, we tested our cocktail in a murine bacteremia model. The results suggested that compared with the single phage, fewer phage-resistant bacteria appeared during the treatment of phage cocktail, thus prolonging the usable time of the phage cocktail and improving its therapeutic effect in phage applications. Importantly, our preparation method of phage cocktail was proved to be generalizable. Because the bacteriophage against the phage-resistant strain is an ideal guard that promptly attacks potential phage resistance, this guard-killer dual-function phage cocktail provides a novel strategy for phage therapy that allows the natural ecology to be sustained.

Apa2H1, the first head domain of Apa2 trimeric autotransporter adhesin, activates mouse bone marrow-derived dendritic cells and immunization with Apa2H1 protects against Actinobacillus pleuropneumoniae infection.

Actinobacillus pleuropneumoniae is the causative pathogen of porcine pleuropneumonia, which results in large economic losses in the pig industry worldwide. There are, however, no effective subunit vaccines are available in the market owing to the various serotypes and the absence of cross-protection against this pathogen. Therefore, the selection of protective components is of great significance for vaccine development. We previously showed that trimeric autotransporter adhesins are important virulence factors of A. pleuropneumoniae. To determine the potential role in vaccine development of the functional head domain (Apa2H1) of Apa2, a trimeric autotransporter adhesin found in A. pleuropneumoniae, we obtained nature-like trimeric Apa2H1 using a prokaryotic expression system and co-culture of Apa2H1 with bone marrow derived dendritic cells (BMDCs) in vitro resulted in maturation of BMDCs, characterised by the up-regulation of CD83, MHC-II, CCR7, ICAM-I and the increased expression of factors related to B lymphoid cells stimulation, such as proliferation-inducing ligand (APRIL), B lymphocyte stimulator (BLyS) and B cell activating factor (BAFF). The in vivo results showed that vaccination with Apa2H1 resulted in the robust production of antigen-specific antibodies, modestly induced mixed Th1 and Th2 immunity, impaired bacterial colonization and dissemination, and improved mouse survival rates. This study is the first to show that Apa2H1 is antigenic and can be used as a component of a subunit vaccine against A. pleuropneumoniae infection, providing valuable reference material for the development of an effective vaccine against A. pleuropneumoniae.