Haemophilus parasuis infection in 3D4/21 cells induces autophagy through the AMPK pathway

Haemophilus parasuis (H. parasuis) is a common commensal in the upper respiratory tract of pigs, but causes Glässer's disease in stress conditions. To date, many studies focused on the immune evasion and virulence of H. parasuis; very few have focused on the role autophagy played in H. parasuis infection, particularly in porcine alveolar macrophages (PAMs). In this study, a PAM cell line, 3D4/21 cells were used to study the role of autophagy in H. parasuis infection. 3D4/21 cells tandemly expressing GFP, mCherry, and LC3 were infected with H. parasuis serovar 5 (Hps5). Western blot analysis and confocal and transmission electron microscopy showed that H. parasuis infection effectively induces autophagy. Using Hps strains of varying virulence (Hps4, Hps5, and Hps7) and UV‐inactivated Hps5, we demonstrated that autophagy is associated with the internalisation of living virulent strains into cells. In 3D4/21 cells pretreated with rapamycin and 3‐MA then infected by Hps4, Hps5, and Hps7, we demonstrated that autophagy affects invasion of H. parasuis in cells. AMPK signal results showed that Hps5 infection can upregulate the phosphorylation level of AMPK, which is consistent with the autophagy development. 3D4/21 cells pretreated with AICAR or Compound C then infected by Hps5 revealed that the autophagy induced by Hps5 infection is associated with the AMPK pathway. Our study contributes to the theoretical basis for the study of H. parasuis pathogenesis and development of novel drugs target for prevention Glässer's disease.     

The Verticillium dahliae Sho1-MAPK pathway regulates melanin biosynthesis and is required for cotton infection

Verticillium dahliae is a soil-borne fungus that causes vascular wilt on numerous plants worldwide. The fungus survives in the soil for up to 14 years by producing melanized microsclerotia. The protective function of melanin in abiotic stresses is well documented. Here, we found that the V. dahliae tetraspan transmembrane protein VdSho1, a homolog of the Saccharomyces cerevisiae Sho1, acts as an osmosensor, and is required for plant penetration and melanin biosynthesis. The deletion mutant ΔSho1 was incubated on a cellophane membrane substrate that mimics the plant epidermis, revealing that the penetration of ΔSho1 strain was reduced compared to the wild-type strain. Furthermore, VdSho1 regulates melanin biosynthesis by a signalling mechanism requiring a kinase-kinase signalling module of Vst50-Vst11-Vst7. Strains, ΔVst50, ΔVst7 and ΔVst11 also displayed defective penetration and melanin production like the ΔSho1 strain. Defects in penetration and melanin production in ΔSho1 were restored by overexpression of Vst50, suggesting that Vst50 lies downstream of VdSho1 in the regulatory pathway governing penetration and melanin biosynthesis. Data analyses revealed that the transmembrane portion of VdSho1 was essential for both membrane penetration and melanin production. This study demonstrates that Vst50-Vst11-Vst7 module regulates VdSho1-mediated plant penetration and melanin production in V. dahliae, contributing to virulence.     

Spontaneous mutations in FgSAD1 suppress the growth defect of the Fgprp4 mutant by affecting tri-snRNP stability and its docking in Fusarium graminearum

FgPrp4, the only kinase in the spliceosome, is not essential for viability, but is important for splicing efficiency in Fusarium graminearum. The Fgprp4 deletion mutant had severe growth defects but often produced spontaneous suppressors with faster growth rate. To better understand the suppression mechanism, we identified and characterized spontaneous mutations in the tri-snRNP-specific protein, FgSad1, which suppressed the growth defects of Fgprp4. The L512P mutation was verified for its suppressive effects on Fgprp4, suggesting that mutations in FgSad1 may have effects involving FgPrp4 phosphorylation on FgSad1. Phosphoproteomics analysis showed that FgSad1 may not be the direct substrate of FgPrp4 kinase. Furthermore, truncation analysis showed that the N-terminal, extra RS-rich region of FgSad1 is critical for its function and is post-translationally modified. The P258S or S269P mutations in FgSad1 increased its interactions with the U5 protein FgPrp8 and the U4/U6 protein FgPrp31, which may result in tri-snRNP stabilization. Additionally, the D76N mutation increased the association of FgSad1 with the U2 snRNP. These data indicate that suppressor mutations in FgSad1 increase the stability of the tri-snRNP and/or the affinity of FgSad1 with U2 snRNP and therefore potentially facilitate the docking of tri-snRNP into the spliceosome.     

The characteristics of antibiotic resistance and phenotypes in 29 outer-membrane protein mutant strains in Aeromonas hydrophila

Although many typical outer-membrane proteins (OMPs) have been well characterized, the biological functions of many OMPs remain largely elusive. In this study, we successfully constructed 29 OMP knockout strains in the pathogen Aeromonas hydrophila, which account for about 50% of all predicted OMPs in this bacterial species. We then further validated the antibiotics' susceptibility characteristics against 20 antimicrobial reagents in these mutants considering several phenotypes. Our results showed that a total of 22 OMP mutants affected the susceptibility to at least one antibiotic. The deletion of some OMPs, such as ΔlamB and ΔbamA, revealed very important roles in the resistance to certain antibiotics. However, not a single OMP mutant presented a constant behaviour to all of the tested antibiotics, suggesting the existence of a complex intercellular regulation mechanism and a protein–protein interaction network underlying the OMP homeostasis in the presence of antibiotics. Meanwhile, some OMP mutants also affected biofilm formation, ECPase and haemolytic activity, and carbon resources utilization. This report demonstrates the biological functions of OMPs on a large scale and most of results have not been reported in A. hydrophila.     

Gut microbiota in patients after surgical treatment for colorectal cancer

Colorectal cancer (CRC) is a common disease worldwide that is strongly associated with the gut microbiota. However, little is known regarding the gut microbiota after surgical treatment. 16S rRNA gene sequencing was used to evaluate differences in gut microbiota among colorectal adenoma patients, CRC patients, CRC postoperative patients and healthy controls by comparing gut microbiota diversity, overall composition and taxonomic signature abundance. The gut microbiota of CRC patients, adenoma patients and healthy controls developed in accordance with the adenoma-carcinoma sequence, with impressive shifts in the gut microbiota before or during the development of CRC. The gut microbiota of postoperative patients and CRC patients differed significantly. Subdividing CRC postoperative patients according to the presence or absence of newly developed adenoma which based on the colonoscopy findings revealed that the gut microbiota of newly developed adenoma patients differed significantly from that of clean intestine patients and was more similar to the gut microbiota of carcinoma patients than to the gut microbiota of healthy controls. The alterations of the gut microbiota between the two groups of postoperative patients corresponded to CRC prognosis. More importantly, we used the different gut microbiota as biomarkers to distinguish postoperative patients with or without newly developed adenoma, achieving an AUC value of 0.72. These insights on the changes in the gut microbiota of CRC patients after surgical treatment may allow the use of the microbiota as non-invasive biomarkers for the diagnosis of newly developed adenomas and to help prevent cancer recurrence in postoperative patients.     

Diversity of the Fusarium pathogens associated with crown rot in the Huanghuai wheat-growing region of China

To investigate the distribution and diversity of the pathogens associated with Fusarium crown rot in the Huanghuai wheat-growing region (HHWGR) of China, we collected wheat samples with symptomatic stem bases from seven provinces in the HHWGR between 2013 and 2016. A total of 1196 isolates obtained from 222 locations were identified as 9 Fusarium species based on morphological and molecular identification. Of these pathogen species, F. pseudograminearum was the dominant species. Furthermore, F. sinensis was isolated from the disease specimens and tested for virulence to wheat. The result of the pathogenicity revealed that an intraspecific differentiation existed in F. pseudograminearum; sequence analysis of the EF-1α gene showed that 194 F. pseudograminearum isolates were differentiated into two distinct clades which closed to the strains from Australia and China respectively, but neither pathogenicity nor EF-1α sequence was related to the geographic origins of these isolates. However, universal rice primers-polymerase chain reaction showed a correlation with the geographical origins of the 194 isolates, which were divided into eight subclusters, the level of genetic diversity was higher within a geographical population than among the different populations. The results of these analyses can be directly used to facilitate disease monitoring and development of control strategies.