Potential virulence factors of Nocardia seriolae AHLQ20-01 based on whole-genome analysis and its pathogenicity to largemouth bass (Micropterus salmoides).

Nocardia seriolae is a major causative agent of fish nocardiosis that results in serious economic losses in the aquaculture industry. However, the virulence factors and pathogenic mechanisms of the bacterium are poorly understood. Here, a new N. seriolae strain AHLQ20-01 was isolated from the diseased Micropterus salmoides and identified by phenotypic examination combined with 16S rRNA sequencing. Subsequently, the potential virulence factors of the strain were analysed at genome level by whole-genome sequencing. The results showed that the whole-genome sequence derived from N. seriolae AHLQ20-01 circular chromosome contains 8,129,380 bp DNA with G + C content of 68.14%, and encompasses 7650 protein-coding genes, 114 pseudo-genes, 3 rRNAs, 66 tRNAs and 36 non-coding RNAs. More importantly, a total of 139 genes, which mainly involved in adhesion, invasion, resistance to oxidative and nitrosative stress, phagosome arresting, iron acquisition system, toxin production and bacterial secretion systems, were identified as core virulence-associated genes. Furthermore, the pathogenicity of N. seriolae AHLQ20-01 to M. salmoides was further investigated through experimental infection. It was found that the LD50 value of the strain to M. salmoides was 9.3 × 106  colony forming unit/fish. Histopathological examination demonstrated typical granuloma with varying sizes in the liver, head kidney, spleen and heart of the experimentally infected fish. Terminal deoxynucleotidyl transferase dUTP nick end labelling assay and 4',6-diamidino-2-phenylindole staining showed that there were distinctly more apoptotic cells in all the tested tissues in the infection group, but not in the control group. Together, these findings provide the foundation to further explore the pathogenic mechanism of N. seriolae, which might contribute to the prevention and treatment of fish nocardiosis.

First identification of Nocardia seriolae GapA adhesion function and its three B-cell epitopes with cell-binding activity.

Fish nocardiosis mainly caused by Nocardia seriolae (N. seriolae) is a serious threat to aquaculture. Bacterial adhesion to host cells mediated by adhesin is an initial step of pathogenesis. But it is not clear whether glyceraldehyde-3-phosphate dehydrogenase (GapA) is an adhesin of N. seriolae. Here, recombinant GapA protein (rGapA) was prokaryotic expressed, and its role in the bacterial adhesion to Ctenopharyngodon idella kidney cells was investigated by indirect immunofluorescence, protein-binding assay and adhesion inhibition assay. The results showed that an obvious green fluorescence was observed on the surface of the cells co-incubated with rGapA protein; the cytomembrane proteins of the cells pretreated with rGapA could react with anti-rGapA antibody; and the antibody significantly inhibited the adhesion ability of the bacteria. Subsequently, B-cell linear epitopes of GapA protein were identified by using a immunoinformatics approach combined with peptide ELISA and Western blot for the first time. It was found that four predicted epitopes (Ep58-69 , Ep139-150 , Ep186-197 , Ep318-329 ) could all react with anti-rGapA antibody and obviously inhibit the immunoreactivity between rGapA and anti-rGapA antibody, and they were confirmed as indeed B-cell linear epitopes of the protein. Furthermore, flow cytometry analysis found the percentage of positive cells co-incubated with FITC-labelled epitope peptides (Ep139-150 , Ep186-197 , Ep318-329 ) was significantly higher than those in the FITC-labelled Ep58-69 , unrelated control peptide and cell control. Collectively, GapA is an adhesin of N. seriolae, and epitope peptides (Ep139-150 , Ep186-197 , Ep318-329 ) possess cell-binding activity, which are potential candidates for developing a multiple epitopes-based adhesin vaccine against fish nocardiosis.

Mammalian sterile 20-like kinase 1/2 inhibits the Wnt/ß-catenin signalling pathway by directly binding casein kinase 1ε.

The Hippo signalling pathway can suppress the Wnt/ß-catenin signalling pathway through the last downstream effectors YAP (Yes-associated protein)/TAZ (tafazzin). MST (mammalian sterile 20-like kinase) 1 functions as the upstream kinase of the Hippo pathway, and CK1ε (casein kinase 1ε) plays roles in the up-stream signal transduction of the Wnt/ß-catenin pathway. In the present study, using tandem affinity purification and MS analysis, CK1ε was identified as a novel partner of MST1. Further analysis showed that the interaction between MST1 and CK1ε was mediated by their kinase domains and enhanced by the activation of MST1. To exclude the interference of the phosphorylated YAP/TAZ, the transduction from MST1 to YAP/TAZ was blocked using anti-WW45 shRNA. In the sh-WW45 cells, MST1 still inhibited the Wnt3A-induced phosphorylation of DVL2 (dishevelled 2) and Wnt/ß-catenin signalling by disturbing the interaction of DVL2 and CK1ε. The growth-suppressive effect of MST1 in the presence of Wnt3A was effectively relieved by the downstream activation of the Wnt/ß-catenin pathway. Moreover, MST2, the close homologue of MST1, also displayed the similar function in suppressing the Wnt/ß-catenin pathway. Therefore the results of the present study revealed that, in addition to the phosphorylated YAP/TAZ, the Hippo pathway can suppress the Wnt/ß-catenin pathway directly through MST1/2.

MicroRNA deregulation in right ventricular outflow tract myocardium in nonsyndromic tetralogy of fallot.

BACKGROUND: Tetralogy of Fallot (TOF) is 1 of the most common heart defects in children, and the underlying mechanisms remain largely elusive. MicroRNAs (miRNAs) are a class of regulators of gene expression and are increasingly recognized for their roles in heart development. METHODS: To identify miRNAs abnormally expressed in TOF, microarrays were used to analyze the miRNA expression profiles of 5 samples of myectomy tissues from right ventricular outflow tract (RVOT) obstruction of infants with nonsyndromic TOF and 3 age-matched normal RVOT tissues. RESULTS: In total, 41 candidate miRNAs were identified. To further validate the microarray results, the 41 miRNAs were detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in a larger independent population of tissue samples, including 21 from patients with TOF and 6 from normal controls; it was found that 18 miRNAs were expressed at significantly different levels. Bioinformatic analysis revealed that these miRNAs targeted a network of genes involved in heart development and human congenital heart diseases. Further in vitro studies indicated that upregulation of miR-424/424* promoted proliferation and inhibited migration of primary embryonic mouse cardiomyocytes, whereas miR-222 promoted cardiomyocyte proliferation and reduced the cardiomyogenic differentiation of P19 cells. The 3'UTR (3' untranslated region) luciferase assay revealed that miR-424/424* suppressed the expression of HAS2 and NF1, and their mRNAs were underexpressed in the RVOT myocardial tissues of TOF. CONCLUSIONS: Eighteen miRNAs were identified as being deregulated in RVOT myocardial tissues from infants with nonsyndromic TOF, and in vitro experiments indicated that miR-424/424* and miR-222 are involved in cardiomyocyte proliferation and migration and the cardiomyogenic differentiation of P19 cells.