Identification and functional characterization of Histone‐like DNA‐binding protein in Nocardia seriolae (NsHLP) involved in cell apoptosis

Nocardia seriolae, a facultative intracellular bacterium, is the main pathogen of fish nocardiosis. Bioinformatic analysis showed that the histone‐like DNA‐binding protein (HLP) gene of N. seriolae (nshlp) encoded a secreted protein and might target the mitochondria in the host cell. To further study the preliminary function of HLP in N. seriolae (NsHLP), the gene cloning, extracellular products identification, subcellular localization, overexpression and apoptosis detection assay were carried out in this study. Mass spectrometry analysis of the extracellular products from N. seriolae showed that NsHLP was a secreted protein. Subcellular localization of HLP‐GFP fusion proteins mainly assembled in the nucleus, which indicated that the NsHLP was co‐located with the nucleus rather than mitochondria in fathead minnow (FHM) cells. Notably, the expression of NsHLP had changed the distribution of mitochondria into lumps in the FHM cell. In addition, apoptotic features were found in the transfected FHM cells by overexpression of NsHLP. Quantitative assays of mitochondrial membrane potential value, caspase‐3 activity and pro‐apoptotic genes mRNA (Bad, Bid and Bax) expression level demonstrated that the cell apoptosis was induced in the transfected FHM cells. All the results presented in this study provided insight on the function of NsHLP, which suggested that it may participate in the cell apoptosis regulation and play an important role in the pathogenesis of N. seriolae.     

一种(鱼师)鱼诺卡氏菌酪氨酸蛋白磷酸酶基因的克隆及功能初步研究

這鱼诺卡氏菌是鱼类诺卡氏菌病的主要病原,可导致鱼类慢性系统性肉芽肿疾病.這鱼诺卡氏菌全基因组序列分析发现了一个酪氨酸蛋白磷酸酶(protein tyrosine phosphtase,PTP)基因,生物信息学分析显示该基因很可能编码一个靶向定位于宿主细胞线粒体的分泌蛋白.本实验对這鱼诺卡氏菌PTP进行了基因克隆、分泌蛋白鉴定、亚细胞定位、过表达和线粒体膜电位检测,结果显示,在這鱼诺卡氏菌胞外产物中质谱鉴定到了PTP肽段,证实其为分泌蛋白.亚细胞定位研究观察到PTP-GFP融合蛋白均匀地分布在FHM细胞中,与线粒体分布不重合,说明這鱼诺卡氏菌PTP蛋白并未靶向定位于线粒体.亚细胞定位和过表达研究都显示PTP蛋白在FHM细胞中表达后,细胞核出现固缩浓染、凋亡小体等明显的细胞凋亡特征.通过线粒体膜电位检测表明,在pcDNA-PTP转染后48 h,线粒体跨膜电位被明显破坏,说明這鱼诺卡氏菌PTP很可能是一种可诱导细胞凋亡的细菌蛋白.通过对這鱼诺卡氏菌PTP开展基因克隆和功能初步研究,为进一步揭示该基因的功能和深入了解這鱼诺卡氏菌的分子致病机理奠定了基础.     

Identification of a cell-wall peptidase (NlpC/P60) from Nocardia seriolae which induces apoptosis in fathead minnow cells

Nocardia seriolae, a Gram-positive bacterium, is the main pathogen of fish nocardiosis. Protein NlpC/P60 is a cell-wall peptidase and a potential virulence factor of N. seriolae. Subcellular localization research revealed that both NlpC/P60-GFP and NlpC/P60Δsig-GFP fusion proteins were evenly distributed in the whole cell of fathead minnow (FHM) cells. Furthermore, typical apoptotic features, such as nuclear pyrosis and apoptotic bodies, were observed in the transfected FHM cells and grouper spleen cells by the overexpression of protein NlpC/P60. Then, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related gene (Bax, BNIP3, TNF1 and TNF6) mRNA expression were conducted. The results showed that ΔΨm was decreased, caspase-3 was significantly activated, and the mRNA expression of pro-apoptotic genes (Bax and BNIP3) and tumour necrosis factors (TNF1 and TNF6) was up-regulated in NlpC/P60-overexpressed cells. Taken together, the results indicated that the protein NlpC/P60 of N. seriolae might involve in apoptosis regulation. This study may lay the foundation for further study on the function of N. seriolae NlpC/P60 and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae.     

Identification of a secreted superoxide dismutase (SOD) from Nocardia seriolae which induces apoptosis in fathead minnow (FHM) cells

Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen. The pathogenesis and virulence factors of N. seriolae are not fully understood. Secreted superoxide dismutase (SOD) may be a virulence factor found by a comparative bioinformatics analysis of the whole genome sequence of N. seriolae and the virulence factor database (VFDB). In order to determine the subcellular localization and study the preliminary function of SOD from N. seriolae (NsSOD), gene cloning, secreted protein identification, subcellular localization in fish cells, and apoptosis detection of NsSOD were carried out in this study. Subcellular localization research revealed that NsSOD‐GFP fusion proteins were evenly distributed in the cytoplasm. Furthermore, apoptotic bodies were observed in the transfected FHM cells by the overexpression of protein NsSOD. Then, assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related genes (Bax, Bid, Bad and Bcl‐2) mRNA expression were conducted. The results showed that ΔΨm was decreased, and caspase‐3 was significantly activated. The mRNA expression of the Bad gene showed significant up‐regulated expression at 24 h.p.t., while Bid and Bax genes showed significant up‐regulated expression at 72 and 96 h.p.t. and anti‐apoptotic gene (Bcl‐2) was down‐regulated in NsSOD overexpressed cells. Taken together, the results indicated that the protein NsSOD might be involved in apoptosis regulation. This study may lay the foundations for further studies on the function of NsSOD and promote the understanding of the virulence factors and the pathogenic mechanisms of N. seriolae.     

Immune response of largemouth bass, <Emphasis Type="Italic">Micropterus salmoides</Emphasis>, to whole cells of different <Emphasis Type="Italic">Nocardia seriolae</Emphasis> strains

Largemouth bass Micropterus salmoides were immunized with four different N. seriolae strains—two α-glucosidase-positive (961113, KU040801) and two α-glucosidase-negative (94260, OTTS) strains—along with Freund’s incomplete adjuvant. After primary immunization (week 0), a booster was administered at weeks 4 and 8. Nonspecific immune responses to multiple immunizations with the different N. seriolae strains were determined based on serum lysozyme activity and nitroblue tetrazolium (NBT)-positive cells in peripheral blood. The serum lysozyme activity and NBT-positive cells in peripheral blood were not significantly increased even after the two booster immunizations. Specific antibody responses against N. seriolae cells were investigated by enzyme-linked immunosorbent assay. At 4 weeks after immunization, all groups immunized with N. seriolae antigens showed significant increases in their specific antibody levels. The sera from fish immunized with different N. seriolae strains exhibited reactivity with N. seriolae sonicated antigens of 28, 30, 36 and 84 kDa by western blot analysis. After two boosters, fish were challenged with live N. seriolae to assess the vaccine’s efficacy; however, multiple injections of the N. seriolae strains did not reduce mortality, irrespective of the bacterin.     

Live vaccine trials against nocardiosis in yellowtail Seriola quinqueradiata

In an attempt to develop a vaccine against Nocardia seriolae, related species of live bacteria N. soli, N. fluminea, and N. uniformis were injected into yellowtail Seriola quinqueradiata. In addition, fish were challenged with a low virulence strain of N. seriolae to model the concept of use of a live vaccine. The fish injected with live N. soli and N. fluminea cells showed slight resistance against an artificial challenge with N. seriolae. On the other hand, the fish that survived the N. seriolae infection showed complete resistance to the N. seriolae challenge. These results suggest that protective immune responses against N. seriolae are induced in yellowtails.     

Dietary soybean β‐conglycinin suppresses growth performance and inconsistently triggers apoptosis in the intestine of juvenile grass carp (Ctenopharyngodon idella) in association with ROS‐mediated MAPK signalling

The current study aimed to investigate the effects of dietary soybean β‐conglycinin on growth performance and intestine apoptosis in juvenile grass carp (Ctenopharyngodon idella). For fish fed with the 80 g β‐conglycinin/kg diet for 7 weeks, the specific growth rate and feed intake were decreased. In the proximal intestine, dietary β‐conglycinin did not induce DNA fragmentation, tended to decrease the reactive oxygen species (ROS) content, and decreased ROS‐generating enzyme (NADPH oxidase [NOX]) activity. Subsequently, in the mid‐intestine, dietary β‐conglycinin caused DNA fragmentation, tended to increase the ROS content, increased caspase‐3, caspase‐8 and caspase‐9 activities, upregulated the mRNA levels of proapoptotic molecules (apoptotic protease‐activating factor‐1 [Apaf1] and Bcl‐2‐associated X protein [BAX]) and mitogen‐activated protein kinase (MAPK)‐related signal molecules (Jun N‐terminal kinase (JNK) and p38 MAPK) and increased the protein levels of p38 MAPK and phospho‐p38 MAPK. Moreover, in the distal intestine, dietary β‐conglycinin induced DNA fragmentation, elevated NOX activity and the ROS content and increased caspase‐3, caspase‐8 and caspase‐9 activities, death ligand (TNF‐α) mRNA expression level, and p38 MAPK and phospho‐p38 MAPK protein levels. In summary, dietary soybean β‐conglycinin suppressed fish growth and inconsistently caused apoptosis among the different intestinal segments which was partially associated with ROS‐mediated MAPK signalling.     

Sequencing of 16S−23S rRNA internal transcribed spacer and its application in the identification of Nocardia seriolae by polymerase chain reaction

A method for the diagnosis of nocardiosis in yellowtail (Seriola quinqueradiata), using polymerase chain reaction (PCR), was developed in this study. Primers specific for Nocardia seriolae were synthesized based on the alignment of 16S?23S rRNA internal transcribed spacer region sequences of N. seriolae. The primers did not amplify specific PCR product from other fish pathogens. However, two and three fishes could be diagnosed as infected with N. seriolae by clinical signs and bacterial isolation. PCR amplification of N. seriolae by specific primers detected six infected fishes. Thus, the primers used in this study are useful in detecting nocardiosis in fish.     

Current knowledge of nocardiosis in teleost fish

Nocardia sp. is the causative agent of nocardiosis, a lethal granulomatous disease of the skin, muscle, and various inner tissues affecting various teleost and shellfish. Four species of Nocardia have been isolated from diseased fish and shellfish, namely Nocardia asteroides, Nocardia seriolae, Nocardia salmonicida and Nocardia crassostreae. Therefore, in fish aquaculture, nocardiosis has caused severe economic losses, especially in the Asian region. Considerable research has been performed, since the first report of identified Nocardia sp. in fish, to characterize Nocardia sp. and identify rapid detection techniques, immune response against infection and prophylactic approaches. In this review, the current state of knowledge about nocardiosis in fish has been presented, including the pathogenesis, diagnosis, host immune response and vaccine development.     

Modified resazurin microtiter assay for in vitro and in vivo assessment of sulfamonomethoxine activity against the fish pathogen <Emphasis Type="Italic">Nocardia seriolae</Emphasis>

Resazurin microtiter assay (REMA) was carried out using four sulfonamides, three culture media, and four inoculum sizes as a first screening step to establish an easy-to-interpret sulfonamides susceptibility testing method for Nocardia seriolae. The in vitro activity of sulfamonomethoxine (SMM) against 190 clinical N. seriolae isolates was then examined, and in vivo experimental treatment was performed. When the culture medium and the inoculum size were considered in tandem, a 0.5× the original concentration of cation-adjusted Mueller–Hinton broth and an inoculum size of 10² CFU/well showed the clearest endpoint reading for all tested drugs, and the REMA-generated data were in excellent agreement with those generated by the reference Etest method. SMM activity showed minimum inhibitory concentration (MIC) values of 4–32 μg/ml against all tested N. seriolae isolates. Treatment of amberjack groups experimentally infected with N. seriolae isolates having SMM MICs of 4 and 32 μg/ml, resulted in survival rates of 100% and 87.5% in the two groups, respectively. In this study, we developed a simple visual method to test SMM activity against N. seriolae.