Search

Vibrio alginolyticus is a common opportunistic pathogen of fish, shrimp, and shellfish, and many diseases it causes can result in severe economic losses in the aquaculture industry. Causing host disease was confirmed by several virulence factors of V. alginolyticus. To date, there have been no reports on the effect of the pstS gene on its virulence regulation of V. alginolyticus. The virulence mechanism of target genes regulating V. alginolyticus is worthy of further study. Previous studies found that Fructus schisandrae (30 mg/mL) inhibited the growth of V. alginolyticus ND-01 (OD600 = 0.5) for 4 h, while the expressions of pstS and pstB were significantly affected by F. schisandrae stress. So, we speculated that pstS and pstB might be the virulence genes of V. alginolyticus, which were stably silenced by RNAi to construct the silencing strains pstS-RNAi and pstB-RNAi, respectively. After the expression of pstS or pstB gene was inhibited, the adhesion capacity and biofilm formation of V. alginolyticus were significantly down-regulated. The chemotaxis and biofilm formation ability of pstS-RNAi was reduced by 33.33% and 68.13% compared with the wild-type strain, respectively. Sequence alignment and homology analysis showed that pstS was highly conserved, which suggested that pstS played a vital role in the secretion system of V. alginolyticus. The pstS-RNAi with the highest silencing efficiency was selected for transcriptome sequencing. The Differentially Expressed Genes (DEGs) and GO terms were mapped to the reference genome of V. alginolyticus, including 1055 up-regulated genes and 1134 down-regulated genes. The functions of the DEGs were analyzed by GO and categorized into different enriched functional groups, such as ribosome synthesis, organelles, biosynthesis, pathogenesis, and secretion. These DEGs were then mapped to the reference KEGG pathways of V. alginolyticus and enriched in commonalities in the metabolic, ribosomal, and bacterial secretion pathways. Therefore, pstS and pstB could regulate the bacterial virulence of V. alginolyticus by affecting its adhesion, biofilm formation ability, and motility. Understanding the relationship between the expressions of pstS and pstB with bacterial virulence could provide new perspectives to prevent bacterial diseases.

Bacterial pathogen-host interactions are highly dynamic, regulated processes that have been primarily investigated using in vitro assays. The dynamics of bacterial pathogen-host interplay in vivo are poorly understood. Using time-resolved dual RNA-seq in a Pseudomonas plecoglossicida-Epinephelus coioides infection model, we observed that bacterial genes encoding classical virulence factors and host genes involved in immune regulation were dynamically expressed during infection. Using network inferencing, we were able to predict interspecies regulatory networks linking bacterial virulence genes to host immune genes. Together with gene co-expression network analysis of the pathogen, secY was predicted to be a key virulence gene for P. plecoglossicida pathogenicity in the host, fliN was predicted to be a less important virulence gene. The results of bioinformatics prediction were confirmed by animal infection experiments. Our work provides the first paradigm to study dynamic alterations of bacterial pathogen and host interactions based on the elucidation of time-resolved interactive transcriptomes in vivo, and may be developed into a novel and universal method for revealing the true complexity of the bacterial infection process.

Pseudomonas plecoglossicida is a Gram-negative aerobic bacterium that causes high mortality and serious economic losses in some commercial marine fish. Expression of secY was found to be significantly upregulated at 18 °C compared to 28 °C by RNA-seq and qRT-PCR. All five tested recombinant vectors (pCM130/tac + shRNA) significantly reduced secY mRNA levels in P. plecoglossicida. The recombinant vector encoding shRNA-1165 exhibited the best gene-silencing efficiency, 82.4% and was used to create an RNAi strain for further studies. Compared with the wildtype strain, infections of Larimichthys crocea with the RNAi strain resulted in a 2-day delay in onset time and a 35% reduction in mortality, as well as the alleviation of spleen symptoms. The spleens of L. crocea infected by the wild type or RNAi strain of P. plecoglossicida were subjected to dual RNA-seq at 2 dpi. Compared with the wildtype strain, infection of P. plecoglossicida with the RNAi strain resulted in significant changes in the transcriptomes of both host and pathogen. KEGG analysis showed that the complement and coagulation cascade and the Toll-like receptor signalling pathway were the most enriched host pathways. In the pathogen, genes of the “Sec secretion system” were significantly downregulated. This downregulation of “Sec secretion system” genes hindered the secretion of bacterial proteins and reduced the virulence of P. plecoglossicida. Thus, it was easier for L. crocea to clear the RNAi strain of P. plecoglossicida, and the immune response was similarly reduced. The results indicated that secY was a virulence gene of P. plecoglossicida and played roles in the host-pathogen interactions of L. crocea and P. plecoglossicida.

As an important pathogen in aquaculture, Pseudomonas plecoglossicida has caused heavy losses. The expression of an ABC transporter gene-L321_23611 of P. plecoglossicida at 18 °C was found significant higher than those at 28 °C by RNA-seq and qRT-PCR. RNAi significantly reduced the content of L321_23611 mRNA in P. plecoglossicida with a maximal decrease of 89.2%. Compared with the wild type strain, the infection of L321_23611-RNAi strain resulted in the reduction in mortality and the onset time delay of a kind of marine teleosts, Epinephelus coioides. The results of dual RNA-seq showed that the RNAi of L321_23611 resulted in a significant change in both pathogen and host transcriptome in the spleens of infected E. coioides. The result of GO and KEGG analysis from dual RNA-seq data showed both host genes of chemokine signaling pathway, coagulation and complement system, hematopoietic cell lineage pathway as well as hemoglobin complex GO term and pathogenic genes of bacterial-type flagellum-dependent cell mortality GO term and flagellar assembly, biosynthesis of amino acids and lysine biosynthesis systems pathways were mainly affected by L321_23611 gene of P. plecoglossicida. The results indicated that: 1. ABC transporter gene-L321_23611 was a virulent gene of P. plecoglossicida. 2. Both the activation of the host immune pathways and depression of pathogenic virulence-related pathways facilitated E. coioides to remove L321_23611-RNAi strain than the wild type strain of P. plecoglossicida.

Several bacteria have been defined as extracellular pathogens; however, in recent years, it has been confirmed that they have the ability to survive and escape the attack of host phagocytes, thus causing further infection. Previous studies have shown that Aeromonas hydrophila could survive in fish macrophages; however, the mechanism remains unknown. In this study, sodA and sodB of the strain A. hydrophila B11 were stable silenced by shRNA. The survival rates of intracellular sodA-RNAi and sodB-RNAi decreased by 91.8% and 74.9% and the immune escape rates decreased by about 32% and 92% respectively. At the same time, reactive oxygen species (ROS) in fish macrophages that phagocytosed sodA-RNAi and sodB-RNAi increased by 40% and 32.6%, respectively, compared to those of macrophages that phagocytosed the wild-type strain. Compared to sodA, the expression of sodB predominates in A. hydrophila without oxidative stress; however, when exposed to oxidative stress, the magnitude of up-regulation of sodA expression is significantly higher than that of sodB. With increased of methyl viologen concentration, the survival rates of sodA-RNAi and sodB-RNAi were significantly decreased. The expressions of sodA and sodB did not affect the growth of A. hydrophila without oxidative stress, but the inhibition of sodA and sodB expression led to a slight decrease in bacterial growth under oxidative stress. These results indicated that (1) sodA and sodB play an important role in the process of bacterial resistance to ROS damage in host phagocytic cells, allowing them to survive or even escape fish macrophages; (2) the sodB expression was dominant in A. hydrophila without oxidative stress, the sodA expression was up-regulated more significantly under oxidative stress, and sodA and sodB contributed equally to the process of bacterial resistance to ROS; (3) sodA and sodB complement each other and cooperate in the process of intracellular survival of bacteria to protect against ROS damage.

Survival in host macrophages is an effective strategy for pathogenic bacteria to spread. Aeromonas hydrophila has been found to survive in fish macrophages, but the mechanisms remain unknown. In this paper, the roles and possible mechanisms of IcmF in bacterial survival in fish macrophages were investigated. First, a stable silencing strain icmF-RNAi was constructed by shRNA and RT-qPCR confirmed the expression of icmF was down-regulated by 94.42%. The expression of Hcp, DotU and VgrG was also decreased in icmF-RNAi. The intracellular survival rate of the wild-type strain was 92.3%, while the survival rate of icmF-RNAi was only 20.58%. The escape rate of the wild-type strain was 20%, while that of the icmF-RNAi was only 7.5%. Further studies indicated that the expression of icmF can significantly affect the adhesion, biofilm formation, motility and acid resistance of A. hydrophila, but has no significant effect on the growth of A. hydrophila even under the stress of H2 O2 . The results indicated that IcmF of A. hydrophila not only acts as a structural protein which participates in virulence-related characteristics such as bacterial motility, adhesion and biofilm formation, but also acts as a key functional protein which participates in the interaction between bacteria and host macrophages.

The incidence of Vibrio alginolyticus infections has increased in recent years due to the influence of climate change and rising sea temperature. Vibrio virulence regulatory RNA 1 (Vvrr1) is a newly found noncoding RNA (ncRNA) predicted to be closely related to the adhesion ability of V. alginolyticus based on the previous RNA-seq. In this study, the target genes of Vvrr1 were fully screened and verified by constructing Vvrr1-overexpressing strains and using the proteome sequencing technology. Pyruvate kinase I (pykF) gene was predicted to be a chief target gene of Vvrr1 involved in virulence regulation. The adhesion ability, biofilm formation and virulence were significantly reduced in the Vvrr1-overexpressing and the pykF-silenced strain compared with the wild strains. Similar to the overexpression of Vvrr1, the silencing of pykF also reduced the expression level of virulence genes, such as ndk, eno, sdhB, glpF, and cysH. Meanwhile, by constructing the "pykF-GFP" fusion expression plasmid and using the GFP reporter gene analysis in Escherichia coli, the fluorescence intensity of the strain containing Vvrr1 whole ncRNA sequence vector was found to be significantly weakened. These indicated that Vvrr1 participated in the virulence regulation mechanism of V. alginolyticus by interacting with the virulence gene pykF.

In the present study, Larimichthys crocea and Pseudomonas plecoglossicida were selected as a host-pathogen interaction model for teleosts and prokaryotic pathogens. Five shRNAs were designed and synthesized to silence the fliA gene, all of which resulted in pronounced reductions in fliA mRNA, the mutant strain with the best silencing efficiency of 92.16% was chosen for subsequent analysis. A significant decrease in motility, intracellular survival and escape was observed for the fliA-RNAi strain of P. plecoglossicida, whereby silencing of the fliA gene led to a 30% decrease in mortality and a four-day delay in the onset of infection in L. crocea. Moreover, silencing of P. plecoglossicida fliA resulted in a significant change in both the pathogen and host transcriptome in the spleens of infected L. crocea. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of pathogen transcriptome data showed that silencing fliA resulted in downregulation of 18 flagellum-related genes, KEGG analysis of host transcriptome data revealed that infection with the fliA-RNAi strain caused upregulation of 47 and downregulation of 106 immune-related genes. These pathogen-host interactions might facilitate clearance of P. plecoglossicida by L. crocea, with a significant decrease in fliA-RNAi P. plecoglossicida strain virulence in L. crocea.

Aryl hydrocarbon receptor (AhR), a ligand-dependent transcriptional factor that responds to environmental chemicals, has been recently found to be closely associated with immune response in mammals. Pseudomonas plecoglossicida (P. plecoglossicida) is a temperature-dependent bacterial pathogen of visceral white spot disease in fish. Using dual RNA-seq, we previously evaluated the expression levels of ahr1a, ahr1b, ahr2 and cyp1a in the spleen of Epinephelus coioides at different time points after infection with P. plecoglossicida. In the present study, the expression levels of ahr1a, ahr1b, ahr2 and cyp1a in different organs of E. coioides and Danio rerio showed similar trends after being infected by P. plecoglossicida. It also was noted that liver, intestine, spleen, and heart were the most obviously affected organs, and ahr2 particularly showed a dramatically increase in the spleen. Subsequently, macrophages of E. coioides were isolated, and then infected by P. plecoglossicida, followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay, which revealed that the expression level of ahr1a in macrophages was significantly down-regulated, while expression levels of ahr1b, ahr2 and cyp1a were noticeably up-regulated. Eventually, it was noted that ahr1b and ahr2 were knocked-down in macrophages, and intracellular survival rate and immune escape rate of P. plecoglossicida were markedly improved. Taken together, ahr1a, ahr1b, ahr2 and cyp1a participate in the immune response to P. plecoglossicida in different organs of fish, while ahr1b and ahr2 may play pivotal roles in the immune response of spleen and macrophages.

Pseudomonas plecoglossicida is a temperature-dependent opportunistic pathogen which is associated with a variety of diseases in fish. During the development of “white nodules” disease, the expression of htpG in P. plecoglossicida was found to be significantly up-regulated at its virulent temperature of 18°C. The infection of htpG-RNAi strain resulted in the onset time delay, reduction in mortality and infection symptoms in spleen of Epinephelus coioides, and affected the bacterial tissue colonization. In order to reveal the effect of htpG silencing of P. plecoglossicida on the virulence regulation in P. plecoglossicida and immune response in E. coioides, dual RNA-seq was performed and a pathogen-host integration network was constructed. Our results showed that infection induced the expression of host genes related to immune response, but attenuated the expression of bacterial virulence genes. Novel integration was found between host immune genes and bacterial virulence genes, while IL6, IL1R2, IL1B, and TLR5 played key roles in the network. Further analysis with GeneMANIA indicated that flgD and rplF might play key roles during the htpG-dependent virulence regulation, which was in accordance with the reduced biofilm production, motility and virulence in htpG-RNAi strain. Meanwhile, IL6 and IL1B were found to play key roles during the defense against P. plecoglossicida, while CELA2, TRY, CPA1, CPA2, and CPB1 were important targets for P. plecoglossicida attacking to the host.

Large yellow croaker (Larimichthys crocea) is an economical important farmed fish in China. "Visceral White Spot Disease" caused by Pseudomonas plecoglossicida is a disease with a high mortality rate in cage-cultured L. crocea in recent years and resulted in heavy economy lossess. The dual RNA-seq results of previous study showed that the expression of clpV gene in P. plecoglossicida was significantly up-regulated during infection. RNAi significantly reduced the expression of clpV in P. plecoglossicida with maximum silencing efficiency of 96.1%. Compared with the wild type strain, infection of clpV-RNAi strain resulted in a delayed onset time and a 25% reduction in mortality of L. crocea, as well as lessening the symptoms of the spleen. The results of dual RNA-seq of L. crocea infected by clpV-RNAi strain of P. plecoglossicida changed considerably, compared with the counterpart infected with the wild strain. The KEGG enrichment analysis showed that Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway and MAPK signaling pathway of L. crocea were most affected by the silence of clpV in P. plecoglossicida. RNAi of clpV resulted in the downregulation of genes in flagella assembly pathway and a weaker immune response of host.

Pseudomonas plecoglossicida is well-known as the cause of viscera granulomas disease in fish. In this study, a cspA1 knock-down strain was constructed and tested in Epinephelus coioides to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the cspA1 knock-down strain caused a significant reduction in the ability of biofilm formation, motility, adhesion and virulence. E. coioides vaccinated with cspA1 knock-down strain were more tolerant of the infection by wild-type P. plecoglossicida. The relative percent survival value of E. coioides vaccinated with cspA1 knock-down strain reached 80% after challenging with wild-type P. plecoglossicida. In the meanwhile, the expression level of genes associated with immunity, including IL-1β, IgM, MHC-I and MHC-II, was up-regulated after vaccination, indicating that the cspA1 knock-down strain can induce effective and durable immune response in E. coioides and it may be an effective attenuated live vaccine candidate for the prevention of infections by P. plecoglossicida.

Interaction between bacterial pathogen and aquatic animal host is exceedingly complex, which involves large dynamic changes in gene expression during different stages of the disease. However, research on identifying key virulence genes based on the dynamics of gene expression changes of a one-sided bacterial pathogen in tissue has not been reported so far across different stages of infectious disease. The clpV for the T6SS of Pseudomonas plecoglossicida was identified for a candidate for key virulence gene based on dynamic changes of gene expression. For the Epinephelus coioides infected using clpV-RNAi strain, no deaths were observed up to 20 dpi. The spleens, kidneys and livers of all the E. coioides that received clpV-RNAi strain failed to develop visible nodules at 5-8 dpi, with the swelling gradually disappearing. The burdens of clpV-RNAi strain in the spleen and blood were greatly reduced at most of the time points after injection, and the burdens of clpV-RNAi strain in the head kidneys and trunk kidneys also had a sharp reduction from 72 to 120 hpi. This paper provides a new insight into the discovery of key virulence genes of pathogens in infected tissue systems.

Macrophage migration inhibitory factor (MIF) is a key pro-inflammatory cytokine in immuno-inflammatory diseases. For the first time, we examined the expression of MIF in Epinephelus awoara ( E. awoara). MIF expressions have been detected in the head kidney, spleen, liver, brain, intestine, gill, heart, stomach, and muscle of E. awoara infected with Vibrio parahaemolyticus. The mRNA levels observed in infected groupers were higher than those in healthy groupers. MIF, tumor necrosis factor-α (TNF-α), and interleukin-1 (IL-1) tissue levels have been measured by ELISA. A significant increase in MIF, TNF-α, and IL-1 tissue levels have been found in the treatment groups compared with those in controls. MIF, TNF-α and IL-1 tissue levels in the spleen, head kidney, intestine, and liver of E. awoara during the challenge trial with V. parahaemolyticus were significantly higher than those in controls. There was evidence of functions of MIF in a positive feedback loop with TNF-α and IL-1 that could perpetuate the inflammatory process in grouper infected with V. parahaemolyticus. In conclusion, these results indicated that MIF was related to pathogen-induced immune response.

Aquaculture suffers from a number of diseases caused by Aeromonas hydrophila. Biofilm can protect bacteria from antibiotic therapy. To identify the genes those play crucial roles in A. hydrophila biofilm formation, a library of mini-Tn10 transposon insertion mutants of A. hydrophila B11 has been constructed, and 10 mutants were subjected to biofilm formation assay. The biofilm formation ability of mutant (B188) was significantly decreased compared with B11. The DNA sequence flanking the mini-Tn10 transposon inserted showed that an ORF of approximately 576 bp of the mutant strain B188 was inserted. This ORF putatively displays the highest identity (92%) with the cytochrome c4 gene (cyt-c4) of A. hydrophila subsp. hydrophila ATCC 7966. Silencing cyt-c4 led to deficiencies in biofilm formation, adhesion, drug resistance and pathogenicity of A. hydrophila, which suggests that cyt-c4 plays crucial role in the biofilm formation and virulence mechanisms of A. hydrophila. ABBREVIATIONS: GEN: gentamycin; SDZ: sulfadiazine; AK: amikacin; P: penicillin; CFP: cefoperazone; LEV: levofloxacin; MH: minocycline; FFC: florfenicol; TE: tetracycline; AMP: ampicillin; KAN: kanamycin; STR: streptomycin; SXT: sulfamethoxazole/trimethoprim; DO: doxycycline; OT: Oxytetracycline.

The success of the pathogenic bacteria is partly attributable to their ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of macrophages. And reactive oxygen species (ROS) is one of the most effective antimicrobial components of macrophages to kill invading bacteria. Our previous studies found that Aeromonas hydrophila can survive in fish macrophages, which suggested that this bacterium might take fish macrophages as their shelters to resist drug killings and other immune damage. But how A. hydrophila survive in host macrophages remains unknown. Since KatG has been reported to have not only catalase activity but also peroxidase and peroxynitritase activity, the amino acid sequence and protein structure of KatG was analyzed in this study, the function of KatG in A. hydrophila survival in and escape from host macrophages was also carried out. The bioinformatics analysis displayed that KatG of A. hydrophila B11 showed >93% homologous to that of KatG in other Aeromonas. KatG of A. hydrophila was stable silenced by shRNA and RT-qPCR confirmed the expression of KatG in KatG-RNAi was significantly reduced. The survival rate of intracellular KatG-RNAi decreased by 80% compared to that of the wild type strain B11, while the intracellular ROS level of the macrophages that phagocytosed KatG-RNAi increased 65.9% when compared to that of the macrophages phagocytosed wild-type strain. The immune escape rate of A. hydrophila decreased by 85% when the expression of KatG was inhibited. These results indicated that (1) The amino acid sequence and protein structure of KatG of A. hydrophila is conserved; (2) KatG helped A. hydrophila to survive in fish macrophages by eliminating the harm of intracellular H2O2 and inhibiting intracellular ROS levels increased; (3) A small portion of intracellular A. hydrophila could escape from host macrophages for further infection, in this process KatG also played important role.

Vibrio aestuarianus is an opportunistic environmental pathogen that has been associated with epidemics in cultured shrimp Penaeus vannamei. Hepatopancreas microsporidian (HPM) and monodon slow growth syndrome (MSGS) have been reported in cultured P. vannamei. In this study, we sequenced and assembled the whole genome of V. aestuarianus strain W‐40, a strain that was originally isolated from the intestines of an infected P. vannamei. The genome of V. aestuarianus strain W‐40 contains two circular chromosomes of 483,7307 bp with a 46.23% GC content. We identified 4,457 open reading frames (ORFs) that occupy 86.35% of the genome. Vibrio aestuarianus strain W‐40 consists primarily of the ATP‐binding cassette (ABC) transporter system and the phosphotransferase system (PTS). CagA is a metabolism system that includes bacterial extracellular solute‐binding protein. Glutathione reductase can purge superoxide radicals (O22−) and hydrogen peroxide (H2O2) damage in V. aestuarianus strain W‐40. The presence of two compete type I restriction‐modification systems was confirmed. A total of 42 insertion sequences (IS) elements and 16 IS elements were identified. Our results revealed a host of virulence factors that likely contribute to the pathogenicity of V. aestuarianus strain W‐40, including the virulence factor genes vacA, clpC, and bvgA, which are important for biofilm dispersion. Several bacitracin and tetracycline antibiotic resistance‐encoding genes and type VI secretion systems were also identified in the genome. The complete genome sequence will aid future studies of the pathogenesis of V. aestuarianus strain W‐40 and allow for new strategies to control disease to be developed.

Adhesion capability to fish mucus, which can be affected by environmental conditions, is considered to be a key virulence factor of Vibrio alginolyticus although the molecular mechanism is still unclear. In the present study, V. alginolyticus was treated with stress conditions including Cu2+ (50 mg/L), Pb2+ (100 mg/L), Hg2+ (50 mg/L) and low pH (pH 5). We found these stress treatments were capable of reducing the adhesion of V. alginolyticus, while the expression levels of multiple genes were significantly changed according to the results of high throughput sequencing. The expression of randomly selected genes was confirmed by QPCR, which reinforced the reliability of the sequencing data. Ontology assignments and KEGG pathway analysis indicated that stress treatments affect pathways that may be related to adhesion. Our results identified genes which might play a key role in the adhesion process of V. alginolyticus, which could lay a foundation for further functional analysis of these genes in the process of adhesion. As these genes were sensitive to environmental factors, this may explain why the adhesion process can be influenced by environmental factors. Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0411-9) contains supplementary material, which is available to authorized users.

In this study, we examined the virulence factors and pathogenesis of Vibrio parahaemolyticus in Epinephelus awoara. The chemotactic motility of V. parahaemolyticus for phagocytosis and intracellular survival in fish macrophages was determined using virulence strains and low-virulence strains of V. parahaemolyticus. We found that the intracellular mean number of virulence strains of V. parahaemolyticus ranged from 0-180 min after co-incubation with macrophages and peripheral leukocytes, was relatively low, and decreased steadily over the observation period. Low-virulence strains of V. parahaemolyticus were unable to survive in peripheral leukocytes and macrophages. Cell viability in response to V. parahaemolyticus was assessed using the MTT assay. Low-virulence V. parahaemolyticus strains exhibited lower cytotoxicity compared to virulent strains. The average percent of live macrophages and peripheral leukocytes infected by V. parahaemolyticus ranged from 13.50-79.20%. These results indicate that V. parahaemolyticus in E. awoara is a facultative intracellular bacterium that may be involved in virulence.

Shrimp is extensively cultured worldwide. Shrimp farming is suffering from a variety of diseases. Probiotics are considered to be one of the effective methods to prevent and cure shrimp diseases. Exiguobacterium arabatum W‐01, a gram‐positive and orange‐pigmented bacterium, was isolated from the intestine of a healthy Penaeus vannamei specimen. Whole‐genome sequencing revealed a genome of 2,914,854 bp, with 48.02% GC content. In total, 3,083 open reading frames (ORFs) were identified, with an average length of 843.98 bp and a mean GC content of 48.11%, accounting for 89.27% of the genome. Among these ORFs, 2,884 (93.5%) genes were classified into Clusters of Orthologous Groups (COG) families comprising 21 functional categories, and 1,650 ORFs were classified into 83 functional Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. A total of 27 rRNA operons and 68 tRNAs were identified, with all 20 amino acids represented. In addition, 91 genomic islands, 68 potential prophages, and 33 tandem repeats, but no clustered regularly interspaced short palindromic repeats (CRISPRs), were found. No resistance genes and only one virulence gene were identified. Among the 150 secreted proteins of E. arabatum W‐01, a variety of transport system substrate‐binding proteins, enzymes, and biosynthetic proteins, which play important roles in the uptake and metabolism of nutrients, were found. Two adherence‐related protein genes and 31 flagellum‐related protein genes were also identified. Taken together, these results indicate potential probiotic functions for E. arabatum W‐01.

The overuse of antimicrobials in aquaculture has promoted the selection of antimicrobial-resistant bacteria. Here we investigated the abundance of antimicrobial-resistance genes and integrons in 108 strains of antibiotic-resistant bacteria isolated from eels and aquaculture ponds in China. Conventional PCR was implemented to examine common antibiotic-resistance genes, integrons, and their gene cassette arrays. The results showed that the antibiotic-resistance genes blaTEM, tetC, sulI, aadA, floR, and qnrB were detected at high percentages, as were a number of other resistance genes. Class I integrons were present in 79.63% of the strains, and 10 out of 108 isolates carried class II integrons. Class III integrons were not detected. Three strains carried both class I and class II integrons, and 73.26% of the class I integron-positive isolates contained the qacEΔ1/sul1 gene. Fourteen types of integron cassette arrays were found among class I integron-positive isolates. A new array, dfrB4-catB3-blaOXA-10-aadA1, was discovered in this study. The gene cassette array dfrA12-orfF-aadA2 was the most widely distributed. In summary, 23 different gene cassettes encoding resistance to 8 classes of antibiotics were identified in the class I integrons, and the main cassettes contained genes encoding resistance to aminoglycosides (aad) and trimethoprim (dfr). All class II integron-positive strains had only a single gene cassette array, viz. dfrA1-catB2-sat2-aadA1. High levels of antimicrobial-resistance genes and integrons in eels and auqauculture ponds suggest that the overuse of antimicrobials should be strictly controlled and that the levels of bacterial antimicrobial-resistance genes in aquaculture should be monitored.

Adhesion to host surface or cells is the initial step in bacterial pathogenesis, and the adhesion mechanisms of the fish pathogenic bacteria Aeromonas hydrophila were investigated in this study. First, a mutagenesis library of A. hydrophila that contained 332 random insertion mutants was constructed via mini-Tn10 Km mutagenesis. Four mutants displayed the most attenuated adhesion. Sequence analysis revealed that the mini-Tn10 insertion sites in the four mutant strains were flgC(GenBank accession numbers KX261880), cytb4(GenBank accession numbers JN133621), rbsR(GenBank accession numbers KX261881) and flgE(GenBank accession numbers JQ974982). To further study the roles of flgC and flgE in the adhesion of A. hydrophila, some biological characteristics of the wild-type strain B11, the mutants M121 and M240, and the complemented strains C121 and C240 were investigated. The results showed that the mutation in flgC or flgE led to the flagellar motility of A. hydrophila significant reduction or abolishment. flgC was not necessary for flagellar biosynthesis but was necessary for the full motility of A. hydrophila, flgE was involved in both flagellar biosynthesis and motility. The flagellar motility is necessary for A. hydrophila to adhere to the host mucus, which suggests flagellar motility plays crucial roles in the early infection process of this bacterium.

Adhesion is an important virulence trait of Vibrio alginolyticus. Bacterial adhesion is influenced by environmental conditions; however, the molecular mechanism underlying this effect remains unknown. The expression levels of flrA, flrB and flrC were significantly downregulated in adhesion-deficient V. alginolyticus strains cultured under Cu(2+), Pb(2+), Hg(2+) and low-pH stresses. Silencing these genes led to deficiencies in adhesion, motility, flagellar assembly, biofilm formation and exopolysaccharide (EPS) production. The expression levels of fliA, flgH, fliS, fliD, cheR, cheV and V12G01_22158 (Gene ID) were significantly downregulated in all of the RNAi groups, whereas the expression levels of toxT, ctxB, acfA, hlyA and tlh were upregulated in flrA- and flrC-silenced groups. These genes play a key role in the virulence mechanisms of most pathogenic Vibrio species. Furthermore, the expression of flrA, flrB and flrC was significantly influenced by temperature, salinity, starvation and pH. These results indicate that (1) flrA, flrB and flrC are important for V. alginolyticus adhesion; (2) flrA, flrB and flrC significantly influence bacterial adhesion, motility, biofilm formation and EPS production by controlling expression of key genes involved in those phenotypes; and (3) flrA, flrB and flrC regulate adhesion in the natural environment with different temperatures, pH levels, salinities and starvation time.

Adhesion is a critical step in the initial stage of Vibrio alginolyticus infection; therefore, it is important to understand the underlying mechanisms governing the adhesion of V. alginolyticus and determine if environmental factors have any effect. A greater understanding of this process may assist in developing preventive measures for reducing infection. In our previous research, we presented the first RNA-seq data from V. alginolyticus cultured under stress conditions that resulted in reduced adhesion. Based on the RNA-seq data, we found that the Tricarboxylic acid cycle (TCA pathway) might be closely related to adhesion. Environmental interactions with the TCA pathway might alter adhesion. To validate this, bioinformatics analysis, quantitative Real-Time PCR (qPCR), RNAi, and in vitro adhesion assays were performed, while V. alginolyticus was treated with various stresses including temperature, pH, salinity, and starvation. The expression of genes involved in the TCA pathway was confirmed by qPCR, which reinforced the reliability of the sequencing data. Silencing of these genes was capable of reducing the adhesion ability of V. alginolyticus. Adhesion of V. alginolyticus is influenced substantially by environmental factors and the TCA pathway is sensitive to some environmental stresses, especially changes in pH and starvation. Our results indicated that (1) the TCA pathway plays a key role in V. alginolyticus adhesion: (2) the TCA pathway is sensitive to environmental stresses.

Influenza virus neuraminidase (NA) plays a crucial role in facilitating the spread of newly synthesized virus in the host and is an important target for controlling disease progression. The NA crystal structure from the 1918 “Spanish flu” (A/Brevig Mission/1/18 H1N1) and that of its complex with zanamivir (Relenza) at 1.65-Å and 1.45-Å resolutions, respectively, corroborated the successful expression of correctly folded NA tetramers in a baculovirus expression system. An additional cavity adjacent to the substrate-binding site is observed in N1, compared to N2 and N9 NAs, including H5N1. This cavity arises from an open conformation of the 150 loop (Gly147 to Asp151) and appears to be conserved among group 1 NAs (N1, N4, N5, and N8). It closes upon zanamivir binding. Three calcium sites were identified, including a novel site that may be conserved in N1 and N4. Thus, these high-resolution structures, combined with our recombinant expression system, provide new opportunities to augment the limited arsenal of therapeutics against influenza.

Adhesion is an important virulence factor of Vibrio alginolyticus. This factor may be affected by environment conditions; however, its molecular mechanism remains unclear. In our previous research, adhesion tender models were obtained by culturing V. alginolyticus under stresses such as Cu2+, Pb2+, Hg2+, and low pH. With high-throughput sequencing and bioinformatics analysis, we found that all of these stress treatments significantly affected the flagellar assembly pathway, which may play an important role in V. alginolyticus adhesion. Therefore, we hypothesized that the environmental stresses of the flagellar assembly pathway may be one way in which environment conditions affect adhesion. To verify our hypothesis, a bioinformatics analysis, QPCR, RNAi, in vitro adhesion assay and motility assay were performed. Our results indicated that 1) the flagellar assembly pathway was sensitive to environmental stresses, 2) the flagellar assembly pathway played an important role in V. alginolyticus adhesion, and 3) motility is not the only way in which the flagellar assembly pathway affects adhesion.

The capability of Vibrio alginolyticus to adhere to fish mucus is a key virulence factor of the bacteria. Our previous research showed that stress conditions, such as Cu(2+), Pb(2+), Hg(2+), and low pH, can reduce this adhesion ability. Non-coding (nc) RNAs play a crucial role in regulating bacterial gene expression, affecting the bacteria's pathogenicity. To investigate the mechanism(s) underlying the decline in adhesion ability caused by stressors, we combined high-throughput sequencing with computational techniques to detect stressed ncRNA dynamics. These approaches yielded three commonly altered ncRNAs that are predicted to regulate the bacterial chemotaxis pathway, which plays a key role in the adhesion process of bacteria. We hypothesized they play a key role in the adhesion process of V. alginolyticus. In this study, we validated the effects of these three ncRNAs on their predicted target genes and their role in the V. alginolyticus adhesion process with RNA interference (i), quantitative real-time polymerase chain reaction (qPCR), northern blot, capillary assay, and in vitro adhesion assays. The expression of these ncRNAs and their predicted target genes were confirmed by qPCR and northern blot, which reinforced the reliability of the sequencing data and the target prediction. Overexpression of these ncRNAs was capable of reducing the chemotactic and adhesion ability of V. alginolyticus, and the expression levels of their target genes were also significantly reduced. Our results indicated that these three ncRNAs: (1) are able to regulate the bacterial chemotaxis pathway, and (2) play a key role in the adhesion process of V. alginolyticus.

Adhesion is a key virulence factor of pathogens and can be affected by the environment. Our previously research with RNA‐seq indicated that mcp, aer, cheB, and cheV might play roles in the regulation of adhesion in Vibrio alginolyticus (ND‐01). In order to determine whether and how environmental factors affect adhesion through these genes, gene silencing was performed followed by quantitative real‐time PCR (qRT‐PCR), RNAi, transmission electron microscopy, and adhesion, capillary, and motility assays to verify how these genes influence adhesion. Silencing these genes led to deficiencies in adhesion, chemotaxis, flagellar assembly, and motility. The expression levels of cheA, cheW, and cheY, which are important genes closely related to the functions of mcp, aer, cheV, and cheB, were significantly downregulated in all of the RNAi groups. The expression of mcp, aer, cheV, and cheB under different gradients of temperature, pH, and salinity and after starvation for various durations was also detected, which showed that these genes were sensitive to certain environmental stresses, particularly pH and starvation. Our results indicated that mcp, aer, cheB, and cheV: (1) are necessary for ND‐01 adhesion; (2) play key roles in the bacterial chemotaxis pathway by controlling the expression of downstream genes; (3) might affect adhesion by impacting motility, though motility is not the only route through which adhesion is affected; and (4) contribute to the regulation of ND‐01 adhesion in natural environments with different temperatures, pH levels, and salinities as well as after various starvation periods.

The interactions of pathogens and phagocytes are complex. Our study demonstrated that Aeromonas hydrophila B11 can survive in the macrophagocytes of Tilapia mossambica. To explore the regulatory processes of A. hydrophila survival in the macrophagocytes, we used the mini‐Tn10 transposon mutagenesis system to build a mutant library by mixing Escherichia coli Sm10 (pLOFKm) and A. hydrophila B11. In total, 102 mutant colonies were detected, and 11 of them showed reduced survival in macrophagocytes. The mutant with the most severe phenotype, AM73, was chosen for further research. The ORF interrupted by mini‐Tn10 in AM73 was approximately 960 bp and was deposited in GenBank with the accession number SRP049226. The 319 amino acid protein encoded by the ORF showed a high degree of identity (89%) with proteins in the histone deacetylase/AcuC/AphA family of A. hydrophila subsp. hydrophila ATCC7966. A strain (AC73) in which the acuC mutation was complemented was constructed by generating the recombinant expression plasmid pACYC184‐acuC and introducing it into the AM73 mutant strain. Our experiments revealed that strain AM73 was deficient in biofilm formation, adhesion, survival in macrophagocytes, and virulence compared with A. hydrophila B11, and all of these biological properties were improved in strain AC73. The expression of 10 significant virulence genes was significantly inhibited in strain AM73. The results indicated that AcuC was an important regulatory protein contributing to the pathogenicity of A. hydrophila.

National Department Public Benefit Research Foundation of China [200903029]; Natural Science Foundation of Fujian Province [2011J06014]