Neiella litorisoli sp. nov., an alginate lyase: producing bacterium from South China Sea, and proposal of Echinimonadaceae fam. nov. in the order Alteromonadales.

A Gram-stain-negative, rod-shaped bacterium, designated HB171785T, was isolated from soil sample collected from Qishui Bay, Hainan, China. The strain grew optimally at pH 7-8, 37-40 °C and with NaCl 3-4%. The predominant isoprenoid quinone was found to be Q-8 and the major fatty acids were C16:0, C16:1 ω7c/C16:1 ω6c, C18:1 ω7c/C18:1 ω6c and C12:0 3OH. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The size of the draft genome was 4.32 Mbp with G + C content 49.7%. Phylogenetic analysis of 16S rRNA gene sequence indicated that the closest phylogenetically related species were Neiella marina j221T, "Neiella holothuriorum" 126 and Echinimonas agarilytica KMM 6351T with the similarities of 98.2, 96.0 and 95.0%, respectively. The phylogenetic tree based on 16S rRNA gene and phylogenomic tree based on core genome showed that strain HB171785T clustered together with N. marina j221T, with the highest values of average nucleotide identity (82.9%) and digital DNA-DNA hybridization (25.4%). The combined phylogenetic relatedness, phenotypic and genotypic features supported the conclusion that strain HB171785T represents a novel species of the genus Neiella, for which the name Neiella litorisoli sp. nov. is proposed. The type strain is HB171785T (= MCCC 1K04625T = KCTC 82319T). In addition, Echinimonadaceae fam. nov. in the order Alteromonadales was proposed.

Polycladospora coralii gen. nov., sp. nov., a novel member of the family Thermoactinomycetaceae isolated from stony coral in the South China Sea.

Two novel filamentous bacteria, designated as IB182353T and IB182357, were isolated from stony coral of the South China Sea. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains IB182353T and IB182357 were closely related to Hazenella coriacea DSM 45707T (with 93.4 and 93.5% similarity, respectively). The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization results showed that the pairwise similarities between isolate IB182353T and the other recognized Thermoactinomycetaceae species were less than 68.9, 60.5 and 21.1 %, respectively. Both strains produced aerial and substrate mycelia, grew optimally at 25-30 °C, pH 8.0-9.0 and with 2-3 % (w/v) NaCl. The cell-wall peptidoglycan type was meso-DAP and the whole-cell hydrolysates contained ribose. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminophospholipid and three unidentified phospholipids. The genomic DNA G+C content was 39.5 mol%. Strain IB182353T was distinguishable from its related type strains by the contents of two fatty acids, iso-C15 : 0 and iso-C17 : 1 ω10c. Based on polyphasic taxonomic characterization, we propose that strains IB182353T and IB182357 represent a novel genus and species within the family Thermoactinomycetaceae, for which the name Polycladospora coralii gen. nov. sp. nov. is proposed. The type strain is IB182353T (=MCCC 1K04631T=JCM 34206T).

The association between vision impairment and cognitive outcomes in older adults: a systematic review and meta-analysis.

OBJECTIVES: To provide a quantitative synthesis of studies on the relationship between vision impairment (VI) and cognitive outcomes in older adults. METHOD: A systematic search was undertaken of relevant databases for original articles published before April 2020. Random effect models were used to obtain pooled estimates of the associations between VI and cognitive outcomes (cognitive impairment and dementia) with subgroup analyses of VI measures, cross-sectional associations of VI with cognitive impairment, and longitudinal associations of baseline VI with incident cognitive impairment and dementia. Potential sources of heterogeneity were explored by meta-regression. Publication bias was evaluated with Egger's test. RESULTS: Sixteen studies including 76,373 participants were included in this meta-analysis, with five cross-sectional studies and eleven longitudinal studies. There was a significantly increased risk of cognitive outcomes with VI identified by subjective measures (odds ratio (OR)=1.63; 95% confidence interval (CI): 1.26-1.99) and objective measures (OR = 1.59; 95% CI: 1.40-1.78). The odds of baseline cognitive impairment were 137% higher in older adults with VI compared with those without VI (OR = 2.37, 95% CI: 1.84-3.03) at baseline. Compared with older adults without VI at baseline, those with baseline VI had a higher relative risk (RR) of incident cognitive impairment (RR = 1.41; 95% CI: 1.31-1.51) and dementia (RR = 1.44, 95% CI: 1.19-1.75). CONCLUSIONS: VI was associated with increased risks of cognitive impairment and dementia across cross-sectional and longitudinal studies. Additional research and randomized clinical trials are warranted to examine the implications of treatment for VI, such as wearing glasses and cataract surgery, to avoid cognitive impairment and dementia.

Paenibacillus sabuli sp. nov., isolated from the South China Sea.

A Gram-positive, rod-shaped, motile, spore-forming bacterium, designated strain IB182496T, was isolated from coastal sand of the South China Sea. The strain grew optimally at pH 7.0-9.0, 20-30 °C, and with NaCl 3.0-5.0 %. The predominant menaquinone was MK-7 and the major cellular fatty acids were anteiso-C15 : 0, iso-C16 : 0 and C16 : 0. The polar lipids in the cell wall included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids and one unidentified lipid. The comparison of 16S rRNA gene sequences indicated that strain IB182496T was most closely related to 'Paenibacillus sambharensis' SMB1 and Paenibacillus tarimensis SA-7-6T with similarities of 95.7 and 95.5 %, respectively. The whole-genome average nucleotide identity values between strain IB182496T and the two reference strains were 70.8 and 70.5%, and the digital DNA-DNA hybridization values were 18.7 and 18.0 %, respectively. Genomic analyses showed that strain IB182496T presented a genome of 6.22 Mbp with chromosomal G+C content of 60.3 %, and a total of 5261 genes were predicted. The combined phylogenetic relatedness, phenotypic and genotypic features supported the conclusion that strain IB182496T should be considered as representing a novel species of the genus Paenibacillus, for which we propose the name Paenibacillus sabuli sp. nov. with the type strain IB182496T (=MCCC 1K04627T=JCM 34216T).

Paenibacillus oceani sp. nov., isolated from surface seawater.

A Gram-stain-positive and motile bacterial strain, designated IB182363T, was isolated from surface seawater of the South China Sea. Cells grew at pH 5.0-9.5 (optimum, pH 7.0-8.0), 20-40 °C (optimum, 30 °C) and with 1-8 % (w/v) NaCl (optimum, 2-4 %). On the basis of 16S rRNA gene sequence analysis, strain IB182363T was affiliated to the genus Paenibacillus and the closest phylogenetically related species was Paenibacillus ginsengarvi DSM18677T with 96.9 % sequence similarity. The values of whole genome average nucleotide identity analysis and digital DNA-DNA hybridization between the isolate and the closely related type strains were less than 86.3 and 25.6 %, respectively. Chemotaxonomic analysis revealed that strain IB182363T possessed meso-diaminopimelic acid in the cell-wall peptidoglycan and contained menaquinone MK-7 as the predominant isoprenoid quinone. The major cellular fatty acids were anteiso-C15 : 0, C16 : 0 and iso-C16 : 0. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified glycolipid, two unidentified aminolipids, two unidentified phospholipids and four unidentified aminophospholipids. The genomic DNA G+C content was 54.5 mol%. On the basis of the above results, strain IB182363T represents a novel species of the genus Paenibacillus, for which we propose the name Paenibacillus oceani sp. nov. with the type strain IB182363T (=MCCC 1K04630T=JCM 34214T).

Evaluation of the efficacy of a novel Vibrio vulnificus vaccine based on antibacterial peptide inactivation in turbot, Scophthalmus maximus.

Tongue sole tissue factor pathway inhibitor 2 (TFPI-2) C-terminus derived peptide, TC38, has previously been shown to kill Vibrio vulnificus cells without lysing the cell membrane; thus, the remaining bacterial shell has potential application as an inactivated vaccine. Therefore, this study aimed to evaluate the immune response induced by the novel V. vulnificus vaccine. The protective potential of TC38-killed V. vulnificus cells (TKC) was examined in a turbot model. Fish were intramuscularly vaccinated with TKC or FKC (formalin-killed V. vulnificus cells) and challenged with a lethal-dose of V. vulnificus. The results showed that compared with FKC, TKC was effective in protecting fish against V. vulnificus infection, with relative percent of survival (RPS) rates of 53.29% and 63.64%, respectively. The immunological analysis revealed that compared with the FKC and control groups, the TKC group exhibited: 1) significantly higher respiratory burst ability and bactericidal activity of macrophages at 7 d post-vaccination; 2) increased alkaline phosphatase, acid phosphatase, lysozyme, and total superoxide dismutase levels post-vaccination; 3) higher serum agglutinating antibody titer with corresponding higher serum bactericidal ability, and a more potent serum agglutination effect, as well as an increased IgM expression level; 4) higher expression of immune relevant genes, which were involved in both innate and adaptive immunity. Taken together, this is the first study to develop a novel V. vulnificus inactivated vaccine based on AMP inactivation, and TKC is an effective vaccine against V. vulnificus infection for aquaculture.

Self-reported vision impairment, vision correction, and depressive symptoms among middle-aged and older Chinese: Findings from the China health and retirement longitudinal study.

OBJECTIVE: To investigate associations between vision impairment (VI), vision correction (VC) and depressive symptoms among middle-aged and older Chinese. METHODS: 22 203 participants aged ≥45 years from China Health and Retirement Longitudinal Study 2011 to 2015 were divided into four self-reported VI categories: no VI, distance VI (DVI) only, near VI (NVI) only, and both distance and near VI (DNVI); and four self-reported VI/VC subgroups: VI(+)/VC(-), VI(+)/VC(+), VI(-)/VC(-) and VI(-)/VC(+). Depressive symptoms were evaluated by 10-item Center for Epidemiological Studies Depression Scale (CESD-10). RESULTS: Compared with no VI, DVI only (OR = 2.12, 95% CI: 1.95, 2.31), NVI only (OR = 1.51, 95% CI: 1.39, 1.63) and DNVI (OR = 2.75, 95% CI: 2.47, 3.07) were associated with higher odds of depressive symptoms. Compared with VI(+)/VC(-), VI(+)/VC(+) (OR = 0.91, 95% CI: 0.83, 0.98), VI (-)/VC(-) (OR = 0.50, 95% CI: 0.48, 0.53) and VI(-)/VC(+) (OR = 0.49, 95% CI: 0.47, 0.54) were associated with lower odds of depressive symptoms. Compared with no VI at baseline, baseline DNVI was significantly associated with higher odds of depressive symptoms after two (OR = 1.48, 95% CI: 1.16, 1.88) and four (OR = 1.32, 95% CI: 1.04, 1.68) years. Baseline depressive symptoms were significantly associated with higher odds of VI after two (OR = 1.53, 95% CI: 1.34, 1.74) and four (OR = 1.54, 95% CI: 1.34, 1.76) years. CONCLUSION: Adults with DNVI were more likely to report depressive symptoms in the future and those with depressive symptoms were more likely to report VI in the future. VC might be a protective factor for preventing depressive symptoms among adults with VI.

Vision impairment, vision correction, and cognitive decline among middle-aged and older Chinese: results from the China health and retirement longitudinal study.

OBJECTIVES: To investigate the associations between vision impairment (VI), vision correction (VC), and cognitive function. METHOD: We included 20,677 participants aged ≥45 years from the China Health and Retirement Longitudinal Study (2011-2015). Participants were grouped into no VI, distance VI (DVI) only, near VI (NVI) only, or both distance and near VI (DNVI), and VI(+)/VC(-), VI(+)/VC(+), VI(-)/VC(-), or VI(-)/VC(+) further at baseline. Cognitive function at baseline and subsequently every two years was applied as a dependent variable in a generalized estimating equation model. RESULTS: DVI only, NVI only, and DNVI had significantly worse cognitive function over time than no VI (all p < .05). DNVI had significantly worse cognitive function over time than DVI only and NVI only (all p < .001). VI(+)/VC(+), VI(-)/VC(-), and VI(-)/VC(+) had significantly better cognitive function over time than VI(+)/VC(-) (all p < .05). VI(-)/VC(+) had significantly better cognitive function over time than VI(+)/VC(+) and VI(-)/VC(-) (all p < .05). CONCLUSION: Cognitive function was worse in middle-aged and older Chinese with VI, especially in those with DNVI. VC was associated with better cognitive function over time regardless of the status of vision.

Amaricoccus solimangrovi sp. nov., isolated from mangrove soil.

Strain HB172011T was isolated from mangrove soil sampled at the Bamenbay mangrove forest, PR China. Cells were easily recognized under the microscope as cocci that were usually arranged in distinctive tetrads. Results of phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate belongs to the genus Amaricoccus and has 95.6-96.3% 16S rRNA gene sequence similarities to the four Amaricoccus type strains. The strain was aerobic or facultatively anaerobic, Gram-stain-negative and non-motile. Cells were found to grow at 10-40 °C (optimum, 30 °C), pH 6.0-9.0 (optimum, pH 7.0) and with 0-9.0% (w/v) NaCl (optimum, 2-4%). Major fatty acids were feature 8 (C18:1 ω7c and/or C18:1 ω6c), C16:0, C19:0 cyclo ω8c and summed feature 2 (C16:1 iso I and/or C14:0-3 OH). Genome sequencing revealed a genome size of 4.87 Mbp and a DNA G+C content of 69.9 mol %. Based on these data, strain HB172011T represents a novel species of Amaricoccus, for which the name Amaricoccus solimangrovi sp. nov. is proposed. The type strain is HB172011T (=CGMCC 1.16728T=JCM 33334T).

Pontibacter mangrovi sp. nov., isolated from mangrove sediment.

A Gram-stain-negative, short-rod-shaped and pink-pigmented bacterial strain (HB172049T) was isolated from mangrove sediment. Cells grew at 10-45 °C (optimum, 30 °C), pH 6.0-9.0 (optimum, pH 7.0) and with 0.5-9.0 % (w/v) NaCl (optimum, 2-5 %). Analysis of the 16S rRNA gene sequence revealed that the isolate had highest sequence similarities to Pontibacter mucosus DSM 100162T (96.5 %) and Pontibacter korlensis X14-1T (96.5 %). The values of average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization between the isolate and its close neighbours were, respectively, less than 80.1, 81.7 and 23.2 %. Chemotaxonomic analysis indicated that the sole respiratory quinone was MK-7 and the predominant cellular fatty acids were summed feature 4 and iso-C15 : 0 (42.2 and 24.6 %, respectively). The major polar lipids consisted of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, one unidentified glycolipid, one unidentified phospholipid, one unidentified aminophospholipid and two unidentified polar lipids. The genomic DNA G+C content was 52.6 mol%. Based on polyphasic taxonomic characterization, it is proposed that strain HB172049T belongs to the genus Pontibacter and represents a novel species, for which the name Pontibacter mangrovi sp. nov. is proposed. The type strain is HB172049T (=CGMCC 1.16729T=JCM 33333T).

Characterization, expression, and antimicrobial activity of histones from Japanese flounder Paralichthys olivaceus.

Histone proteins are not only structurally important for chromosomal DNA packaging but also involved in the regulation of gene expression and the immune response of host against pathogens. Japanese flounder (Paralichthys olivaceus) as one of the most important marine flatfish, suffered from widespread outbreaks of diseases, and its immunological functioning remained to be elucidated. In the present study, we reported the expression patterns of four histones (H1, H2A, H3, and H3.3) and functional characterization of the histone H3.3 from flounder. Quantitative real time RT-PCR (RT-qPCR) analysis showed that expression of the four histones occurred in multiple tissues, but their levels of expression were relatively high in immune organs, and inducible in response to pathogens infection. Infection with extracellular and intracellular bacterial pathogens and viral pathogen regulated the expression of histones in a manner that depended on tissue type, pathogen, and infection stage. Specifically, H1 expression was highly induced by intracellular viral pathogens; H2AX and H3 expressions were highly induced by intracellular bacterial pathogen; dissimilarly, H3.3 expression was slightly induced by extracellular bacterial pathogen, but was inhibited by intracellular bacterial and viral pathogens. To further investigate H3.3 function, recombinant H3.3 (rH3.3) was obtained, and in vitro experiments showed rH3.3 possessed the capability of binding to both Gram-negative and Gram-positive bacteria and inhibiting the growth of some target bacteria. Consistently, In vivo results showed that overexpression of H3.3 promoted the host defense against invading pathogenic microorganism and regulated the expressions of several cytokines. These results suggested that flounder histones exhibit different expression patterns in response to the infection of different microbial pathogens, and H3.3 serves as an immune-related protein and plays an important role in antimicrobial immunity of Japanese flounder. Taken together, this study is the first report about the expression profile of different histones upon different kind of pathogens and anti-infectious immunity of H3.3 in teleost, which offered new insights into the immunological function of histones in teleost.

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida.

Edwardsiella piscicida is a severe fish pathogen. Haem utilization systems play an important role in bacterial adversity adaptation and pathogenicity. In this study, a speculative haem utilization protein, HutZEp, was characterized in E. piscicida. hutZEp is encoded with two other genes, hutW and hutX, in an operon that is similar to the haem utilization operon hutWXZ identified in V. cholerae. However, protein activity analysis showed that HutZEp is probably not related to hemin utilization. To explore the biological role of HutZEp, a markerless hutZEp in-frame mutant strain, TX01ΔhutZ, was constructed. Deletion of hutZEp did not significantly affect bacterial growth in normal medium, in iron-deficient conditions, or in the presence of haem but significantly retarded bacterial biofilm growth. The expression of known genes related to biofilm growth was not affected by hutZEp deletion, which indicated that HutZEp was probably a novel factor promoting biofilm formation in E. piscicida. Compared to the wild-type TX01, TX01ΔhutZ exhibited markedly compromised tolerance to acid stress and host serum stress. Pathogenicity analysis showed that inactivation of hutZEp significantly impaired the ability of E. piscicida to invade and reproduce in host cells and to infect host tissue. In contrast to TX01, TX01ΔhutZ was defective in blocking host macrophage activation. The expression of hutZEp was directly regulated by the ferric uptake regulator Fur. This study is the first functional characterization of HutZ in a fish pathogen, and these findings suggested that HutZEp is essential for E. piscicida biofilm formation and contributes to host infection.

Thioredoxin H (TrxH) contributes to adversity adaptation and pathogenicity of Edwardsiella piscicida.

Thioredoxins (Trxs) play an important role in defending against oxidative stress and keeping disulfide bonding correct to maintain protein function. Edwardsiella piscicida, a severe fish pathogen, has been shown to encode several thioredoxins including TrxA, TrxC, and TrxH, but their biological roles remain unknown. In this study, we characterized TrxH of E. piscicida (named TrxHEp) and examined its expression and function. TrxHEp is composed of 125 residues and possesses typical thioredoxin H motifs. Expression of trxHEp was upregulated under conditions of oxidative stress, iron starvation, low pH, and during infection of host cells. trxHEp expression was also regulated by ferric uptake regulator (Fur), an important global regulatory of E. piscicida. Compared to the wild type TX01, a markerless trxHEp in-frame mutant strain TX01∆trxH exhibited markedly compromised tolerance of the pathogen to hydrogen peroxide, acid stress, and iron deficiency. Deletion of trxHEp significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis shows that the inactivation of trxHEp significantly impaired the ability of E. piscicida to invade host cells, reproduce in macrophages, and infect host tissues. Introduction of a trans-expressed trxH gene restored the lost virulence of TX01∆trxH. There is likely to be a complex relationship of functional complementation or expression regulation between TrxH and another two thioredoxins, TrxA and TrxC, of E. piscicida. This is the first functional report of TrxH in fish pathogens, and the findings suggest that TrxHEp is essential for coping with adverse circumstances and contributes to host infection of E. piscicida.

Global profiling and characterization of Japanese flounder (Paralichthys olivaceus) kidney microRNAs regulated by Edwardsiella tarda infection in a time-dependent fashion.

Japanese flounder (Paralichthys olivaceus) is an important economic fish species farmed in China and other countries. It is susceptible to infection by Edwardsiella tarda, a severe fish pathogen with a broad host range. In this study, we employed high-throughput deep sequencing technology to identify, in a global scale, flounder kidney microRNAs (miRNAs) induced by E. tarda at different stages of infection. Differentially expressed miRNAs (DEmiRNAs) and mRNAs (DEmRNAs) exhibiting significantly altered expression levels before and after E. tarda infection were examined. A total of 96 DEmiRNAs were identified, for which 2779 target genes were predicted. Eighty-seven miRNA-mRNA pairs, involving 29 DEmiRNAs and 86 DEmRNAs, showed negative correlations in their expression patterns. GO and KEGG enrichment analysis revealed that the putative target genes of the DEmiRNAs were associated with diverse biological processes, cellular components, and molecular functions. One of the DEmiRNAs, pol-miR-182-5p, was demonstrated to regulate sphingosine-1-phosphate receptor 1 (PoS1PR1) negatively in a manner that depended on the specific interaction between the seed sequence of pol-miR-182-5p and the 3'-UTR of PoS1PR1. Overexpression of pol-miR-182-5p in flounder cells promoted apoptosis and inhibited cellular viability. Knockdown of PoS1PR1 in flounder enhanced E. tarda invasion and dissemination in fish tissues. These results provide new insights into miRNA-mediated anti-bacterial immunity in flounder.

Granulocyte colony stimulating factor (GCSF) of Japanese flounder (Paralichthys olivaceus): Immunoregulatory property and anti-infectious function.

Granulocyte colony stimulating factor (GCSF) is a key regulator of neutrophil production, and plays a vital role in immune response of mammals and teleost against pathogen. Sequences of GCSF were identified in several teleost species, however, the function and activity of GCSF in teleost remain largely unknown. In this study, we examined the biological activity and the immunomodulatory property of a GCSF homologue, PoGCSF, from Japanese flounder (Paralichthys olivaceus). Structural analysis showed that PoGCSF possesses conserved structural characteristics of GCSF proteins, including a signal peptide and a typical IL-6 domain. The expression of PoGCSF was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoGCSF increased the capability of host cells to defense against pathogen infection and enhanced the expression of immune related genes. The knockdown of PoGCSF attenuated the ability of host cells to eliminate pathogenic bacteria. In vivo results showed that overexpression of PoGCSF promoted the host defense against invading pathogenic microorganism. Collectively, this study is the first report about the immunoregulatory property and anti-infectious immunity of GCSF in teleost. These findings suggested that PoGCSF serves as an immune-related cytokine and plays an important role in the immune defense system of Japanese flounder.

Universal stress proteins contribute Edwardsiella piscicida adversity resistance and pathogenicity and promote blocking host immune response.

Universal stress proteins (Usps) exist ubiquitously in bacteria and other organisms. Usps play an important role in adaptation of bacteria to a variety of environmental stresses. There is increasing evidence that Usps facilitate pathogens to adapt host environment and are involved in pathogenicity. Edwardsiella piscicida (formerly included in E. tarda) is a severe fish pathogen and infects various important economic fish including tilapia (Oreochromis niloticus). In E. piscicida, a number of systems and factors that are involved in stress resistance and pathogenesis were identified. However, the function of Usps in E. piscicida is totally unknown. In this study, we examined the expressions of 13 usp genes in E. piscicida and found that most of these usp genes were up-regulated expression under high temperature, oxidative stress, acid stress, and host serum stress. Particularly, among these usp genes, usp13, exhibited dramatically high expression level upon several stress conditions. To investigate the biological role of usp13, a markerless usp13 in-frame mutant strain, TX01Δusp13, was constructed. Compared to the wild type TX01, TX01Δusp13 exhibited markedly compromised tolerance to high temperature, hydrogen peroxide, and low pH. Deletion of usp13 significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis showed that the inactivation of usp13 significantly impaired the ability of E. piscicida to invade into host cell and infect host tissue. Introduction of a trans-expressed usp13 gene restored the lost virulence of TX01Δusp13. In support of these results, host immune response induced by TX01 and TX01Δusp13 was examined, and the results showed reactive oxygen species (ROS) levels in TX01Δusp13-infected macrophages were significantly higher than those in TX01-infected cells. The expression level of several cytokines (IL-6, IL-8, IL-10, TNF-α, and CC2) in TX01Δusp13-infected fish was significantly higher than that in TX01-infected fish. These results suggested that the deletion of usp13 attenuated the ability of bacteria to overcome the host immune response to pathogen infection. Taken together, our study indicated Usp13 of E. piscicida was not only important participant in adversity resistance, but also was essential for E. piscicida pathogenicity and contributed to block host immune response to pathogen infection.

Major histocompatibility complex class I (MHC Iα) of Japanese flounder (Paralichthys olivaceus) plays a critical role in defense against intracellular pathogen infection.

The major histocompatibility complex (MHC) is a highly polymorphic region of the vertebrate genome that plays a critical role in initiating immune responses towards invading pathogens. It is well known that MHC I molecules play a central role in the immune response to viruses. However, rare literatures were reported the role of MHC I in the resistance to intracellular bacteria. Sequences of MHC Iα were identified in multiple teleost species, including Japanese flounder (Paralichthys olivaceus), however, the immunological function of MHC Iα remain largely unknown. In this study, we examined the expression profile and biological activity of an MHC Iα homologue, PoMHC Iα, from P. olivaceus. Structural analysis showed that PoMHC Iα possesses conserved structural characteristics of MHC Iα proteins, including MHC_I domain, IGc1 domain, transmembrane region. Expression of PoMHC Iα was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen infection. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoMHC Iα increased the capability of host cells to defense against intracellular pathogen Edwardsiella tarda infection and enhanced the expression of immune related genes. The knockdown of PoMHC Iα attenuated the ability of cells to eliminate E. tarda, which was sustained by the in vivo results that overexpression of PoMHC Iα promoted the host defense against invading E. tarda. Antigen uptake assay indicated PoMHC Iα participated in cells antigen presentation. Collectively, this study is the first report that MHC Iα plays an important role in immune defense against intracellular bacterial pathogen in teleost. Taken together, these findings add new insights into the biological function of teleost MHC Iα and emphasize the importance of MHC I gene products for the control of E. tarda infection.

Characterization, expression, enzymatic activity, and functional identification of cathepsin S from black rockfish Sebastes schlegelii.

Cathepsin S belong to the cathepsin L-like family of cysteine cathepsins. It is well known that Cathepsin S participate in various physiological processes and host immune defense in mammals. However, in teleost fish, the function of cathepsin S is less investigated. In the present study, a cathepsin S homologue (SsCTSS) from the teleost fish black rockfish (Sebastes schlegelii) were identified and examined at expression and functional levels. In silico analysis showed that three domains, including signal peptide, cathepsin propeptide inhibitor I29 domain, and functional domain Pept_C1, were existed in the cathepsin. SsCTSS possesses a peptidase domain with three catalytically essential residues (Cys25, His162, and Asn183). Phylogenetic profiling indicated that SsCTSS are evolutionally close to the cathepsin S of other teleost fish. The expression of SsCTSS in immune-related tissues was upregulated in a time-dependent manner upon bacterial pathogen infection. Purified recombinant SsCTSS (rSsCTSS) exhibited apparent peptidase activity, which was remarkably declined in the presence of the cathepsin inhibitor E-64. rSsCTSS showed strong binding ability to LPS and PGN, the major constituents of the outer membranes of Gram-negative and Gram-positive bacteria, respectively. rSsCTSS also exhibited the capability of agglutination to different bacteria. The knockdown of SsCTSS attenuated the ability of host to eliminate pathogenic bacteria. Taken together, our results suggested that SsCTSS functions as cysteine protease which might be involved in the antibacterial immunity of black rockfish.

A novel calreticulin-related molecule that interacts with bacteria and enhances host resistance against bacterial infection in black rockfish, Sebastes schlegeli.

Calreticulin (CRT) is a highly conserved and multi-functional protein with diverse localizations. CRT has lectin-like properties and possesses important immunological activities in mammalian. In teleost, very limited studies on CRT immunologic function have been documented. In the present study, a CRT homologue (SsCRT) was cloned, identified and characterized from black rockfish, Sebastes schlegeli, an important aquaculture species in East Asia. The full length of SsCRT cDNA is 2180 bp and encoded a polypeptide of 425 amino acids. SsCRT contains a signal peptide, three distinct structural and functional domains (N-, P- and C-domains), and an endoplasmic reticulum (ER) retrieval signal sequence (KDEL). The deduced amino acid sequence of SsCRT shares 89-92% overall sequence identities with the CRT proteins of several fish species. SsCRT was distributed ubiquitously in all the detected tissues and was highly expressed in the spleen, muscle and liver. After the infection of fish extracellular bacterial pathogen Vibrio anguillarum and intracellular bacterial pathogen Edwardsiella tarda, the mRNA transcripts of SsCRT in spleen, liver, and head kidney were significantly up-regulated. The expression patterns were time-dependent and tissue-dependent. Recombinant SsCRT (rSsCRT) exhibited apparent binding activities against different bacteria and PAMPs. In vivo studies showed that the expressions of multiple immune-related genes such as TNF13B, IL-1ß, IL-8, SAA, Hsp70, and ISG15 in head kidney were significantly enhanced when black rockfish were treated with rSsCRT. Furthermore, rSsCRT reduced pathogen dissemination and replication in fish kidney and spleen. These results indicated that SsCRT served as an immune receptor to recognize and eliminate the invading pathogens, which played a vital role in the immune response of Sebastes schlegeli. These findings provide new insights into understanding the roles of CRT proteins in immune response and pathogen infection in teleost.

High mobility group box 2 of black rockfish Sebastes schlegelii: Gene cloning, immunoregulatory properties and antibacterial effect.

High-mobility group box 2 (HMGB2) is a non-histone chromosomal protein that involved diverse functions such as transcriptional regulation and innate immune responses in mammalian. In teleost, very limited studies on HMGB2 proteins have been documented. Black rockfish (Sebastes schlegelii) is an economic fish species and cultured worldwide. However, the study of black rockfish about immunology is very scarce. In the present study, a HMGB2 homologue gene (SsHMGB2) was identified and characterized in black rockfish. The open reading frame of SsHMGB2 is 648 bp, and the deduced amino acid sequence of SsHMGB2 shares 74.4%-91.2% overall sequence identities with the HMGB2 proteins of several fish species. In silico analysis identified several conserved features, including two basic HMG boxes and an acidic C-terminal tail composed of 24 Asp/Glu residues. Expression of SsHMGB2 occurred in multiple tissues and was upregulated during pathogens infection. Recombinant SsHMGB2 (rSsHMGB2) exhibited apparent binding activities against DNA. In vivo studies showed that the expressions of multiple immune-related genes in head kidney were significantly enhanced when black rockfish were treated with rSsHMGB2. Furthermore, rSsHMGB2 reduced pathogen dissemination and replication in fish kidney and spleen. Taken together, these results suggest that SsHMGB2 possesses apparent immunoregulatory properties and played a role in fighting bacterial infection.