Differential expression of microRNAs in shrimp Marsupenaeus japonicus in response to Vibrio alginolyticus infection

Till date numerous microRNAs (miRNAs) have been discovered from various organisms, including mammals, plants, insects, nematodes and viruses. They are known to have antiviral functions in crustaceans such as shrimp Marsupenaeus japonicas. However, little is known about the role of miRNAs against bacterial infection in this shrimp caused by Vibrio alginolyticus. We performed small RNA sequencing to characterize the differentially expressed microRNAs in V. alginolyticus challenged shrimp, in comparison to that in control uninfected shrimp, at 24 h and 48 h. In total, 55 host miRNAs were differentially expressed in response to the infection and most of these were downregulated at both the time-points. TargetScan and miRanda algorithms showed that the target genes of these down-regulated miRNAs were related to innate immune functions such as production of phenoloxidase enzyme, apoptosis and phagocytosis. Further, gene ontology analysis revealed that many immune signaling pathways were mediated by these miRNAs. This study is one of the earliest attempts at characterizing shrimp miRNAs that respond to V. alginolyticus infection, and will help unravel the miRNA pathways involved in antibacterial action in shrimp.     

Dual oxidases participate in the regulation of intestinal microbiotic homeostasis in the kuruma shrimp Marsupenaeus japonicus

The metazoan gut lumen harbors numerous microbial communities. Tolerance for high bacterial counts and maintenance of microbiota homeostasis remain insufficiently studied. In this study, we identified a novel dual oxidase (MjDUOX2) involved in reactive oxygen species (ROS) production in the kuruma shrimp Marsupenaeus japonicus. MjDUOX2 is a transmembrane protein with an N-signal peptide region (19 aa) and a peroxidase homology domain (PHD, 554 aa) in the extracellular region; seven transmembrane regions; and three EF (calcium-binding region) domains (110 aa), a FAD-binding domain (104 aa), and a NAD-binding domain (156 aa) in the intracellular region. The novel MjDUOX2 exhibits a relatively low similarity (26.84% identity) to a previously reported DUOX in the shrimp (designated as MjDUOX1). The mRNA of MjDUOXs was widely distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Oral infection of the shrimp with pathogenic bacteria upregulated the mRNA expression of MjDUOXs and increased the ROS level in the intestine. However, High ROS level could inhibit the expression of MjDUOXs in shrimp after Vibrio anguillarum infection. Knockdown of MjDUOXs by RNA interference (RNAi) decreased the ROS level, increased the bacterial count in the intestine, and decreased the survival rate of the MjDUOX-RNAi shrimp infected with V. anguillarum. These results suggest that MjDUOXs play an important role for microbiota homeostasis in intestine of shrimp.     

The role of crustins in Litopenaeus vannamei in response to infection with shrimp pathogens: An in vivo approach

Crustin antimicrobial peptides, identified in crustaceans, are hypothesized to have both antimicrobial and protease inhibitor activity based on their primary structure and in vitro assays. In this study, a reverse genetic approach was utilized to test the hypothesis that crustins are antimicrobial in vivo in response to bacterial and fungal challenge. Injection of double-stranded RNA specific to a 120-bp region of LvABP1, one of the most prominent crustin isoforms, yielded a significant reduction in the expression of both crustin mRNA and protein within the hemocytes. To test the role of crustins in the shrimp immune response, RNAi was first used to suppress crustin expression and animals were subsequently injected with low pathogenic doses of either Vibrio penaeicida or Fusarium oxysporum. A significant increase in mortality in crustin-depleted animals was observed in animals infected with V. penaeicida as compared to controls, whereas no significant change in shrimp mortality was observed following infection with F. oxysporum.     

Immunization with the Haemophilus ducreyi Hemoglobin Receptor HgbA with Adjuvant Monophosphoryl Lipid A Protects Swine from a Homologous but Not a Heterologous Challenge

Haemophilus ducreyi, the etiological agent of chancroid, has a strict requirement for heme, which it acquires from its only natural host, humans. Previously, we showed that a vaccine preparation containing the native hemoglobin receptor HgbA purified from H. ducreyi class I strain 35000HP (nHgbA_I) and administered with Freund''s adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbA_I dispensed with monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, offered protection against a challenge with H. ducreyi strain 35000HP expressing either class I or class II HgbA (35000HPhgbA_I and 35000HPhgbA_II, respectively). Pigs immunized with the nHgbA_I/MPL vaccine were protected against a challenge from homologous H. ducreyi strain 35000HPhgbA_I but not heterologous strain 35000HPhgbA_II, as evidenced by the isolation of only strain 35000HPhgbA_II from nHgbA_I-immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbA_I-immunized animals challenged with strains 35000HPhgbA_I but not those challenged with strain 35000HPhgbA_II. Mock-immunized pigs were not protected from a challenge by either strain. The enzyme-linked immunosorbent assay (ELISA) activity of the nHgbA_I/MPL antiserum was lower than the activity of antiserum from animals immunized with the nHgbA_I/Freund''s vaccine; however, anti-nHgbA_I from both studies bound whole cells of 35000HPhgbA_I better than 35000HPhgbA_II and partially blocked hemoglobin binding to nHgbA_I. In conclusion, despite eliciting lower antibody ELISA activity than the nHgbA_I/Freund''s, the nHgbA_I/MPL vaccine provided protection against a challenge with homologous but not heterologous H. ducreyi, suggesting that a bivalent HgbA vaccine may be needed.Chancroid is one of the genital ulcer diseases and is transmitted through sexual contact. Lesions caused by chancroid initially appear as papules, which evolve within several days into pustules. If left untreated, chancroid pustules develop into painful, bleeding ulcers with soft, irregular borders. Chancroid is prevalent in certain developing countries but is rarely found in the United States (47, 62). Several studies have shown that chancroid serves as an important independent cofactor in the heterosexual transmission of HIV where both diseases are endemic (29, 35, 48, 51, 66). Commercial sex workers serve as the reservoir of chancroid, and control of disease in this population strikingly reduces the number of cases of chancroid in their male clients (58). Thus, one possible approach to control chancroid is to implement a limited vaccination program to control infection in this reservoir; however, no vaccine for chancroid currently exists.Haemophilus ducreyi, the etiologic agent of chancroid, is a fastidious Gram-negative bacterium and a strict human pathogen. An interesting biologic feature of H. ducreyi is its obligate requirement for heme. Heme (for H. ducreyi) and iron are critical nutrients required for most pathogenic bacteria. Many Gram-negative bacteria obtain heme/Fe through systems that include TonB-dependent outer membrane receptors specific for heme/Fe compounds. The relatively small genome of prototypical H. ducreyi strain 35000HP encodes only three TonB-dependent receptors; in comparison, other bacterial genomes can encode more than 30 (14). Using isogenic mutants, the Spinola and Elkins laboratories surveyed the ability of H. ducreyi TonB-dependent receptor mutants to initiate infection in the human experimental model of chancroid. An H. ducreyi mutant that does not express the gene encoding the hemoglobin (Hb) receptor, hgbA, did not establish human infection (3). In contrast, an isogenic double mutant lacking the genes encoding the two other TonB-dependent receptors of H. ducreyi, tdhA and tdX, was fully virulent, indicating that HgbA is the only TonB-dependent receptor of H. ducreyi to be a virulence factor in the human experimental model of chancroid (38). Since HgbA is required for the utilization of heme from Hb by H. ducreyi (17), these data suggest that Hb is the most important source of heme in the early stages of the human experimental model of chancroid and that HgbA is a potential vaccine candidate.HgbA is a large, 100-kDa outer membrane protein that has a complex structure similar to that of other TonB-dependent receptors whose structure has been solved (10, 12, 20, 21, 40). HgbA is believed to contain 22 transmembrane beta sheets and 11 putatively surface-exposed loops. A recent study in our laboratory using H. ducreyi mutants expressing single loop deletions in HgbA provided evidence for surface exposure of loops 4, 5, 6, and 7 (44). Moreover, deletions of loops 5 and 7 but not of the other 9 loops of HgbA abrogated the binding of human Hb to HgbA. We also found that IgG from pigs immunized with native HgbA (nHgbA) bound loops 4, 5, and 7 and that antibodies directed at loops 4 and 5 partially blocked Hb binding to HgbA in vitro. Thus, a central domain of the primary sequence of HgbA is immunogenic, required for binding Hb, and surface exposed.H. ducreyi strains exist in two groups, designated class I and class II, based on striking primary sequence differences in certain outer membrane proteins, such as DsrA (ducreyi serum resistance A) and NcaA (necessary for collagen adhesion A), and on their lipooligosaccharide (LOS) structures (49, 50, 53, 67). In contrast, the HgbA proteins of different classes of H. ducreyi strains are more than 95% identical. Prototypical H. ducreyi strain 35000HP, a class I isolate, is the strain used for most studies, including isogenic mutant construction and the experimental human model of chancroid. 35000HP is the only H. ducreyi strain whose genome has been sequenced.Previously, we showed that immunization of swine with native HgbA from class I strain 35000HP (nHgbA_I) in Freund''s adjuvant provided complete protection from a homologous challenge infection with H. ducreyi strain 35000HP (1). The antibodies elicited by nHgbA_I/Freund''s showed modest bactericidal activity, bound to the cell surface of both class I and class II H. ducreyi strains, and partially blocked Hb binding to nHgbA (1). nHgbA_I antisera did not recognize the surface of, nor did they show bactericidal activity against the isogenic hgbA mutant, demonstrating specificity of the humoral response to HgbA.In the current study, we pursued two objectives. First, we investigated the effectiveness of monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, to elicit an immune response to the nHgbA_I vaccine that is protective against an H. ducreyi challenge in the experimental swine model of chancroid. Second, we examined the ability of this vaccine to protect swine from a challenge infection with H. ducreyi strain 35000HP expressing either class I hgbA (hgbA_I) or class II hgbA (hgbA_II) from H. ducreyi strain DMC111 (homologous versus heterologous challenge, respectively).     

Complete genome sequence of goose parvovirus Y strain isolated from Muscovy ducks in China

A goose parvovirus (GPV) Y strain was isolated from Muscovy ducks in Anhui Province of China. By polymerase chain reaction method, its complete genomic sequence was found to be 5,106 bp in length, consisting of 444-bp inverted terminal repeat, 1,844-bp non-structural protein and 2,199-bp capsid protein (VP) regions. Then its sequence was aligned with the sequences of GPV and Muscovy duck parvovirus published in the GenBank using the neighbor-joining method. The phylogenetic analyses based on the VP3 gene sequences revealed that the GPV Y strain along with those from Taiwan belonged to the subgroup IIb, while other GPV strains from Muscovy ducks belonged to the subgroup Ib and most of other GPV strains isolated in China mainland were clustered in the subgroup IIa. The absence of the deduced 703–705NRT glycosylation site in VP region may explain the host specificity of the GPV Y strain. The complete genomic sequence of the GPV Y strain from Muscovy ducks will help to understand the molecular and evolutionary characteristics of GPV.     

Isolation of the structural polypeptides of foot-and-mouth disease virus and analysis of their C-terminal sequences

The three polypeptides—VP₁, VP₂ and VP₃—which comprise approximately 91% of the protein of foot-and-mouth disease virus, type A₁₂-119, were separated by polyacrylamide gel electrophoresis (PAGE) in sufficient purity and quantity for determination of their amino acid compositions as well as for analysis of their C-terminal sequences and molecular weights by reaction with carboxypeptidase-A. At least seven distinct differences in amino acid compositions were observed among the three polypeptides, but their average composition was nearly identical to that of the total protein of the virus, VP_0–4. C-Terminal amino acid sequences for VP₁ and VP₃ appear to to be (—serine-glutamine) and (—glutamine-alanine-leucine), respectively, and the C-terminal amino acid of VP₂ is probably glutamic acid. Molecular weights for VP₁ and VP₃ calculated from C-terminal data were in the 27,500–31,000 dalton range compared with values of 28,500 determined previously by PAGE.     

Leptospira interrogans Catalase Is Required for Resistance to H2O2 and for Virulence

Pathogenic Leptospira spp. are likely to encounter higher concentrations of reactive oxygen species induced by the host innate immune response. In this study, we characterized Leptospira interrogans catalase (KatE), the only annotated catalase found within pathogenic Leptospira species, by assessing its role in resistance to H₂O₂-induced oxidative stress and during infection in hamsters. Pathogenic L. interrogans bacteria had a 50-fold-higher survival rate under H₂O₂-induced oxidative stress than did saprophytic L. biflexa bacteria, and this was predominantly catalase dependent. We also characterized KatE, the only annotated catalase found within pathogenic Leptospira species. Catalase assays performed with recombinant KatE confirmed specific catalase activity, while protein fractionation experiments localized KatE to the bacterial periplasmic space. The insertional inactivation of katE in pathogenic Leptospira bacteria drastically diminished leptospiral viability in the presence of extracellular H₂O₂ and reduced virulence in an acute-infection model. Combined, these results suggest that L. interrogans KatE confers in vivo resistance to reactive oxygen species induced by the host innate immune response.     

Transcriptome profiling of zebrafish infected with Streptococcus suis

Streptococcus suis is an important pathogen in swine, and it also represents an emerging zoonotic agent. Zebrafish as a model for the evaluation of virulence of S. suis has been demonstrated before. Here, an Affymetrix Zebrafish GeneChip was used to identify alterations in gene expression of zebrafish injected with S. suis serotype 2 strain HA9801. The results showed that 189 genes were differentially expressed, of which 125 genes were upregulated and 64 genes were downregulated. Gene Ontology category and KEGG pathway were analyzed for differentially expressed genes. Upregulated genes were involved in response to bacterium, immune response, inflammatory response, complement activation, defense response. Three genes (encoding serum amyloid protein A, matrix metalloproteinase 9 and apoptosis-related cysteine protease) and genes involved in the regulation of IL-6 biosynthetic process, which have previously been implicated in the response to S. suis infection in other organisms, were also upregulated. Downregulated genes played roles in glycolysis, carbohydrate metabolic process, amino acids metabolism, behavior and muscle. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR on 12 representative genes. The data may provide further validation of this model, which will contribute to understanding of S. suis pathogenic mechanisms.     

Identification and characterization of Cynoglossus semilaevis microRNA response to Vibrio anguillarum infection through high-throughput sequencing

MicroRNAs (miRNA) play key regulatory roles in diverse biological processes. Cynoglossus semilaevis is an important commercial mariculture fish species in China. To identify miRNAs and investigate immune-related miRNAs of C. semilaevis, we performed high-throughput sequencing on three small RNA libraries prepared from C. semilaevis immune tissues (liver, head kidney, spleen, and intestine). One library was prepared under normal conditions (control, CG); two were prepared during Vibrio anguillarum infection, where vibriosis symptoms were obvious and non-obvious (HOSG and NOSG, respectively). We obtained 11,216,875, 12,313,404, and 11,398,695 clean reads per library, respectively. Bioinformatic analysis identified 452 miRNAs, including 24 putative novel miRNAs. We analyzed differentially expressed miRNAs between two libraries using pairwise comparison. For NOSG–CG, there was significant differential expression of 175 (38.72%) miRNAs. There was significant differential expression of 215 (47.57%) miRNAs between HOSG and CG. Compared with CG, The HOSG–NOSG comparison revealed significantly different expression of 122 (26.99%) miRNAs respectively. Real-time quantitative PCR (RT-qPCR) experiments were performed for 10 miRNAs of the three samples, and agreement was found between the sequencing and RT-qPCR data. For miRNAs that were significantly differentially expressed, functional annotation of target genes by Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that a set of miRNAs that were expressed highly abundantly and significantly differentially were might involved in immune system development and immune response. To our understanding, this is the first report of comprehensive identification of C. semilaevis miRNAs being differentially regulated in immune tissues (liver, head kidney, spleen, and intestine) in normal conditions relating to V. anguillarum infection. Many miRNAs were differentially regulated upon pathogen exposure. This work provides an opportunity for further understanding of the molecular mechanisms of miRNA regulation in C. semilaevis host–pathogen interactions.     

Induction of cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza virus infection in mice by inoculation of apathogenic H5N1 influenza virus particles inactivated with formalin

We investigated whether a vaccine derived from an apathogenic reassortant type A H5N1 influenza strain could induce immune responses in vivo that mediated protection from highly pathogenic avian influenza virus infection in mice. After two subcutaneous immunizations with formalin-inactivated H5N1 whole virus particles (whole particle vaccine), significant killing specific for cells presenting a nucleoprotein peptide from the vaccine strain of the virus was observed. Similar vaccination with viruses treated with ether and formalin, which are commonly used for humans as ether-split vaccines, induced little or no cytotoxic T-cell response. Furthermore, whole particle vaccines of the apathogenic H5N1 strain were more effective than ether-split vaccines at inducing antibody production able to neutralize a highly pathogenic H5N1 strain. Finally, whole particle vaccines of H5N1 protected mice against infection by an H5N1 highly pathogenic avian influenza virus more effectively than did ether-split vaccines. These results suggest that formalin-inactivated virus particles of apathogenic strains are effective for induction of both cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza viruses in vivo, resulting in protection from infection by a highly pathogenic H5N1 virus.     

A global assembly of cotton ESTs

Approximately 185,000 Gossypium EST sequences comprising >94,800,000 nucleotides were amassed from 30 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including drought stress and pathogen challenges. These libraries were derived from allopolyploid cotton (Gossypium hirsutum; A_T and D_T genomes) as well as its two diploid progenitors, Gossypium arboreum (A genome) and Gossypium raimondii (D genome). ESTs were assembled using the Program for Assembling and Viewing ESTs (PAVE), resulting in 22,030 contigs and 29,077 singletons (51,107 unigenes). Further comparisons among the singletons and contigs led to recognition of 33,665 exemplar sequences that represent a nonredundant set of putative Gossypium genes containing partial or full-length coding regions and usually one or two UTRs. The assembly, along with their UniProt BLASTX hits, GO annotation, and Pfam analysis results, are freely accessible as a public resource for cotton genomics. Because ESTs from diploid and allotetraploid Gossypium were combined in a single assembly, we were in many cases able to bioinformatically distinguish duplicated genes in allotetraploid cotton and assign them to either the A or D genome. The assembly and associated information provide a framework for future investigation of cotton functional and evolutionary genomics.     

YbcL of Uropathogenic Escherichia coli Suppresses Transepithelial Neutrophil Migration

Uropathogenic Escherichia coli (UPEC) strains suppress the acute inflammatory response in the urinary tract to ensure access to the intracellular uroepithelial niche that supports the propagation of infection. Our understanding of this initial cross talk between host and pathogen is incomplete. Here we report the identification of a previously uncharacterized periplasmic protein, YbcL, encoded by UPEC that contributes to immune modulation in the urinary tract by suppressing acute neutrophil migration. In contrast to wild-type UPEC, an isogenic strain lacking ybcL expression (UTI89 ΔybcL) failed to suppress transepithelial polymorphonuclear leukocyte (PMN) migration in vitro, a defect complemented by expressing ybcL episomally. YbcL homologs are present in many E. coli genomes; expression of the YbcL variant encoded by nonpathogenic E. coli K-12 strain MG1655 (YbcL_MG) failed to complement the UTI89 ΔybcL defect, whereas expression of the UPEC YbcL variant (YbcL_UTI) in MG1655 conferred the capacity for suppressing PMN migration. This phenotypic difference was due to a single amino acid difference (V78T) between the two YbcL homologs, and a majority of clinical UPEC strains examined were found to encode the suppressive YbcL variant. Purified YbcL_UTI protein suppressed PMN migration in response to live or killed MG1655, and YbcL_UTI was detected in the supernatant during UPEC infection of bladder epithelial cells or PMNs. Lastly, early PMN influx to murine bladder tissue was augmented upon in vivo infection with UTI89 ΔybcL compared with wild-type UPEC. Our findings demonstrate a role for UPEC YbcL in suppression of the innate immune response during urinary tract infection.