Expression characterization and activity analysis of a cathepsin B from Pacific abalone Haliotis discus hannai

Cathepsin B (EC 3.4.22.1) is a member of lysosomal cysteine protease and has a papain-like fold. In mammals, it is involved in protein degradation and other physiological processes including immune response. However, little is known about the function of cathepsin B in mollusks. In this study, we identified and analyzed a cathepsin B homolog (HdCatB) from Pacific abalone (Haliotis discus hannai), an economically important mollusk species cultured in East Asia. HdCatB is composed of 336 amino acid residues and its mature form is predicted to start at residue 86. HdCatB possesses typical domain architecture of cathepsin B and contains a propeptide region and a cysteine protease domain, the latter containing the four active site residues (Q108, C114, H282, and N302) that are conserved in many different organisms. HdCatB shares 40–60% overall sequence identities with the cathepsin Bofa number of vertebrates and invertebrates and is phylogenetically very close to mollusk cathepsin B. Quantitative real time RT-PCR analysis revealed that HdCatB expression occurred in multiple tissues and was upregulated by bacterial infection. Recombinant HdCatB purified from Escherichia coli exhibited apparent protease activity, which was optimal at 45 °C and pH 6.0. These results indicate that HdCatB is a bioactive protease that is likely to be implicated in the immune response of abalone during bacterial infection.     

Cytogenetic Analysis and Chromosomal Characteristics of the Polymorphic 18S rDNA of Haliotis discus hannai from Fujian,China

We report on novel chromosomal characteristics of Haliotis discus hannai from a breeding population at Fujian, China. The karyotypes of H. discus hannai we obtained from an abalone farm include a common type 2n = 36 = 10M + 8SM (82%) and two rare types 2n = 36 = 11M + 7SM (14%) and 2n = 36 = 10M + 7SM + 1ST (4%). The results of silver staining showed that the NORs of H. discus hannai were usually located terminally on the long arms of chromosome pairs 14 and 17, NORs were also sometimes located terminally on the short arms of other chromosomes, either metacentric or submetacentric pairs. The number of Ag-nucleoli ranged from 2 to 8, and the mean number was 3.61 ± 0.93. Among the scored interphase cells, 41% had 3 detectable nucleoli and 37% had 4 nucleoli. The 18S rDNA FISH result is the first report of the location of 18S rDNA genes in H. discus hannai. The 18S rDNA locations were highly polymorphic in this species. Copies of the gene were observed in the terminal of long or/and short arms of submetacentric or/and metacentric chromosomes. Using FISH with probe for vertebrate-like telomeric sequences (CCCTAA)₃ displayed positive green FITC signals at telomere regions of all analyzed chromosome types. We found about 7% of chromosomes had breaks in prophase. A special form of nucleolus not previously described from H. discus hannai was observed in some interphase cells. It consists of many small silver-stained nucleoli gathered together to form a larger nucleolus and may correspond to prenucleolar bodies.     

Immunization with Heat Shock Protein A and γ-Glutamyl Transpeptidase Induces Reduction on the Helicobacter pylori Colonization in Mice

The human gastric pathogen Helicobacter pylori (H. pylori) is a successful colonizer of the stomach. H. pylori infection strongly correlates with the development and progression of chronic gastritis, peptic ulcer disease, and gastric malignances. Vaccination is a promising strategy for preventing H. pylori infection. In this study, we evaluated the candidate antigens heat shock protein A (HspA) and H. pylori γ-glutamyl transpeptidase (GGT) for their effectiveness in development of subunit vaccines against H. pylori infection. rHspA, rGGT, and rHspA-GGT, a fusion protein based on HspA and GGT, were constructed and separately expressed in Escherichia coli and purified. Mice were then immunized intranasally with these proteins, with or without adjuvant. Immunized mice exhibited reduced bacterial colonization in stomach. The highest reduction in bacterial colonization was seen in mice immunized with the fusion protein rHspA-GGT when paired with the mucosal adjuvant LTB. Protection against H. pylori colonization was mediated by a strong systemic and localized humoral immune response, as well as a balanced Th1/Th2 cytokine response. In addition, immunofluorescence microscopy confirmed that rHspA-GGT specific rabbit antibodies were able to directly bind H. pylori in vitro. These results suggest antibodies are essential to the protective immunity associated with rHspA-GGT immunization. In summary, our results suggest HspA and GGT are promising vaccine candidates for protection against H. pylori infection.     

Contribution of Many Charged Residues at the Stator-Rotor Interface of the Na+-Driven Flagellar Motor to Torque Generation in Vibrio alginolyticus

In torque generation by the bacterial flagellar motor, it has been suggested that electrostatic interactions between charged residues of MotA and FliG at the rotor-stator interface are important. However, the actual role(s) of those charged residues has not yet been clarified. In this study, we systematically made mutants of Vibrio alginolyticus whose charged residues of PomA (MotA homologue) and FliG were replaced by uncharged or charge-reversed residues and characterized the motilities of those mutants. We found that the members of a group of charged residues, 7 in PomA and 6 in FliG, collectively participate in torque generation of the Na⁺-driven flagellar motor in Vibrio. An additional specific interaction between PomA-E97 and FliG-K284 is critical for proper performance of the Vibrio motor. Our results also reveal that more charged residues are involved in the PomA-FliG interactions in the Vibrio Na⁺-driven motor than in the MotA-FliG interactions in the H⁺-driven one. This suggests that a larger number of conserved charged residues at the PomA-FliG interface contributes to the robustness of the Vibrio motor against mutations. The interaction surfaces of the stator and rotor of the Na⁺-driven motor seem to be more complex than those previously proposed in the H⁺-driven motor.     

Comparative analysis of oyster (Crassostrea gigas) immune responses under challenge by different Vibrio strains and conditions

Screening a virulent Vibrio strain to study the interactions between bacterial infection and host immune defence is important to resolve large-scale summer oyster mortalities. Using adductor muscle injection we tested the oyster responses to phosphate-buffered saline (PBS), lipopolysaccharide (LPS), or Vibrio strains. Mortalities and stress gene expressions were used to characterize oyster immune responses. At 5 days post injection, the cumulative mortality rates in PBS, LPS, Vibrio tubiashii, Vibrio anguillarum, Vibrio alginolyticus and Vibrio aestuarianus groups were 8%, 16%, 36%, 40%, 40% and 76%, respectively, indicating that V. aestuarianus induced the highest death rate. Two-factor analyses of variance revealed that expression of SOD, CAT, GPX and HSP70 was influenced by bacterial injections in a time-dependent manner. The expression of all genes increased and reached their peak 3 or 12 h after bacterial injection and then decreased. These genes could be applied as immune responsive biomarkers to monitor early changes in oysters in response to bacterial infection. The greatest changes were observed in the V. aestuarianus-injected group, which may indicate that V. aestuarianus could be used as one of the more virulent strains for experimental infections.     

Exoproteome and Secretome Derived Broad Spectrum Novel Drug and Vaccine Candidates in Vibrio cholerae Targeted by Piper betel Derived Compounds

Vibrio cholerae is the causal organism of the cholera epidemic, which is mostly prevalent in developing and underdeveloped countries. However, incidences of cholera in developed countries are also alarming. Because of the emergence of new drug-resistant strains, even though several generic drugs and vaccines have been developed over time, Vibrio infections remain a global health problem that appeals for the development of novel drugs and vaccines against the pathogen. Here, applying comparative proteomic and reverse vaccinology approaches to the exoproteome and secretome of the pathogen, we have identified three candidate targets (ompU, uppP and yajC) for most of the pathogenic Vibrio strains. Two targets (uppP and yajC) are novel to Vibrio, and two targets (uppP and ompU) can be used to develop both drugs and vaccines (dual targets) against broad spectrum Vibrio serotypes. Using our novel computational approach, we have identified three peptide vaccine candidates that have high potential to induce both B- and T-cell-mediated immune responses from our identified two dual targets. These two targets were modeled and subjected to virtual screening against natural compounds derived from Piper betel. Seven compounds were identified first time from Piper betel to be highly effective to render the function of these targets to identify them as emerging potential drugs against Vibrio. Our preliminary validation suggests that these identified peptide vaccines and betel compounds are highly effective against Vibrio cholerae. Currently we are exhaustively validating these targets, candidate peptide vaccines, and betel derived lead compounds against a number of Vibrio species.     

Isolation and Characterization of Two Lytic Bacteriophages, φSt2 and φGrn1; Phage Therapy Application for Biological Control of Vibrio alginolyticus in Aquaculture Live Feeds

Bacterial infections are a serious problem in aquaculture since they can result in massive mortalities in farmed fish and invertebrates. Vibriosis is one of the most common diseases in marine aquaculture hatcheries and its causative agents are bacteria of the genus Vibrio mostly entering larval rearing water through live feeds, such as Artemia and rotifers. The pathogenic Vibrio alginolyticus strain V1, isolated during a vibriosis outbreak in cultured seabream, Sparus aurata, was used as host to isolate and characterize the two novel bacteriophages φSt2 and φGrn1 for phage therapy application. In vitro cell lysis experiments were performed against the bacterial host V. alginolyticus strain V1 but also against 12 presumptive Vibrio strains originating from live prey Artemia salina cultures indicating the strong lytic efficacy of the 2 phages. In vivo administration of the phage cocktail, φSt2 and φGrn1, at MOI = 100 directly on live prey A. salina cultures, led to a 93% decrease of presumptive Vibrio population after 4 h of treatment. Current study suggests that administration of φSt2 and φGrn1 to live preys could selectively reduce Vibrio load in fish hatcheries. Innovative and environmental friendly solutions against bacterial diseases are more than necessary and phage therapy is one of them.