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Protective efficacy of recombinant hemolysin co-regulated protein (Hcp) of Aeromonas hydrophila in common carp (Cyprinus carpio)
Accepted Manuscript Protective Efficacy of Recombinant Hemolysin Co-regulated Protein (Hcp) of Aeromonas hydrophila in Common carp (Cyprinus carpio) Nannan Wang, Yafeng Wu, Maoda Pang, Jin Liu, Chengping Lu, Yongjie Liu PII:
S1050-4648(15)30047-4
DOI:
10.1016/j.fsi.2015.06.019
Reference:
YFSIM 3508
To appear in:
Fish and Shellfish Immunology
Received Date: 12 March 2015 Revised Date:
12 June 2015
Accepted Date: 15 June 2015
Please cite this article as: Wang N, Wu Y, Pang M, Liu J, Lu C, Liu Y, Protective Efficacy of Recombinant Hemolysin Co-regulated Protein (Hcp) of Aeromonas hydrophila in Common carp (Cyprinus carpio), Fish and Shellfish Immunology (2015), doi: 10.1016/j.fsi.2015.06.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Protective Efficacy of Recombinant Hemolysin Co-regulated Protein
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(Hcp) of Aeromonas hydrophila in Common carp (Cyprinus carpio) Nannan Wang, Yafeng Wu, Maoda Pang, Jin Liu, Chengping Lu, Yongjie Liu*
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College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095,
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China
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* Corresponding author. Tel./fax: 0086-25-84398606
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E-mail address: [email protected] (Y. Liu)
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Abstract
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Motile aeromonad septicemia (MAS) caused by Aeromonas hydrophila is one of the
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common bacterial causes of fish mortalities. Prophylactic vaccination against this and
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other diseases is essential for continued growth of aquaculture. The type VI secretion
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system (T6SS) plays a crucial role in the virulence of A. hydrophila. The hemolysin
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co-regulated protein (Hcp) is an integral component of the T6SS apparatus and is
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considered a hallmark of T6SS function. Here, the T6SS effector Hcp was expressed
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and characterized, and its immunogenicity and protective efficacy were evaluated in
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common carp (Cyprinus carpio). Hcp secretion was found to be strongly induced by
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low temperature in A. hydrophila. Immunoblot analysis demonstrated that Hcp is
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conserved among A. hydrophila strains of different origins. The vaccination with
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recombinant Hcp resulted in an increased survival (46.67%) in common carp during a
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10-day challenge time compared to non-vaccinated fish (7.14%). The vaccinated fish
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also showed the significantly increased levels of IgM antibody in serum and cytokines
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such as inerleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in kidney, spleen
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and gills. The recombinant Hcp shows promise as a vaccine candidate against A.
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hydrophila.
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Keywords: Aeromonas hydrophila; Hemolysin co-regulated protein (Hcp); Protective
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efficacy; Fish; Vaccine
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1. Introduction
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Aeromonas hydrophila is a Gram-negative bacterium widely present in 2
ACCEPTED MANUSCRIPT freshwater habitats and causes infections in humans and animals such as amphibians
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and fish [1-3]. A. hydrophila is associated with many fish diseases like hemorrhagic
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septicemia and dropsy, and leads to significant economic losses worldwide. It has
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been accepted that vaccination is an effective method to protect fish from the
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infectious bacterial diseases [4]. Several studies have demonstrated that different
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types of vaccines such as heat-killed cells, heat or formalin-inactivated bacterial
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extracts and live cells of A. hydrophila stimulate an effective response in fish that
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protects against the bacterial infection [5-8]. However, it should be noted that such
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vaccines are not always effective, especially when the expected immune response is
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directed against specific antigens. And they contain complex mixtures and undefined
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molecules that have been evidenced to interact synergistically or antagonistically and
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that can stimulate, cross-react with, inhibit or even suppress the immune response to
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specific antigens [9]. Recently, the development of the recombinant subunit vaccine
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has caught a lot of attention. This vaccine contains fragments of pathogenic
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microorganisms, which are highly purified and immunogenic antigens. This ensures
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that the antigen has a well-defined composition. Also, vaccination with a protein
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present in a range of serotypes would overcome some of the limitations of antigenic
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diversity in A. hydrophila strains. Some previous studies targeted subunit vaccine
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candidates against A. hydrophila have mainly paid attention to the outer membrane
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proteins (OMPs) [10-12]. However, in the case of A. hydrophila, vaccination with
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extracellular secreted proteins may be particularly important, because its
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pathogenicity appears to be closely related to the production of extracellular products,
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which are lost, partially, in conventional bacterin preparations. Bacterial pathogenicity critically relies on various secretion systems to deliver
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toxic molecules from the cytoplasm to the outer space [13]. Lately, a novel secretion
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system named the type VI secretion system (T6SS) was reported for several
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pathogens [14-17] and characterized as the most common secretion system of
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Gram-negative bacteria [18]. Two typical proteins of T6SS, hemolysin co-regulated
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protein (Hcp) and valine-glycine repeat protein G (VgrG) have been proved to
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function not only as structural elements of the T6SS device but also effector proteins
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[17, 19, 20]. Furthermore, many Gram-negative pathogens have been found to secrete
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these two conserved effector proteins to the outer space through T6SS [16, 17, 21-23].
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Of late, however, Hcp which used to be a static tubule was proven to be a chaperone
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and receptor of type VI secretion substrates in Pseudomonas aeruginosa [24]. T6SS
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components have been reported to concern with virulence-related mechanism of
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various bacteria. During V. cholerae infection, T6SS genes were proved to be
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essential for toxicity in Dictyostelium amoebae and mammalian J774 macrophages
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[17]. In association with P. aeruginosa, Hcp1 was detected in cystic fibrosis sufferers
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[16]. Burtnick et al. [25] found that the recombinant Hcp2 protein provided mice with
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good protection (80%) against Burkholderia pseudomallei challenge. The above
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reports led us to speculate that Hcp may be a suitable vaccine candidate to prevent A.
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hydrophila infection.
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The previous study from our group showed that Hcp could be recognized by
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immunized rabbit sera on 2-D immunoblots that were performed to evaluate the 4
ACCEPTED MANUSCRIPT extracellular proteins of A. hydrophila [26]. In the present study, we demonstrated that
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there was a functionally active T6SS in A. hydrophila and investigated the
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immunogenicity and protective efficacy of the T6SS effector Hcp in common carp
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(Cyprinus carpio).
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2. Materials and methods
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2.1. Ethics Statement
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Animal experiments were conducted according to the Animal Welfare Council of
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China with approval for the experimental protocols from the Animal Ethics
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Committee of Nanjing Agricultural University.
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2.2. Bacterial strains, plasmids and growth conditions
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A total of 24 A. hydrophila isolates were used in the present study. A. hydrophila
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J-1 [26] and NJ-35 [27] were responsible for Aeromonad septicaemia in Jiangsu
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Province of China in 1989 and 2010, respectively. The environmental isolate A.
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hydrophila ATCC 7966 is the type strain for this species. The whole genome
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sequences of strains NJ-35 (accession number CP006870.1), J-1 (CP006883.1) and
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ATCC 7966 (CP000462.1) have been deposited in GenBank.
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The other A. hydrophila strains used were obtained from five different areas in
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China. All strains were isolated from aquatic animals of the following species:
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Common carp (Cyprinus carpio) (n = 15), Crucian carp (Carassius carassius) (n = 2),
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Silver carp (Hypophthalmichthys molitrix) (n = 2), Soft-shell turtle (Trionyx Sinensis)
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(n = 1), Eel (Monopterus albus) (n = 1). 5
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A. hydrophila and E. coli used in this study were cultured in Luria-Bertani (LB)
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medium at 28°C and 37°C, respectively.
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2.3. Animals
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New Zealand white rabbits weighing about 1.5 kg were supplied by Jiangsu
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Academy of Agricultural Sciences. Common carp weighing about 10 g were obtained
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from the Freshwater Fisheries Research Center, Chinese Academy of Fishery
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Sciences in China, and maintained at 28°C with ideal conditions of feeding, aeration
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and water exchange.
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2.4. Expression and purification of the recombinant Hcp (rHcp) and polyclonal
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antibody preparation
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The primer sets were designed based on the sequence of the hcp gene of A.
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hydrophila ATCC 7966 available in GenBank (accession no. CP000462.1). The
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primer sequences hcp-F (5'-GGAATTCATGCCAACTCCATGTTATATCAG-3') and
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hcp-R (5'-CCG CTCGAGTTAGGCCTCGATCGGC-3'), contained the EcoR I and
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Xho I restriction enzyme sites (underlined), respectively. Cloning of hcp gene from A.
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hydrophila NJ-35 was conducted as described previously [2].
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Hcp proteins were expressed in E. coli BL21 harboring the recombinant plasmid
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pET28a-hcp. Purification of the recombinant Hcp (rHcp) was achieved using
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HisTrapTM HP (GE Healthcare, USA). The purified protein was analyzed by sodium
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dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to verify the identity.
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The Bradford Protein Quant Kit (Tiangen, China) was used to determine the protein 6
ACCEPTED MANUSCRIPT concentration and the samples were stored at -20°C. Approximately 10 µg of purified
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rHcp was subjected to SDS-PAGE, and transferred to PVDF membranes. Western
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blot analysis was conducted with convalescent serum from fish challenged with A.
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hydrophila NJ-35 as the primary antibody.
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Rabbits were immunized subcutaneously with one milliliter (1 ml) of purified
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rHcp (0.5 mg/ml) emulsified with an ISA 206 adjuvant (SEPPIC, France) at a ratio of
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1:1 on days 0, 14 and 28. Sera were obtained prior to injection and 7 days after the
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final immunization. ELISA titres of sera to rHcp were determined as described by Ni
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et al. [26].
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2.5. PCR detection and Western blot analysis PCR
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primers,
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(5'-ATTCCGTCGGCAACATCTTC-3') and hcp-R' (5'-GGATCAGTTGGGTGAAG
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TCAGAC-3'), to determine the distribution of hcp gene in A. hydrophila strains. PCR
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conditions included an initial denaturation at 95°C for 5 min followed by 30 cycles of
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denaturation at 95°C for 30 s, annealing at 57°C for 30 s and extension at 72°C for 25
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s and a final extension at 72°C for 10 min. Genomic DNA of ATCC 7966 and sterile
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deionized water were used as the templates for positive and negative controls,
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respectively.
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Western blot was performed to explore the levels of Hcp protein expression and
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secretion in A. hydrophila strains of different origins as described elsewhere [28, 29].
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Briefly, all A. hydrophila strains were grown to OD600 ~ 2.0 in LB medium.
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One-millilliter bacteria cultures were collected and centrifuged at 10, 000 × g for 5 7
ACCEPTED MANUSCRIPT min. The cell pellets were re-suspended in 160 µl 1 × phosphate-buffered saline (PBS)
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and 40 µl 5 × protein sample buffer. The supernatant was filtered using a 0.22-µm
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membrane filter and mixed with 5 × protein sample buffer. After boiled for 10
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minutes, equal volumes of whole-cell and supernatant samples from the strains were
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used for SDS-PAGE immunoblot analysis. Anti-Hcp polyclonal antiserum (prepared
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in our laboratory using the recombinant Hcp from this study) or anti-OmpA
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polyclonal antiserum [30] was used as the primary antibody and HRP-conjugated goat
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anti-rabbit IgG was used as the second antibody. The blots were then developed using
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the DAB kit.
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2.6. LD50 determination in fish
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Fish used in this study were maintained and cared for following established
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protocols (Pearl River Fishery Research Institute, Chinese Academy of Fishery
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Science). Fish challenge experiment with A. hydrophila NJ-35 was conducted as
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previously described for the zebrafish model [31]. Overnight cultures of A. hydrophila
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NJ-35 were harvested at late-log phase by centrifugation and washed twice in PBS
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(pH 7.4). Common carp were anesthetized by immersion with 100 mg/l tricaine
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methanesulfonate (MS-222) (Hangzhou Animal Medicine Factory, China). Eight
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groups of 10 fish were intraperitoneally (i.p.) injected with 0.1 ml of 10-fold serially
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diluted suspensions of bacteria (102 to 109 colony forming units (CFU)) in sterile PBS.
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The control group was injected i.p. with 0.1 ml sterile PBS. Mortality was recorded
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daily until 7 days post-infection. The 50% lethal dose (LD50) values were calculated
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as described by the Reed and Muench method [32].
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2.7. Fish vaccination and challenge Fish were reared in the laboratory for two weeks before experimental procedures
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and then randomized in three groups: two vaccination groups (NJ-35 group and Hcp
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group) and one control group (Control group). Each treatment group included two
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tanks with 50 fish per tank (100 total fish). Before vaccination, the fish were fasted
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for 24 h. Fish in the Hcp group were injected intraperitoneally (i.p.) with 100 µl of
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purified rHcp (500 µg/ml) emulsified with ISA 763 adjuvant (SEPPIC, France) at a
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ratio of 1:1, and therefore each fish received 25 µg of rHcp. The concentration of
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rHcp used in this study was selected on the basis of a preliminary sighting study, in
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which the immunization dose was screened from the fixed levels of 15 µg, 25 µg and
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35 µg of rHcp for each fish expected to elicit a higher level of antibody following
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low-dose immunization. Fish in the NJ-35 group were injected i.p. with 100 µl of
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formaldehyde-inactivated whole cells of A. hydrophila NJ-35 (1 × 109 CFU/ml)
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emulsified with the same adjuvant as the Hcp group. After 14 days, the fish received a
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second vaccination with the same dose of antigen. Fish in the control group inoculated
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with 100 µl PBS at the same time points.
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At 45 days after the first vaccination, 30 fish from each treatment group were
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divided into two subgroups (15 for each subgroup) and the fish were challenged i.p.
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with 50 LD50 of log-phase A. hydrophila NJ-35 strain in 0.1 ml of PBS. Mortality was
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recorded several times a day up to 10 days post-challenge, and the relative percentage
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survival (RPS) was calculated based on the formula of Amend: RPS = (1- [mortality
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of vaccinated group/mortality of unvaccinated control group]) × 100. The RPS results 9
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were calculated from the cumulative mortalities of two subgroups.
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2.7.1. Sample collection For sampling, the common carp were anaesthetized using MS-222. On 0, 14, 21,
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28, 35, 50 days post-vaccination, blood of four fish taken randomly from each tank
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was drawn from the vena caudalis using a 1-ml plastic syringe. Then the blood clotted
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at 37°C for 1 h to collect the serum. The serum samples from four fish in each tank
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were pooled and kept at -70 °C until they were processed for ELISA titres.
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At the same time points, spleen, kidney and gill tissues were taken from four fish
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per tank for the evaluation of cytokine levels. The samples from four fish were pooled,
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snap-frozen in liquid nitrogen instantly after dissection, and then stored at -70 °C.
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2.7.2. Antibody titres
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ELISA was performed with all collected sera for the titres of specific IgM
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antibodies to rHcp or inactivated vaccine as described by previous studies [12, 26].
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Fish sera (1:10 dilutions) from immunization groups were added to duplicate wells of
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ELISA plates, which were firstly coated with cognate antigens, followed by
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mouse-anti-common carp IgM antibodies (1:2,000) which were prepared as described
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in our previous study [12]. Color development occurs by reaction of the horseradish
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peroxidase (HRP)-conjugated goat-anti-mouse IgG (Dingguo, China) and 3,3',5,5'-
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tetramethylbenzidine (TMB) substrate. The plates were read at 450 nm using a
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microtiter plate reader. Each serum sample was assessed in duplicate. The negative
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control used PBS to replace the primary antibody.
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2.7.3. Cytokine levels To evaluate the effect of vaccination on the expression levels of cytokines, an
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ELISA assay was performed using the Fish Tumor necrosis factor α ELISA Kit and
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Fish interleukin 1β ELISA Kit (RB, USA) according to manufacturer’s instructions.
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The spleen, kidney and gill tissues were homogenized in appropriate 10 mM PBS (pH
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7.4), respectively. After centrifugation at 3 000 × g for 20 min, the supernatants of the
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tissue extracts were used for ELISA. Then tissue supernatant is added to each well
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followed by adding HRP-labeled cytokine antibodies. The reaction was developed by
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TMB substrate with H2O2. The cytokine levels in the tissues were then determined
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from the OD450 values relative to the standard curve.
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2.8. Statistical analysis
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GraphPad Prism version 5 was employed to analyze the data. Analysis of
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variance (ANOVA) was applied for comparison of the differences among
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experimental groups. A significant difference was considered at P < 0.05.
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3. Results
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3.1. Identification of T6SS in the genome of A. hydrophila NJ-35
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In search for evidence of the presence of a complete T6SS gene cluster in A.
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hydrophila NJ-35, we performed the genome sequences of this bacterial strain and
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another two strains of A. hydrophila, J-1 and ATCC 7966. The organization of the
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T6SS gene cluster in A. hydrophila NJ-35 resembled to that in J-1 and ATCC 7966.
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Specifically, we focused on the organization of effector proteins Hcp and VgrG. Three
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copies of hcp (designed hcp-1, hcp-2 and hcp-3) and four copies of vgrG genes 11
ACCEPTED MANUSCRIPT (vgrG-1, vgrG-2, vgrG-3 and vgrG-4) were identified in A. hydrophila NJ-35. Among
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them, hcp-3, vgrG-3 and vgrG-4 are located in the T6SS gene cluster, while the other
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ones located outside of the cluster (Fig.1). It’s interesting to be noted that hcp-1 and
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vgrG-1 genes were genetically linked through a region less than 500 bp, as was seen
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in A. hydrophila SSU [28]. This phenomenon also appeared in hcp-2 and vgrG-2
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genes, and hcp-3 and vgrG-3 genes. A. hydrophila J-1 and ATCC 7966 had similar
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organizations of Hcp and VgrG with NJ-35, except that only one hcp-vgrG gene pair
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was found outside of the T6SS cluster in the two strains (Fig.1).
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3.2. Distribution of the hcp gene in different A. hydrophila isolates
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The hcp gene sequence from ATCC 7966 was searched in all the 31 A.
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hydrophila genome published in the databases (http://www.ncbi.nlm.nih.gov/genome/
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genomes/1422). The results revealed the presence of the hcp gene in 30/31 of A.
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hydrophila strains with the identity >90%. Further, the hcp fragment was PCR
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amplified from all the A. hydrophila strains (24/24) tested here, suggesting that hcp is
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widespread in the A. hydrophila strains.
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3.3. Expression and antigenicity of Hcp
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The hcp gene product was cloned into the pET28a vector and transformed into E.
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coli BL21 host cells. The expression of a 24-kDa recombinant protein was observed
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in the induced E. coli harboring pET28a-hcp, while no protein was found at the same
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position in the non-induced E. coli harboring pET28a-hcp or pET28a by SDS-PAGE
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(Fig. 2). And the purified rHcp protein was shown to have ability to react with
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convalescent serum from fish clinically infected with A. hydrophila NJ-35 in Western 12
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blot, indicating that Hcp was expressed during naturally occurring infections and
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could be recognized by the immune system. To prepare polyclonal antibodies against rHcp, the purified rHcp protein was
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used to vaccinate rabbits and after three injections, the animals showed an apparent
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ELISA titer of 1:12 800.
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3.4. Hcp secretion in various A. hydrophila strains
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To assess the expression and secretion of Hcp, the polyclonal antibody against
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rHcp was used to detect the levels of Hcp in the supernatants and total cellular
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fractions of nine A. hydrophila strains of different origins or serotypes. Hcp was
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detected in the whole cell samples of all strains, and supernatants showed a greater
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variation (Fig. 3). Hcp secretion was more pronounced in strains J-1, BSK10, FBS35
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and HA50, compared to strains NJ-35, PEG14, L316 and AH9617. Strain ATCC 7966
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showed minimal Hcp secretion. The outer membrane protein OmpA was not
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detectable in all supernatant samples prepared from theses strains, indicating that the
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presence of Hcp in this fraction was not the result of bacterial cell lysis. Further, the
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supernatants of 15 A. hydrophila isolates from diseased Common carp were subjected
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to Western blot analysis using rabbit polyclonal anti-rHcp, and all were demonstrated
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to be positive for Hcp.
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Then we investigated whether different growth temperatures influenced the
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secretion of Hcp from A. hydrophila NJ-35. As shown in Fig. 4A, Hcp could be
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secreted into the culture supernatants at 16°C, 20°C and 28°C, but not at 37°C. To test
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whether the temperature-dependent secretion of Hcp was specific to A. hydrophila 13
ACCEPTED MANUSCRIPT NJ-35 or whether it might also happen to other A. hydrophila strains, we measured the
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secretion levels of Hcp in A. hydrophila J-1 and ATCC7966. The results showed that
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the effect of the temperature on Hcp production in the two strains was similar to that
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in A. hydrophila NJ-35 (Fig. 4B and C). The only exception is that at 37°C, Hcp
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production could be observed in the total cellular proteins of NJ-35 and J-1, but not in
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ATCC 7966. Additionally, it is notable that Hcp secretion levels were elevated when
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the bacteria were cultured at the lower temperature. These results suggested that the
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secretion feature of Hcp under low-temperature conditions may be common to A.
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hydrophila strains.
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3.5. Vaccine protective efficacy
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The LD50 value of A. hydrophila NJ-35 was determined to be approximately 1.0
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× 104 CFU in Common carp (Table 1). At 45 days post-vaccination, 30 fish from each
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group were i.p. challenged with 5.0 × 105 CFU (50 LD50) of A. hydrophila NJ-35. As
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shown in Fig. 5, fish vaccinated with rHcp protein and inactivated whole cell vaccine
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were well protected when challenged with A. hydrophila, and exhibited a survival of
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46.67 and 53.33%, respectively; whereas only 7.14% survival was observed in the
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control group. The RPS values were 42.86 and 50.00 for rHcp protein and inactivated
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vaccine groups, respectively. Symptoms of hemorrhagic septicemia were observed in
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the dying fish. A. hydrophila was isolated from kidneys of all dead fish, suggesting
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that mortality was caused by the experimental challenge.
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3.6. Antibody levels
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The specific IgM antibody was determined in the serum of vaccinated fish by 14
ACCEPTED MANUSCRIPT ELISA. Compared with the control group, significantly higher antibody levels (P <
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0.05) were detected in the vaccinated groups after the injection, while there was no
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significant difference in antibody titres between fish vaccinated with the inactivated
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vaccine and rHcp protein (P > 0.05) (Fig. 6). After 21 days post-vaccination, the
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specific IgM of the immunized groups reached the peak, and subsequently a little
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decreased, but still kept at a high level.
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3.7. Cytokine levels
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Compared with the control group, IL-1β levels in all three examined tissues of
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vaccinated groups were upregulated throughout 50 days post-vaccination. In the
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kidney, significant higher levels of IL-1β (P < 0.05) were observed at 14 days in the
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vaccinated groups than that in the control group (Fig. 7A). By 35 and 50 days
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post-vaccination, the spleen IL-1β levels were significantly elevated (P < 0.05) in the
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vaccinated groups compared to the controls (Fig. 7B). The gills in vaccinated groups
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also had remarkable higher levels (P < 0.05) of IL-1β on days 21 and 50 than that in
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the controls (Fig. 7C). As shown in Figures 7D, E and F, TNF-α levels in all three
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examined tissues were significantly increased (P < 0.05) in the vaccinated groups at
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most time points in comparison with the control group. At the remaining times,
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TNF-α levels in the vaccinated groups had increased but not significantly (P > 0.05)
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when compared with the controls.
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4. Discussion
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Hcp is an important T6SS effector protein and plays crucial roles in the
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ACCEPTED MANUSCRIPT pathogenicity of some Gram negative bacterial pathogens. It is also the hallmark
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protein of a functional T6SS in all bacteria that own this system [33]. The present
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study indicated that under standard laboratory conditions, Hcp expression is
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conserved among A. hydrophila strains. However, the secretion levels of Hcp protein
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varied among isolates. Moreover, our experiment showed that this difference in the
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detection of Hcp by Western blot analysis was due to varying amounts of Hcp present
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in the culture supernatants of different strains. The major reason for this is attributed
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to the diversity in expression levels of hcp gene and secretion of the corresponding
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protein and/or its possible rates of degradation in different strains. Additionally, we
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observed the effect of growth temperature on Hcp secretion. When A. hydrophila
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strains were grown at 16°C, 20°C and 28°C, Hcp was secreted. However, this protein
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was not secreted when the bacteria were grown at 37°C, indicating that Hcp secretion
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was strongly induced by low temperature. Similar results were also seen in V.
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cholerae [29] and V. parahaemolyticus [34]. Different from our result, studies
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conducted by Suarez et al. [35] showed that Hcp in A. hydrophila SSU could be also
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secreted at 37°C. The difference might be due to the fact that the bacterial strains were
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isolated from different ecological niches. A. hydrophila SSU was a human isolate,
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while the strains used in this study were mostly isolated from aquatic animals, in
344
which body temperature follows the surrounding environment. Another very notable
345
phenomenon is that in contrast to the fish isolates NJ-35 and J-1, the environmental
346
isolate ATCC 7966 did not produce Hcp at 37°C. In agreement with this finding,
347
Grim et al. [36] reported that Hcp synthesis and secretion could not be observed in
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strain ATCC 7966. Further studies will be necessary to investigate the factors that
349
influence Hcp production and secretion. The report by Mougous et al. [16] showed that in the patients with P. aeruginosa
351
infection, a high level of antibody against Hcp was tested. During Burkholderia mallei
352
infections, Hcp was expressed in several hosts and was immunogenic [37]. In A.
353
hydrophila SSU, the Hcp protein was demonstrated to provide good protective
354
efficacy for the immunized mice [35]. With the purpose of exploring the potential
355
application of Hcp as a vaccine candidate against fish bacterial pathogens, the current
356
study evaluates its protective efficacy in common carp. The results from the challenge
357
experiment revealed that common carp vaccinated with the recombinant Hcp in this
358
study were well protected relative to the non-vaccinated ones, and the RPS of Hcp
359
protein-vaccinated fish was 42.86, which is in accordance with a report conducted in
360
mice by Suarez et al. [35]. We also notice that the RPS of the inactivated A.
361
hydrophila NJ-35-vaccinated fish was slightly higher than that of the Hcp
362
protein-vaccinated fish during infection, although the difference was not significant
363
(P > 0.05). The small difference may be due to the fact that the inactive whole cell
364
vaccines usually present multiple antigenic components, which contribute to a certain
365
degree of immune synergy against homogenous challenge.
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Resistance to and recovery from bacterial infections are the results of an efficient
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and complex immune response by a combination of the innate and acquired immune
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system [38,39]. An adaptive immune system in fish is mediated by lymphocytes that
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exert their effects mainly by means of antibodies [40]. The antibody type in most fish 17
ACCEPTED MANUSCRIPT is IgM tetramers [41]. In the study, the recombinant Hcp showed good
371
immunogenicity in eliciting humoral immune responses in common carp. In
372
comparison with the control group, IgM antibody titers in Hcp protein- or whole
373
bacterial cell-vaccinated groups were significantly increased (P < 0.05), but there was
374
no statistically significant difference between these two vaccinated groups, indicating
375
the immunogenicity of Hcp is equivalent to whole cell antigen. This result led us to
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speculate that the increased antibody level may contribute to the higher survival rates
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in both the two vaccinated groups.
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As modulators of the immune responses, cytokines are related to both innate and
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adaptive immune systems [42]. The main pro-inflammatory cytokines IL-1β [43] and
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TNF-α [44] are the commonly investigated immune-regulatory cytokines in fish.
381
TNF-α has been proved to induce the inflammatory response by regulating the
382
expression of other cytokines including IL-1β, and both of them can enhance a variety
383
of cellular responses such as leukocyte migration and phagocytosis [45, 46]. In this
384
work, we collected three tissues, including kidney with both renal and immune
385
functions [47, 48], spleen as a major secondary organ [43] and gill as one of the main
386
mucosal surfaces and immune barriers [49], to analyze cytokine levels. The enhanced
387
expression of these cytokines in fish vaccinated with rHcp suggested that both innate
388
immunity and adaptive immunity were induced to some extent. In other words, Hcp
389
protein stimulates the fish to upregulate the immune response against exogenous
390
antigen. Dash et al. [50] reported that the expression of IL-1β in fish vaccinated with
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recombinant outer membrane protein R-based vaccine of A. hydrophila was increased
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in spleens, which is consistent with our findings. Aeromonas species have a wide range of serotypes and a variety of strains [51],
394
which is a major problem in developing vaccines against this pathogen. Some studies
395
have demonstrated that O9 and O5 were the main serotypes causing MAS outbreaks
396
in China [52, 53]. A recent study from our laboratory [54] showed that there exists an
397
identical O-antigen gene cluster in strains NJ-35 and J-1 (O5 serotype), which were
398
responsible for the MAS outbreaks in Jiangsu Province, in 2010 and 1989,
399
respectively, suggesting little change on the epidemic serotypes in these years in
400
China. In the present study, serotypes O9 and O5 as well as some unknown serotypes
401
of A. hydrophila strains were investigated for the presence of Hcp. Our results
402
obtained from PCR and Western blot analyses that Hcp could be detected in the
403
supernatants from all of the A. hydrophila strains detected , indicated that rHcp has
404
the potential to be a common protective antigen of A. hydrophila strains of different
405
origins. The protective efficacy of the rHcp against heterologous challenge strains of
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A. hydrophila will be evaluated in the future study.
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In conclusion, our results demonstrate that Hcp is conserved among A.
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hydrophila strains studied here, and the expressed Hcp of strain NJ-35 induces a
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valuable protective response against this bacterial infection. The recombinant Hcp
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shows promise as a vaccine candidate against A. hydrophila.
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Author Contributions
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Conceived and designed the experiments: NW YL. Performed the experiments:
412 413
NW
YW
MP
JL.
Analyzed
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data:
NW
YW.
Contributed
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reagents/materials/analysis tools: CL YL. Wrote the paper: NW YW MP JL CL YL.
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Acknowledgments
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This research was funded by National Nature Science Foundation (31072151, 31372454) and Aquatic Three New Projects in Jiangsu Province (D2013-5-4).
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[52] Qian D, Chen Y, Shen J, Shen Z. Serogroups, virulence and hemolytic activity of
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Aeromonas hydrophila which caused fish bacterial septicaemia. Wei Sheng Wu
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Xue Bao 1995;35:460-4.(in Chinese) [53] Dong C, Lin T, Yu F, Chen R, Gong H, Chen Z. Isolation and identification of
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Figure Legends
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Fig. 1. Genetic organization of the T6SS gene cluster of Aeromonas hydrophila NJ-35,
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J-1 and ATCC 7966. Blue and orange arrows indicate hcp and vgrG genes,
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respectively.
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Fig. 2. SDS-PAGE and Western blot analysis of Hcp expression. Lane M, molecular
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weight marker; lane 1, pET28a-hcp in E. coli BL21, non-induced; lane 2, pET28a-hcp
599
in E. coli BL21, 1 mM IPTG-induced for 5 h; lane 3, pET28a in E. coli BL21,
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non-induced; lane 4, pET28a in E. coli BL21, 1 mM IPTG-induced for 4.5 h; lane 5,
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purified recombinant Hcp; lane 6, Western blot of purified Hcp using convalescent
602
serum from challenged fish with A. hydrophila J-1 as the primary antibody.
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Fig. 3. Hcp secretion in A. hydrophila isolates of different serotypes. The bacteria
605
were cultured at 28 °C in LB medium to an OD of 2.0. Lane M, molecular weight
606
marker; lanes 1–9, culture supernatants from AhJ-1 (O5), BSK10 (O5), FBS35 (O9),
607
HA50 (O9), NJ-35 (unknown), PEG14 (O9), L316 (unknown), AH9617 (O9) and
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ATCC 7966 (unknown). The arrows indicate immunoblot reactivity to Hcp and
609
OmpA. As no OmpA protein was detected in the supernatants, we concluded that
610
there was no significant cell lysis in the procedure.
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Fig. 4. Effects of growth temperature on the expression (whole cells) and secretion 28
ACCEPTED MANUSCRIPT 613
(supernatants) of Hcp in A. hydrophila NJ-35 (A), J-1 (B) and ATCC 7966 (C).
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Bacteria strains were cultured under indicated temperature to an OD of 2.0. Western
615
blot analyses were performed with anti-Hcp serum and anti-OmpA antibodies.
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Fig. 5. Survival rates of common carp challenged with A. hydrophila. Fish vaccinated
618
with rHcp protein or inactivated A. hydrophila vaccine were challenged (i.p.) by A.
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hydrophila strain NJ-35 after 45 days post-vaccination (i.p.), and the mortalities were
620
recorded for 10 days.
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Fig. 6. Antibody levels in common carp following vaccination. Serum collected was
623
assayed by ELISA for the presence of antibodies to immunized antigens. Each bar
624
represents the mean of three replicates and errors bars represent the standard deviation.
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Significant differences at P < 0.05 among different groups at the same time point are
626
indicated by different letters (a, b, c).
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Fig. 7. Cytokine levels in kidney, spleen and gills of common carp at 14, 21, 28, 35
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and 50 days post-vaccination. Each bar represents the mean of three replicates and
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errors bars represent the standard deviation. Significant differences at P < 0.05 among
631
different groups at the same time point are indicated by different letters (a, b, c).
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Tables
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Table 1. Mortalities recorded for LD50 calculations of the Aeromonas hydrophila strain No. of fish that died after inoculation witha:
(CFU)
Expt 1
109
10
10
108
10
10
107
10
9
106
8
9
105
8
104
5
103
2
102
0 1.5×104
10 fish per group were inoculated.
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9
8
7
6
5
2
3
0
0
1.0×104
1.2×104
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ACCEPTED MANUSCRIPT Highlights Hcp secretion was found to be strongly induced by low temperature in A. hydrophila. Hcp is conserved among A. hydrophila strains of different origins.
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The Hcp vaccination provided relative efficient protection against A. hydrophila infection.
The Hcp vaccination could enhance both the innate and adaptive immune responses in
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fish.
S1050-4648(15)30047-4
DOI:
10.1016/j.fsi.2015.06.019
Reference:
YFSIM 3508
To appear in:
Fish and Shellfish Immunology
Received Date: 12 March 2015 Revised Date:
12 June 2015
Accepted Date: 15 June 2015
Please cite this article as: Wang N, Wu Y, Pang M, Liu J, Lu C, Liu Y, Protective Efficacy of Recombinant Hemolysin Co-regulated Protein (Hcp) of Aeromonas hydrophila in Common carp (Cyprinus carpio), Fish and Shellfish Immunology (2015), doi: 10.1016/j.fsi.2015.06.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Protective Efficacy of Recombinant Hemolysin Co-regulated Protein
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(Hcp) of Aeromonas hydrophila in Common carp (Cyprinus carpio) Nannan Wang, Yafeng Wu, Maoda Pang, Jin Liu, Chengping Lu, Yongjie Liu*
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College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095,
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China
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* Corresponding author. Tel./fax: 0086-25-84398606
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E-mail address: [email protected] (Y. Liu)
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Abstract
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Motile aeromonad septicemia (MAS) caused by Aeromonas hydrophila is one of the
24
common bacterial causes of fish mortalities. Prophylactic vaccination against this and
25
other diseases is essential for continued growth of aquaculture. The type VI secretion
26
system (T6SS) plays a crucial role in the virulence of A. hydrophila. The hemolysin
27
co-regulated protein (Hcp) is an integral component of the T6SS apparatus and is
28
considered a hallmark of T6SS function. Here, the T6SS effector Hcp was expressed
29
and characterized, and its immunogenicity and protective efficacy were evaluated in
30
common carp (Cyprinus carpio). Hcp secretion was found to be strongly induced by
31
low temperature in A. hydrophila. Immunoblot analysis demonstrated that Hcp is
32
conserved among A. hydrophila strains of different origins. The vaccination with
33
recombinant Hcp resulted in an increased survival (46.67%) in common carp during a
34
10-day challenge time compared to non-vaccinated fish (7.14%). The vaccinated fish
35
also showed the significantly increased levels of IgM antibody in serum and cytokines
36
such as inerleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in kidney, spleen
37
and gills. The recombinant Hcp shows promise as a vaccine candidate against A.
38
hydrophila.
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Keywords: Aeromonas hydrophila; Hemolysin co-regulated protein (Hcp); Protective
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efficacy; Fish; Vaccine
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1. Introduction
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Aeromonas hydrophila is a Gram-negative bacterium widely present in 2
ACCEPTED MANUSCRIPT freshwater habitats and causes infections in humans and animals such as amphibians
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and fish [1-3]. A. hydrophila is associated with many fish diseases like hemorrhagic
45
septicemia and dropsy, and leads to significant economic losses worldwide. It has
46
been accepted that vaccination is an effective method to protect fish from the
47
infectious bacterial diseases [4]. Several studies have demonstrated that different
48
types of vaccines such as heat-killed cells, heat or formalin-inactivated bacterial
49
extracts and live cells of A. hydrophila stimulate an effective response in fish that
50
protects against the bacterial infection [5-8]. However, it should be noted that such
51
vaccines are not always effective, especially when the expected immune response is
52
directed against specific antigens. And they contain complex mixtures and undefined
53
molecules that have been evidenced to interact synergistically or antagonistically and
54
that can stimulate, cross-react with, inhibit or even suppress the immune response to
55
specific antigens [9]. Recently, the development of the recombinant subunit vaccine
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has caught a lot of attention. This vaccine contains fragments of pathogenic
57
microorganisms, which are highly purified and immunogenic antigens. This ensures
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that the antigen has a well-defined composition. Also, vaccination with a protein
59
present in a range of serotypes would overcome some of the limitations of antigenic
60
diversity in A. hydrophila strains. Some previous studies targeted subunit vaccine
61
candidates against A. hydrophila have mainly paid attention to the outer membrane
62
proteins (OMPs) [10-12]. However, in the case of A. hydrophila, vaccination with
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extracellular secreted proteins may be particularly important, because its
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pathogenicity appears to be closely related to the production of extracellular products,
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which are lost, partially, in conventional bacterin preparations. Bacterial pathogenicity critically relies on various secretion systems to deliver
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toxic molecules from the cytoplasm to the outer space [13]. Lately, a novel secretion
68
system named the type VI secretion system (T6SS) was reported for several
69
pathogens [14-17] and characterized as the most common secretion system of
70
Gram-negative bacteria [18]. Two typical proteins of T6SS, hemolysin co-regulated
71
protein (Hcp) and valine-glycine repeat protein G (VgrG) have been proved to
72
function not only as structural elements of the T6SS device but also effector proteins
73
[17, 19, 20]. Furthermore, many Gram-negative pathogens have been found to secrete
74
these two conserved effector proteins to the outer space through T6SS [16, 17, 21-23].
75
Of late, however, Hcp which used to be a static tubule was proven to be a chaperone
76
and receptor of type VI secretion substrates in Pseudomonas aeruginosa [24]. T6SS
77
components have been reported to concern with virulence-related mechanism of
78
various bacteria. During V. cholerae infection, T6SS genes were proved to be
79
essential for toxicity in Dictyostelium amoebae and mammalian J774 macrophages
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[17]. In association with P. aeruginosa, Hcp1 was detected in cystic fibrosis sufferers
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[16]. Burtnick et al. [25] found that the recombinant Hcp2 protein provided mice with
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good protection (80%) against Burkholderia pseudomallei challenge. The above
83
reports led us to speculate that Hcp may be a suitable vaccine candidate to prevent A.
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hydrophila infection.
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The previous study from our group showed that Hcp could be recognized by
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immunized rabbit sera on 2-D immunoblots that were performed to evaluate the 4
ACCEPTED MANUSCRIPT extracellular proteins of A. hydrophila [26]. In the present study, we demonstrated that
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there was a functionally active T6SS in A. hydrophila and investigated the
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immunogenicity and protective efficacy of the T6SS effector Hcp in common carp
90
(Cyprinus carpio).
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2. Materials and methods
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2.1. Ethics Statement
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Animal experiments were conducted according to the Animal Welfare Council of
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China with approval for the experimental protocols from the Animal Ethics
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Committee of Nanjing Agricultural University.
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2.2. Bacterial strains, plasmids and growth conditions
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A total of 24 A. hydrophila isolates were used in the present study. A. hydrophila
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J-1 [26] and NJ-35 [27] were responsible for Aeromonad septicaemia in Jiangsu
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Province of China in 1989 and 2010, respectively. The environmental isolate A.
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hydrophila ATCC 7966 is the type strain for this species. The whole genome
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sequences of strains NJ-35 (accession number CP006870.1), J-1 (CP006883.1) and
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ATCC 7966 (CP000462.1) have been deposited in GenBank.
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The other A. hydrophila strains used were obtained from five different areas in
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China. All strains were isolated from aquatic animals of the following species:
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Common carp (Cyprinus carpio) (n = 15), Crucian carp (Carassius carassius) (n = 2),
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Silver carp (Hypophthalmichthys molitrix) (n = 2), Soft-shell turtle (Trionyx Sinensis)
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(n = 1), Eel (Monopterus albus) (n = 1). 5
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A. hydrophila and E. coli used in this study were cultured in Luria-Bertani (LB)
110
medium at 28°C and 37°C, respectively.
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2.3. Animals
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New Zealand white rabbits weighing about 1.5 kg were supplied by Jiangsu
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Academy of Agricultural Sciences. Common carp weighing about 10 g were obtained
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from the Freshwater Fisheries Research Center, Chinese Academy of Fishery
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Sciences in China, and maintained at 28°C with ideal conditions of feeding, aeration
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and water exchange.
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2.4. Expression and purification of the recombinant Hcp (rHcp) and polyclonal
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antibody preparation
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The primer sets were designed based on the sequence of the hcp gene of A.
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hydrophila ATCC 7966 available in GenBank (accession no. CP000462.1). The
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primer sequences hcp-F (5'-GGAATTCATGCCAACTCCATGTTATATCAG-3') and
122
hcp-R (5'-CCG CTCGAGTTAGGCCTCGATCGGC-3'), contained the EcoR I and
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Xho I restriction enzyme sites (underlined), respectively. Cloning of hcp gene from A.
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hydrophila NJ-35 was conducted as described previously [2].
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Hcp proteins were expressed in E. coli BL21 harboring the recombinant plasmid
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pET28a-hcp. Purification of the recombinant Hcp (rHcp) was achieved using
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HisTrapTM HP (GE Healthcare, USA). The purified protein was analyzed by sodium
128
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to verify the identity.
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The Bradford Protein Quant Kit (Tiangen, China) was used to determine the protein 6
ACCEPTED MANUSCRIPT concentration and the samples were stored at -20°C. Approximately 10 µg of purified
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rHcp was subjected to SDS-PAGE, and transferred to PVDF membranes. Western
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blot analysis was conducted with convalescent serum from fish challenged with A.
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hydrophila NJ-35 as the primary antibody.
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Rabbits were immunized subcutaneously with one milliliter (1 ml) of purified
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rHcp (0.5 mg/ml) emulsified with an ISA 206 adjuvant (SEPPIC, France) at a ratio of
136
1:1 on days 0, 14 and 28. Sera were obtained prior to injection and 7 days after the
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final immunization. ELISA titres of sera to rHcp were determined as described by Ni
138
et al. [26].
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2.5. PCR detection and Western blot analysis PCR
was
performed
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with
the
pair
of
primers,
hcp-F'
(5'-ATTCCGTCGGCAACATCTTC-3') and hcp-R' (5'-GGATCAGTTGGGTGAAG
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TCAGAC-3'), to determine the distribution of hcp gene in A. hydrophila strains. PCR
143
conditions included an initial denaturation at 95°C for 5 min followed by 30 cycles of
144
denaturation at 95°C for 30 s, annealing at 57°C for 30 s and extension at 72°C for 25
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s and a final extension at 72°C for 10 min. Genomic DNA of ATCC 7966 and sterile
146
deionized water were used as the templates for positive and negative controls,
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respectively.
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Western blot was performed to explore the levels of Hcp protein expression and
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secretion in A. hydrophila strains of different origins as described elsewhere [28, 29].
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Briefly, all A. hydrophila strains were grown to OD600 ~ 2.0 in LB medium.
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One-millilliter bacteria cultures were collected and centrifuged at 10, 000 × g for 5 7
ACCEPTED MANUSCRIPT min. The cell pellets were re-suspended in 160 µl 1 × phosphate-buffered saline (PBS)
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and 40 µl 5 × protein sample buffer. The supernatant was filtered using a 0.22-µm
154
membrane filter and mixed with 5 × protein sample buffer. After boiled for 10
155
minutes, equal volumes of whole-cell and supernatant samples from the strains were
156
used for SDS-PAGE immunoblot analysis. Anti-Hcp polyclonal antiserum (prepared
157
in our laboratory using the recombinant Hcp from this study) or anti-OmpA
158
polyclonal antiserum [30] was used as the primary antibody and HRP-conjugated goat
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anti-rabbit IgG was used as the second antibody. The blots were then developed using
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the DAB kit.
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2.6. LD50 determination in fish
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Fish used in this study were maintained and cared for following established
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protocols (Pearl River Fishery Research Institute, Chinese Academy of Fishery
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Science). Fish challenge experiment with A. hydrophila NJ-35 was conducted as
165
previously described for the zebrafish model [31]. Overnight cultures of A. hydrophila
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NJ-35 were harvested at late-log phase by centrifugation and washed twice in PBS
167
(pH 7.4). Common carp were anesthetized by immersion with 100 mg/l tricaine
168
methanesulfonate (MS-222) (Hangzhou Animal Medicine Factory, China). Eight
169
groups of 10 fish were intraperitoneally (i.p.) injected with 0.1 ml of 10-fold serially
170
diluted suspensions of bacteria (102 to 109 colony forming units (CFU)) in sterile PBS.
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The control group was injected i.p. with 0.1 ml sterile PBS. Mortality was recorded
172
daily until 7 days post-infection. The 50% lethal dose (LD50) values were calculated
173
as described by the Reed and Muench method [32].
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2.7. Fish vaccination and challenge Fish were reared in the laboratory for two weeks before experimental procedures
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and then randomized in three groups: two vaccination groups (NJ-35 group and Hcp
177
group) and one control group (Control group). Each treatment group included two
178
tanks with 50 fish per tank (100 total fish). Before vaccination, the fish were fasted
179
for 24 h. Fish in the Hcp group were injected intraperitoneally (i.p.) with 100 µl of
180
purified rHcp (500 µg/ml) emulsified with ISA 763 adjuvant (SEPPIC, France) at a
181
ratio of 1:1, and therefore each fish received 25 µg of rHcp. The concentration of
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rHcp used in this study was selected on the basis of a preliminary sighting study, in
183
which the immunization dose was screened from the fixed levels of 15 µg, 25 µg and
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35 µg of rHcp for each fish expected to elicit a higher level of antibody following
185
low-dose immunization. Fish in the NJ-35 group were injected i.p. with 100 µl of
186
formaldehyde-inactivated whole cells of A. hydrophila NJ-35 (1 × 109 CFU/ml)
187
emulsified with the same adjuvant as the Hcp group. After 14 days, the fish received a
188
second vaccination with the same dose of antigen. Fish in the control group inoculated
189
with 100 µl PBS at the same time points.
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At 45 days after the first vaccination, 30 fish from each treatment group were
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divided into two subgroups (15 for each subgroup) and the fish were challenged i.p.
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with 50 LD50 of log-phase A. hydrophila NJ-35 strain in 0.1 ml of PBS. Mortality was
193
recorded several times a day up to 10 days post-challenge, and the relative percentage
194
survival (RPS) was calculated based on the formula of Amend: RPS = (1- [mortality
195
of vaccinated group/mortality of unvaccinated control group]) × 100. The RPS results 9
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were calculated from the cumulative mortalities of two subgroups.
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2.7.1. Sample collection For sampling, the common carp were anaesthetized using MS-222. On 0, 14, 21,
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28, 35, 50 days post-vaccination, blood of four fish taken randomly from each tank
200
was drawn from the vena caudalis using a 1-ml plastic syringe. Then the blood clotted
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at 37°C for 1 h to collect the serum. The serum samples from four fish in each tank
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were pooled and kept at -70 °C until they were processed for ELISA titres.
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At the same time points, spleen, kidney and gill tissues were taken from four fish
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per tank for the evaluation of cytokine levels. The samples from four fish were pooled,
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snap-frozen in liquid nitrogen instantly after dissection, and then stored at -70 °C.
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2.7.2. Antibody titres
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ELISA was performed with all collected sera for the titres of specific IgM
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antibodies to rHcp or inactivated vaccine as described by previous studies [12, 26].
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Fish sera (1:10 dilutions) from immunization groups were added to duplicate wells of
210
ELISA plates, which were firstly coated with cognate antigens, followed by
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mouse-anti-common carp IgM antibodies (1:2,000) which were prepared as described
212
in our previous study [12]. Color development occurs by reaction of the horseradish
213
peroxidase (HRP)-conjugated goat-anti-mouse IgG (Dingguo, China) and 3,3',5,5'-
214
tetramethylbenzidine (TMB) substrate. The plates were read at 450 nm using a
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microtiter plate reader. Each serum sample was assessed in duplicate. The negative
216
control used PBS to replace the primary antibody.
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2.7.3. Cytokine levels To evaluate the effect of vaccination on the expression levels of cytokines, an
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ELISA assay was performed using the Fish Tumor necrosis factor α ELISA Kit and
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Fish interleukin 1β ELISA Kit (RB, USA) according to manufacturer’s instructions.
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The spleen, kidney and gill tissues were homogenized in appropriate 10 mM PBS (pH
222
7.4), respectively. After centrifugation at 3 000 × g for 20 min, the supernatants of the
223
tissue extracts were used for ELISA. Then tissue supernatant is added to each well
224
followed by adding HRP-labeled cytokine antibodies. The reaction was developed by
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TMB substrate with H2O2. The cytokine levels in the tissues were then determined
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from the OD450 values relative to the standard curve.
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2.8. Statistical analysis
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GraphPad Prism version 5 was employed to analyze the data. Analysis of
229
variance (ANOVA) was applied for comparison of the differences among
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experimental groups. A significant difference was considered at P < 0.05.
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3. Results
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3.1. Identification of T6SS in the genome of A. hydrophila NJ-35
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In search for evidence of the presence of a complete T6SS gene cluster in A.
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hydrophila NJ-35, we performed the genome sequences of this bacterial strain and
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another two strains of A. hydrophila, J-1 and ATCC 7966. The organization of the
236
T6SS gene cluster in A. hydrophila NJ-35 resembled to that in J-1 and ATCC 7966.
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Specifically, we focused on the organization of effector proteins Hcp and VgrG. Three
238
copies of hcp (designed hcp-1, hcp-2 and hcp-3) and four copies of vgrG genes 11
ACCEPTED MANUSCRIPT (vgrG-1, vgrG-2, vgrG-3 and vgrG-4) were identified in A. hydrophila NJ-35. Among
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them, hcp-3, vgrG-3 and vgrG-4 are located in the T6SS gene cluster, while the other
241
ones located outside of the cluster (Fig.1). It’s interesting to be noted that hcp-1 and
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vgrG-1 genes were genetically linked through a region less than 500 bp, as was seen
243
in A. hydrophila SSU [28]. This phenomenon also appeared in hcp-2 and vgrG-2
244
genes, and hcp-3 and vgrG-3 genes. A. hydrophila J-1 and ATCC 7966 had similar
245
organizations of Hcp and VgrG with NJ-35, except that only one hcp-vgrG gene pair
246
was found outside of the T6SS cluster in the two strains (Fig.1).
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3.2. Distribution of the hcp gene in different A. hydrophila isolates
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The hcp gene sequence from ATCC 7966 was searched in all the 31 A.
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hydrophila genome published in the databases (http://www.ncbi.nlm.nih.gov/genome/
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genomes/1422). The results revealed the presence of the hcp gene in 30/31 of A.
251
hydrophila strains with the identity >90%. Further, the hcp fragment was PCR
252
amplified from all the A. hydrophila strains (24/24) tested here, suggesting that hcp is
253
widespread in the A. hydrophila strains.
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3.3. Expression and antigenicity of Hcp
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The hcp gene product was cloned into the pET28a vector and transformed into E.
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coli BL21 host cells. The expression of a 24-kDa recombinant protein was observed
257
in the induced E. coli harboring pET28a-hcp, while no protein was found at the same
258
position in the non-induced E. coli harboring pET28a-hcp or pET28a by SDS-PAGE
259
(Fig. 2). And the purified rHcp protein was shown to have ability to react with
260
convalescent serum from fish clinically infected with A. hydrophila NJ-35 in Western 12
ACCEPTED MANUSCRIPT 261
blot, indicating that Hcp was expressed during naturally occurring infections and
262
could be recognized by the immune system. To prepare polyclonal antibodies against rHcp, the purified rHcp protein was
264
used to vaccinate rabbits and after three injections, the animals showed an apparent
265
ELISA titer of 1:12 800.
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3.4. Hcp secretion in various A. hydrophila strains
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To assess the expression and secretion of Hcp, the polyclonal antibody against
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rHcp was used to detect the levels of Hcp in the supernatants and total cellular
269
fractions of nine A. hydrophila strains of different origins or serotypes. Hcp was
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detected in the whole cell samples of all strains, and supernatants showed a greater
271
variation (Fig. 3). Hcp secretion was more pronounced in strains J-1, BSK10, FBS35
272
and HA50, compared to strains NJ-35, PEG14, L316 and AH9617. Strain ATCC 7966
273
showed minimal Hcp secretion. The outer membrane protein OmpA was not
274
detectable in all supernatant samples prepared from theses strains, indicating that the
275
presence of Hcp in this fraction was not the result of bacterial cell lysis. Further, the
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supernatants of 15 A. hydrophila isolates from diseased Common carp were subjected
277
to Western blot analysis using rabbit polyclonal anti-rHcp, and all were demonstrated
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to be positive for Hcp.
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Then we investigated whether different growth temperatures influenced the
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secretion of Hcp from A. hydrophila NJ-35. As shown in Fig. 4A, Hcp could be
281
secreted into the culture supernatants at 16°C, 20°C and 28°C, but not at 37°C. To test
282
whether the temperature-dependent secretion of Hcp was specific to A. hydrophila 13
ACCEPTED MANUSCRIPT NJ-35 or whether it might also happen to other A. hydrophila strains, we measured the
284
secretion levels of Hcp in A. hydrophila J-1 and ATCC7966. The results showed that
285
the effect of the temperature on Hcp production in the two strains was similar to that
286
in A. hydrophila NJ-35 (Fig. 4B and C). The only exception is that at 37°C, Hcp
287
production could be observed in the total cellular proteins of NJ-35 and J-1, but not in
288
ATCC 7966. Additionally, it is notable that Hcp secretion levels were elevated when
289
the bacteria were cultured at the lower temperature. These results suggested that the
290
secretion feature of Hcp under low-temperature conditions may be common to A.
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hydrophila strains.
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3.5. Vaccine protective efficacy
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The LD50 value of A. hydrophila NJ-35 was determined to be approximately 1.0
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× 104 CFU in Common carp (Table 1). At 45 days post-vaccination, 30 fish from each
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group were i.p. challenged with 5.0 × 105 CFU (50 LD50) of A. hydrophila NJ-35. As
296
shown in Fig. 5, fish vaccinated with rHcp protein and inactivated whole cell vaccine
297
were well protected when challenged with A. hydrophila, and exhibited a survival of
298
46.67 and 53.33%, respectively; whereas only 7.14% survival was observed in the
299
control group. The RPS values were 42.86 and 50.00 for rHcp protein and inactivated
300
vaccine groups, respectively. Symptoms of hemorrhagic septicemia were observed in
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the dying fish. A. hydrophila was isolated from kidneys of all dead fish, suggesting
302
that mortality was caused by the experimental challenge.
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3.6. Antibody levels
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The specific IgM antibody was determined in the serum of vaccinated fish by 14
ACCEPTED MANUSCRIPT ELISA. Compared with the control group, significantly higher antibody levels (P <
306
0.05) were detected in the vaccinated groups after the injection, while there was no
307
significant difference in antibody titres between fish vaccinated with the inactivated
308
vaccine and rHcp protein (P > 0.05) (Fig. 6). After 21 days post-vaccination, the
309
specific IgM of the immunized groups reached the peak, and subsequently a little
310
decreased, but still kept at a high level.
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3.7. Cytokine levels
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Compared with the control group, IL-1β levels in all three examined tissues of
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vaccinated groups were upregulated throughout 50 days post-vaccination. In the
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kidney, significant higher levels of IL-1β (P < 0.05) were observed at 14 days in the
315
vaccinated groups than that in the control group (Fig. 7A). By 35 and 50 days
316
post-vaccination, the spleen IL-1β levels were significantly elevated (P < 0.05) in the
317
vaccinated groups compared to the controls (Fig. 7B). The gills in vaccinated groups
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also had remarkable higher levels (P < 0.05) of IL-1β on days 21 and 50 than that in
319
the controls (Fig. 7C). As shown in Figures 7D, E and F, TNF-α levels in all three
320
examined tissues were significantly increased (P < 0.05) in the vaccinated groups at
321
most time points in comparison with the control group. At the remaining times,
322
TNF-α levels in the vaccinated groups had increased but not significantly (P > 0.05)
323
when compared with the controls.
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4. Discussion
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Hcp is an important T6SS effector protein and plays crucial roles in the
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ACCEPTED MANUSCRIPT pathogenicity of some Gram negative bacterial pathogens. It is also the hallmark
327
protein of a functional T6SS in all bacteria that own this system [33]. The present
328
study indicated that under standard laboratory conditions, Hcp expression is
329
conserved among A. hydrophila strains. However, the secretion levels of Hcp protein
330
varied among isolates. Moreover, our experiment showed that this difference in the
331
detection of Hcp by Western blot analysis was due to varying amounts of Hcp present
332
in the culture supernatants of different strains. The major reason for this is attributed
333
to the diversity in expression levels of hcp gene and secretion of the corresponding
334
protein and/or its possible rates of degradation in different strains. Additionally, we
335
observed the effect of growth temperature on Hcp secretion. When A. hydrophila
336
strains were grown at 16°C, 20°C and 28°C, Hcp was secreted. However, this protein
337
was not secreted when the bacteria were grown at 37°C, indicating that Hcp secretion
338
was strongly induced by low temperature. Similar results were also seen in V.
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cholerae [29] and V. parahaemolyticus [34]. Different from our result, studies
340
conducted by Suarez et al. [35] showed that Hcp in A. hydrophila SSU could be also
341
secreted at 37°C. The difference might be due to the fact that the bacterial strains were
342
isolated from different ecological niches. A. hydrophila SSU was a human isolate,
343
while the strains used in this study were mostly isolated from aquatic animals, in
344
which body temperature follows the surrounding environment. Another very notable
345
phenomenon is that in contrast to the fish isolates NJ-35 and J-1, the environmental
346
isolate ATCC 7966 did not produce Hcp at 37°C. In agreement with this finding,
347
Grim et al. [36] reported that Hcp synthesis and secretion could not be observed in
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strain ATCC 7966. Further studies will be necessary to investigate the factors that
349
influence Hcp production and secretion. The report by Mougous et al. [16] showed that in the patients with P. aeruginosa
351
infection, a high level of antibody against Hcp was tested. During Burkholderia mallei
352
infections, Hcp was expressed in several hosts and was immunogenic [37]. In A.
353
hydrophila SSU, the Hcp protein was demonstrated to provide good protective
354
efficacy for the immunized mice [35]. With the purpose of exploring the potential
355
application of Hcp as a vaccine candidate against fish bacterial pathogens, the current
356
study evaluates its protective efficacy in common carp. The results from the challenge
357
experiment revealed that common carp vaccinated with the recombinant Hcp in this
358
study were well protected relative to the non-vaccinated ones, and the RPS of Hcp
359
protein-vaccinated fish was 42.86, which is in accordance with a report conducted in
360
mice by Suarez et al. [35]. We also notice that the RPS of the inactivated A.
361
hydrophila NJ-35-vaccinated fish was slightly higher than that of the Hcp
362
protein-vaccinated fish during infection, although the difference was not significant
363
(P > 0.05). The small difference may be due to the fact that the inactive whole cell
364
vaccines usually present multiple antigenic components, which contribute to a certain
365
degree of immune synergy against homogenous challenge.
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Resistance to and recovery from bacterial infections are the results of an efficient
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and complex immune response by a combination of the innate and acquired immune
368
system [38,39]. An adaptive immune system in fish is mediated by lymphocytes that
369
exert their effects mainly by means of antibodies [40]. The antibody type in most fish 17
ACCEPTED MANUSCRIPT is IgM tetramers [41]. In the study, the recombinant Hcp showed good
371
immunogenicity in eliciting humoral immune responses in common carp. In
372
comparison with the control group, IgM antibody titers in Hcp protein- or whole
373
bacterial cell-vaccinated groups were significantly increased (P < 0.05), but there was
374
no statistically significant difference between these two vaccinated groups, indicating
375
the immunogenicity of Hcp is equivalent to whole cell antigen. This result led us to
376
speculate that the increased antibody level may contribute to the higher survival rates
377
in both the two vaccinated groups.
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As modulators of the immune responses, cytokines are related to both innate and
379
adaptive immune systems [42]. The main pro-inflammatory cytokines IL-1β [43] and
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TNF-α [44] are the commonly investigated immune-regulatory cytokines in fish.
381
TNF-α has been proved to induce the inflammatory response by regulating the
382
expression of other cytokines including IL-1β, and both of them can enhance a variety
383
of cellular responses such as leukocyte migration and phagocytosis [45, 46]. In this
384
work, we collected three tissues, including kidney with both renal and immune
385
functions [47, 48], spleen as a major secondary organ [43] and gill as one of the main
386
mucosal surfaces and immune barriers [49], to analyze cytokine levels. The enhanced
387
expression of these cytokines in fish vaccinated with rHcp suggested that both innate
388
immunity and adaptive immunity were induced to some extent. In other words, Hcp
389
protein stimulates the fish to upregulate the immune response against exogenous
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antigen. Dash et al. [50] reported that the expression of IL-1β in fish vaccinated with
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recombinant outer membrane protein R-based vaccine of A. hydrophila was increased
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in spleens, which is consistent with our findings. Aeromonas species have a wide range of serotypes and a variety of strains [51],
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which is a major problem in developing vaccines against this pathogen. Some studies
395
have demonstrated that O9 and O5 were the main serotypes causing MAS outbreaks
396
in China [52, 53]. A recent study from our laboratory [54] showed that there exists an
397
identical O-antigen gene cluster in strains NJ-35 and J-1 (O5 serotype), which were
398
responsible for the MAS outbreaks in Jiangsu Province, in 2010 and 1989,
399
respectively, suggesting little change on the epidemic serotypes in these years in
400
China. In the present study, serotypes O9 and O5 as well as some unknown serotypes
401
of A. hydrophila strains were investigated for the presence of Hcp. Our results
402
obtained from PCR and Western blot analyses that Hcp could be detected in the
403
supernatants from all of the A. hydrophila strains detected , indicated that rHcp has
404
the potential to be a common protective antigen of A. hydrophila strains of different
405
origins. The protective efficacy of the rHcp against heterologous challenge strains of
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A. hydrophila will be evaluated in the future study.
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In conclusion, our results demonstrate that Hcp is conserved among A.
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hydrophila strains studied here, and the expressed Hcp of strain NJ-35 induces a
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valuable protective response against this bacterial infection. The recombinant Hcp
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shows promise as a vaccine candidate against A. hydrophila.
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Author Contributions
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Conceived and designed the experiments: NW YL. Performed the experiments:
412 413
NW
YW
MP
JL.
Analyzed
the 19
data:
NW
YW.
Contributed
ACCEPTED MANUSCRIPT 414
reagents/materials/analysis tools: CL YL. Wrote the paper: NW YW MP JL CL YL.
415
Acknowledgments
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This research was funded by National Nature Science Foundation (31072151, 31372454) and Aquatic Three New Projects in Jiangsu Province (D2013-5-4).
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Figure Legends
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Fig. 1. Genetic organization of the T6SS gene cluster of Aeromonas hydrophila NJ-35,
594
J-1 and ATCC 7966. Blue and orange arrows indicate hcp and vgrG genes,
595
respectively.
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Fig. 2. SDS-PAGE and Western blot analysis of Hcp expression. Lane M, molecular
598
weight marker; lane 1, pET28a-hcp in E. coli BL21, non-induced; lane 2, pET28a-hcp
599
in E. coli BL21, 1 mM IPTG-induced for 5 h; lane 3, pET28a in E. coli BL21,
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non-induced; lane 4, pET28a in E. coli BL21, 1 mM IPTG-induced for 4.5 h; lane 5,
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purified recombinant Hcp; lane 6, Western blot of purified Hcp using convalescent
602
serum from challenged fish with A. hydrophila J-1 as the primary antibody.
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Fig. 3. Hcp secretion in A. hydrophila isolates of different serotypes. The bacteria
605
were cultured at 28 °C in LB medium to an OD of 2.0. Lane M, molecular weight
606
marker; lanes 1–9, culture supernatants from AhJ-1 (O5), BSK10 (O5), FBS35 (O9),
607
HA50 (O9), NJ-35 (unknown), PEG14 (O9), L316 (unknown), AH9617 (O9) and
608
ATCC 7966 (unknown). The arrows indicate immunoblot reactivity to Hcp and
609
OmpA. As no OmpA protein was detected in the supernatants, we concluded that
610
there was no significant cell lysis in the procedure.
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Fig. 4. Effects of growth temperature on the expression (whole cells) and secretion 28
ACCEPTED MANUSCRIPT 613
(supernatants) of Hcp in A. hydrophila NJ-35 (A), J-1 (B) and ATCC 7966 (C).
614
Bacteria strains were cultured under indicated temperature to an OD of 2.0. Western
615
blot analyses were performed with anti-Hcp serum and anti-OmpA antibodies.
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Fig. 5. Survival rates of common carp challenged with A. hydrophila. Fish vaccinated
618
with rHcp protein or inactivated A. hydrophila vaccine were challenged (i.p.) by A.
619
hydrophila strain NJ-35 after 45 days post-vaccination (i.p.), and the mortalities were
620
recorded for 10 days.
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Fig. 6. Antibody levels in common carp following vaccination. Serum collected was
623
assayed by ELISA for the presence of antibodies to immunized antigens. Each bar
624
represents the mean of three replicates and errors bars represent the standard deviation.
625
Significant differences at P < 0.05 among different groups at the same time point are
626
indicated by different letters (a, b, c).
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Fig. 7. Cytokine levels in kidney, spleen and gills of common carp at 14, 21, 28, 35
629
and 50 days post-vaccination. Each bar represents the mean of three replicates and
630
errors bars represent the standard deviation. Significant differences at P < 0.05 among
631
different groups at the same time point are indicated by different letters (a, b, c).
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Tables
2
Table 1. Mortalities recorded for LD50 calculations of the Aeromonas hydrophila strain No. of fish that died after inoculation witha:
(CFU)
Expt 1
109
10
10
108
10
10
107
10
9
106
8
9
105
8
104
5
103
2
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0 1.5×104
10 fish per group were inoculated.
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9
8
7
6
5
2
3
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0
1.0×104
1.2×104
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ACCEPTED MANUSCRIPT Highlights Hcp secretion was found to be strongly induced by low temperature in A. hydrophila. Hcp is conserved among A. hydrophila strains of different origins.
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The Hcp vaccination provided relative efficient protection against A. hydrophila infection.
The Hcp vaccination could enhance both the innate and adaptive immune responses in
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fish.