Recombinant Aeromonas Hydrophila Research Articles

Effect of a new recombinant Aeromonas hydrophila vaccine on the grass carp intestinal microbiota and correlations with immunological responses

Intestinal microbiota has become an integral component of the fish, and plays a key role in host metabolism, immunity and health maintenance. However, information on the immune responses after vaccine administration in relation to the intestinal microbiota is absent in fish. The present study focused on the effect of a new recombinant Aeromonas hydrophila vaccine (Aera) by using a novel functionalized, single-walled carbon nanotubes (SWCNTs) as a delivery vehicle on the intestinal microbiota of grass carp (Ctenopharyngodon idella) through the bath immunization, and further explored the immunological responses in intestine, kidney and spleen. By performing deep sequencing, a total of 81,979 valid reads and 609 OTUs obtained from 4 intestine samples were analyzed. We detected 141 genuses, most of which belonged to Firmicutes, Fusobacteria and Proteobacteria. Of note, the quantity of Aeromonas in library Aera (after 6 h Aera vaccine pretreatment, fish were transferred to tanks without SWCNTs-Aera for 28 d) and Aera-GD (6 h Aera vaccine pretreated prior to the group injected by A. hydrophila) was declined 6.5% and 14.6% compared with the control, respectively. Moreover, the expression of seven immune-related genes (IFN-I, TNF-α, CRP, IL-8, IgM, MHC I and CD8α) in the intestine, kidney and spleen of Aera treated fish was significantly enhanced, which indicated that a better tissue immune response in grass carp was induced by the SWCNTs-Aera vaccine. Therefore, a new recombinant SWCNT-Aera vaccine may represent potentially efficient and immunological role in grass carp intestine to resist A. hydrophila infection.

Single-walled carbon nanotubes as delivery vehicles enhance the immunoprotective effects of a recombinant vaccine against Aeromonas hydrophila

To reduce the economic losses caused by diseases in aquaculture industry, more efficient and economic prophylactic measures should be urgently investigated. In this research, the effects of a novel functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for recombinant Aeromonas hydrophila vaccine administration via bath or injection in juvenile grass carp were studied. The results showed that SWCNT as a vector for the recombinant protein aerA, augmented the production of specific antibodies, apparently stimulated the induction of immune-related genes, and induced higher level of survival rate compared with free aerA subunit vaccine. Furthermore, we compared the routes of bath and intramuscular injection immunization by SWCNTs-aerA vaccine, and found that similar antibody levels induced by SWCNTs-aerA were observed in both immunization routes. Meanwhile, a similar relative percentage survival (approximately 80%) was found in both a 40 mg/L bath immunization group, and a 20 μg injection group. The results indicate that functionalized SWCNTs could be a promising delivery vehicle to potentiate the immune response of recombinant vaccines, and might be used to vaccinate juvenile fish by bath administration method.

Recombinant Aeromonas hydrophila outer membrane protein 48 (Omp48) induces a protective immune response against Aeromonas hydrophila and Edwardsiella tarda

The gene coding for an outer membrane protein Omp48 of Aeromonas hydrophila isolated from an infected fish was cloned and sequenced. Analysis of nucleotide sequence showed the omp48 gene to be an adhesin encoding a protein of 426 amino acids with high identity to the omp48 gene of Aeromonas veronii, another fish pathogen. The gene belonged to the maltoporin group of porins and had high similarity to LamB porins of A. hydrophila, Aeromonas salmonicida and Vibrio parahaemolyticus. The expressed purified recombinant protein had an estimated molecular weight of 48 kDa. Further, rabbit hyperimmune sera against the recombinant protein reacted with A. hydrophila, Aeromonas sobria and A. veronii whole cell proteins at the region of 48 kDa, in western blotting. The recombinant protein was immunogenic in the fish Labeo rohita Hamilton. Fish immunized with recombinant protein, when challenged with virulent A. hydrophila and another bacterial fish pathogen, Edwardsiella tarda, showed relative percent survivals of 69 and 60, respectively. Our results suggest that Omp48 of A. hydrophila could be used as a potential vaccine candidate for protection not only against A. hydrophila infection, but also against the fish pathogen E. tarda.

Metabolic engineering for microbial production of polyhydroxyalkanoates consisting of high 3-hydroxyhexanoate content by recombinant Aeromonas hydrophila

Polyhydroxyalkanoate synthase gene phaCah in Aeromonas hydrophila strain 4AK4 was deleted and its function was replaced by phaC1ps cloned from Pseudomonas stutzeri strain 1317 which favors 3-hydroxyhexanoate (3HHx) and longer chain length monomers. Genes fadD and fadL encoding Escherichia coli acyl-CoA synthase and Pseudomonas putida KT2442 fatty acid transport protein, respectively, were introduced into the recombinant with new phaC1ps. Accumulation of a series of novel medium-chain-length polyhydroxyalkanoates (mcl-PHA) consisting of 80–94mol% 3HHx were observed. The recombinant accumulated 54% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) in cell dry weight consisting of 94.5mol% 3HHx or 51% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) consisting of 82mol% 3HHx and 16mol% of 3HO during a two-step cultivation process under nitrogen limitation when grown on sodium hexanoate or sodium octanoate. The two polyesters containing high percentage of 3HHx are physically characterized. They could be used as biodegradable pressure sensitive adhesives, coatings, polymer binding agents in organic-solvent-free paints or a source for chiral R-3-hydroxyhexanoate.

Production and characterization of homopolymer poly(3-hydroxyvalerate) (PHV) accumulated by wild type and recombinant Aeromonas hydrophila strain 4AK4

Aeromonas hydrophila 4AK4 normally produces copolyesters (PHBHHx) consisting of 3-hydroxybutyrate (C4) and 3-hydroxyhexanoate (C6). Wild type and recombinant A. hydrophila 4AK4 (pSXW02) expressing vgb and fadD genes encoding Vitreoscilla haemoglobin and Escherichia coli acyl-CoA synthase respectively, were found able to produce homopolyester poly(3-hydroxyvalerate) (PHV) (C5) on undecanoic acid as a single carbon source. The recombinant grew to 5.59 g/L cell dry weight (CDW) containing 47.74 wt% PHV in shake flasks when growth was conducted in LB medium and PHV production in undecanoic acid. The cells grew to 47.12 g/L CDW containing 60.08 wt% PHV in a 6 L fermentor study. Physical characterization of PHV produced by recombinant A. hydrophila 4AK4 (pSXW02) in fermentor showed a weight average molecular weight ( M w) of 230,000 Da, a polydispersity of 3.52, a melting temperature of 103 °C and a glass transition temperature of −15.8 °C. The degradation temperature at 5% weight loss of the PHV was around 258 °C.

Biocompatibility of poly(3-hydroxybutyrate- co-3-hydroxyvalerate- co-3-hydroxyhexanoate) with bone marrow mesenchymal stem cells

As a new member of the polyhydroxyalkanoate (PHA) family, poly(3-hydroxybutyrate- co-3-hydroxyvalerate- co-3-hydroxyhexanoate) (PHBVHHx) was produced by recombinant Aeromonas hydrophila 4AK4. PHBVHHx showed a rougher surface and had higher hydrophobicity than the well-studied polymers poly( l-lactic acid) (PLA) and poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (PHBHHx). Human bone marrow mesenchymal stem cells (MSCs) adhered better on PHBVHHx film than on tissue culture plates (TCPs), PLA film and PHBHHx film. The cell number on the PHBVHHx film was 115% higher than that on the TCPs, 66% higher than on the PHBHHx film and 263% higher than on the PLA film ( p<0.01). PHBVHHx also supported the osteogenic differentiation of MSCs. Previous studies have shown that all PHA polymers tested were either poorer than or equal to TCPs for supporting cell growth. PHBVHHx is the only PHA polymer to significantly increase cell numbers compared with TCPs. These data demonstrate that PHBVHHx could be a promising biomaterial for bone tissue engineering.

Influence of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) on growth and osteogenic differentiation of human bone marrow‐derived mesenchymal stem cells

As a new member of polyhydroxyalkanoate (PHA) family, the novel polyester poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-4HB-3HHx)) was produced by recombinant Aeromonas hydrophila 4AK4 and used for the first time to test its biocompatibility. It was shown that P(3HB-4HB-3HHx) had higher hydrophobicity, surface energy, and rougher surface than the well-studied polymers poly(L-lactic acid) (PLA) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Human mesenchymal stem cells (MSCs) attached better on P(3HB-4HB-3HHx) film than on tissue culture plates (TCPs), PLA film, and PHBHHx film. MSC proliferation on P(3HB-4HB-3HHx) film was 126% higher than that on TCPs, 84% higher than that on PHBHHx film, and 312% higher than that on PLA film (p < 0.01). P(3HB-4HB-3HHx) also supported osteogenic differentiation of MSCs. Previous studies found that all PHA materials tested were either less than or equal to TCPs for supporting cell growth. Among all PHA materials tested, P(3HB-4HB-3HHx) was the only PHA material to significantly promote cell proliferation compared with TCPs. P(3HB-4HB-3HHx) could be exploited for applications in bone tissue engineering.

Study on the biocompatibility of novel terpolyester poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate‐co‐3‐hydroxyhexanoate)

Terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) containing 5.3 mol % 3-hydroxyvalerate (3-HV) and 10.2 mol % 3-hydroxyhexanoate was obtained via microbial synthesis using recombinant Aeromonas hydrophila. For the first time in vitro biocompatibility of the terpolyester was evaluated in comparison with poly(L-lactic acid), poly (3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Scanning electron microscopy showed whorls-like morphology only on PHBVHHx film surface. Methylthiazol tetrazolium assay demonstrated that PHBVHHx was better than the above four materials in promoting cell attachment and proliferation of fibroblast cell line L929 and osteoblast cell line MC3T3, respectively. Histological study using rabbits proved that PHBVHHx was a fairly harmless implantable biomaterial. Thus, PHBVHHx with an adjustable mechanical properties, combined with its biocompatibility, are in the process of developing into a new generation of bioimplant material.

Production and characterization of terpolyester poly(3-hydroxybutyrate- co-4-hydroxybutyrate- co-3-hydroxyhexanoate) by recombinant Aeromonas hydrophila 4AK4 harboring genes phaPCJ

Terpolyester consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyhexanoate (3HHx) abbreviated as P(3HB- co-4HB- co-3HHx), was produced by recombinant Aeromonas hydrophila 4AK4 harboring polyhydroxyalkanoate (PHA) synthesis genes phaPCJ. The monomer content of 4HB in the terpolyesters was dependent on the concentration of 1,4-butanediol, ranging from 4.3 to 8.5 mol% in the presence of 5–25 g/l 1,4-butanediol, respectively, accompanying by a decreasing 3HHx content from 22 to 17.6 mol% and a constant 3HB content of 74 mol% during growth in lauric acid containing mineral medium. Number average molecular weights of the terpolyesters were 440,000 to 550,000, much higher than 220,000 of the copolyester consisting of 3HB and 12 mol%3HHx commonly produced by wild type A. hydrophila 4AK4. Thermal and mechanical properties of the above terpolyesters were better than those of homopolyester poly(3-hydroxybutyrate) (PHB) and its copolymers P(3HB- co-7 mol%4HB), P(3HB- co-12 mol%3HHx). Moreover, physical properties of the terpolyesters changed with variations of monomer composition. The terpolyesters were characterized using nuclear magnetic resonance (NMR), gas chromatography (GC), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and stress–strain measurements. The introduction of 4HB and 3HHx monomers into PHB enhanced the thermal stability, changed its crystallinity and flexibility compared with homopolyester PHB and their two monomer containing copolyesters.

Production and characterization of terpolyester poly(3-hydroxybutyrate- co-3-hydroxyvalerate- co-3-hydroxyhexanoate) by recombinant Aeromonas hydrophila 4AK4 harboring genes phaAB

Recombinant Aeromonas hydrophila 4AK4 harboring phbA and phbB ( phaAB) genes encoding β-ketothiolase and acetoacetyl-CoA reductase of Ralstonia eutropha produced a terpolyester of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB- co-3HV- co-3HHx)] from mixtures of dodecanoic acid and propionic acid. Depending on the concentration of propionic acid in bacterial cultures, cell growth represented by cellular dry weight (CDW), P(3HB- co-3HV- co-3HHx) contents in dry cells and 3HV molar percentage in the terpolyester ranged from 0.43 g l −1 to 3.29 g l −1, 20.7% to 35.6%, 2.3 mol% to 7.1 mol%, respectively. Number average molecular ( M n) weights of the terpolyesters were 303,000–800,000, independent from monomer fraction content. This terpolyester was characterized by nuclear magnetic resonance (NMR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and stress–strain measurement studies. Results showed that the terpolyester had higher thermal stability and elongation at break compared with that of homopolymer poly(3-hydroxybutyrate) (PHB) and its copolymers P(3HB- co-5 mol%3HV) or P(3HB- co-12 mol%3HHx). In addition, the terpolyester had lower melting ( T m) temperatures and enthalpy of fusions (Δ H m) than PHB did.

Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Gluconate and Glucose by Recombinant Aeromonas hydrophila and Pseudomonas putida

Aeromonas hydrophila 4AK4 and Pseudomonas putida GPp104 were genetically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) using gluconate and glucose rather than fatty acids. A truncated tesA gene, encoding cytosolic thioesterase I of Escherichia coli which catalyzes the conversion of acyl-ACP into free fatty acids, was introduced into A. hydrophila 4AK4. When grown in gluconate, the recombinant A. hydrophila 4AK4 synthesized 10% (w/w) PHBHHx containing 14% (mol/mol) 3-hydroxyhexanoate. If additional PHBHHx synthesis genes, phaPCJ, were over-expressed with the truncated tesA in A. hydrophila 4AK4, the PHBHHx content increased to 15% (w/w) and contained 19% (mol/mol) 3-hydroxyhexanoate. Recombinant P. putida GPp104 harboring phaC encoding PHBHHx synthase of A. hydrophila, phaB encoding acetoacetyl-CoA reductase of Wautersia eutropha and phaG encoding 3-hydroxyacyl-ACP-CoA transferase of P. putida, synthesized 19% (w/w) PHBHHx containing 5% (mol/mol) 3-hydroxyhexanoate from glucose. The results suggest that the engineered pathways were applicable to synthesize PHBHHx from unrelated carbon sources such as gluconate and glucose.