Oral vaccination of mice with attenuated Salmonella encoding Trichinella spiralis calreticulin and serine protease 1.1 confers protective immunity in BALB/c mice.

BACKGROUND: Trichinella spiralis is a foodborne parasitic nematode which is a serious risk to meat safety. Development of anti-Trichinella vaccine is needed to control Trichinella infection in food animals. In this study, two novel T. spiralis genes (calreticulin and serine protease 1.1) in combination were used to construct oral DNA vaccines, and their induced protective immunity was evaluated in a murine model. METHODOLOGY/PRINCIPAL FINDINGS: TsCRT+TsSP1.1, TsCRT and TsSP1.1 DNA were transformed into attenuated Salmonella typhimurium ΔcyaSL1344. Oral vaccination of mice with TsCRT+TsSP1.1, TsCRT and TsSP1.1 DNA vaccines elicited a gut local mucosal sIgA response and systemic Th1/Th2 mixed response. Oral vaccination with TsCRT+TsSP1.1 induced obviously higher level of serum specific antibodies, mucosal sIgA and cellular immune response than either of single TsCRT or TsSP1.1 DNA vaccination. Oral vaccination of mice with TsCRT+TsSP1.1 exhibited a 53.4% reduction of enteral adult worms and a 46.05% reduction of muscle larvae, conferred a higher immune protection than either of individual TsCRT (44.28 and 42.46%) or TsSP1.1 DNA vaccine (35.43 and 29.29%) alone. Oral vaccination with TsCRT+TsSP1.1, TsCRT and TsSP1.1 also obviously ameliorated inflammation of intestinal mucosa and skeletal muscles of vaccinated mice after challenge. CONCLUSIONS: TsCRT and TsSP1.1 might be regarded the novel potential targets for anti-Trichinella vaccines. Attenuated Salmonella-delivered DNA vaccine provided a prospective approach to control T. spiralis infection in food animals.

Oral immunization with attenuated Salmonella encoding an elastase elicits protective immunity against Trichinella spiralis infection.

Elastase belongs to the serine protease family. Previous studies showed that Trichinella spiralis elastase (TsE) was highly expressed in intestinal infective larvae (IIL). Recombinant TsE (rTsE) promoted the larval intrusion of enteral epithelium cells (IECs), whereas anti-rTsE antibodies and siRNA impeded larval intrusion. Subcutaneous vaccination of mice with rTsE showed a partial protective immunity, suggesting that TsE might be a promising vaccine target against Trichinella infection. In this study, complete TsE cDNA sequence was cloned into pcDNA3.1, and the rTsE DNA was transformed into attenuated S. typhimurium strain ΔcyaSL1344. Oral vaccination of mice with TsE DNA elicited a systemic Th1/Th2/Treg mixed immune response and gut local mucosal sIgA response. Immunized mice exhibited a significant immune protection against T. spiralis larval challenge, as demonstrated by a 52.48% reduction of enteral adult worms and a 69.43% reduction of muscle larvae. The protection might be related to the TsE-induced production of intestinal mucus, specific anti-TsE sIgA and IgG, and secretion of IFN-γ, IL-2, IL-4 and IL-10, which protected gut mucosa from larval intrusion, suppressed worm development and impeded female reproduction. The results demonstrated that attenuated Salmonella-delivered TsE DNA vaccine provided a prospective strategy for the control of Trichinella infection in food animals.

A metalloproteinase Tsdpy31 from Trichinella spiralis participates in larval molting and development.

Trichinellosis is a serious food-borne zoonotic parasitic disease with global distribution, causing serious harm to public health and food safety. Molting is prerequisite for intestinal larval development in the life cycle of T. spiralis. Metalloproteinases play an important role in the molting process of T. spiralis intestinal infective larvae (IIL). In this study, the metalloproteinase Tsdpy31 was cloned, expressed and characterized. The results revealed that the Tsdpy31 was expressed at various T. spiralis stages and it was principally located in cuticle, hypodermis and embryos of the nematode. Recombinant Tsdpy31 (rTsdpy31) had the catalytic activity of natural metalloproteinase. Silencing of Tsdpy31 increased the permeability of larval new cuticle. When the mice were orally challenged with dsRNA treated- muscle larvae, the burden of intestinal adult and muscle larvae in Tsdpy31 dsRNA treatment group was significantly reduced, compared with the control green fluorescent protein (GFP) dsRNA and PBS groups (P < 0.05). Tsdpy31 may play a major role in the new cuticle synthesis and old cuticle shedding. Tsdpy31 also participates in T. spiralis embryonic development. We conclude that Tsdpy31 could be a candidate vaccine target molecule against intestinal T. spiralis ecdysis and development.

Proteomic analysis of hydrolytic proteases in excretory/secretory proteins from Trichinella spiralis intestinal infective larvae using zymography combined with shotgun LC-MS/MS approach.

The critical step of Trichinella spiralis infection is that the muscle larvae (ML) are activated to intestinal infective larvae (IIL) which invade the intestinal columnar epithelium to further develop. The IIL excretory/secretory (ES) proteins play an important role in host-parasite interaction. Proteolytic enzymes are able to mediate the tissue invasion, thereby increasing the susceptibility of parasites to their hosts. The aim of the current study was to screen and identify the natural active proteases in T. spiralis IIL ES proteins using Western blot and gel zymography combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). The T. spiralis ML and IIL ES proteins were collected from the in vitro cultures and their enzymatic acitvities were examined by gelatin zymography and azocasein degradation. The protease activities were partially inhibited by PMSF, E-64 and EDTA. Three protein bands (45, 118 and 165 kDa) of T. spiralis IIL ES proteins were identified by shotgun LC-MS/MS because they have hydrolytic activity to gelatin compared to the ML ES proteins. Total of 30 T. spiralis proteins were identified and they are mainly serine proteinases (19), but also metalloproteinases (7) and cysteine proteinases (3). The qPCR results indicated that transcription levels of four T. spiralis protease genes (two serine proteases, a cathepsin B-like cysteine proteinase and a zinc metalloproteinase) at IIL stage were obviously higher than at the ML stage. These proteolytic enzymes are directly exposed to the host intestinal milieu and they may mediate the worm invasion of enteral epithelium and escaping from the host's immune responses. The results provide the new insights into understanding of the interaction of T. spiralis with host and the invasion mechanism.

Molecular characterization of a Trichinella spiralis aspartic protease and its facilitation role in larval invasion of host intestinal epithelial cells.

BACKGROUND: T. spiralis aspartic protease has been identified in excretion/secretion (ES) proteins, but its roles in larval invasion are unclear. The aim of this study was to characterize T. spiralis aspartic protease-2 (TsASP2) and assess its roles in T. spiralis invasion into intestinal epithelial cells (IECs) using RNAi. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant TsASP2 (rTsASP2) was expressed and purified. The native TsASP2 of 43 kDa was recognized by anti-rTsASP2 serum in all worm stages except newborn larvae (NBL), and qPCR indicated that TsASP2 transcription was highest at the stage of intestinal infective larvae (IIL). IFA results confirmed that TsASP2 was located in the hindgut, midgut and muscle cells of muscle larvae (ML) and IIL and intrauterine embryos of the female adult worm (AW), but not in NBL. rTsASP2 cleaved several host proteins (human hemoglobin (Hb), mouse Hb, collagen and IgM). The proteolytic activity of rTsASP2 was host-specific, as it hydrolyzed mouse Hb more efficiently than human Hb. The enzymatic activity of rTsASP2 was significantly inhibited by pepstatin A. The expression levels of TsASP2 mRNA and protein were significantly suppressed by RNAi with 5 µM TsASP2-specific siRNA. Native aspartic protease activity in ML crude proteins was reduced to 54.82% after transfection with siRNA. Larval invasion of IECs was promoted by rTsASP2 and inhibited by anti-rTsASP2 serum and siRNA. Furthermore, cell monolayer damage due to larval invasion was obviously alleviated when siRNA-treated larvae were used. The adult worm burden, length of adult worms and female fecundity were clearly reduced in mice challenged using siRNA-treated ML relative to the PBS group. CONCLUSIONS: rTsASP2 possesses the enzymatic activity of native aspartic protease and facilitates T. spiralis invasion of host IECs.