Oral vaccination with recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase elicited a protective immunity in BALB/c mice.

BACKGROUND: Trichinellosis is a serious zoonotic disease distributed around the world. It is needed to develop a safe, effective and feasible anti-Trichinella vaccine for prevention and control of trichinellosis. The aim of this study was to construct a recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase (TsPPase) and investigate its immune protective effects against T. spiralis infection. METHODOLOGY/PRINCIPAL FINDINGS: The growth of recombinant L. plantarum was not affected by TsPPase/pSIP409-pgsA' plasmid, and the recombinant plasmid was inherited stably in bacteria. Western blot and immunofluorescence assay (IFA) indicated that the rTsPPase was expressed on the surface of recombinant L. plantarum. Oral vaccination with rTsPPase induced higher levels of specific serum IgG, IgG1, IgG2a and mucosal secretory IgA (sIgA) in BALB/c mice. ELISA analysis revealed that the levels of IFN-γ and IL-4 released from spleen, mesenteric lymph nodes and Peyer's patches were evidently increased at 2-4 weeks following vaccination, compared to MRS (De Man, Rogosa, Sharpe) medium control group (P < 0.05). Immunization of mice with rTsPPase exhibited a 67.18, 54.78 and 51.91% reduction of intestinal infective larvae, adult worms and muscle larvae at 24 hours post infection (hpi), 6 days post infection (dpi) and 35 dpi, respectively (P < 0.05), and the larval molting and development was significantly inhibited by 45.45% at 24 hpi, compared to the MRS group. CONCLUSIONS: TsPPase plays a crucial role in T. spiralis molting and development, oral vaccination with rTsPPase induced a significant local mucosal sIgA response and systemic Th1/Th2 immune response, and immune protection against T. spiralis infection in BALB/c mice.

Identification of a novel PYP-1 gene in Sarcoptes scabiei and its potential as a serodiagnostic candidate by indirect-ELISA.

Scabies is a parasitic disease caused by the ectoparasite Sarcoptes scabiei, affecting different mammalian species, including rabbits, worldwide. In the present study, we cloned and expressed a novel inorganic pyrophosphatase, Ssc-PYP-1, from S. scabiei var. cuniculi. Immunofluorescence staining showed that native Ssc-PYP-1 was localized in the tegument around the mouthparts and the entire legs, as well as in the cuticle of the mites. Interestingly, obvious staining was also observed on the fecal pellets of mites and in the integument of the mites. Based on its good immunoreactivity, an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant Ssc-PYP-1 (rSsc-PYP-1) as the capture antigen was developed to diagnose sarcoptic mange in naturally infected rabbits; the assay had a sensitivity of 92·0% and specificity of 93·6%. Finally, using the rSsc-PYP-1-ELISA, the Ssc-PYP-1 antibody from 10 experimentally infected rabbits could be detected from 1 week post-infection. This is the first report of S. scabiei inorganic pyrophosphatase and the protein could serve as a potential serodiagnostic candidate for sarcoptic mange in rabbits.

Presence of an isoform of H+-pyrophosphatase located in the alveolar sacs of a scuticociliate parasite of turbot: physiological consequences.

H+-pyrophosphatases (H+-PPases) are integral membrane proteins that couple pyrophosphate energy to an electrochemical gradient across biological membranes and promote the acidification of cellular compartments. Eukaryotic organisms, essentially plants and protozoan parasites, contain various types of H+-PPases associated with vacuoles, plasma membrane and acidic Ca+2 storage organelles called acidocalcisomes. We used Lysotracker Red DND-99 staining to identify two acidic cellular compartments in trophozoites of the marine scuticociliate parasite Philasterides dicentrarchi: the phagocytic vacuoles and the alveolar sacs. The membranes of these compartments also contain H+-PPase, which may promote acidification of these cell structures. We also demonstrated for the first time that the P. dicentrarchi H+-PPase has two isoforms: H+-PPase 1 and 2. Isoform 2, which is probably generated by splicing, is located in the membranes of the alveolar sacs and has an amino acid motif recognized by the H+-PPase-specific antibody PABHK. The amino acid sequences of different isolates of this ciliate are highly conserved. Gene and protein expression in this isoform are significantly regulated by variations in salinity, indicating a possible physiological role of this enzyme and the alveolar sacs in osmoregulation and salt tolerance in P. dicentrarchi.

Presence of a plant-like proton-translocating pyrophosphatase in a scuticociliate parasite and its role as a possible drug target.

The proton-translocating inorganic pyrophosphatases (H(+)-PPases) are primary electrogenic H(+) pumps that derive energy from the hydrolysis of inorganic pyrophosphate (PPi). They are widely distributed among most land plants and have also been found in several species of protozoan parasites. Here we describe, for the first time, the molecular cloning and functional characterization of a gene encoding an H(+)-pyrophosphatase in the protozoan scuticociliate parasite Philasterides dicentrarchi, which infects turbot. The predicted P. dicentrarchi PPase (PdPPase) consists of 587 amino acids of molecular mass 61.7 kDa and an isoelectric point of 5.0. Several motifs characteristic of plant vacuolar H(+)-PPases (V-H(+)-PPases) were also found in the PdPPase, which contains all the sequence motifs of the prototypical type I V-H(+)-PPase from Arabidopsis thaliana vacuolar pyrophosphatase type I (AVP1) plant. The PdPPase has a characteristic residue that determines strict K(+)-dependence, but unlike AVP1, PdPPase contains an N-terminal signal peptide (SP) sequence. Antibodies generated by vaccination of mice with a genetic or recombinant protein containing a partial sequence of the PdPPase and a common motif with the polyclonal antibody PABHK specific to AVP1 recognized a single band of about 62 kDa in western blots. These antibodies specifically stained both vacuole and the alveolar membranes of trophozoites of P. dicentrarchi. H+ transport was partially inhibited by the bisphosphonate pamidronate (PAM) and completely inhibited by NaF. The bisphosphonate PAM inhibited both H+-translocation and gene expression. PdPPase and PAM also inhibited in vitro growth of the ciliates. The apparent lack of V-H(+)-PPases in vertebrates and the parasite sensitivity to PPI analogues may provide a molecular target for developing new drugs to control scuticociliatosis.

Development and evaluation of enzyme-linked immunosorbent assay based on recombinant inorganic pyrophosphatase gene antigen for the detection of Mycoplasma suis antibodies.

The recombinant ppa protein of Mycoplasma suis migrated to 21 kDa. Using this antigen, an ELISA system to detect the antibody against M. suis infection in swine was established. The rELISA demonstrated 98.5% specificities among negative samples and 96.9% sensitivity among positive samples with M. suis infection. A comparison of this ELISA system with an indirect hemagglutination assay (IHA) test using 132 swine samples revealed that the positive rate was 34.0% in ELISA and 28.0% in IHA. Compared with IHA, the present rELISA system using recombinant ppa antigen significantly improves the specificity, sensitivity, and stability for serodiagnosis of M. suis infection in swine.

Expression of new human inorganic pyrophosphatase in thyroid diseases: its intimate association with hyperthyroidism.

Inorganic pyrophosphatase (PPase) controls the level of inorganic pyrophosphate produced by biosynthesis of protein, RNA, and DNA. Thus, PPase is essential for life. PPase expression is unclear in the thyroid. We cloned a new human PPase, phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPPase), and established a rabbit polyclonal anti-LHPPase antibody. This is the first study to determine the PPase expression by immunohistochemistry and Western blot. Intranuclear LHPPase expression of thyrocytes was enhanced most prominently in Graves' disease and autonomously functional thyroid nodule. To estimate a regulating factor of subcellular localization of LHPPase, we examined its expression of Graves' disease-derived thyrocytes in vitro with the disease-originated serum. Nuclear expression of LHPPase was lost in cultured thyrocytes even with the serum, while its cytoplasmic expression was retained. The data suggest that increased expression of LHPPase is associated with hyperthyroidism. Intranuclear expression of LHPPase may not be regulated by Graves' disease-derived serum factors.