Identification of a cysteine-rich antimicrobial peptide from salivary glands of the tick Rhipicephalus haemaphysaloides

The presence of an effective immune response in the hemocoel of ticks is crucial for survival, as it prevents the invasion of pathogens throughout the animal's body. Antimicrobial peptides (AMPs) play an important role in this response by rapidly killing invading microorganisms. In this study, a subtraction hybridization cDNA library was constructed from the salivary glands of the unfed and fed female tick Rhipicephalus haemaphysaloides, and a novel cysteine-rich AMP designated Rhamp (R. haemaphysaloides antimicrobial peptide) was isolated and identified. The Rhamp was encoded by a gene with an open reading frame of 303 bp which encoded a mature peptide with 8 kDa molecular weight. No identity was found by BLAST search to any database entries. The sequence encoding the Rhamp was subcloned into the pGEX-4T vector and expressed in Escherichia coli. The recombinant protein of Rhamp showed chymotrypsin and elastase-inhibitory activity and markedly inhibited the growth of Gram-negative bacteria, including Pseudomonas aeruginosa, Salmonella typhimurium, and E. coli. Moreover, the recombinant protein also exerted low hemolytic activity. These results indicate the Rhamp is a novel antimicrobial peptide with proteinase activity from the tick R. haemaphysaloides.     

Proteomic analysis of calcium-dependent secretion in Toxoplasma gondii

Toxoplasma gondii is an intracellular protozoan parasite that invades a wide range of nucleated cells. In the course of intracellular parasitism, the parasite releases a large variety of proteins from three secretory organelles, namely, micronemes, rhoptries and dense granules. Elevation of intracellular Ca(2+) in the parasite causes microneme discharge, and microneme secretion is essential for the invasion. In this study, we performed a proteomic analysis of the Ca(2+)-dependent secretion to evaluate the protein repertoire. We found that Ca(2+)-mobilising agents, such as thapsigargin, NH(4)Cl, ethanol and a Ca(2+) ionophore, A23187, promoted the secretion of the parasite proteins. The proteins, artificially secreted by A23187, were used in a comparative proteomic analysis by 2-DE followed by PMF analysis and/or N-terminal sequencing. Major known microneme proteins (MICs), such as MIC2, MIC4, MIC6 and MIC10 and apical membrane antigen 1 (AMA1), were identified, indicating that the proteomic analysis worked accurately. Interestingly, new members of secretory proteins, namely rhoptry protein 9 (ROP9) and Toxoplasma SPATR (TgSPATR), which was a homologue of a Plasmodium secreted protein with an altered thrombospondin repeat (SPATR), were detected in Ca(2+)-dependent secretion. Thus, we succeeded in detecting Ca(2+)-dependent secretory proteins in T. gondii, which contained novel secretory proteins.     

Engorgement of Rhipicephalus haemaphysaloides ticks blocked by silencing a protein inhibitor of apoptosis

Inhibitors of apoptosis (IAPs) are regulators of cell death and may play a role in the salivary glands of ticks during blood-feeding. We cloned the open reading frame (ORF) sequence of the IAP gene in Rhipicephalus haemaphysaloides (RhIAP). The RhIAP ORF of 1887 bp encodes a predicted protein of 607 amino acids, which contains three baculovirus IAP repeat domains and a RING finger motif. A real-time PCR assay showed that RhIAP mRNA was expressed in all the tick developmental stages (eggs, larvae, nymphs, and adults) and in all tissues examined (midgut, ovary, salivary glands, fat body, and hemolymph). Western blot showed that the protein level of RhIAP in salivary glands increased during tick blood-feeding and decreased towards the end of tick engorgement. RhIAP gene silencing in vitro experiments with salivary glands demonstrated that RhIAP could be effectively knocked down within 48 h after dsRNA treatment, and as a consequence, salivary glands displayed apoptotic morphology. RhIAP gene silencing also inhibited tick blood-feeding and decreased the engorgement rate. These data suggest that RhIAP might be a suitable RNAi target for tick control.

     

Protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing TgAMA1 as vaccines against Toxoplasma gondii infection in mice

A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection.     

Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method

Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity. In this study, we used a conserved sequence in the 200- to 300-fold repetitive 529 bp gene of Toxoplasma gondii to design primers for LAMP test. Detection limit of T. gondii LAMP assay with the primers is 1 pg/μL of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. Furthermore, LAMP and conventional PCR methods were applied for amplification of the T. gondii DNA extracted from the lymph nodes taken from pigs which were suspected to be Toxoplasma infection. As a result, 76.9% (70/91) and 85.7% (78/91) of the samples were positive on PCR and LAMP analyzes, respectively. Therefore, the LAMP has a potential to be applied as an alternative molecular diagnostic tool for detection of T. gondii infection from veterinary samples. This is the first study, which applies the LAMP method to diagnose Toxoplasma from veterinary samples.     

Construction of Toxoplasma gondii bradyzoite expressing the green fluorescent protein

The bradyzoite stage of Toxoplasma gondii is a key step in the parasite life cycle. For a better understanding of this stage, a sensitive system to detect the tissue cysts would be required. In this study, we generated the T. gondii cyst-forming strain PLK expressing green fluorescent protein (GFP) under control of the dense granule protein 1 promoter, which works at both the tachyzoite and the bradyzoite stages. The bradyzoites with GFP fluorescence within both small and large cysts were detectable in the brain of mice infected with the recombinant PLK. Indeed, the bradyzoites expressing GFP had infectivity to mice. This study shows that transfection of the cyst-forming strain with GFP gene under control of the GRA1 promoter could be a useful approach for the study of the bradyzoite stage of T. gondii.