Houshuang Zhang

Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis

Loop-mediated isothermal amplification (LAMP) is a simple method that can amplify DNA with high specificity, sensitivity, and rapidity. In this study, we compared the performance of LAMP and real-time PCR assays for diagnosis of toxoplasmosis. We designed a real-time PCR assay targeting a 529 bp element repeated 200-300 times in the Toxoplasma gondii genome. The detection limits of the LAMP and real-time PCR assays were 10 fg/μL and 1 fg/μL of T. gondii DNA, respectively. Conventional PCR, LAMP, and real-time PCR methods were applied to detect T. gondii DNA in blood samples from 284 pigs and 292 sheep. Positive results were obtained with 0.4%, 3.2%, and 4.2% of the pig samples and 3.8%, 17.1%, and 17.8% of the sheep samples with conventional PCR, LAMP, and real-time PCR analyses, respectively. The real-time PCR assay provided the most sensitive diagnosis of toxoplasmosis, but the LAMP assay has potential as an alternative tool for detection of T. gondii in the field.

Characterization of the anticoagulant protein Rhipilin-1 from the Rhipicephalus haemaphysaloides tick

To understand the molecular mechanism of tick blood feeding, an anticoagulant protein, Rhipilin-1, was identified in the tick Rhipicephalus haemaphysaloides. The cDNA sequence of Rhipilin-1 is 620bp, and it encodes a deduced 164 amino acid protein with a size of 18kDa. Bioinformatic analysis shows that Rhipilin-1 belongs to the Kunitz-type family of inhibitors, containing one Kunitz domain with high homology to the tissue factor pathway inhibitor (TFPI). The recombinant protein expressed in Escherichia coli delayed normal clotting of rabbit plasma both in the recalcification time (RT) and the activated partial thromboplastin time (APTT) tests. Using RT-PCR, mRNA transcripts of Rhipilin-1 were detected in fed but not in unfed ticks. Disruption of the Rhipilin-1 gene with RNAi led to a 52.7% decrease in the tick attachment rate 24h after introduction in the rabbit ears and a 21.9% decrease in the average engorged body weight of ticks. These results indicate that Rhipilin-1 is a novel anticoagulant protein involved in tick blood feeding with possible future application as a vaccine candidate. The discovery of Rhipilin-1 is the first report on anticoagulant genes in this species of tick.

Isolation and characterization of two novel serpins from the tick Rhipicephalus haemaphysaloides.

Two novel serpins with anti-chymotrypsin activity, RHS-1 and RHS-2, were identified in the tick Rhipicephalus haemaphysaloides. The complementary cDNA sequence of RHS-1 was 1286 base pairs (bp) and encoded a deduced 403-amino acid protein with a signal peptide, whereas that of RHS-2 was 1682bp and encoded a deduced 380-amino acid protein with no signal peptide. Although both RHS-1 and RHS-2 exhibited high sequence similarities to known serpins from other ticks, the level of similarity at the amino acid level between the 2 serpins characterized here was only 32.5%. Salivary gland-specific expression of RHS-1 and midgut-specific expression of RHS-2 were found by Western blot using the relevant antiserum. We tested the ability of purified recombinant rRHS-1 and rRHS-2 to inhibit various serine proteases and found that both significantly inhibited chymotrypsin (95.6% and 94.2%, respectively). We further demonstrated that RHS-1, but not RHS-2 exhibited anticoagulation activity, based on activated partial thromboplastin time (APTT). Disruption of the genes encoding the 2 serpins with RNA interference (RNAi) led to a significant decrease in tick attachment and engorgement rates. These results indicate that RHS-1 and RHS-2 are 2 novel serpins with anti-chymotrypsin activity that are involved in blood feeding of R. haemaphysaloides.

Distinctive microRNA profiles in the salivary glands of Haemaphysalis longicornis related to tick blood-feeding

The salivary glands are vital to the biological success of ticks and they are a major route of pathogen transmission. Tick salivary glands undergo remarkable growth and differentiation during the blood-feeding period. MicroRNAs (miRNAs) are noncoding small RNA molecules found in diverse organisms that regulate gene expression at the post-transcriptional level. To explore transcriptional differences in the miRNAs of fed and unfed tick (Haemaphysalis longicornis) salivary glands, we investigated small RNA (sRNA) transcriptomes derived from the salivary glands and made a comparative analysis of miRNA profiles related to tick blood-feeding in the salivary glands. We generated two small RNA libraries from the salivary glands of unfed and fed H. longicornis, and obtained 14.8 and 10.3 million reads of 18–30xa0nt, respectively. The unfed-specific sRNAs were clearly richer than the fed-specific sRNAs in terms of the unique and total sRNAs. Overall, 769 conserved miRNA families were found in unfed samples, whereas 440 conserved miRNA families were found in fed samples. Six of the ten most abundant miRNA were found in both the unfed and fed tick salivary glands, i.e., miR-1, miR-375, bantam, miR-184, miR-739, and miR-263a. We found that known miRNA homologs displayed a wide variety of expression profiles in unfed and fed tick salivary glands. After blood-feeding, 162 known miRNAs were upregulated. The six main upregulated miRNAs were mir-1810, mir-2138, mir-2140, mir-425*, mir-429, and mir-516*. Likewise, 231 known miRNAs were downregulated after blood-feeding. The six main downregulated miRNAs were miR-2941-1*, miR-10-5p, miR-2973, miR-1183, miR-4006b-5p, and miR-881. We found that distinct microRNA profiles in the salivary glands of H. longicornis were relating to tick blood feeding. The differential expression of miRNAs in unfed and fed tick salivary glands supported their involvement at new levels in the regulation of tick blood-feeding. Our data provide an important resource for a more detailed functional analysis of miRNAs in this species.

CHARACTERIZATION OF A NEW KUNITZ-TYPE SERINE PROTEASE INHIBITOR FROM THE HARD TICK Rhipicephalus hemaphysaloides

A new Kunitz-type serine protease inhibitor, Rhipilin-2, was identified in the tick Rhipicephalus hemaphysaloides. The cDNA sequence of Rhipilin-2 is 693 bp, and it encodes a deduced 195 amino acid protein with a size of 22 kDa. Bioinformatic analysis shows that Rhipilin-2 belongs to the Kunitz-type family of inhibitors, containing one Kunitz domain with homology to the tissue factor pathway inhibitor. Using Real time polymerase chain reaction (Real time-PCR), Rhipilin-2 mRNA transcripts were detected in tick salivary glands and midgut. Blood feeding induced transcript expression. The recombinant protein was expressed in insect Sf9 cells and confirmed by immunofluorescence test and Western blot analysis with an anti-His antibody. The purified recombinant Rhipilin-2 inhibited serine protease trypsin and elastase, but not thrombin. The anticoagulant activity of Rhipilin-2 was shown by delaying normal clotting of rabbit plasma in the activated partial thromboplastin time tests. These results indicate that Rhipilin-2 is a novel Kunitz-type serine protease inhibitor involved in tick blood feeding.

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 animals 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.

RNA interference and the vaccine effect of a subolesin homolog from the tick Rhipicephalus haemaphysaloides

Subolesin is a well-characterized protective antigen in many ticks and, thus, it is potentially useful in the development of a broad-spectrum vaccine or an autocidal gene silencing strategy to control tick infestations. A subolesin homolog was cloned from the tick Rhipicephalus haemaphysaloides, which is widespread in China, by rapid amplification of complementary DNA (cDNA) ends. Its full-length cDNA was 1386 base pairs (bp), containing a 483xa0bp open reading frame with a predicted molecular mass of 18.7 kilodaltons and an isoelectric point of 9.26. The subolesin protein had a typical nuclear localization signal in its amino-terminus. The full-length cDNA of R. haemaphysaloides showed 52 and 80xa0% identities to those from Ixodes scapularis and R. microplus, respectively, whereas amino acid sequence alignments showed 80 and 97xa0% identities, respectively. Native subolesin was recognized in the unfed tick midgut by an antibody against recombinant subolesin. Transcriptional analysis showed that subolesin was expressed in the tick’s four developmental stages and in all of the tissues examined, except for the synganglion. The pathogen Babesia microti induced the subolesin transcript by fourfold. Subolesin gene silencing by RNA interference significantly decreased the larval engorgement rate, the attachment rate and body weight of engorged nymphs, and the body weight and attachment and engorgement rates of adults, as well as the egg weight per female tick. Vaccinating mice and rabbits with recombinant subolesin induced a significant protective effect, resulting in a reduction of blood feeding and oviposition. These results encourage further studies of using subolesin to control tick infestations in China.

Characterization of a secreted cystatin from the tick Rhipicephalus haemaphysaloides.

A novel cystatin, designated RHcyst-2, was isolated from the tick Rhipicephalus haemaphysaloides. The full-length cDNA of RHcyst-2 is 773xa0bp, including an intact open reading frame encoding an expected protein of 139 amino acids and consisting of a 23 amino acids signal peptide. Predicted RHcyst-2 mature protein molecular weight is about 13xa0kDa, isoelectric point is 4.96. A sequence analysis showed that it has significant homology with the known type 2 cystatins. The recombinant protein of RHcyst-2 was expressed in a glutathione S-transferase-fused soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, C, H, and S, as well as papain, was identified by fluorogenic substrate analysis. The results showed that rRHcyst-2 can effectively inhibit the six cysteine proteases’ enzyme activities. An investigation of the RHcyst-2 genes’ expression profile by quantitative reverse transcription-PCR demonstrated that it was more richly transcribed in the embryo (egg) stage and mainly distributed in the mid-gut of adult ticks. Western blot analysis confirmed that RHcyst-2 was secreted into tick saliva.

Identification and anticoagulant activity of a novel Kunitz-type protein HA11 from the salivary gland of the tick Hyalomma asiaticum

Kunitz/bovine pancreatic trypsin inhibitor proteins are abundant in the salivary glands of ticks and perform multiple functions in blood feeding, including inhibiting blood coagulation, regulating host blood supply and disrupting host angiogenesis. In this study, we identified a novel gene designated HA11 (Hyalomma asiaticum 11xa0kDa protein) from the salivary gland of the tick H. asiaticum. HA11 is encoded by a gene with an open reading frame of 306xa0bp that is translated into a deduced 101 amino acid 11xa0kDa protein that shares 27% sequence identity with a Kunitz-like protease inhibitor precursor in Amblyomma variegatum. Bioinformatic analysis confirmed HA11 as a member of the Kunitz-type family of inhibitors. Real time-PCR detected HA11 mRNA transcripts in tick larvae and nymphae stages, with levels highest in salivary gland tissue, and transcription was induced by blood feeding. HA11 anticoagulant activity was demonstrated by its ability to delay normal clotting of rabbit plasma in an activated partial thromboplastin time assay. Furthermore, RNA interference confirmed that HA11 influences H. asiaticum development and blood feeding, and the recombinant protein exerted low hemolytic activity. These results suggest HA11 is a novel Kunitz-type anticoagulant protein involved in tick blood feeding that may have potential as an anticoagulant drug or vaccine.

Babesia microti thioredoxin 3 is an effective antioxidant and involved in the response to antiprotozoal drugs

The intra-erythrocytic apicomplexan Babesia microti is the predominant pathogen that causes human babesiosis, an infectious disease that occurs worldwide. B. microti relies on the antioxidant including thioredoxin system to maintain the redox balance during the erythrocytic stage. In the present study, the full-length B. microti thioredoxin 3 (BmTrx3) gene was cloned, expressed in vitro, and its response to antiprotozoal drugs were tested. The full-length BmTrx3 was 663u202fbp and contained an intact open reading frame of 567u202fbp. The encoded polypeptide was 188 amino acids and the predicted molecular weight of the protein was 21.7u202fkDa. A conserved thioredoxin-like family domain was found in BmTrx3. The expression of BmTrx3 was upregulated on both the third and eighth day post-infection in mice, whereas expression was downregulated during the beginning and later stages. Western blot analysis showed that mouse anti-BmTrx3 serum could recognize the native BmTrx3 in parasite lysates and that the mouse anti-B. microti serum could recognize the recombinant BmTrx3 protein. Immunofluorescence microscopy showed that BmTrx3 localized in the cell cytoplasm of B. microti merozoites in B. microti-infected red blood cells. The results of bovine insulin reduction assay indicated the enzyme activity of the purified recombinant BmTrx3 protein. The anti-malaria drug chloroquine significantly inhibited the expression of BmTrx3, however, another anti-malaria drug qunine, and a known anti-babesiosis drug clindamycin, induced significantly higher upregulation of BmTrx3 mRNA. The results of the present study demonstrate that BmTrx3 is a functional enzyme with antioxidant activity and may be involved in the response of B. microti to anti-parasite drugs.