Search Results (7)

Leeches hold significant medical and pharmaceutical value for antithrombotic treatments, yet their genetic diversity patterns remain poorly understood. We performed population genetic analyses on seven Hirudinaria manillensis populations from southern China using mitochondrial protein-coding genes (MitPCGs). Complete sequences of all 13 MitPCGs were obtained from 74 individuals. Haplotype diversity exhibited a logarithmic relationship with the gene length (R² = 0.858, p < 0.001), while nucleotide diversity showed a near-perfect alternating low-high pattern (Z = 2.938, p = 0.003). Concatenated sequence analyses indicated high haplotype diversity (>0.5) and low nucleotide diversity (<0.005) across all populations, suggesting a historical bottleneck followed by rapid expansion and mutation accumulation. The haplotype network, haplotype phylogenetics, and genetic structure analyses revealed moderate genetic differentiation across populations, dividing them into three clades: a basal Yunnan population (YNHH), sub-basal Guangxi populations (GXGG, GXLZ, and GXYL), and distal Guangdong/Hainan populations (GDMM, GDZJ, and HNDA). Analysis of historical population demography revealed five phases from ancient to recent times (P1–5): growth, prolonged stability, rapid decline, rapid growth, and secondary decline. These phases correlate strongly with past climatic events, demonstrating that glacial–interglacial cycles profoundly impacted the leech’s effective population size. This study provides a key scientific basis for H. manillensis resource conservation and utilization. Full article

Thrombotic diseases represent a significant global health burden, particularly for middle-aged and elderly populations. Medicinal leeches, such as Hirudinaria manillensis and Whitmania pigra, have been traditionally used for their anticoagulant properties. The genomes of these leeches each harbor three lefaxin genes, which are designated lefaxin_Hman1–3 and lefaxin_Wpig1–3, respectively. We conducted genomic and transcriptomic sequencing on wild populations of both species. Bioinformatics tools were employed to analyze intraspecific variation, molecular evolution, and protein structures. We expressed recombinant lefaxin proteins in Pichia pastoris and assessed their anticoagulant activities using in vitro coagulation assays. H. manillensis exhibited greater genetic diversity and stability, whereas W. pigra showed higher expression levels and hydrophilicity. Both species exhibited purifying selection, indicating conserved function, and their lefaxin structures are similar to the archetypal lefaxin (UniProt No. P86681.1). W. pigra lefaxins bound Factor Xa more effectively. W. pigra lefaxins exhibited more robust anticoagulant activity in vitro compared to those from H. manillensis. W. pigra, a non-hematophagous leech, shows potent anticoagulant activity through lefaxins, challenging traditional views on leech efficacy. This study underscores the potential of lefaxins as therapeutic targets for thrombotic diseases and highlights the need to reconsider the use of various leech species in medicine. Full article

With the growing severity of antibiotic resistance, antimicrobial peptides demonstrate significant potential for medical applications. Here, we performed genome and transcriptome sequencing of 30 Asian buffalo leech (Hirudinaria manillensis) individuals and integrated data from three other leech species (Whitmania pigra, Hirudo nipponia, and Hirudo medicinalis) to investigate genetic variation and gene expression of H. manillensis macins. Three macins (Hman1, Hman2, and Hman3), along with their encoding genes (Hman1, Hman2, and Hman3), were identified in H. manillensis. Hman1 exhibited the highest similarity (63.5 ± 12.0%) to macins from other leeches, followed by Hman2 (57.8 ± 7.4%) and Hman3 (30.0 ± 3.5%). Both amino acid and codon sequences of Hman1 were conserved within the species, whereas Hman2 and Hman3 exhibited markedly higher variability. All Hman1 sequences were translatable, while four Hman2 and 28 Hman3 sequences had degenerated into pseudogenes. Transcripts per million (TPM) values for Hman1, Hman2, and Hman3 were 2196.63, 242.35, and 1.22, respectively. Total macin expression in H. manillensis was less than 1/20 of that in W. pigra. Based on sequence variation and expression patterns, we propose that Hman1 retains functionality while Hman2 and Hman3 have lost or are losing their antibacterial functions. Full article

Despite being a non-hematophagous leech, Whitmania pigra is widely used in traditional Chinese medicine for the treatment of antithrombotic diseases. In this study, we provide a high quality genome of W. pigra and based on which, we performed a systematic identification of the potential antithrombotic genes and their corresponding proteins. We identified twenty antithrombotic gene families including thirteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. Unexpectedly, a total of 79 antithrombotic genes were identified, more than a typical blood-feeding Hirudinaria manillensis, which had only 72 antithrombotic genes. In addition, combining with the RNA-seq data of W. pigra and H. manillensis, we calculated the expression levels of antithrombotic genes of the two species. Five and four gene families had significantly higher and lower expression levels in W. pigra than in H. manillensis, respectively. These results showed that the number and expression level of antithrombotic genes of a non-hematophagous leech are not always less than those of a hematophagous leech. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a non-hematophagous leech to date and will significantly enhance the investigation and utilization of leech derivatives in thrombosis therapy research and pharmaceutical applications. Full article

Leeches are well-known annelids due to their obligate blood-feeding habits. Some leech species secrete various biologically active substances which have important medical and pharmaceutical value in antithrombotic treatments. In this study, we provided a high-quality genome of the Asian buffalo leech (Hirudinaria manillensis), based on which we performed a systematic identification of potential antithrombotic genes and their corresponding proteins. Combining automatic and manual prediction, we identified 21 antithrombotic gene families including fourteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. A total of 72 antithrombotic genes, including two pseudogenes, were identified, including most of their corresponding proteins forming three or more disulfide bonds. Three protein families (LDTI, antistasin, and granulin) had internal tandem repeats containing 6, 10, and 12 conserved cysteines, respectively. We also measured the anticoagulant activities of the five identified hirudins (hirudin_Hman1 ~ hirudin_Hman5). The results showed that three (hirudin_Hman1, hirudin_Hman2, and hirudin_Hman5), but not the remaining two, exhibited anticoagulant activities. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a leech to date. These results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes in the treatment of thrombotic diseases. Full article

Staphylococcus aureus (S. aureus) infections are a leading cause of morbidity and mortality, which are compounded by drug resistance. By manipulating the coagulation system, S. aureus gains a significant advantage over host defense mechanisms, with hypercoagulation induced by S. aureus potentially aggravating infectious diseases. Recently, we and other researchers identified that a higher level of LL-37, one endogenous antimicrobial peptide with a significant killing effect on S. aureus infection, resulted in thrombosis formation through the induction of platelet activation and potentiation of the coagulation factor enzymatic activity. In the current study, we identified a novel antimicrobial peptide (RK22) from the salivary gland transcriptome of Hirudinaria manillensis (H. manillensis) through bioinformatic analysis, and then synthesized it, which exhibited good antimicrobial activity against S. aureus, including a clinically resistant strain with a minimal inhibitory concentration (MIC) of 6.25 μg/mL. The RK22 peptide rapidly killed S. aureus by inhibiting biofilm formation and promoting biofilm eradication, with good plasma stability, negligible cytotoxicity, minimal hemolytic activity, and no significant promotion of the coagulation system. Notably, administration of RK22 significantly inhibited S. aureus infection and the clinically resistant strain in vivo. Thus, these findings highlight the potential of RK22 as an ideal treatment candidate against S. aureus infection. Full article

Kazal-type serine proteinase inhibitors are found in a large number of living organisms and play crucial roles in various biological and physiological processes. Although some Kazal-type serine protease inhibitors have been identified in leeches, none has been reported from Hirudinaria manillensis, which is a medically important leech. In this study, a novel Kazal-type trypsin inhibitor was isolated from leech H. manillensis, purified and named as bdellin-HM based on the sequence similarity with bdellin-KL and bdellin B-3. Structural analysis revealed that bdellin-HM was a 17,432.8 Da protein and comprised of 149 amino acid residues with six cysteines forming three intra-molecular disulfide bonds. Bdellin-HM showed similarity with the Kazal-type domain and may belong to the group of “non-classical” Kazal inhibitors according to its Cys^I-Cys^II disulfide bridge position. Bdellin-HM had no inhibitory effect on elastase, chymotrypsin, kallikrein, Factor (F) XIIa, FXIa, FXa, thrombin and plasmin, but it showed a potent ability to inhibit trypsin with an inhibition constant (K_i) of (8.12 ± 0.18) × 10⁻⁹ M. These results suggest that bdellin-HM from the leech of H. manillensis plays a potent and specific inhibitory role towards trypsin. Full article