Molecular based identification and phylogenetic relationship of the leech Hirudinaria manillensis from India by using mitochondrial cytochrome c oxidase subunit I gene.

BACKGROUND: A molecular approach for the identification of unknown species by the using mitochondrial cox1 gene is an effective and reliable as compared with morphological-based identification. Hirudinaria manillensis referred to as Asian Buffalo Leech, is found in South Asia and traditionally used as medicine owing to its medicinal properties. METHODS AND RESULTS: The study aimed to isolate and identify the leech species using cox1 gene sequencing and their phylogenetic relationships. The nucleotide sequences of cytochrome c oxidase subunit I (cox1) mitochondrial genes were analyzed for species identification and the phylogenetic relationship of crucial therapeutic leech Hirudinaria manillensis. The isolated DNA from the leech sample was amplified with cox1 gene-specific primers. BLAST results with the H. manillensis sequence showed 89.24% homology with H. manillensis and phylogenetic tree analysis revealed the genetic relationship with other GenBank submitted sequences. CONCLUSION: The present study concluded that the cox1 gene could be an effective way to identify the leech H. manillensis and provided sufficient phylogenetic information to distinguish H. manillensis indicating a significant mtDNA-based approach to species identification.

Identification and Characterization of RK22, a Novel Antimicrobial Peptide from Hirudinaria manillensis against Methicillin Resistant Staphylococcus aureus.

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.

Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis.

Haematophagous leeches express a broad variety of secretory proteins in their salivary glands, among them are hirudins and hirudin-like factors. Here, we describe the identification, molecular and initial functional characterization of Tandem-Hirudin (TH), a novel salivary gland derived factor identified in the Asian medicinal leech, Hirudinaria manillensis. In contrast to the typical structure of hirudins, TH comprises two globular domains arranged in a tandem-like orientation and lacks the elongated C-terminal tail. Similar structures of thrombin inhibitors have so far been identified only in kissing bugs and ticks. Expression of TH was performed in both cell-based and cell-free bacterial systems. A subsequent functional characterization revealed no evidence for a thrombin-inhibitory potency of TH.

Hirudins of the Asian medicinal leech, Hirudinaria manillensis: same same, but different.

Blood coagulation in vertebrates is a complex mechanism that involves the precisely coordinated and regulated action of a cascade of factors in order to prevent excessive blood loss upon wounding. Any blood sucking ectoparasite, however, has to circumvent this mechanism to ensure the uptake of an adequate blood meal. Inhibitors of blood coagulation in the saliva are hence widespread among these animals. Thrombin as a key factor of blood coagulation is a prominent target of such inhibitors, and hirudin is probably the best known among the thrombin inhibitors. Hirudin was originally described in the genus Hirudo, but occurs in other leech genera like Hirudinaria and Macrobdella as well. Besides several isoforms of hirudin, a new class of putative leech saliva components, the hirudin-like factors (HLFs), was identified in both genera Hirudo and Hirudinaria. Here, we describe the expression, purification, and functional characterization of three HLFs (HLF5, 6, and 8, respectively) and two additional hirudins (HM3 and HM4) of Hirudinaria manillensis. While HLF6 lacked any inhibitory activity on thrombin, HLF5 as well as HLF8 clearly exhibited anticoagulatory properties. The inhibitory activity of HLF5 and HLF8, however, was much lower compared with both HM3 and HM4 of Hirudinaria manillensis as well as the hirudin variants 1 (HV1) and 2 (HV2) of Hirudo medicinalis. Neither an inhibition of trypsin nor a platelet aggregation was caused by HLF8. Our data indicates the presence of two classes (rather than isoforms) of hirudins in Hirudinaria manillensis with markedly different inhibitory activity on human thrombin.

Analysis of codon usage patterns in Hirudinaria manillensis reveals a preference for GC-ending codons caused by dominant selection constraints.

BACKGROUND: Hirudinaria manillensis is an ephemeral, blood-sucking ectoparasite, possessing anticoagulant capacities with potential medical applications. Analysis of codon usage patterns would contribute to our understanding of the evolutionary mechanisms and genetic architecture of H. manillensis, which in turn would provide insight into the characteristics of other leeches. We analysed codon usage and related indices using 18,000 coding sequences (CDSs) retrieved from H. manillensis RNA-Seq data. RESULTS: We identified four highly preferred codons in H. manillensis that have G/C-endings. Points generated in an effective number of codons (ENC) plot distributed below the standard curve and the slope of a neutrality plot was less than 1. Highly expressed CDSs had lower ENC content and higher GC content than weakly expressed CDSs. Principal component analysis conducted on relative synonymous codon usage (RSCU) values divided CDSs according to GC content and divided codons according to ending bases. Moreover, by determining codon usage, we found that the majority of blood-diet related genes have undergone less adaptive evolution in H. manillensis, except for those with homologous sequences in the host species. CONCLUSIONS: Codon usage in H. manillensis had an overall preference toward C-endings and indicated that codon usage patterns are mediated by differential expression, GC content, and biological function. Although mutation pressure effects were also notable, the majority of genetic evolution in H. manillensis was driven by natural selection.

In vitro anticancer activity of Hirudinaria manillensis methanolic extract and its validation using in silico molecular docking approach.

Cancer has emerged as a potentially lethal illness, which recently upsurged in the mortality rate. Animal-derived compounds could be promising targets with higher efficacy and low toxicity in anticancer therapy. The present study aimed to explore the presence of anticancer potential compounds in Hirudinaria manillensis methanolic extract and their anticancer potential against various cancer cell types and target identification by Auto dock method. Initially, the identification of bioactive compounds was achieved by GC-MS analysis followed by the anticancer activity by MTT assay against A549, HeLa, MDA-MB-231, MG-63, and MOLT-4. Further, the effect of a lead compound on the cancer cell target was analyzed by the Auto dock method. GC-MS analysis results revealed the presence of 25 different bioactive compounds including anticancer potential compounds, such as Lupeol, Carvacrol, and Demecolcine. Interestingly, MTT assay results demonstrated the anticancer potential of Hirudinaria manillensis extract (LE) against various cancer cell lines, such as A549 (54.60 µg/ml), HeLa (19.93 µg/ml), MDA-MB-231 (20.23 µg/ml), MG-63 (20.04 µg/ml), and MOLT-4 (171.8 µg/ml), respectively. Among these cell types, the maximum inhibition was observed against HeLa with the IC50 concentration of 19.93 µg/ml. Furthermore, Demecolcine compound was docked with the EGFR tyrosine kinase showed the binding affinity of the docked complex was predicted to be - 6.2 kcal/mol. Thus, we conclude that H. manillensis has a significant anticancer effect on human cancer cell lines and could be used as a natural target which paves the way for further studies on biomedical applications in cancer therapeutics.

Hirudotherapy in Wound Healing.

Leeches are hermaphrodite, bloodsucking parasitic worms usually found in places with fresh water. Leech therapy existed 3000 years, and it is being used at a different scope. Several species of leeches have been used in medicine, and the most common species used is Hirudo medicinalis. Leeches suck the excess blood, reduce the swelling in the tissues, and promote healing by allowing fresh oxygenated blood to reach the area until normal circulation can be restored. Pain relief from leech therapy is rapid, effective, and long-lasting in many conditions. The aim of this study was to evaluate the efficacy and duration of healing utilizing sterile medicinal leeches, Hirudinaria manillensis, in the management of pain and wound healing. Leech was taken out from its sterile tube by using a pair of non-tooth sterile plastic forceps and gloved hands. Each leech was left in place for as long as it was feeding. Leeches were removed only after they became detached from the patient. The specimen jars containing the used leeches were sealed in either a biohazard bag or in a small yellow clinical waste bin liner securely fastened with a cable tie. The leech was killed by using 70% alcohol prior to disposal into a yellow hazard bin, which undergoes incineration. All 3 patients had improvements in their condition, especially in terms of reduction in the pain and improvement in their sense of balance. All the wounds healed well. Therefore, leech therapy is effective in reducing pain and increasing perfusion to allow the wounds to heal quickly. However, a more robust trial is needed to show significance as the sample size is small.

Comprehensive Transcriptome Sequencing Analysis of Hirudinaria manillensis in Different Growth Periods.

Medical leeches are widely been used in biochemical and clinical medical studies, helping to restore blood circulation to grafted or severely injured tissue. Mostly, adult leeches are being used in the traditional pharmacopeia, but the gene expression profiling of leeches in different growth periods is not well-reported. So, in this study, we used transcriptome analysis to analyze the comparative gene expression patterns of Hirudinaria manillensis (H. manillensis) in different growth periods, including larval, young, and adult stages. We constructed 24 cDNA libraries from H. manillensis larval, young, and adult stages, and about 54,639,118 sequences were generated, 18,106 mRNA transcripts of which 958 novel mRNAs and 491 lncRNAs were also assembled as well. Furthermore, the results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially upregulated genes from the larval to adult stages were enriched in pathways such as cilium, myofibril, contractile fiber, cytoskeleton proteins, dilated cardiomyopathy, adrenergic signaling in cardiomyocytes, etc. Moreover, in the adult stages, a significant increase in the expression of the Hirudin-HM (HIRM2) genes was detected. In addition, our comparative transcriptome profiling data from different growth stages of H. manillensis also identified a large number of DEGs and DElncRNAs which were tentatively found to be associated with the growth of H. manillensis; as it grew, the muscle-related gene expression increased, while the lipid metabolism and need for stimulation and nutrition-related genes decreased. Similarly, the higher expression of HIRM2 might attribute to the high expression of protein disulfide isomerase gene family (PDI) family genes in adulthood, which provides an important clue that why adult leeches rather than young leeches are widely used in clinical therapeutics and traditional Chinese medicine.

A new species of mammalian trypanosome, Trypanosoma (Megatrypanum) bubalisi sp. nov., found in the freshwater leech Hirudinaria manillensis.

Leeches have long been considered potential vectors for the aquatic lineage of trypanosomes, while bloodsucking insects are generally considered as the vectors for the terrestrial lineage of trypanosomes. The freshwater leech, Hirudinaria manillensis, is a widely distributed species in southern China and could potentially act as the vector for trypanosomes. Prior to this study, no trypanosomes had been reported from this leech. However, in this study, leeches were collected from three different places in Guangdong province, China, and a large number of flagellates were isolated and successfully cultured in vitro. Based on morphology, these flagellates looked like a typical trypanosome species. Analysis was carried out on the molecular sequences of the 18S rRNA gene and the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. To our surprise, these flagellates were identified as likely to be a mammalian trypanosome belonging to the clade containing Trypanosoma (Megatrypanum) theileri but they are significantly different from the typical TthI and TthII stocks. Analyses of blood composition indicated that the source of the blood meal in these leeches was from the water buffalo (Bubalus bubalis). To further test if this flagellate from the freshwater leech was indeed a mammalian trypanosome, we transferred the trypanosomes cultured at 27-37 °C and they were able to successfully adapt to this mammalian body temperature, providing further supporting evidence. Due to the significant genetic differences from other related trypanosomes in the subgenus Megatrypanum, we propose that this flagellate, isolated from H. manillensis, is a new species and have named it Trypanosoma bubalisi. Our results indicate that freshwater leeches may be a potential vector of this new mammalian trypanosome.

Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis.

A large number of protease inhibitors have been found from leeches, which are essential in various physiological and biological processes. In the curret study, a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis, which was named HMEI-A. Primary structure analysis showed that HMEI-A belonged to a new family of proteins. HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant (Ki) of 1.69 × 10-8 mol·L-1. Further study showed that HMEI-A inhibited the formation of neutrophil extracellular trap (NET). These results suggested that HMEI-A from the leech of H. manillensis is a novel elastase inhibitor which can suppress NET formation. It may play a significant role in blood-sucking of leeches and is a potential candidate as an anti-inflammatory agent.

A new species of buffalo leech in the genus Hirudinaria Whitman, 1886 (Arhynchobdellida, Hirudinidae) from Thailand.

Hirudinaria manillensis (Lesson, 1842), commonly known as the buffalo leech, shows a polymorphism of two ventral colorations. The green color morph has a plain green ventral surface and the red color morph has a brick-red ventral surface with two black submarginal stripes. Based on molecular and morphological evidence in the present study, these two color morphs were revealed as two different species. The red color morph fits well with the description of H. manillensis, while the green color morph showed some distinctions, and therefore is described herein as Hirudinaria thailandica Jeratthitikul & Panha, sp. nov. The new species can be distinguished from its congeners by the dark greenish or dark olive ventral surface and a round atrium with ventral insertion of ejaculatory ducts in the male reproductive organ. A phylogenetic tree based on concatenated data of COI and 28S genes supported the new species and further indicated it as a sister species to H. bpling Phillips, 2012.

Draft Genome of the Asian Buffalo Leech Hirudinaria manillensis.

The Asian Buffalo leech, Hirudinaria manillensis, is an aquatic sanguivorous species distributed widely in Southeast Asia. H. manillensis has long been used clinically for bloodletting and other medical purposes. Recent studies have focused on artificial culturing, strain optimization, and the identification and development new drugs based on the anticoagulant effects of H. manillensis bites; however, data regarding its genome remain unclear. This study aimed to determine the genome sequence of an adult Asian Buffalo leech. We generated a draft assembly of 151.8 Mb and a N50 scaffold of 2.28 Mb. Predictions indicated that the assembled genome contained 21,005 protein-coding genes. Up to 17,865 genes were annotated in multiple databases including Gene Ontology. Sixteen anticoagulant proteins with a Hirudin or Antistasin domain were identified. This study is the first to report the whole-genome sequence of the Asian Buffalo leech, an important sanguivorous leech of clinical significance. The quality of the assembly is comparable to those of other annelids. These data will help further the current understanding of the biological mechanisms and genetic characteristics of leeches and serve as a valuable resource for future studies.

Purification and characterization of a novel anti-coagulant from the leech Hirudinaria manillensis.

Protease inhibitors have been reported rarely from the leech Hirudinaria manillensis. In this study, we purified a novel protease inhibitor (bdellin-HM-2) with anticoagulant properties from H. manillensis. With a molecular weight of 1.4x104, bdellin-HM-2 was also characterized with three intra-molecular disulfide bridges at the N-terminus and multiple HHXDD and HXDD motifs at the C-terminus. cDNA cloning revealed that the putative nucleotide-encoding protein of bdellin-HM-2 contained 132 amino acids and was encoded by a 399 bp open reading frame (ORF). Sequence alignment showed that bdellin-HM-2 shared similarity with the "non-classical" Kazal-type serine protease inhibitors, but had no inhibitory effect on trypsin, elastase, chymotrypsin, kallikrein, factor XIIa (FXIIa), factor XIa (FXIa), factor Xa (FXa), thrombin, or plasmin. Bdellin-HM-2 showed anticoagulant effects by prolonging the activated partial thromboplastin time (aPTT), indicating a role in enabling H. manillensis to obtain a blood meal from its host. Our results suggest that bdellin-HM-2 may play a crucial role in blood-sucking in this leech species and may be a potential candidate for the development of clinical anti-thrombotic drugs.

Purification and Characterization of a Novel Kazal-Type Trypsin Inhibitor from the Leech of Hirudinaria manillensis.

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 (Ki) 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.

In vivo anti-hyperglycemic activity of saliva extract from the tropical leech Hirudinaria manillensis.

The anticoagulant effect of leech saliva was traditionally employed in the treatment of diabetes mellitus complications such as peripheral vascular complications. This study was carried out to examine the effect of leech saliva extract (LSE) on blood glucose levels in alloxan-induced diabetic rats. First, LSE was collected from leeches which were fed on a phagostimulatory solution. Second, total protein concentration was estimated using the Bradford assay. Third, diabetic rats were injected subcutaneously (sc) with LSE at doses of 500 and 1 000 µg·kg(-1) body weight (bw). Other diabetic rats were injected sc with insulin at doses of 10 and 20 U·kg(-1) bw. Another group was injected simultaneously with LSE (250 µg·kg(-1) bw) and insulin (10 U·kg(-1) bw). Fasting blood glucose (FBG) concentrations were monitored during a study period of eight hours at regular intervals. Findings showed that both doses of LSE resulted in a significant and gradual decrease in FBG starting from 10%-18% downfall after two hours of injection reaching the maximal reduction activity of 58% after eight hours. Remarkably, LSE was sufficient to bring the rats to a near norm-glycemic state. The high dose of insulin induced a severe hypoglycemic condition after 2-4 h of injection. The lower dose was able to decline FBG for 2-6 h in rats which became diabetic again after 8 h. On the other hand, the concurrent injection of low doses of LSE and insulin produced a hypoglycemic effect with all rats showing normal FBG levels. Taken together, these findings indicated that the subcutaneous injection of LSE of the medicinal Malaysian leech was able to provide better glycemic control compared with insulin. Moreover, the synergism between LSE and insulin suggests that LSE could be utilized as an adjuvant medication in order to reduce insulin dosage or to achieve better control of blood glucose.