3.10 | Identifying anthelmintic resistance-related gene family and drug targets
We use the HMMER3 software to scan several detoxification-related gene families at the whole genome scale, including ATP dependent (ABC ), cytochrome P450 (CYP), glutathione s-transferase (GST), glycoside hydrolase family 18 (CHIA), patched family (PTCHD) and protein tyrosine phosphatase family (PTP) (Fig S11d). A total of 97 ABCtransporters, multipass membrane proteins, were identified in B. schroederi , and the average number of ABC transporter genes in roundworms was greater than that in C. elegans (60)(Schumacher & Benndorf, 2017). The high-quality genome data of B. schroederiprovides an opportunity to identify biologically active anthelmintic compounds. On the one hand, it enables identification of the targets of existing anthelmintics, on the other hand, it also enables identification of new potential targets for compounds from other areas of drug discovery. All compound related proteins were searched against target proteins from ChEMBL v26(Anna et al.) using BLASTP (E≤1×10−10), and a total of 4,554 small molecules with recorded biological activities were identified. By blasting against the ChEMBL(Anna et al.) databases, a total of 90 known genes, which encode specific drug targets were identified. The corresponding drugs (13 drugs used to treat humans with World Health Organization (WHO) ATC code P02 ‘WHO anthelmintics’ and 10 drugs from DrugBank(S et al., 2017)) were further collated by searching DrugBank databases and the literature (Supplementary Data 4a). Some of these drugs have been proven to be effective against B. schroederi , such as albendazole(Fu et al., 2011), mebendazole(Bourne, Cracknell, & Bacon, 2010), pyrantel(Xie et al.) and ivermectin(C. Wang et al., 2015). Many existing anthelmintics are compromised by the increase of resistance in roundworm populations(D. Li et al., 2015). In addition to known drugs, we were committed to identifying new potential drug targets. We focused on single protein ChEMBL targets that may be easier to develop drugs against than protein complexes(International et al., 2018). By blasting against target proteins (similarities > 80%) in the single protein database ChEMBL, we identified 95 genes encoding single proteins. Then we set a score of ‘0/1’ considering six main factors to evaluate the potential of the protein as a drug target (see methods; Fig. 8). Finally, we located the position of all the drug target encoding genes on superscaffolds (Fig. 8). Since the existing Phase III and above drugs have greater potential for development into new anthelmintics, we searched for commercially available compounds against each target protein although these compounds were not originally designed as anthelmintics. Among all the proteins, we found that three target genes (cmd-1 , Ap2s1 ,HRAS ) have available compounds with Phase III/IV approvals (Supplementary Data 4b). These potential drug targets and compounds will provide references for the development of new anthelmintics.
4 | DISCUSSION
B. schroederi exhibits strong environmental adaptability and wide distribution, and is a threat to the health of giant pandas (Zou et al., 1998). The in-depth studies of B. schroederi have been hampered by the lack of a high-quality genome. The scaffold N50s of publishedA. suum , P. univalens and T. canis genomes are 290 kb, 1,825 kb and 375 kb, respectively (Table 1). In this study, we present the first chromosome-scale genome assembly of the B. schroederi with the scaffold N50 of 12.69 Mb, representing a genome assembly with the best contiguity in Ascarididae. We envisage that this genome will provide a valuable and useful genetic resource for future research on roundworms, as well as drug development for expulsion.
Roundworms have special characteristics that are different from free-living nematodes reflecting the adaptation to the parasitic life. Eggs of roundworms have a tough and elastic polysaccharide chitin shell, which enables eggs to persist in the soil for up to ten years(FAIRBAIRN, 1970). We have observed a significant expansion of the chitin-binding protein CPG-2 family in roundworm branches, which may be related to the formation of the roundworm egg shell, thereby prolonging the survival of roundworms even in a harsh environment. In addition, in the parasitic stage, larvae enter the intestine, penetrate the intestinal wall, migrate among tissues and organs(K. Kazacos & W. M. Boyce, 1989), molt and develop, finally return to the small intestine to develop into adults and lay eggs again. Some genes potentially involved in tissue invasion and immune evasion have been significantly expanded in roundworms, including genes homologous to metallopeptidase and serine/threonine-protein kinase, respectively. Previous studies have shown that metallopeptidase(s) in the secretory products of astacins in the nematode epidermis can digest collagen in host tissues, and thus be involved in the migration of larvae in viscera(Hanns et al., 2011; Williamson et al.).
Although the morphological characteristics of Ascarididae roundworms are similar, the B. schroederi still shows unique molecular evolutionary traits. The giant panda has gradually evolved in response to the bamboo diet during millions of years of evolution(Zhou, Hu, Yuan, & Wei, 1997). However, the giant panda has maintained the intestinal structure of carnivores, with short and thick small intestines(Guibo Yang, 1995). Due to the low digestibility of bamboo, giant pandas have to ingest large amounts of bamboo to meet their nutrient needs(Sims et al., 2007), and a large amount of feces is produced and discharged every day. Accordingly, B. schroederi needs to absorb nutrients as much as possible. In the B. schroederi genome, several unique gene families of B. schroederi were found to be involved in the metabolism of essential amino acids, especially the degradation of valine, leucine and isoleucine (KO00280; P <0.01), which is likely to enhance the ability of B. schroederi to absorb nutrients. In addition, B. schroederi needs stronger motor ability than A. suum , P. univalens and T. canis to survive in the small intestine because of its much smaller body size, and at the same time ensure that they can avoid expulsion. The muscle tissue of the roundworm plays a key role in motility and the extreme expansion and positive selection of the actin family may have provided the driving force for muscle contraction and cell movement(Hall, 1998). Actin promotes muscle contraction and plays a very important role in the movement and migration of B. schroederi in the host. Studies have shown that actin is involved in the repair of nematode epidermis damage(Suhong & Chisholm, 2012), which is of great significance to the migration of B. schroederi in the giant panda. The expansion of the actin gene family may, at least to some extent, explain the genetic basis of stronger locomotion ability of B. schroederi than other roundworms.
According to a previous investigation, the cause of death of giant pandas in recent decades has shifted from starvation and poaching to VLM-related deaths(J.-S. Zhang et al., 2008). Frequent use of drugs may drive the increasing frequency of genes related to drug resistance in the population, leading to widespread drug resistance in the B. schroederi population. Furthermore, there have been reports of side effects in giant pandas after the administration of existing anthelmintic drugs(C. Wang et al., 2015). We identified the ABC , CYP , GST , CHIA ,PTCHD and PTP gene families in the B. schroederigenome. These genes may be involved in the metabolism of drugs and other xenobiotics and/or biosynthesis and metabolism of endogenous compounds. We observed a recent significant positive selection of ABC andCYP family members and other resistance-related genes (glc-1 , nrf-6 , pgp-3 and bre-4 ) in captive (SC) populations. Although wild and captive populations were obtained from two different regions (Qinling and Sichuan), natural selection analysis mainly considers recent changes in gene frequency. The two populations are facing completely different selection pressures for deworming, and thus, offer an option for evaluating natural selection trends of a few resistance-related genes. The results indicated an increased frequency of drug resistance-related genes in captive populations. This may be related to the frequent use of drugs in recent decades. Although the degree of natural selection in the current resistance areas cannot be quantified, it is possible that the gene frequency of these genes is still increasing, and it may cause the emergence and increase of resistant individuals. Studies have shown that some new sources of infection may even evolve into potential antibiotic-resistant pathogens(Zumla & Hui, 2019). Therefore, the identification of drug-resistance genes and the detection of drug-resistant individuals are still essential in future works.
There is an urgent need for new anthelmintic drugs for intestinal expulsion of roundworms(James, Hudson, & Davey, 2009; Jia, Melville, Utzinger, King, & Zhou, 2012). Specifically, there is a pressing need for new anthelmintic drugs to protect the giant panda, since existing drugs suffer from low efficacy, serious side effects or rising drug resistance in parasite populations due to increased frequency of use(C. Wang et al., 2015) . The chromosome-scale genome of B. schroederiprovides a reference for the development of species-specific drugs, and drug targets can be screened from the whole genome level. We identified a total of 90 known drug targets and 95 potential drug targets, providing a basis for the development of follow-up drugs and vaccines. We searched four compounds (lonafarnib, haloperidol, trifluoperazine and chlorpromazine; Supplementary Data 4b) that have a phase 3/4 approval. These compounds could be considered for repurposing as novel anthelmintics, which would save considerable effort and expense. Nevertheless, the anthelmintic activity of these compounds and other potential target compounds needs further testing. We envision that such works will provide new modalities for the prevention and treatment of baylisascariasis and other parasitic diseases.