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-binding cassette (ABC) transporters, cytochrome P450 (CYP), glutathione S-transferase (GST), glycoside hydrolase family 18 (CHIA), patched family (PTCHD) and protein tyrosine phosphatase family (PTP) (Fig S12d). A total of 97 ABC transporters, 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 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 databases (Anna et al., 2012), 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 (Wishart 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 againstB. 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 (A. Coghlan 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 sat 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 being developed 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 at possible starting point 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). In-depth studies of B. schroederi have been hampered by the lack of a high-quality genome sequence. The scaffold N50s of published A. suum , P. univalens and T. canisgenomes are 290 kb, 1,825 kb and 375 kb, respectively (Table 1). In this study, we present the first chromosome-scale genome assembly of theB. schroederi with the scaffold N50 of 12.32 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 eggshell, 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, mate, and lay eggs. 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 (Soblik et al., 2011; Williamson et al., 2006).
Although the morphological characteristics of Ascarididae species 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). 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. Actin promotes muscle contraction and plays a very important role in the movement and migration of B. schroederi in the host. The significant expansion and positive selection of the actin family may have provided the driving force for muscle contraction and cell movement (Hall, 1998). 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 ofB. 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 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 at 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. 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 expenses. 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.