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.