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.