Combating Parasitic Nematode Infections, Newly Discovered Antinematode Compounds from Marine Epiphytic Bacteria

Parasitic nematode infections cause debilitating diseases and impede economic productivity. Antinematode chemotherapies are fundamental to modern medicine and are also important for industries including agriculture, aquaculture and animal health. However, the lack of suitable treatments for some diseases and the rise of nematode resistance to many available therapies necessitates the discovery and development of new drugs. Here, marine epiphytic bacteria represent a promising repository of newly discovered antinematode compounds. Epiphytic bacteria are ubiquitous on marine surfaces where they are under constant pressure of grazing by bacterivorous predators (e.g., protozoans and nematodes). Studies have shown that these bacteria have developed defense strategies to prevent grazers by producing toxic bioactive compounds. Although several active metabolites against nematodes have been identified from marine bacteria, drug discovery from marine microorganisms remains underexplored. In this review, we aim to provide further insight into the need and potential for marine epiphytic bacteria to become a new source of antinematode drugs. We discuss current and emerging strategies, including culture-independent high throughput screening and the utilization of Caenorhabditis elegans as a model target organism, which will be required to advance antinematode drug discovery and development from marine microbial sources.


Introduction
Humans are vulnerable to infectious diseases caused by parasitic helminths (nematodes) resulting in morbidity and mortality within the population [1][2][3]. Approximately 24% of the global human population, corresponding to 1.5 billion people, suffer from parasitic helminth infections [4]. There are almost 300 nematodes associated with zoonotic diseases that are able to infect humans, including some of the most devastating parasites such as Ascaris lumbricoides (roundworm), Ancylostoma duodenale (hookworm), Gnathostoma spinigerum, Halicephalobus gingivalis and Trichinella spiralis (Trichina worm) [1,5,6]. While some parasitic nematodes (e.g., Ancylostoma duodenale, Strongyloides stercoralis and Halicephalobus gingivalis) can penetrate human skin or invade through existing skin lesions [7,8], several parasites infect humans via ingestion of food products contaminated with the embryonated eggs (e.g., Ascaris lumbricoides and Trichuris trichiura) [9,10] or from eating raw or undercooked freshwater fish, birds, frogs or reptiles contaminated with the parasitic nematode larvae (e.g., Gnathostoma spinigerum, Dracunculus medinensis, Eustrongylides sp. and Trichinella spiralis) [11][12][13]. Apart from causing diseases, Extensive exploration of terrestrial microbial compounds for therapeutic drug development was initiated in the 20th century [53]. Since then, more than 50,000 beneficial bioactive metabolites have been successfully identified [58,59] some of which have potent nematotoxicity via distinct modes of action (MOAs) ( Table 1). Unfortunately, after almost 50 years of drug screening, few newly discovered compounds have been identified from terrestrial-borne microorganisms [59] hence the requirement for a new source of antinematode drug discovery to combat the rapidly growing nematode resistance. Here, the underexplored marine ecosystem, with highly diverse unidentified macro-and microorganisms, represents a new repository for novel nematicidal drugs [59]. In fact, marine bioactive compounds have been acknowledged as having substantial chemical novelty compared to terrestrial metabolites [60].
The marine environment represents the largest biome on the earth (70% of the earth surface; 361 million km 2 with average depth of 3730 m) and provides a habitat for a wide diversity of life that outnumbers terrestrial environments [61][62][63][64]. Microorganisms are abundant in this ecosystem (10 5 to 10 6 of cells per milliliter), reaching an average 10 28 to 10 29 cells either in the open ocean, deep sea, sediment or on the subsurface [64][65][66]. However, the majority of marine bacteria remain unrecognized or uncultivable [67]. Given the enormous diversity and untapped bioactive potential, marine microorganisms are likely to produce newly discovered bioactive compounds with a well-defined molecular architecture and biological function including nematicidal activities [68,69].

Surface Associated Marine Bacteria: A Reservoir for Novel Antimicrobial and Antinematode Drug Discovery
Marine inhabitants, particularly microorganisms, are continuously exposed to multiple detrimental interactions imposed by competitors and predators [83,84] and different physical-chemical variables such as fluctuating temperature, pH, UV exposure, salinity, toxic compounds and desiccation, particularly in the intertidal zone [85][86][87]. As a survival strategy, some marine bacteria adhere to each other and/or surfaces and are embedded in enclosed matrices to form a biofilm ( Figure 1) [88,89]. Surface-associated marine bacteria live on the nutrient-rich marine surfaces (for example macroalgae or cnidaria) in the form of biofilms. These marine biofilms are exposed to biotic (intraand/or inter-species interaction with other microorganisms or predators i.e., protozoa and nematodes) and abiotic physical-chemical stressors. The predator-prey interactions lead to the production of nematicidal metabolites by the surface-associated marine bacteria, while the harsh environmental conditions enhance the chemical, molecular and functional properties of the produced microbial compounds. Image created with BioRender.com.

Figure 1.
Surface-associated marine bacteria live on the nutrient-rich marine surfaces (for example macroalgae or cnidaria) in the form of biofilms. These marine biofilms are exposed to biotic (intra-and/or inter-species interaction with other microorganisms or predators i.e., protozoa and nematodes) and abiotic physical-chemical stressors. The predator-prey interactions lead to the production of nematicidal metabolites by the surface-associated marine bacteria, while the harsh environmental conditions enhance the chemical, molecular and functional properties of the produced microbial compounds. Image created with BioRender.com.
The continuous development of biofilm on marine surfaces leads to epibiosis, which involves multispecies biofilm formation [90,91]. Macroalgal surfaces, for example, are a hot-spot for colonization by opportunistic epibionts such as algal spores, invertebrate larvae, diatoms, fungi and other bacteria [85,92], mostly due to the accumulation of nutrients and macroalgal exudates composed of organic carbon and nitrogen particles [93][94][95]. Consequently, the competition among marine microorganisms to reserve a space within the biofilm community is tremendously intense and bacterial strains that are equipped with broad-spectrum inhibitory phenotypes are likely to be successful epibiotic colonizers ( Figure 1) [83,96].
In addition, predation by heterotrophic protozoa and bacterivorous nematodes represents another biotic stress resulting in major mortality for both planktonic and surface-associated bacteria in the marine habitat ( Figure 1) [85,97]. Protozoans, for example Rhynchomonas nasuta and Cafeteria roenbergensis, are among the most abundant ubiquitous species in the ocean and are the major controllers of the food web in the marine environment through their function as bacterial predators [98][99][100]. Nematodes such as Pareudiplogaster pararmatus are among the natural consumers of organic biomass in benthic habitats actively grazing bacterial mats and the biofilms of biotic surfaces [101,102].
The omnipresence of inter-and intra-species interactions supports the evolution of diverse defense strategies by surface-associated marine bacteria. Such defense mechanisms include the production of bioactive compounds showing antibacterial, antifungal, antitumor, antifouling, antiprotozoal and antinematode activities (Table 2) [103,104]. Interestingly, the physical-chemical properties, molecular structure and functional features of those marine microbial compounds are believed to be shaped by the naturally harsh conditions of the marine environment [105]. Moreover, it is speculated that bioactive metabolites originally attributed to marine invertebrates such as sponges, tunicates, bryozoans and molluscs are actually produced by their associated microorganisms [106,107]. For example, the antibiotic peptides andrimid and trisindoline isolated from the sponge Hyatella sp. and Hyrtios altum are believed to be produced by symbiotic Vibrio sp. [108,109]. An antitumor cyclic peptide leucamide A isolated from the sponge Leucetta microraphis is closely related to compounds produced by cyanobacterial symbionts [110]. In addition, a commercialized antitumor drug Didemnin B initially isolated from the tunicate Trididemnum solidum [111] was recently demonstrated to be produced by symbiotic bacteria Tistrella mobilis and T. bauzanensis [112,113]. These observations also hold true for numerous antinematode compounds that have been successfully isolated from marine eukaryotes, with production of many now attributed to host-associated microorganisms [51,114].
The potential for marine surface-associated microorganisms to be repositories of unusual gene functions and bioactivities is further corroborated by global biodiversity studies such as the Tara Oceans [69,115] and the Global Ocean Sampling (GOS) expedition [116,117]. These and other studies continue to reveal unprecedented levels of information on microbial diversity, which has subsequently led to investigations on underexplored bacterial diversity in various marine ecosystems. Furthermore, abundant and newly discovered biosynthetic gene clusters encoding rare non-ribosomal peptides (NRPS), polyketides (PKS) and NRPS-PKS hybrids have been discovered from marine biofilm samples, further strengthening the concept of the marine environment as a rich source of newly discovered bioactive compounds [68,69].

Caenorhabditis elegans as a Model Organism for Antinematode Drug Discovery and Development
Antinematode drug research is impeded by several challenges. These include (i) similarity of biochemical reactions between parasitic nematodes and the infected host, (ii) complex parasite life cycles that involve infections in multiple hosts, (iii) different parasite geographical locations and (iv) rapid development of resistant phenotypes. Therefore, an easily maintained nematode model with the capability for rapid screening of potential antinematode compounds represents a solution to some of these challenges [128,129]. Sydney Brenner and colleagues first introduced the free-living soil nematode C. elegans in 1965 as an animal model for research including anti-infective and antinematode drug studies [130][131][132][133][134][135].
Owing to its small size (1-1.5 mm-adult length, 80 µm-diameter), transparency, rapid life cycle (~3 days) and diet based on a simple bacterial culture of Escherichia coli OP50, C. elegans has emerged as a valuable animal model [134,136,137]. C. elegans possess several features which make the organism an efficient and low-cost surrogate organism for antinematode drug discovery. Unlike parasitic nematodes which require vertebrate hosts for reproduction and maintenance [48,138], synchronized C. elegans can be easily propagated at the desired life stage on Nematode Growth Media (NGM) ready to be used for antinematode drug studies [139]. The earliest antinematode drug testing using synchronized C. elegans individuals was performed by exposing the animals to nematicidal agents incorporated into the agar media [140]. After a few years, the screening protocol evolved rapidly with the development of high throughput screening methods employing micro-fluidic systems or high-content screening (HCS) technologies, allowing for fast and large-scale drug testing (~14,000 to 360,000 of compounds) using C. elegans as the model organism ( Figure 2) [141,142]. Today, important drug discoveries, such as benzimidazoles [143], ivermectin and its analogues, moxidectin, milbemycin oxime, doramectin, selamectin, abamectin, eprinomectin [144] and the nematicidal activity of crystal protein insecticide Cry5B, Cry21A from Bacillus thuringiensis [72,145], can be attributed to the use of C. elegans as an effective animal model [48].

Caenorhabditis elegans as a Model Organism for Antinematode Drug Discovery and Development
Antinematode drug research is impeded by several challenges. These include (i) similarity of biochemical reactions between parasitic nematodes and the infected host, (ii) complex parasite life cycles that involve infections in multiple hosts, (iii) different parasite geographical locations and (iv) rapid development of resistant phenotypes. Therefore, an easily maintained nematode model with the capability for rapid screening of potential antinematode compounds represents a solution to some of these challenges [128,129]. Sydney Brenner and colleagues first introduced the free-living soil nematode C. elegans in 1965 as an animal model for research including anti-infective and antinematode drug studies [130][131][132][133][134][135].
Owing to its small size (1-1.5 mm-adult length, 80 µm-diameter), transparency, rapid life cycle (~3 days) and diet based on a simple bacterial culture of Escherichia coli OP50, C. elegans has emerged as a valuable animal model [134,136,137]. C. elegans possess several features which make the organism an efficient and low-cost surrogate organism for antinematode drug discovery. Unlike parasitic nematodes which require vertebrate hosts for reproduction and maintenance [48,138], synchronized C. elegans can be easily propagated at the desired life stage on Nematode Growth Media (NGM) ready to be used for antinematode drug studies [139]. The earliest antinematode drug testing using synchronized C. elegans individuals was performed by exposing the animals to nematicidal agents incorporated into the agar media [140]. After a few years, the screening protocol evolved rapidly with the development of high throughput screening methods employing micro-fluidic systems or highcontent screening (HCS) technologies, allowing for fast and large-scale drug testing (~14,000 to 360,000 of compounds) using C. elegans as the model organism ( Figure 2) [141,142]. Today, important drug discoveries, such as benzimidazoles [143], ivermectin and its analogues, moxidectin, milbemycin oxime, doramectin, selamectin, abamectin, eprinomectin [144] and the nematicidal activity of crystal protein insecticide Cry5B, Cry21A from Bacillus thuringiensis [72,145], can be attributed to the use of C. elegans as an effective animal model [48].  C. elegans can be used as a surrogate nematode for the initial screening against the potential compounds or microorganisms with nematotoxic properties either via the conservative agar plate method or through high throughput screening technologies. C. elegans-based research offers several advantages including nematode genetic manipulation, determination of drug MOA, evaluation of the resulting nematode responses and a large-scale initial drug screening due to easy nematode maintenance and propagation in the laboratory. Image created with BioRender.com.
C. elegans shares many conserved genes and protein functions with parasitic nematodes. Analysis of the intestinal parasite Strongyloides stercoralis genetic sequences showed 85% of protein homologs to C. elegans genes. The infective stage of S. stercoralis (L3i/dauer) also shows an increased proportion of protein homologs to C. elegans dauer larvae [146]. Genetic manipulation and RNA interference (RNAi) studies have been widely performed on C. elegans to provide a better understanding of the nematode response against the nematicidal drugs at the molecular level ( Figure 2). Quantitative polymerase chain reaction (qPCR) and "omic" technologies, e.g., transcriptomic profiling and proteomics, are also being used to provide a global snapshot of the molecular response of C. elegans to drug exposure [147,148]. Given the conserved gene homologs and protein function among the members of phylum Nematoda, these studies provide insight into the possible mechanisms used by parasitic nematodes against similar drug exposure [48].
The majority of the current drugs used to treat parasitic nematode infections target proteins that regulate neuromuscular activity including neurotransmitter receptors and ion channels [48]. Utilization of C. elegans as a model organism confers a better understanding of the mode of action (MOA) of potential nematicidal drugs (Figure 2). Studies have shown that C. elegans' neuromuscular system, its major neurotransmitters GABA (4-aminobutyric acid) and glutamate and its enzyme choline acetyltransferase, responsible for the synthesis of the neurotransmitter acetylcholine, display strong similarities to parasitic roundworms Ascaris suum and Ascaris lumbricoides [48, [149][150][151][152]. Therefore, exposure of C. elegans to antinematode compounds which target the neurotransmitter receptor and ion channels has enabled the discovery of the target binding molecule and the resulting toxicity to parasitic nematodes [153]. For example, observations of body muscle contraction and spastic paralysis in C. elegans exposed to levamisole, led Lewis and colleagues [154] to determine that binding to the muscle acetylcholine receptors was key to its activity. The initiation of amino-acetonitrile derivative (AAD) toxic activity against nematodes by binding to a nicotinic acetylcholine receptor was also revealed via forwards genetic screening using C. elegans mutants [155]. More recently, the MOA of paraherquamide, a broad-spectrum nicotinic nematicidal alkaloid isolated from Penicillium paraherquei [156], and antinematode plant-derived compounds [157] were also elucidated using C. elegans [157][158][159]. While using C. elegans to identify specific targets is promising, assessing side effects related to these newly discovered compounds will be important considering many of the neurotransmitter targets will also be present in the host organism [48]. Nevertheless, in the event that novel drugs negatively impact non-target organisms, derivatives may be developed with reduced side effects [160,161].

Conclusions
The increasing prevalence of parasitic nematode infection and the emergence of antinematode drug resistance represent critical global issues, impacting human wellbeing and economic development. Moreover, increasing temperatures and changing weather patterns, moisture and rainfall as a result of global climate change may also escalate the prevalence of parasitic nematode diseases worldwide [40][41][42][43][44]. Although for decades many anthelmintic/nematicidal drugs have been successfully derived from terrestrial microorganisms (see examples in Table 1), the paucity of new antinematode drug classes and the challenges now presented with drug resistance underline the importance of discovering new antinematode bioactive metabolites from natural sources [4,50]. This review highlighed the potential of marine epiphytic bacteria as a new platform for novel antinematode drug development. Marine epiphytic bacteria are highly diverse, harboring unique genes expressing newly discovered bioactive metabolites with commercially or pharmaceutically relevant biological potentials including antinematode activities [118][119][120]. Owing to the extraordinary molecular structure and physical/chemical properties, marine bioactive compounds are regarded as "blue gold from the ocean" and are believed to be a promising source for future novel antinematode drugs. The ability to uncover these novel marine-derived antinematode drugs will be dependent on the successful implementation of innovative cultures, culture-independent techniques and high-throughput bioassays, for which the model nematode C. elegans is well suited.