TECHNICAL FIELD

The present invention generally relates to treatment of saprolegniasis in aquatic animals.

BACKGROUND

The oomycete pathogen Saprolegnia parasitica is an opportunistic facultative parasite causing saprolegniasis in various fish species, amphibians, crustaceans and other aquatic animals, and insects. Virulent strains of S. parasitica cause infection in eggs, juvenile and adult fish leading to huge losses worldwide in fishery industry. If untreated, S. parasitica causes death by hemodilution and osmoregulatory failure. Malachite green was used effectively for the treatment of saprolegniasis until it was banned in 2002 by the United States of America and several countries due to undesirable effects (carcinogenic and mutagenic) on human and animal health. Following the ban, there has been a re- emergence of Saprolegnia infections in aquaculture. With the ban of malachite green efforts have been made to find suitable alternatives.

Several chemicals have been tested with different degrees of effectiveness for the treatment of Saprolegnia infections in aquaculture. Sodium chloride at high concentrations is effective but impractical for use in freshwater. Formaldehyde has been used as an alternative compound for the treatment of saprolegniasis in eggs and larval stages but is harmful to the environment. Hydrogen peroxide is promising with minimal impact on environment and has been used to treat Saprolegnia infection in catfish. Peracetic acid, a strong disinfectant with wide antimicrobial activity, has been suggested to be effective in controlling Saprolegnia infection. Boric acid has been shown to inhibit germination and colonization of spores and mycelial growth by provoking mitochondrial dysfunction, and, thus, has been suggested for prophylaxis and control of saprolegniasis. By affecting multiple biological functions including protein and energy biogenesis, copper sulfate has been shown to inhibit the growth of Saprolegnia, but this salt is detrimental to the environment.

Alternate chemical based approaches have been attempted with varying success to control infection. Antifungal azoles like clotrimazole, which inhibits the CYP51 protein, a 14a-demethylase (SpCYP51), has been shown to be as potent as malachite green in inhibiting Saprolegnia growth by affecting sterol metabolism, suggesting its potential use against Saprolegnia infection. Amphotericin B, a commonly used antifungal agent, has also been reported to be effective in controlling the growth of Saprolegnia in catfish. Fungicidal activity of modified chitosans (methylpyrrolidinone chitosan, N-carboxymethyl chitosan and N-phosphonomethyl chitosan) against Saprolegnia infection has been reported. Bronopol (2-bromo-2-nitropropane-1 ,3-diol), a thiol-containing dehydrogenase inhibitor, and its methyl derivative, 2-methyl-4-isothiazolin-3-one (MT), are also promising in controlling infection in adult fish and eggs with lower toxicity. Even though bronopol is effective, tolerant strains of Saprolegnia have been reported recently. This led to the development of novel oxyalkylated derivatives of 2',4'-dihydroxychalcone (2- hydroxy,4-farnesyloxychalcone), which are suggested to be effective against the tolerant strains. Saprolmycins A and E derived from the spr gene cluster in Streptomyces sp., belong to a new angucycline-type of antibiotics and have been shown to be very potent against S. parasitica infection, with weak or no activity against fungi, bacteria and other microalgae or zooplankton. Similarly, a derivative of oridamycin A found in the fermentation broth of Streptomyces sp. exhibited anti- Saprolegnia activity and shows promising results in controlling S. parasitica infection. Dioscin, an ether-ethyl acetate-methanol derivative from the plant Dioscorea collettii considerably inhibits the growth of Saprolegnia by effectively damaging the mycelium and leading to the accumulation of reactive oxygen species by increasing total antioxidant and superoxide dismutase activity. Cladomarine and cladosporin analogs recently isolated from a deep-sea fungus ( Penicillium coralligerum YK-247) have been reported as novel anti -Saprolegnia compounds and are potential promising candidates with low toxicity to control saprolegniasis.

Lawhavinit et al., Studies on Fungus Diseases of Pejerry Odonthestes bonariensis (C & V.), Bull. Nippon. Vet. Zootech. Coll. 35: 135-140 (1986) discloses that pejerrey with Saprolegniasis also suffered from bacterial infection caused by Aeromonas hydrophilia strain NJB 8501. The sensitivity of the bacterial strain against chloramphenicol, oxolinic acid and oxytetracycline was tested in vitro and the bacterial strain was determined to be susceptible to all these three compounds. Pejerry was also treated with oxolinic acid and oxytetracycline. Almost all fish died with treatment of oxytetracycline, whereas oxolinic acid treatment was efficient. The authors concluded that if A. hydrophilia is a primary infection in pejerrey with Saprolegniasis, then oxolinic acid will be suitable for preventing Saprolegniasis in pejerrey.

Despite these reports of bioactive agents, there is still a need for agents effective in treating or controlling saprolegniasis, and in particular for agents that can be safely used in the treatment of the disease.

SUMMARY

It is a general objective to provide agents effective in treating or controlling saprolegniasis. This and other objectives are met by embodiments of the present invention.

The present invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

An embodiment relates to a compound for use in the treatment of saprolegniasis in an aquatic animal. The compound is an inhibitor of a non-homologous essential protein in Saprolegnia. The non- homologous essential protein is essential for a Saprolegnia oomycete and is not homologous to any protein in the proteome of the aquatic animal. The compound is selected from the group consisting of acetohydroxamic acid, azelaic acid, sulfamethoxazole, chloramphenicol, and a combination thereof.

Another embodiment relates to a composition for use in the treatment of saprolegniasis in an aquatic animal. The composition comprises a first compound and a second compound and the second compound is different from the first compound. The first compound is an inhibitor of a non-homologous essential protein in Saprolegnia. The non-homologous essential protein is essential for a Saprolegnia oomycete and is not homologous to any protein in the proteome of the aquatic animal. The first compound is selected from the group consisting of acetohydroxamic acid, azelaic acid, sulfamethoxazole, chloramphenicol, and a combination thereof. The second compound is selected from the group consisting of triclosan; benzoic acid; acetohydroxamic acid; azelaic acid; sulfamethoxazole; chloramphenicol; NaCI; hydrogen peroxide; peracetic acid; boric acid; an antifungal azole, such as clotrimazole; a saprolmycin, such as saprolmycin A-E; a chitosan; an ordidamycin; bronopol; a 2’, 4’- dihyxychalcone; dioscin; amphotericin B; cladomarine; cladosporin; nikkomycin; and a combination thereof.

A further embodiment relates to a method for treating saprolegniasis. The method comprises contacting an aquatic animal with a compound and/or composition as defined above.

Yet another embodiment relates to a system for fish farming. The system comprises a farming system comprising marine or fresh water and a compound and/or a composition as defined above.

The present invention provides compounds useful for controlling Saprolegnia oomycetes and thereby useful for the treatment of saprolegniasis in aquatic animals, including fish. The compounds of the invention are safe to use for the aquatic animals and also for humans. BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:

Fig. 1 - Schematic representation of the subtractive proteomics approach used for identification of potential effective compounds interacting with the proteins from Saprolegnia parasitica.

Fig. 2 - Effects of selected compounds on the growth of S. parasitica in liquid Machlis medium. A: Contrail , B: Control, C: Triclosan, D: Benzoic Acid, E: Acetohydroxamic Acid, F: Sulfamethoxazole, G: Chloramphenicol, H: Azelaic Acid, I: Glycerol, J: Citric acid, K: Albendazole, L: Thiabendazole, M: Copper sulfate, N: Boric Acid, 0: Malachite Green.

Fig. 3 - Representative images of growth of S. parasitica on PDA (Potato Dextrose Agar) plates in the presence of selected compounds after 72 hours of growth at 24°C. A: No Drug, B: Triclosan (4 mg/ml), C: Benzoic acid (400 mg/ml), D: Acetohydroxamic acid (600 mg/ml), E: Sulfamethoxazole (800 mg/ml), F: Chloramphenicol (800 mg/ml), G: Azelaic acid (800 mg/ml), H: Copper sulfate (400 mg/ml), I: Boric acid (400 mg/ml), J: Malachite green (0.8 mg/ml). Fig. 4 - Relative growth of S. parasitica on PDA plates at sub-inhibitory concentrations of the tested compounds after 72 hours of growth at 24°C. In parenthesis are the lower concentrations of the drugs used for radial growth of S. parasitica on PDA.

Fig. 5 - Effect of selected compounds on growth of S. parasitica in liquid Machlis culture medium after 24 hours of growth at 24°C as observed under at 20X magnification (optical microscopy). A: No Drug, B: Triclosan, C: Benzoic Acid, D: Acetohydroxamic Acid, E: Sulfamethoxazole, F: Chloramphenicol, G: Azelaic acid, H: Copper sulfate, I: Boric Acid, J: Malachite Green.

Fig. 6 - Homology modelling and docking of compounds effective against Saprolegnia growth with their respective target proteins.

DETAILED DESCRIPTION

The present invention generally relates to the treatment of saprolegniasis in aquatic animals. Saprolegniasis, also referred to as cotton wool disease, cotton mold or water mold, is a fungal disease of aquatic animals, including fish and fish eggs. Saprolegniasis is usually caused by oomycetes in the genus Saprolegnia and is characterized by white to brownish cotton-like patches on the surface of the skin and/or gills of the aquatic animal. Early lesions consist of pale foci with peripheral areas of erythema and a central zone of lifted scales, which frequently becomes ulcerated, exposing underlying musculature. Systemic infections are characterized by mycelial masses in the gut and surrounding viscera causing peritonitis with extensive hemorrhage, necrosis and adhesions. If untreated, the disease causes death by haemodilution and osmoregulatory failure. The previous golden standard in treatment of saprolegniasis was malachite green. However, malachite green is currently banned in several countries due to undesirable effects in terms of carcinogenic and mutagenic activity in humans and animals. There is therefore a need for agents effective in treating or controlling saprolegniasis, and in particular for such agents that can be safely used in the treatment of the disease.

An aspect of the embodiments relates to a compound for use in the treatment of saprolegniasis in an aquatic animal. The compound is an inhibitor of a non-homologous essential protein in Saprolegnia. The non-homologous essential protein is essential for a Saprolegnia oomycete and is not homologous to any protein in the proteome of the aquatic animal. According to an embodiment, the compound is selected from the group consisting of acetohydroxamic acid, azelaic acid, sulfamethoxazole, chloramphenicol, and a combination thereof.

Hence, the compound for use according to the invention is an inhibitor of an essential protein in Saprolegnia. This essential protein is, however, not homologous to any protein in the proteome of the aquatic animal to be treated with the compound. As a consequence, the compound is an inhibitor of a non-homologous essential protein in Saprolegnia.

The non-homologous essential protein is regarded as not being homologous to any protein in the proteome of the aquatic animal if a sequence similarity or comparison between the non-homologous essential protein and proteins of the aquatic animal results in an E-value that is more 1.0E-03 (1.0x10 ³ ) and a bit score equal to or less than 100. The E-value provides information about the likelihood that a given sequence match is purely by chance. The lower the E-value, the less likely the match is a result of random chance and therefore the more significant the match is. The bit score measures sequence similarity independently of query sequence length and database size and is normalized based on the raw pairwise alignment score. The bit score (SB) is determined by the following formula:

InK) / In2, wherein l is the Gumble distribution constant, SA is the raw alignment score, and K is a constant associated with the scoring matrix used. Clearly, the bit score is linearly related to the raw alignment score. Thus, the higher the bit score, the more highly significant the match is. The bit score provides a constant statistical indicator for searching different databases of different sizes or for searching the same database at different times as the database enlarges.

The non-homologous essential protein is indispensable and therefore essential for the cellular life of Saprolegnia. As a consequence, inhibition of the non-homologous essential protein by the compound of the embodiments will cause a reduction in the growth of Saprolegnia oomycetes. Saprolegnia may cause saprolegniasis in various aquatic animals including various fish species (van West, 2006), amphibians (Blaustain et al., 1994; Fernandez-Beneitez et al., 2008), crustaceans (Dieguez-Uriveondo et al., 1994; Soderhall et al., 1991) and aquatic insects (Sarowar et al., 2013; Sarowar et al., 2014). In an embodiment, the aquatic animal is selected from the group consisting of a fish, an amphibian, a crustacean and an aquatic insect. In a particular embodiment, the aquatic animal is a fish. Fish as used herein includes various developmental stages of fishes, such as fish egg, juvenile fish, fry, fingerling and adult fish.

In a particular embodiment, the fish is selected from the group consisting of a brown trout, an Atlantic salmon, a rainbow trout, a coho salmon, a catfish, a pike, an arctic car, an eel, a roach, a carp, a sturgeon, a kissing gourami, a guppy, a swordfish, a tilapia, a cod, a platyfish, a zebrafish, a torafugu, a spotted gar, a medaka, a tetra, a tongue sole, and a killifish.

Saprolegniasis is most often caused by oomycetes of the Saprolegnia order. Hence, in an embodiment, saprolegniasis in the aquatic animal is caused by an oomycete of a Saprolegnia species. In a particular embodiment, the Saprolegnia species is selected from the group consisting of S. diclina and S. parasitica. In a preferred embodiment, the Saprolegnia species is S. parasitica.

The non-homologous essential protein, which the compound of the embodiments targets and inhibits, is preferably not homologous to any protein in the proteome of the aquatic animal and in the proteome of humans. Hence, the compound preferably does not target or inhibit any proteins present in humans. As a consequence, an aquatic animal treated for saprolegniasis by the compound of the embodiments can safely be used as human food. In an embodiment, the non-homologous essential protein is selected from the group consisting of SPRG_06801 , SPRG_19504, SPRG_35021 and SPRG_13682.

SPRG_06801 is annotated as an urease (EC 3.5.1.5), which is a nickel-dependent amidohydrolase catalyzing the hydrolysis of urea into carbon dioxide and ammonia. The hydrolysis of urea occurs in two stages. In the first stage, ammonia and carbamate are produced. The carbamate spontaneously and rapidly hydrolyzes to ammonia and carbonic acid. Ammonia is an important nitrogen source in bacteria, fungi and plants. SPRG_19504 is a hypothetical protein containing 7,8-dihydro-6-hydroxymethylpterin- pyrophosphokinase (HPPK) and a pterin-binding domain, which belong to the dihydropteroate synthase (DHPS) family. In several bacteria and lower eukaryotes, the enzymes HPPK (EC 27.6.3) and DHPS (EC 2.5.1.15) catalyze the sequential reactions in the folic acid biosynthetic pathway. Higher eukaryotes obtain folate from dietary sources and lack necessary enzymes for folate biosynthesis, whereas eubacteria and lower eukaryotes synthesize tetrahydrofolate.

SPRG_35021 is predicted to be a 50S ribosomal protein L16 protein.

SPRG_13682 is a hypothetical protein with 5'-3' exonuclease activity and an N-terminal resolvase-like domain.

According to the embodiments, the compound is selected from the group consisting of acetohydroxamic acid, azelaic acid, sulfamethoxazole, chloramphenicol, and a combination thereof. For instance, the compound could be acetohydroxamic acid; azelaic acid; sulfamethoxazole; or chloramphenicol. Alternatively, the compound is in the form of a combination or mixture of acetohydroxamic acid and azelaic acid; acetohydroxamic acid and sulfamethoxazole; acetohydroxamic acid and chloramphenicol; azelaic acid and sulfamethoxazole; azelaic acid and chloramphenicol; or sulfamethoxazole and chloramphenicol. Also combinations or mixtures of more than two of the compounds could be used, such as a combination or mixture of acetohydroxamic acid, azelaic acid and sulfamethoxazole; acetohydroxamic acid, azelaic acid and chloramphenicol; acetohydroxamic acid, sulfamethoxazole and chloramphenicol; azelaic acid, sulfamethoxazole and chloramphenicol; or acetohydroxamic acid, azelaic acid, sulfamethoxazole and chloramphenicol. In an embodiment, the compound is sulfamethoxazole. In such an embodiment, the sulfamethoxazole is preferably used in a concentration of at least 100 mg/ml, preferably at least 200 mg/ml, more preferably at least 400 mg/ml, such as at least 600 mg/ml or at least 800 mg/ml. In another embodiment, the compound is chloramphenicol. In such an embodiment, the chloramphenicol is used at a concentration of at least 200 mg/ml, preferably at least 400 mg/ml, more preferably at least 600 mg/ml, such as at least 800 mg/ml.

In a particular embodiment, the compound is selected from the group consisting of acetohydroxamic acid, azelaic acid, and a combination thereof.

In an embodiment, the compound could be acetohydroxamic acid. In such an embodiment, the acetohydroxamic acid is preferably used at a concentration of at least 100 mg/ml, preferably at least 200 mg/ml, more preferably at least 400 mg/ml, such as at least 600 mg/ml.

In another embodiment, the compound is azelaic acid. In such an embodiment, the azelaic acid is preferably used at a concentration of at least 200 mg/ml, preferably at least 400 mg/ml, more preferably at least 600 mg/ml, such as at least 800 mg/ml. The above presented examples of preferred concentrations for the various compounds are final concentrations of the compound in a solvent or marine or fresh water volume, in which the aquatic animal to be treated is present.

The compound of the embodiments is preferably provided dissolved or dispersed in a solvent. In an embodiment, the solvent is selected from the group consisting of water, ethanol, acetone and dimethyl sulfoxide (DMSO), preferably water. For instance, water is a preferred solvent for acetohydroxamic acid and azelaic acid, whereas DMSO may be used for sulfamethoxazole and ethanol, such as 1 % ethanol, for chloramphenicol. Another aspect of the embodiments relates to a composition for use in the treatment of saprolegniasis in an aquatic animal. The composition comprises a first compound and a second compound that is different from the first compound. The first compound is an inhibitor of a non-homologous essential protein in Saprolegnia. The non-homologous essential protein is essential for a Saprolegnia oomycete and is not homologous to any protein in the proteome of the aquatic animal. In an embodiment, the first compound is selected from the group consisting of acetohydroxamic acid, azelaic acid, sulfamethoxazole, chloramphenicol, and a combination thereof. In an embodiment, the second compound is selected from the group consisting of triclosan; benzoic acid; acetohydroxamic acid; azelaic acid; sulfamethoxazole; chloramphenicol; NaCI; hydrogen peroxide; peracetic acid; boric acid; an antifungal azole, such as clotrimazole; a saprolmycin, such as saprolmycin A-E; a chitosan; an ordidamycin; bronopol; a 2’,4’-dihyxychalcone; dioscin; amphotericin B; cladomarine; cladosporin; nikkomycin; and a combination thereof.

The previously described embodiments with regard to examples of aquatic animals, examples of Saprolegnia oomycetes, examples of solvents and examples of concentrations of the first compounds also apply to this aspect of the embodiments.

An example of the second compound is triclosan. Triclosan is preferably used in a concentration of at least 1 mg/ml, preferably at least 2 mg/ml, such as at least 6 mg/ml and more preferably at least 6 mg/ml, or more, such as at least 8 g/ml, at least 10 mg/ml, at least 20 mg/ml, at least 40 mg/ml, at least 60 mg/ml or at least 80 mg/ml. Triclosan may advantageously be dissolved in acetone, such as 1% acetone.

Another example of the second compound is benzoic acid. Benzoic acid is preferably used in a concentration of at least 200 mg/ml, preferably at least 400 mg/ml, and more preferably at least 600 mg/ml, or more, such as 800 mg/ml or 1000 mg/ml. Water may advantageously be used as solvent for benzoic acid.

Further examples of the second compound include NaCI, which is preferably used at a concentration of at least 10 mg/ml, such as at least 15 mg/ml; boric acid, which is preferably used at a concentration of at least 0.1 mg/ml, such as at least 0.5 mg/ml; peracetic acid, which is preferably used at a concentration of at least 1 mg/ml, such as at least 4 mg/ml, including 4-10 mg/ml; hydrogen peroxide, which is preferably used at a concentration of at least 25 nl/ml, such as at least 50 nl/ml, including 5075 nl/ml; clotrimazole, which is preferably used at a concentration of at least 0.5 mg/ml, such as at least 1 mg/ml, including 1-2 mg/ml; bronopol, which is preferably used at a concentration of at least 15 mg/ml, such as at least 30 mg/ml; saprolmycin, which is preferably used at a concentration of at least 2.5 ng/ml, such as at least 3.9 ng/ml, including 3.9-7.8 ng/ml; chitosan, including a chitosan derivative, which is preferably used at a concentration of at least 0.1 mg/ml, such as at least 0.4 mg/ml; oridamycin, which is preferably used at a concentration of at least 1 mg/ml, such as at least 3.0 mg/ml; dioscin, which is preferably used at a concentration of at least 0.5 mg/ml, such as at least 2.0 mg/ml; amphotericin B, which is preferably used at a concentration of at least 1 mg/ml, such as at least 5.0 mg/ml; cladomarine, which is preferably used at a concentration of at least 5 mg/ml, preferably at least 13 mg/ml, including 13-23 mg/ml; and nikkomycin, which is preferably used at a concentration of at least 10 mg/ml, such as at least 25.0 mg/ml.

A further aspect of the embodiments relates to a method for treating saprolegniasis. The method comprises contacting an aquatic animal with a compound of the embodiments and/or a composition of the embodiments.

In an embodiment, the method comprise dissolving the compound in a solvent, or the first and second compounds of the composition in a solvent, or the first compound in a first solvent and the second compound in a second solvent, to provide one or more solutions. In such a case, the contacting step preferably comprises contacting the aquatic animal with the solution(s). For instance, the compound(s) or solvent(s) may be added to fresh or marine water in which the aquatic animal is present. Alternatively, the compound(s) or solvent(s) could first be added to the fresh or marine water and then the aquatic animal is submersed into the treated fresh or marine water containing the compound(s) or solvent(s). In an embodiment, the method may also comprise removing the aquatic animal from the fresh or marine water comprising the compound(s) or solvent(s).

Treating saprolegniasis involves the application or administration of a compound, composition or solvent of the embodiments to the aquatic animal, which suffers from saprolegniasis, has a symptom of saprolegniasis or a predisposition toward saprolegniasis, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect saprolegniasis, the symptoms of saprolegniasis, or the predisposition toward saprolegniasis. For example, treatment of an aquatic animal, in which no symptoms or clinically relevant manifestations of saprolegniasis have been identified is preventive or prophylactic therapy, whereas clinical, curative, or palliative treatment of an aquatic animal, in which symptoms or clinically relevant manifestations of saprolegniasis have been identified generally does not constitute preventive or prophylactic therapy. Treating thereby encompasses inhibiting or preventing saprolegniasis in an aquatic animal.

Treatment of saprolegniasis in an aquatic animal comprises inhibiting or preventing colonization of infectious stages of Saprolegnia or killing of infectious and/or multiplying stages of Saprolegnia like hyphae. Treating saprolegniasis in an aquatic animal and preventing saprolegniasis progression may include alleviating or preventing symptoms, saprolegniasis associated with Saprolegnia infections, thereby curing an infection and restituting the health of the aquatic animal or through prophylactic treatment, preventing clinical manifestation of saprolegniasis to occur. The previously described embodiments with regard to examples of aquatic animals, examples of Saprolegnia oomycetes, examples of solvents and examples of concentrations of the compounds also apply to this aspect of the embodiments.

Yet another aspect of the embodiments includes a system for fish farming. The system comprises a farming system comprising marine or fresh water and a compound and/or composition of the embodiments.

In an embodiment, system also comprises fishes present in the marine or fresh water in the farming system.

The system is preferably a fish farming or pisciculture system used for raising fish commercially in tanks or enclosures, such as fish ponds, usually for food. The farming system may, thus, be in the form of a tank, a fish pond, a fish cage in a lake, bayous, pond, river or ocean, and the like. The previously described embodiments with regard to examples of aquatic animals, examples of Saprolegnia oomycetes, examples of solvents and examples of concentrations of the compounds also apply to this aspect of the embodiments.

EXAMPLE

In the present example, the whole proteome of Saprolegnia parasitica CBS 223.65 was analyzed to identify non-homologous essential proteins that could be targeted with inhibitors with an aim to control saprolegniasis in aquaculture. Selected inhibitors were tested in vitro for their efficacy in arresting the growth of S. parasitica and several inhibitors with high in vitro efficacy in controlling the growth of S. parasitica were reported.

MATERIALS AND METHODS

A subtractive proteomics approach (Fig. 1) was used for the identification of probable drug targets among the non-homologous proteins within the proteome of S. parasitica. Selection of protein databases

The complete proteome of Saprolegnia parasitica CBS 223.65 (assembly ASM15154v2) was retrieved from the NCBI database. The conserved domains in the proteins were predicted using pfam v31.0 (Finn et al., 2016) and Conserved Domain Database (CDD) (Marchler-Bauer et al., 2017). Protein sequences from human ( Homo sapiens GRCh38.p11) and 15 fish genomes (completed to the Chromosome assembly level) (Table 1) were compared with the proteins from S. parasitica using blastp (BLAST 2.2.31 +).

Table 1 - List of proteomes screened for the identification of non-homologous proteins in Saprolegnia parasitica

Identification of non-homologous essential proteins

Saprolegnia proteins without any significant hits (E-value 1.0E-03, bitscore >100) against any of the human or fish proteomes were considered non-homologous and therefore used for further analysis. Essential proteins from S. parasitica were predicted by searching against the Database of Essential Genes (DEG) v15.2 (Luo et al., 2014). This database consists of indispensable genes from various organisms (from Archaea, Bacteria and Eukarya domains) essential for their cellular life. This essentiality criterion was based on the assumption that the proteins essential to one organism were likely to be essential in another organism. Proteins from S. parasitica similar to any of the proteins from the DEG database (E-value 1.0E-05, bitscore >100) were selected. These non-homologous essential proteins from S. parasitica were considered as potential targets.

Druggability potential of non-homologous essential proteins

An effective way to identify druggability of a protein is the identification of similar proteins binding to the drug-like compounds. The DrugBank database (Wishart et al., 2018) is a unique collection of bioinformatics and cheminformatics resources combining drug data along with the target information. Potential target proteins (non-homologous essential proteins) from S. parasitica were searched against the DrugBank database (v5.1.1) using blastp (E-value 1.0E-05, bitscore >100). Proteins from S. parasitica with significant hits against the DrugBank database were further considered. Among these, only Food and Drug Administration (FDA) approved compounds were selected for in vitro testing (Table 2).

Table 2 - List of FDA-approved compounds obtained from the DrugBank database after initial screening using non-homologous essential proteins from S. parasitica

Homology modeling and docking

The homology models of the predicted drug targets in S. parasitica were generated by comparative modeling using CPHmodels-3.2 Server (Nielsen et al., 2010). The details about the proteins and templates used are listed in Table 3.

Table 3 - List of target proteins used for homology modelling and docking of their respective interacting compounds

The generated models were further minimized through Steepest Descent followed by the Conjugate gradient method in the Discovery Studio 2.5 software (BIOVIA, 2018). These models were also used for molecular docking with selected candidate drugs using AudoDock Vina (Trott and Olson, 2010) in PyRx (Dallakyan, 2008) with default parameters. Three dimensional structures of drug molecules were obtained from the DrugBank database and minimized in PyRx before docking. Docked complexes were further visualized and analyzed using PyMol (Seeliger and de Groot, 2010).

Saprolegnia strain and culture conditions S. parasitica (CBS223.65) cultures were maintained at 24°C by inoculating Potato Dextrose Agar (PDA) with infected agar plugs and transferring them to a fresh plate every 15 days. Saprolegnia was also sub-cultured and grown in liquid Machlis medium (Machlis, 1953). Screening of potential inhibitors

Screening of compounds for their effects on Saprolegnia growth was performed using sesame seeds as baits. First, sesame seeds were sterilized (121 °C for 20 minutes) and sprinkled over the PDA plates. These plates were inoculated with a Saprolegnia infected agar plug at the center and were incubated at 24°C for 5 days until seeds were infected. Subsequently, Saprolegnia- colonized sesame seeds were transferred to a 24-well flat bottom tissue culture plate containing 1 ml Machlis medium with appropriate dilutions of the compounds and controls. The dose-dependent effect of each compound was tested at various concentrations. Copper sulfate, boric acid, and malachite green, reported earlier as effective treatments against S. parasitica were used as positive controls. Sterilized solvents (water, dimethyl sulfoxide (DMSO), ethanol and acetone) were used as negative controls. Varying concentrations of each solvent (0.1%, 1.0%, 2.0%, and 10.0%) were also tested in triplicates for their effect on Saprolegnia growth. The plates were incubated at 24°C for 4 days and mycelium growth was checked daily for the growth in each well. Minimum inhibitory concentration (MIC100) was calculated for the growth of S. parasitica on Machlis medium with little or no growth among four replicates (lane A, B, C, and D). Herein, MIC100 was defined as the lowest concentration of the compounds in which growth remains completely inhibited at 24°C for 4 days.

The effect of different compounds on the growth of S. parasitica on PDA plates was also examined. The PDA plates were prepared with the minimum inhibitory concentration of compounds and controls as inferred from the results of growth inhibition in liquid Machlis medium. Radial growth of S. parasitica was measured (diameter in mm) at 0, 24, 48, and 72 h at 24°C on PDA plates inoculated with an infected agar plug (5 mm).

Optical microscopy

The effects of selected compounds on the growth of S. parasitica was observed on 24-well flat bottom tissue culture plates with 1 ml Machlis medium containing appropriate dilutions of the compounds, and controls. Optical microscopy was performed on mycelium grown in plates after 24 h incubation in the presence of appropriate dilutions of the drug at 24°C. Observations at 20X magnification were made using an optical microscope (Leica Microsystems Ltd. DFC295) with Leica Application Suite v4.1.0. RESULTS AND DISCUSSION

In the present example, effectiveness of several compounds was identified and tested using a subtractive proteomics approach (Fig. 1) for the identification of probable druggable targets and FDA- approved effective compounds among the non-homologous essential proteins within the proteome of S. parasitica. Review of literature revealed various compounds to be explored with the aim of preventing the growth of S. parasitica (Table 4).

Table 4 - List of few known compounds effective against S. parasitica growth in aquaculture

Identification of non-homologous essential proteins in S. parasitica

With an aim to identify pathogen specific-proteins, thus, minimizing undesirable cross-reactivity of the drug molecule to the active sites of host homologous proteins, proteins were subjected to blast against the proteins from human and 15 fish genomes (see Table 1). Among 20,121 predicted proteins from S. parasitica, a total of 5,990 proteins were homologous to either human or fish proteins and, thus, were removed. The remaining 14,131 non-homologous proteins were considered for further analysis.

All 20,121 proteins were searched against the DEG database. Among these, 630, 3,134 and 7,774 proteins were found similar to the essential proteins from Archaea, Bacteria and Eukarya, respectively, totaling to 8,458 unique predicted essential proteins in S. parasitica. Among these, at least 480 proteins were common to all three domains of life. The rest of the 11 ,663 proteins did not show significant similarity to the essential proteins from either of the domains and were thus considered non-essential. Among the 14,131 non-homologous proteins, 3,012 proteins were found to have significant hits against the essential proteins in the DEG database and, thus, were considered essential for S. parasitica. These 3,012 non-homologous essential proteins were further considered for prioritization.

Selection of compounds for in vitro testing

Among the total of 3,012 non-homologous essential proteins from S. parasitica, a total of 117 proteins were found to be similar to the target sequences (130 proteins) in the DrugBank database, yielding 231 unique DrugBank entries. Among these, a total of 45 compounds were FDA-approved. The DrugBank entries with low probability of being inhibitors were removed (DB03147, DB03247, DB00140, DB09510, DB09513), resulting in a total of 40 compounds (Table 2). These compounds were further sub-divided into two groups: group I contains the compounds interacting with two or more proteins and group II contains the compounds interacting with one protein from S. parasitica.

Compounds interacting with two or more proteins from S. parasitica - Group I in Table 2

In S. parasitica, echinocandins, i.e., caspofungin, anidulafungin and micafungin, were predicted to interact with at least nine of the b-1 ,3-D-glucan synthases required for the synthesis of b-1 ,3-D-glucan. Echinocandins are well characterized antifungal agents, predicted to interact with proteins containing the 1 ,3- b-glucan synthase domain required for the biosynthesis of b-1 ,3-D-glucan, a major component of oomycete cell wall similar to callose from plants. The protein 1 ,3-b-glucan synthase being unique to fungal cell wall with no mammalian homologue is a favored target for these antifungal drugs. CDD analysis of the whole proteome led to the identification of at least ten proteins that belong to the glucan synthase superfamily, including SPRG_05460, which was not identified in the in silico screening as interacting partner with echinocandins. Another compound, picric acid has been used as antiseptic for a very long time and was predicted to inhibit S. parasitica by binding at least eight proteins from the pathogen.

Citric acid was predicted to interact with at least four proteins from S. parasitica, including two uncharacterized proteins (SPRG_06530 (probable FtsZ), SPRG_11850 (phosphoribosylaminoimidazole carboxylase (NCAIR synthetase)) and with tryptophan synthase (SPRG_05079) and the cell division protein FtsZ (SPRG_11942). Tryptophan synthase, which catalyzes the final step in tryptophan biosynthesis, is absent in mammals, which makes it an attractive target for other pathogens. The cell division protein FtsZ has been reported as potential drug target in various bacterial pathogens. The role of phosphoribosylaminoimidazole carboxylase as a novel target for antifungal drug therapy has also been proposed previously for Candida albicans. Citric acid under our experimental conditions did not inhibit up to a final concentration of 1000 mg/ml (data not shown).

Formic acid was predicted to interact with at least three chorismate binding enzymes, the anthranilate synthase proteins SPRG_06062, SPRG_12737, and SPRG_16397. Antimicrobial activity of formic acid has also been reported previously on various bacterial and yeast pathogens.

Artenimol is predicted to interact with at least two proteins, namely SPRG_00366 and SPRG_09913, which are a pyridoxal biosynthesis lyase PdxS and a negative regulator of GroEL domains, respectively. Artemisinin has been shown to be active against certain tumor cells, virus, malarial protozoa and various plant pathogenic fungi.

Compounds interacting with at most one protein from S. parasitica - Group II in Table 2

Sulfonamides, such as sulfamethoxazole, were predicted to interact with the protein SPRG_19504 in S. parasitica, which corresponds to a 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK) with a pterin binding domain, which belong to the dihydropteroate synthase (DHPS) family. In several bacteria and lower eukaryotes, the enzymes HPPK (EC 2.7.6.3) and DHPS (EC 2.5.1.15) catalyze sequential reactions in the folic acid biosynthetic pathway. Higher eukaryotes obtain folate from dietary sources and lack the necessary enzymes for folate biosynthesis, whereas eubacteria and lower eukaryotes synthesize tetrahydrofolate. Therefore, the folate pathway is an ideal target for the anti- infective agents. Dihydropteroate synthase (DHPS) is a well-known target of the sulfonamide class of antibacterial agents. CDD analysis of the proteins from S. parasitica showed the presence of two proteins SPRG_10565 and SPRG_17339 as dihydrofolate reductase (DHFR), which is the final enzyme in the folic acid biosynthetic pathway. Trimethoprim, a selective inhibitor of DHFR, in combination with sulfonamides, is a proven potent broad spectrum antibacterial agent.

Quaternary ammonium compounds like cetrimonium and didecyldimethylammonium were predicted to interact with SPRG_00820, a hypothetical protein with mycolic acid cyclopropane synthetase domain. Antimycotic activity of cetrimide, without showing pathological effect to the eye corneal tissues, was reported against fungal keratitis caused by Fusarium solani. Similarly, didecyldimethylammonium chloride (DDAC) was able to inactivate photosystem II (PSII) efficiency and disintegrate marine phytoplankton species and it was also found effective against Escherichia coli by bleb formation in the cell membrane causing leakage of the intracellular molecules and subsequent death of the cells.

Triclosan and triclocarban are commonly used broad-spectrum antibacterial and antifungal agents that block fatty acid synthesis by inhibiting enoyl-ACP reductase. They were predicted to interact with the protein SPRG_20215 in S. parasitica, an uncharacterized protein with a short-chain dehydrogenases/reductases (SDR) domain. Triclosan has been found to be effective against various gram-negative, gram-positive bacteria, as well as Mycobacteria, Plasmodium falciparum, wherein it is also targeting dihydrofolate reductase, and Leishmania panamensis with no toxicity towards mammalian cells up to 200 mg/ml. Benzimidazoles (albendazole and thiabendazole) were predicted to interact with the protein SPRG_07119, which contains an FAD-binding domain and is structurally similar to the fumarate reductase 2 (PDB 5GLG) from Saccharomyces cerevisiae. The protein is also the target of the FDA- approved inhibitors albendazole and thiabendazole. Benzimidazoles are commonly used fungicides and anthelminthic drugs that specifically inhibit microtubule assembly.

Urease inhibitors (acetohydroxamic acid and ecabet) were predicted to interact with the protein SPRG_06801 , annotated as urease (EC 3.5.1.5), which is a nickel-dependent amidohydrolase that catalyzes the decomposition of urea into carbamate and ammonium ion, constituting an important nitrogen source in bacteria, fungi and plants. Urease activity has been reported in Cryptococcus gattii and the recently discovered novel systemic fungal pathogen Emergomyces africanus. Urease is considered critical and is explored as a crucial target for antibacterial agents. This protein is a known target and has two FDA-approved inhibitors, acetohydroxamic acid and ecabet. Urea assimilation has been observed with mixed results in the genus Saprolegnia. Azelaic acid was predicted to interact with SPRG_13682, a hypothetical protein with 5'-3' exonuclease activity and an N-terminal resolvase-like domain. Azelaic acid has been found effective against various gram-positive and gram-negative pathogens by inhibiting several oxidoreductive enzymes and impeding glycolysis.

Chloramphenicol was predicted to inhibit the SPRG_35021 (50S Ribosomal protein L16) protein. Chloramphenicol is a common anti-bacterial agent that acts by inhibiting protein synthesis and is considered fungistatic. It has been recently reported to have variable degree of inhibitory effect in Phytophthora oomycetes. Even though effective, chloramphenicol resistant strains of Phytophthora have been reported.

Tromethamine was predicted to interact with a hyptothetical protein SPRG_04175, which contains Biotin and Thiamin Synthesis associated domain. Glycerin was predicted to interact with the SPRG_01876 protein, a histidinol dehydrogenase. Glycerin has been explored previously for its antibacterial activity.

Benzoic acid, a commonly used food preservative to prevent growth of yeast and molds, was predicted to interact with SPRG_02663, a hypothetical protein with an X-Pro dipeptidyl-peptidase C-terminal non- catalytic domain.

Effects of selected compounds on the growth of S. parasitica in liquid media

Among these DrugBank compounds, which were predicted to interact/inhibit non-homologous proteins from S. parasitica, 10 compounds were tested in vitro for their effectiveness (Table 5), along with copper sulfate, boric acid and malachite green as positive controls and solvents as negative control (see Material and Methods). Solvents used for preparing stock solutions of the selected compounds, i.e., water, DMSO, ethanol and acetone, were also tested for their effect and did not show any growth inhibition of S. parasitica up to 1% v/v solvent concentration (Figs. 2A and 2B). Growth inhibition by DMSO and ethanol was observed at a solvent concentration higher than 2% v/v, but no inhibition was observed for water and acetone up to solvent concentrations of 10% v/v.

Table 5 - In vitro screening of the selected compounds on the growth of S. parasitica

Triclosan and triclocarban were predicted to target the protein SPRG_20215. Triclosan was found to be very effective when tested on liquid Machlis medium with the lowest concentration (MIC100) of 4 mg/ml (Fig. 2C) and significant levels of inhibition were even observed even at a lower concentration (2 mg/ml). Benzoic acid (targeting hypothetical protein SPRG_02663) and acetohydroxamic acid (targeting urease SPRG_06801) were found effective in arresting growth at 600 and 800 mg/ml respectively with significant growth inhibition at lower concentrations of 400 and 600 mg/ml, respectively (Figs. 2D and 2E). Other compounds, i.e., sulfamethoxazole (800 mg/ml), chloramphenicol (800 mg/ml), and azelaic acid (800 mg/ml), showed growth inhibition at less than 1000 mg/ml, with significant inhibition at lower concentrations (Figs. 2F to 2H). Glycerol, citric acid, albendazole, and thiabendazole did not show significant inhibitory effect up to 1000 mg/ml or less (Figs. 2I-2L). Compounds significantly inhibiting growth of S. parasitica at a concentration less than 1000 mg/ml were considered effective. Copper sulfate (400 mg/ml), boric acid (400 mg/ml) and malachite green (2 mg/ml) (Figs. 2N to 20) were found effective in arresting the growth of the mycelium in liquid Machlis medium.

Fig. 2 shows the effect of all the compounds tested on the growth of S. parasitica at various concentrations in liquid Machlis medium. Effect of the selected compounds on S. parasitica radial growth

There was no growth on PDA plates at the minimum inhibitory concentrations for any of the compounds as determined using liquid Machlis medium culture. Therefore, a lower (sub-inhibitory) concentration of each drug was used to test the effect on growth on PDA plates. Excessive growth was observed in PDA plate with negative control (no drug), and glycerol (1000 mg/ml) showing no inhibition of growth over 72 hours at 24°C (Figs. 3 and 4). Significant level of inhibition was observed when treated with triclosan (4 mg/ml), benzoic acid (400 mg/ml), acetohydroxamic acid (600 mg/ml), sulfamethoxazole (800 mg/ml), azelaic acid (1000 mg/ml), chloramphenicol (800 mg/ml), copper sulfate (400 mg/ml), boric acid (400 mg/ml), and malachite green (0.8 mg/ml). Fig. 3 shows the representative images of the growth of S. parasitica on PDA plates. Fig. 4 shows relative growth of S. parasitica on PDA plates at sub-inhibitory concentrations of the tested compounds after 72 hours of growth at 24°C.

Effect of the selected compounds on growth morphology in S. parasitica

Optical microscopy was performed at a lower concentration than MIC100 to observe the effect of the selected compounds on the growth of S. parasitica. Fig. 5 shows the effect of selected compounds on the growth of S. parasitica in liquid Machlis medium after 24 hours of growth at 24°C as observed by optical microscopy at 20X magnification compared to the controls. Normally, untreated mycelium appears as transparent elongated structures (Fig. 5A) under optical microscopy. Hyper-branching of mycelium appears to be a common phenomenon in the presence of some of the drugs arresting the growth of S. parasitica as apparent in Fig. 5. However, hyper-branching towards the tip of the mycelium is neither very pronounced nor observed frequently in an untreated hypha (Fig. 5A). The degree of hyper-branching varied for different compounds and appeared to be dose dependent (data not shown). A pronounced hyper-branching of the mycelium was observed for triclosan (4 mg/ml), benzoic acid (400 mg/ml), sulfamethoxazole (400 mg/ml), and boric acid (400 mg/ml). A lower degree of hyper-branching was observed for acetohydroxamic acid (600 mg/ml), chloramphenicol (800 mg/ml), azelaic acid (800 mg/ml), copper sulfate (200 mg/ml), and malachite green (0.8 mg/ml). Interestingly, hyper-branching did not appear to be pronounced in the case of malachite green as compared to the other compounds that arrest the growth of S. parasitica (Fig. 5J). Homology modeling and docking

Homology modeling or comparative modeling is the process of generating a three-dimensional (3D) model of a protein sequence using a homologous protein structure as a template. CPHmodels server uses profile-profile alignment guided by protein secondary structure and exposure predictions for template identification (Nielsen et al., 2010). Homology modeling and docking studies were performed with six compounds shown to be effective in in vitro testing along with their respective predicted target proteins. The six target proteins were SPRG_20215, SPRG_02663, SPRG_06801 , SPRG_19504, SPRG_35021, and SPRG_13682. The 3D template structure for each selected proteins and the corresponding percent sequence identity are mentioned in Table 2. Except for protein SPRG_02663, where the sequence identity was 25.5%, all other proteins had significant sequence identity of >30%, allowing the production of reliable 3D protein structures.

The docking results showed an approximate binding pattern of the drug molecules with the target proteins. Analysis of the docked complexes, i.e., compounds with their respective proteins, showed considerable number of hydrogen bonding (H-bond) within the protein cavity or the active sites, which supports the binding with their respective proteins (Fig. 6). The docking results further supported the initial screening results and provided evidences for probable mechanism of actions of the six drug- proteins interactions as apparent in the in vitro testing of compounds against S. parasitica. In the present example, at least 40 compounds along with their probable target proteins were predicted using the whole genome subtractive genomics approach and 10 of these compounds were tested in vitro for their efficacy against the growth of S. parasitica. Among these, triclosan was found to be most effective with the lowest MIC100 (4-6 mg/ml), followed by benzoic acid (400-600 mg/ml), acetohydroxamic acid (600-800 mg/ml), sulfamethoxazole (800 mg/ml), chloramphenicol (800 mg/ml), and azelaic acid (800 mg/ml). Hence, several compounds, with an MIC100 lower than 1000 mg/ml against S. parasitica growth were found to be effective in the in vitro screening.

The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. The scope of the present invention is, however, defined by the appended claims. REFERENCES

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