Introduction

Vibrio harveyi is a negative-gram bacteria that lives in aquatic environments, particularly in tropical and warm waters (Zhang et al. 2018; Firmino et al. 2019). Many warm-water fish and invertebrates have been linked to V. harveyi-related diseases, including grouper (Shen et al. 2017; Zhu et al. 2018), sea bream (Haldar et al. 2010), barramundi (Dong et al. 2017), tiger puffer (Mohi et al. 2010), seahorses (Raj et al. 2010; Qin et al. 2017), abalone (Wang et al. 2018), rock lobster (Diggles et al. 2000), shrimp (Manilal et al. 2010; Zhou et al. 2012; Muthukrishnan et al. 2019), and sea cucumber (Becker et al. 2004). This bacterium has been linked to a variety of fish symptoms, including opercula nodules, scaling, skin ulcers, tail rot, vasculitis, necrotizing enteritis, and gastroenteritis are all symptoms (Zhang et al. 2020). Muscular necrosis caused white or opaque lesions in the tails of the affected shrimp (Zhou et al. 2012). V. harveyi caused significant mortality in captivity seahorses and was identified by white spots on the surface and anorexia (Raj et al. 2010). The presence of white spot was also detected in abalone, which was followed by pustules and foot muscle atrophy (Wang et al. 2018). In immunosuppressed organisms, the severity of disease-related V. harveyi appears to be very high.

The virulence of a V. harveyi strain has been shown to be highly dependent on the host species (Vera et al. 1992), dose, duration of exposure, host species age (Jun and Huai-Shu 1998), and pathogenic factors of the bacterial strain (Gomez-Gil et al. 1998; Esteve and Herrera 2000). Extracellular proteases, hemolysin, outer membrane protein, phospholipase, and the secretion system, all played important roles in V. harveyi pathogenesis (Austin and Zhang 2006; Natrah et al. 2011). Hemolysin is a toxin that contributes significantly to the virulence of V. harveyi (Chattopadhyay and Banerjee 2003; Qiao et al. 2012; Zhao et al. 2021). Protein hemolysin has a molecular weight of 47.3 kDa and 419 amino acids (Zhong et al. 2006; Zhao et al. 2021).

Some microalgal allelopathic chemicals have piqued the interest of researchers due to their roles in algal community succession and potential as biocontrol agents. Tohmola et al. (2011) reported that microalgae produce and secrete metabolites into the medium during growth. Carbohydrates are the most common constituent of microalgal exudates, followed by nitrogenous compounds and vitamins (Watanabe et al. 2008). Extensive research will be required to determine the extracellular products of microalgae's chemical composition and mode of action. Several microalgae have been reported to have antimicrobial activity. Chlorella sp. produced the extracellular compound cysteine protease (ECPI-2) to protect cells from threats such as viruses and herbivorous animals (Ishihara et al. 2006). Chlorella sp. extracellular fluid can also be used against Pseudokirchneriella subcapitata (DellaGreca et al. 2010). Natrah et al. (2011) discovered that extracts from Chlorella vulgaris CCAP211/12 and C. saccharophila CCAP211/48 inhibited QS-regulated violacein production in CV026 indicating the presence of N-hexanoyl homoserine lactone.

Chlorella sp. CHS1 has been identified morphologically and molecularly as a species of microalgae indigenous to shrimp ponds. The potential biocontrol of vibriosis caused by V. harveyi by an extracellular metabolite of Chlorella sp. CHS1 was investigated. Knowing how a candidate for vibriosis biocontrol interacts with the virulence protein is important when considering a candidate for vibriosis biocontrol. The goal of this study was to evaluate the in-vitro effect of a Chlorella sp. CHS1 extracellular metabolite on V. harveyi and the interaction of those with the hemolysin protein from V. harveyi using a molecular docking study.

Materials and Methods

Culture of microalgae

Chlorella sp. CHS1 from ponds in the Situbondo area was used in this study for microalgae culture. Cultures of Chlorella sp. CHS1 have been identified morphologically and molecularly. Chlorella sp. CHS 1 was grown in Walne medium (Jayasankar and Valsala 2008) at 25°C, pH 7, 25 ppt salinity, and a light intensity of 4,500 lux.

Extraction of extracellular metabolites of Chlorella sp. CHS1

The method of Natrah et al. (2011) for preparing of algal supernatant extract for microalgae was used. During the late stationary period, algae were harvested. The culture was centrifuged for 5 min at 5000 rpm, and the supernatant was collected at -20°C. A 10 mL of the Chlorella sp. supernatant was thoroughly mixed with 10 mL of ethyl acetate. The solution was centrifuged at 3000 rpm for 10 min, and the ethyl acetate fraction was collected. This extraction was carried out twice. The sample was then evaporated at 30°C dissolved in 100 µL of 100% acetonitrile, and finally diluted with 300 µL of ddH₂O. All samples should be stored at -20°C in glass sample vials until use.

In vitro analysis

This study was conducted to evaluate antimicrobial growth against V. harveyi, which was cultured for 24 h in 10 mL of TSB containing 2% of NaCl at 30℃. The broth was washed and suspended with PBS after being centrifuged at 2000 rpm (pH 7, 2). This bacterial suspension (1 mL) was placed in a TSA plate containing 2% NaCl. TSA wells were formed, and 10 μL of Chlorella sp. extracellular metabolites was added. The plates were incubated at 30°C for 24 and 48 h, and the zones of inhibition around the wells were measured at both times.

GC-MS analysis

Gas chromatography-mass spectrometry (GC-MS) analysis was performed using an Agilent Technologies-7890A gas chromatograph with a mass-selective detector (5975C NICI-MS). A capillary column DB-5HT was used in the chromatography system (30 mm ´ 0.320 mm, film thickness 0.10 m). The injection was carried out automatically at a temperature of 250°C in the injector. The programmed temperature was applied with column starting at 180°C, and gradually increasing by 50°C per min until it reached 325°C and held there for 1 min. In split-less mode, helium gas (99.99% purity) is used as the carrier gas, with a constant flow rate of 3.5 mL.min^-1 (1 L).

In silico analysis

Homology modeling of hemolysin protein: The SWISS-MODEL template library was searched for template using BLAST (Camacho et al. 2009; Bienert et al. 2017). BLAST was used to search the target sequence of the V. harveyi (AAG25957.1) against the primary amino acid sequence in the database (Zhang et al. 2001). A total of eight templates were discovered. Models were created using the target-template alignment. The QMEANDisCo scoring function was used to evaluate the global and per-residue model (Studer et al. 2020).

Preparation of the ligand: In the current docking study, several identified compounds from Chlorella sp. CHS1 extracellular metabolite were used. PubChem (http://pubchem.ncbi.nlm.nih.gov) was used to download the 3D structures. The inhibitor molecule was given the Gasteiger and Kollman united atom charges. The 3D structures were minimized to obtain the most stable energy and then converted to *.pdb format with open babel oftware (O’Boyle et al. 2011) and compiled in Pyrx 0.8.

Molecular docking and ligan interaction: The Auto Dock Vina program (Trott and Olson 2010) compiled in Pyrx software was used for molecular docking. To evaluate the ligand binding energies across the conformational search space, the Lamarckian genetic algorithm was used. A polar hydrogen atom was added to the receptor protein. The active site of the protein accommodated in the docking was defined by a grid box region of 25 ´ 25 ´ 25 with a 0.375 Å span and center coordinates of x (204.54), y (15.42), z (34.74). The interaction of Ligan protein was investigated using Discovery Studio tools. SwissADME web-based platform (http://www.swissadme.ch/) was used to analyze the physicochemical properties and fulfillment of Lipinski’s (Daina et al. 2017).

Results

In vitro analysis

The antimicrobial properties of Chlorella sp. CHS1 extracellular metabolites were tested against V. harveyi using a well diffusion assay. Table 1 shows the results of the antagonism assays used in this study. The zone inhibition increased as the concentration of extracellular metabolites of Chlorella sp. CHS1 increased, according to the well diffusion assay. As a control, there was no zone of inhibition. After 48 h, the inhibitory zones in all concentrations were reduced by 5.86–29.76%.

GM-MS analysis

The GC-MS analysis of Chlorella sp. extracellular metabolites CHS1 was successful in detecting 41 different of active compounds. The active compounds were identified using a comparison of retention time and mass spectrum in the GC-MS library (Fig. 1). Only eight putative compounds shared more than 80% similarity with the C-MS library database (Table 2). Following that, the eight compounds were chosen for molecular docking analysis.

Homology modeling of protein hemolysin V. harveyi

Hemolysin, which play an important role in the virulence of V. harveyi, has the ability to lyse red blood cells by creating pores in the cytoplasmic membrane. The amino acid sequence of the V. harveyi hemolysin protein, accession number of AAG25957.1, was obtained from the Gen Bank (Zhang et al. 2001). Three-dimensional (3D) structure modeling was performed using the Swiss model (https://swissmodel.expasy.org/) and a template from V. vulnificus protein hemolysin. There were eight protein structures from a protein database (pdb) with sequence identities ranging from 75.36 to 76.79% (data were not shown). Model 7 was chosen as the best model based on the highest QMEANDisCoGlobal value (0.90) among the other models. The alignment of the amino acid sequence of V. harveyi hemolysin protein and the template model (PDB ID 6jl2.2.A) is shown in Fig. 2. The Ramachandran plot was used to assess the structure quality of the modeled hemolysin protein (model 7). Fig. 3a–b show the 3D structure of the modeled hemolysin protein as well as the Ramachadran plot result. According to the Ramachandran plot analysis, 97.37% of the amino acids in that protein were in the favored region.

The molecular docking and ligand interaction

The docking analysis revealed that seven (7) compounds and the naturally occurring ligand (hexa-ethylene glycol) could bind to the same active site of the hemolysin protein. However, butylated hydroxytoluene (ID11) was unable to bind to the same active site. Fig. 4 depicts the binding positions of eight (8) putative compounds and the natural ligand for the hemolysin protein. Table 3 shows the binding affinity of eight putative compounds and natural ligands with hemolysin V. harveyi. There were five compounds with lower binding affinity values than natural ligands (hexa-ethylene glycol); (1) Hexadecanoic acid, methyl ester, (2) Heneicosane, (3) Docosane, (4) Tetracosane and (5) Tricosane. This suggested that those five compounds are potential inhibitors of V. harveyi protein hemolysin. The ADME (adsorption, distribution, metabolism and elimination) related physicochemical and pharmacokinetic properties were evaluated using SwissADME (Daina et al. 2017) (http://www.swissadme.ch/). Table 4 shows the psychochemical and pharmacokinetic properties of the eight putative compounds. The scoring was done based on the value of binding energy, Lipinski violation, bioavailability, and potential for chemical synthesis to select the compound with high potential (Table 5). The scoring results ranged from 24.84 to 26.97. (1) 1,2,3-propanetricarboxylic acid, 2-hydroxy-, triethyl ester, (2) hexadecanoic acid, (3) tricosane were the top three candidates for anti-vibriosis compounds. Various chemical interactions can cause a ligan to bind to the target protein. To discover this relationship, the discovery studio was used to analyze how ligands and receptors interact. Previous research suggested that the three chemicals might have anti-vibrio properties. Fig 5 depicts the interactions of these three compounds with the hemolysin protein of V. harveyi.

Discussion

The zone inhibition increased as the amount of Chlorella sp. extracellular metabolites increased. This decrease demonstrated that Chlorella sp. extracellular metabolites CHS1 is bacteriostactic (suppresses growth) rather than bacteriocidal (kills bacteria). High cell density microalgae cultures were discovered to excrete growth inhibitors into supernatant, as previously reported in Nannochloropsis sp (Richmond and Zou 1999) and C. vulgaris (Javanmardian and Palsson 1991). Diatom extracellular polyunsaturated aldehydes also influenced bacterial development (Ribalet et al. 2008). However, depending on the type of bacteria, these chemicals have varying effects on bacterial growth, either suppressing or stimulating. Natrah et al. (2011) discovered no evidence of inhibitory growth activity caused by the cell-free supernatant of microalgal cultures. The chemicals, on the other hand, inhibited acyl-homoserine lactone-regulated violacein production and bioluminescence in the aquaculture pathogen V. harveyi. Haloperoxidase enzyme Table 1: Extracellular metabolites of Chlorella sp CHS1 were evaluated in vitro

Concentration of Chlorella sp extracellular metabolites (µg.mL^-1)	Inhibitory zone (mm) at
	24 h	48 h
1	0.93±0.10	0.66±0.17
10	3.03±0.06	2.85±0.06
100	3.97±0.26	3.67±0.25
1000	5.89±0.21	5.41±0.22
DMSO	0.00±0.00	00.0±0.00

Mean ± standard deviation

Table 2: The putative compounds derived from Chlorella sp. CHS1extracellular metabolites

No	ID	RT	Area (%)	Library/ID	Quality	MF	MW (g.mol^-1)
1	33	17.805	3.44	Heneicosane	96	C₂₁H₄₄	296.6
2	40	21.449	4.95	Docosane	98	C₂₂H₄₆	310.6
3	22	11.337	9.97	Tricosane	99	C₂₃H₄₈	324.6l
4	34	18.715	11.91	Tetracosane	97	C₂₄H₅₀	338.7
5	28	13.828	2.8	Hexadecanoic acid, methyl ester	90	C₁₇H₃₄O₂	270.5
6	11	9.454	4.22	Butylated Hydroxytoluene	98	C₁₅H₂₄O	220.35
7	26	13.093	20	1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester	86	C₁₆H₂₂O₄	278.34
8	17	10.639	6.38	1,2,3-Propanetricarboxylic acid, 2 -hydroxy-, triethyl ester	86	C₁₂H₂₀O₇	276.28

RT: retention time, MF: molecular formula, MW: molecular weight

Timeline

Description automatically generated

Fig. 1: Chlorella sp. CHS1 extracellular metabolites GC-MS analysis

Graphical user interface, application

Description automatically generated

Fig. 2: The amino acid sequences alignment of V. harveyi and template V. vulnificus hemolysins (6jl2.2.A)

from diatoms also interfered with quorum sensing by halogenating the acyl side chain and preventing AHL binding to the quorum sensing regulator (Amin et al. 2012).

Molecular docking, using either structure-based or ligand-based methods, has played an important role in the development of therapeutically important small molecules (Sliwoski et al. 2014). When the crystal structure of a protein cannot be determined empirically, homology modelling is used. Schmidt et al. (2014) demonstrated how modeling could result in precise target prediction in drug designing activity. Virtual screening of bioactive molecules is essential for weeding out potentially effective drugs in preclinical development.

According to Lipinski's rule, drug compounds that meet the criteria can penetrate cell membranes and be absorbed in the body: (1) a molecular weight (MW) <500 Table 3: The putative compounds derived from Chlorella sp. CHS1 extracellular metabolites

No	Compound ID	Molecular Formula	Affinity (kkal/mol^-1)
1	28	Hexadecanoic acid, methyl ester	−7.1
2	33	Heneicosane	−6.5
3	40	Docosane	−6.4
4	34	Tetracosane	−6.3
5	22	Tricosane	−6.2
6	17	1,2,3-Propanetricarboxylic acid, 2 -hydroxy-, triethyl ester	−5.1
7	26	1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester	−5.1
8	11	Butylated Hydroxytoluene	−3.9
9	Natural ligand	Hexaethylene glycol	−5.4

Table 4: The psychochemical properties of eight potential Chlorella sp. Compounds SwissADME-based CHS1

ID	Molecular Formula	MW	H-bond acceptors	H-bond donors	Consensus Log P	ESOL Log S	Violation of Lipinski	Bioavailability	Synthetic Accessibility
11	C₁₅H₃₀O	249.58	1	1	3.71	-4.32	0	0.55	4.19
17	C₁₂H₂₀O₇	295.43	7	1	0.80	-0.99	0	0.55	3.63
22	C₂₃H₄₈	324.63	0	0	9.00	-8.14	1	0.55	3.08
26	C₁₆H₂₈O₄	306.57	4	0	3.19	-3.85	0	0.55	4.51
28	C₁₇H₃₄O₂	304.72	2	0	5.42	-5.39	1	0.55	3.89
33	C₂₁H₄₄	340.92	0	0	8.08	-7.69	1	0.55	4.66
34	C₂₄H₅₀	338.65	0	0	9.35	-8.50	1	0.55	3.20
40	C₂₂H₄₆	310.60	0	0	8.64	-7.78	1	0.55	2.96

Fig. 3: (a) The 3D structure of V. harveyi hemolysin protein model (b). The Ramachandran plot’s outcome

Fig. 4: (a) The natural ligand’s pose and binding position (hexa-ethylene glycol), (b) the seven putative compounds from Chlorella sp. extracellular metabolites of CHS1

grams.mol⁻¹, (2) a hydrogen bond proton donor group <5, (3) a hydrogen bond proton acceptor group <10, and (4) the logarithm value of the partition coefficient in water and 1-octanol <5 (Lipinski et al. 2012). Table 4 shows that the chemicals with the ID numbers 11, 17 and 26 did not violate Lipinski’s role. Meanwhile, the other chemicals violated Lipinski's role (ID 22, 28, 33, 34 and 40). The ability of a drug compound to absorb and circulate in the body is referred to as bioavailability (Daina et al. 2017). The SwissADME predictions revealed that all compounds identified in this study have a bioavailability value of 0.55, indicating a high possibility of absorption in the body. Synthetic accessibility (SA) score is based on the assumption that the frequency of molecular fragments in “truly” attainable compounds correlates with the ease of synthesis (Daina et al. 2017). Furthermore, the SA score should be between 1 to 10. The higher the value, the more difficult it is for compound to be chemically synthesized. The SA values of the eight putative Chlorella sp. compounds CHS1 ranged between 2.96 to 4.66. Docosane (ID 40) was the simplest to synthesize, while Heneicosane was the most difficult (ID 33).

The biological activity was thought to be caused by both H-bond and hydrophobic interactions between the ligands/inhibitors and the active regions of the receptor (Madeswaran et al. 2012). The interactions that occurred between the ligand and the hemolysin protein in this study were hydrogen bond (H-bond) and hydrophobic. According to Fig. 5, the compound 1,2,3-Propanetricarboxylic acid, 2-hydroxy, triethyl ester, and protein hemolysin were able to form five hydrogen bonds and ten hydrophobic interactions. ASN248, HIS393, THR392 and PRO394 amino acids were used to form hydrogen bonds. The methyl ester of hexadecenoic acid could form five hydrogen bonds and seven hydrophobic interactions. ASN248, ASN 252, TYR368, THR392 and ASN252 were the active sites of the residues involved. Furthermore, 28 hydrophobic interactions were found for tricosane, indicating that ligan can bind to the hemolysin protein. Small compounds bound to the target protein pocket via H-bonds and hydrophobic interactions may be able to prevent the conformational changes that result in fusion (Sivakumar et al. 2021). Although larger conformational changes degrade performance, smaller ligand-induced protein movements appear to have little effect on rigid docking performance (Verdonk et al. 2008). It was discovered that a flavonoid (found in papaya, apple and lemon) with anti-dengue activity could halt the dengue virus fusion process by impeding the movement of the hinge area and obstructing the conformational rearrangement in envelope protein (Mir et al. 2016).

The drug for neutralizing a target protein of Vibrio spp. was predicted using a molecular docking study against vibriosis. Sivakumar et al. (2021) used a compound from Ulva fasciata extract in a molecular docking study against the hemolysin protein V. harveyi. They discovered that methyl dehydroabietate had the highest binding affinity on the active pocket of hemolysin protein. Arunkumar et al. (2017) investigated several natural substances for their ability to suppress Vibrio hemolysin. Cyanidin and Bergapten have been identified as potential compounds for the development of novel and potentially effective drugs to treat vibriosis. Boronic acid derivatives bind to the binding site of the LuxP protein, according to V. harveyi, Rajamanikandan and Jeyakanthan (2017). The chemical inhibitor of V. harveyi biofilm development was discovered to be [2.2.1] hept-5-ene-2,3-dicarboxylic acid-2,6-dimethylpyridine 1-oxide.

Conclusion

An in vitro study revealed that the extracellular metabolites of Chlorella sp. CHS1 is bacteriostactic (growth suppressing) in a dose dependent manner. According to the virtual screening analysis using molecular docking, physiochemistry criteria, and Lipinski's drug similarity rule, three potential anti-vibrio compounds exist; 1,2,3-Propanetricarboxylic acid, 2 -hydroxy-, triethyl ester), Hexadecenoic acid, methyl ester), and Tricosane. The potent compounds interact with the hemolysin protein V. harveyi via hydrogen bond and hydrophobic interactions.

Acknowledgements

University of Brawijaya funded this study with grant number: 90/UN1/DITLIT/DIT-LIT/LT/2017.

Author Contributions

ATY designed the experiment and wrote the paper. MD carried out the in-silico analysis. The microalgae culture, extracellular metabolites, GC-MS analysis, and in-vitro study were prepared by NBA and RY. AMH reviewed the manuscript and analyzed the data.

Conflicts of Interest

This research has no potential conflict of interest.

Data Availability

The data described in this work will be made available upon reasonable request to the corresponding author.

Ethics Approval

Not applicable in this study.

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Received 24 January 2023; Accepted 02 March 2023; Published 17 March 2023

Abstract