Larvicidal and Cytotoxic Activities of Exopolysaccharides Produced by Thermophilic Bacteria

doi:10.21203/rs.3.rs-5304452/v1

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Larvicidal and Cytotoxic Activities of Exopolysaccharides Produced by Thermophilic Bacteria

https://doi.org/10.21203/rs.3.rs-5304452/v1

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EPSs synthesized by thermophilic bacteria are natural biopolymers that have recently garnered attention due to their potential applications in areas such as pharmaceuticals and biomedicine. In this study, EPSs produced by five distinct thermophilic bacterial isolates from hot springs in Turkey were purified using ion exchange and gel chromatography, and the larvicidal and cytotoxic effects of these EPSs were examined. While Geobacillus thermodenitrificans HBB111 produces the highest quantity (650,9 µg/mL) of EPS, the protein content of crude EPS samples ranges from 0.3–1.5%. EPS111 and EPS261 showed the most effective larvicidal action, eliminating 72% and 62.7% of Ae. albopictus larvae after 48 hours, respectively. Among the purified samples, EPS111 exhibited the most significant effect on the proliferation of PC3 cells, resulting in a 68% inhibition (IC₅₀ of 0,23 mg/mL) followed by EPS106 in a 55% (IC₅₀ of 0,45 mg/mL). According to the results of our study, thermophilic EPSs demonstrate potential due to their insecticidal and anticancer properties.

Exopolysaccharides

Geobacillus

Anoxybacillus

Parageobacillus

thermophilic bacteria

larvicidal activity

cytotoxicity

Exopolysaccharides (EPSs) are high molecular weight carbohydrate biopolymers composed of sugar residues [1]. They are secreted into the environment by producing microorganisms and can offer various beneficial properties and functions to these microorganisms, including protection against predation, biofilm formation, the creation of a biocompatible extracellular environment, adhesion, molecular recognition, and intercellular signal transmission [2, 3, 4, 5].

Thermophilic microorganisms can thrive at elevated temperatures, and EPS production is proposed as one of their adaptation mechanisms for survival in extreme conditions. Thermophilic bacteria are particularly beneficial for producing industrially significant EPSs, owing to their non-pathogenic nature and rapid growth rate [6]. EPSs synthesized by thermophiles are highly thermostable when compared to mesophilic ones and can display various biological activities, including antioxidant properties, non-cytotoxicity, anti-diabetic effects, prebiotic and fibrinolytic activities, as well as anti-viral and immunostimulant effects [7, 8, 9, 10, 11].

Members of the genera Geobacillus, Parageobacillus, and Anoxybacillus are natural inhabitants of geothermal areas, such as hot springs and hydrothermal vents, and they typically grow rapidly in relatively simple and cost-effective media [12, 10]. While numerous studies focus on mesophilic producers, research on thermophilic exopolysaccharides is rarely reported in the literature. Therefore, studies on the production and biological characterization of EPSs from thermophilic bacilli will yield more insights into their biotechnological applications.

In this study, EPSs produced by five thermophilic bacterial strains were purified and assessed for their biological characterization. The larvicidal activities and cytotoxic effects of thermophilic EPSs were examined. To our knowledge, there are no studies in the literature regarding the larvicidal activity of EPSs produced by species from the Geobacillus, Parageobacillus, and Anoxybacillus genera. Furthermore, our report is the first to evaluate thermophilic EPSs in the PC3 and RWPE1 cell lines.

Taxonomic analysis of bacterial isolates

Thermophilic bacterial strains, HBB-20, HBB-74, HBB-106, HBB-111 and HBB-261 were previously isolated from hot springs and sediment samples [13] and used for EPS production. 16S rRNA analysis based molecular identification of isolates was done [9] and the sequence data were deposited in GenBank, under accession numbers OR896930, OR896946, OR896962, OR896966, OR897006 for HBB-20, HBB-74, HBB-106, HBB-111 and HBB-261, respectively.

The 16S rRNA gene sequences of strains HBB 20, 74, 106, 111, and 261, along with those of twenty-five closely related strains obtained from GenBank, were aligned using MUSCLE. A phylogenetic tree for the isolates was then constructed using the neighbor-joining method with 1000 bootstrap replicates in MEGA11: Molecular Evolutionary Genetics Analysis Version 11 software.

Extraction and purification of exopolysaccharides

EPSs of five thermophilic bacteria were extracted by the following method. Firstly, thermophilic bacteria were cultured in basal media (0.1% (NH₄)₂HPO₄, 0.01% MgSO₄, 1% yeast extract, 0.02% KCl, 0.00001% thiamine, 2% sucrose, pH 7.2) at 55°C, 180 rpm for 18–24 h [14]. Then, cells were removed by centrifugation at 5000 rpm for 10 min, and 10 mL of concentrated supernatants were treated with trichloroacetic acid (4% w/v) to remove proteins. After shaking at 180 rpm for 30 min at room temperature supernatants were precipitated by adding two volumes of ethanol (95%) at 4°C. The precipitates were separated by centrifugation at 5000 rpm for 10 min and then dissolved in 10 mL of distilled water. After the determination of total carbohydrates was completed, samples were lyophilized and stored at -80 ℃ [15, 16].

To purify the EPSs ion exchange chromatography with the DEAE Cellulose (30477, Sigma-Aldrich) was used. The EPSs were eluted with a linear gradient of 0.1–1 mol/L NaCl with a flow rate of 1 mL/min. Both size separation and desalination were applied to the fractions collected by gel filtration chromatography. For this purpose, Sephadex G-100 column was used and the flow rate was set at 0.5 mL/min [17, 18].

Determination of protein and sugar content

The Bradford method was used to determine the protein concentration in crude EPS samples [19]. Bovine Serum Albumin (BSA) was prepared as a standard. Protein sample (25µL) was added into 1 mL Bradford solution and, after mixing it was kept at room temperature for 10 min. Two hundred microliters of the mixture were transferred to the microplate and distilled water was added to the dye instead of the blank protein. Optical density (OD) was measured at 595 nm [20].

The method for determining total carbohydrate amounts in the samples was adapted from Masuko et al. [21]. Briefly, 250 µL of the sample was mixed with 750 µL of concentrated sulfuric acid, followed by the addition of 150 µL of phenol solution (5% w/v). After incubating the mixture at 90°C for 5 minutes, the optical density (OD) was measured at 491 nm. The total carbohydrate content was determined using a standard calibration curve prepared with glucose.

Larvicidal (Insecticidal) activities of exopolysaccharides

Larvicidal activities of EPSs extracted from thermophilic bacterial strains were investigated against Aedes albopictus in 24 well plates [22]. Eggs were collected from adult Ae. albopictus kept in cages (30 × 30 × 30 cm) in a controlled temperature room at 30°C and 12 h light: 12 h dark photoperiod and hatched in tap water. Larvae were fed on crushed ground fish food until they reached 3rd stage [23, 24].

In the experiments, 3rd stage larvae were distributed into wells in 24-well plates and then one mL of extracted EPS diluted in distilled water was added to wells. At first the effects of the EPS at 100 µg/mL were evaluated and then the most effective two EPSs were further diluted to concentrations of 50, 20 and 10 µg/mL. Distilled water alone was used as a negative control. Larvae were fed with ground fish food. The study was conducted twice with at least 10 replicates per treatment. The plates were incubated at 26 ± 1°C. Larval mortality was larvae after 24 and 48 h exposures [22].

Cell culture

Human prostate cancer cell line (PC-3) (CRL-1435, ATCC) and the normal prostate epithelial cell line (RWPE1) (CRL-11609, ATCC) were kindly provided by Prof. Dr. Sarhan Sakarya. PC3 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin and 100 µg/mL streptomycin in a humidified incubator (5% CO₂ at 37 ^oC). RWPE1 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium with 20% (v/v) FBS, 1% penicillin and streptomycin in a humidified atmosphere containing 5% CO₂ at 37 ^oC. The cell lines were cultured in the culture plate, and the experiment was carried out when the cells entered the logarithmic growth period. H₂O₂ was dissolved in DMEM (serum-free) and stock solutions were prepared before treatment.

Cytotoxicity test

The inhibitory effects of both crude and purified EPSs on normal and cancer cell lines were investigated using the MTT assay. Both RWPE1 and PC3 (5x10³ cells/well) were incubated in their own culture media described above and in a humidified atmosphere containing 5% CO₂ for 24 h at 37°C. The cytotoxic effect was determined using a 96 well microtiter plate assay with five different EPS samples prepared at different concentrations (250, 500, 1000 and 2000 µg/mL) with serum-free medium [25]. The medium was subsequently replaced with 100 µL of EPS added to each well and incubated at 37°C for 24 h. After treatment, the cells were incubated with 10 µL MTT reagent (5 mg/mL) for 4 h at 37ºC under 5% CO₂. Supernatants were removed and the formazan crystals were dissolved in 150 µL of DMSO. The mixture was shaken up in the dark for 15 min, the absorption value of each well was determined at 490 nm by microplate reader. DMSO was used as a control. All the experiments were performed three times and the percentage of cell viability is calculated by using the Eq. (1₎ [26].

Cell viability %= (OD_sample / OD_control) × 100 ₍₁₎

Statistical analysis

Statistical analyses were conducted in the SPSS software program. ANOVA was used to analyze data on the effects of EPS on mosquito larvae and the differences among the averages were grouped using Tukey’s test at the level of P = 0.05. Arcsine transformation was performed on larval mortality before statistical analyses. Probit analysis was used to calculate LC₅₀ values.

Taxonomic analysis of thermophilic isolates

According to 16S rDNA sequence analysis results, HBB-20, HBB-111 and HBB-261 showed the highest homology ratios (94,35%-100%) with Geobacillus thermodenitrificans strains. At the same time, HBB-74 and HBB-106 were identified as Anoxybacillus suryakundensis and Parageobacillus thermoglucosidasius with 100% and 99,21% similarity rates, respectively [8, 9].

Neighbor-joining method with 1000 bootstrap replicates in MEGA11 was applied to 16S rRNA gene sequences of isolates and selected similar thermophilic sequences to construct a phylogenetic tree (Fig. 1). As can be seen from the phylogenetic tree, HBB-20, HBB-111 and HBB-261 constitute branches in G. thermodenitrificans cluster. On the other hand, while HBB-74 forms a branch with A. suryakundensis, HBB-106 forms a branch with P. thermoglucosidasius.

Isolation and purification of EPSs

Among the crude culture supernatants, the highest EPS content was detected in that of HBB-111 with 650,9 mg/L, followed by HBB-20 (419,4 mg/L), HBB-261 (357,6 mg/L), HBB-106 (352,2 mg/L) and HBB-74 (269,7 mg/L). Purification percentages and plots of EPSs are detailed in publications of Aytar et al [8, 9]. While the highest loss was recorded for EPS111 (37.1%), pure EPS concentrations ranged from 193.9 to 377.3 mg/mL (Fig. 2). The protein content of crude EPS samples was found to vary between 1,3 and 5,2 mg/L (Fig. 3).

Larvicidal activities of exopolysaccharides

Significant differences were observed in the larval susceptibility to EPSs extracted from different thermophilic bacteria. After 24h, EPS 111 and EPS 261 were the most effective, killing 67 and 56% of Ae. albopictus larvae respectively, while the effects of the other EPSs varied between 5 and 41% (F = 22.401; df = 5,102; P < 0.001). After 48 hours, effects of EPS111 and EPS261 increased to 72 and 62 percent up from 67 and 56% respectively. EPS 111 and 261 were statistically more effective than those of EPS20, EPS74 and EPS106 (F = 29.126; df = 5,107; P < 0.001) (Fig. 4).

A downward trend was noted in the mortality rate of Ae. albopictus larvae treated with different concentrations (100, 50, 20, and 10 µg/mL) of the two most effective EPSs, namely EPS111 and EPS261 (Fig. 5). After 24 and 48 hours, 10 µg/mL EPS26 was notably less effective than higher concentrations (100, 50, and 20 µg/mL) (F_{24 h}: =20.706, df = 3,50, P < 0.001; F_{48 h}: =22.084, df = 3,50, P < 0.001). Conversely, for EPS111, concentrations ranging from 20–100 µg/mL were significantly higher than the 10 µg/mL concentration after both 24 and 48 hours (P < 0.001) (Fig. 7). After 24-hours exposure, the LC₅₀ of EPS261 and EPS111 were 38 µg/mL and 95,5 µg/mL.

Cytotoxicity of EPSs

When crude EPSs were tested on RWPE1 and PC3 cell lines at concentrations ranging from 25 to 1000 µg /ml, no significant inhibitory effect was observed in either cell line (Fig. 6). Among the EPS samples at a concentration of 2000 µg/ml, the least cytotoxic to healthy RWPE1 cells are EPS111, EPS261, EPS74, EPS20, and EPS106, respectively. When EPSs were tested at the same concentration on the PC3 cell line, the most significant inhibitory effect was observed for EPS20, while EPS111 showed the least effectiveness. At a concentration of 2000 mg/ml, EPS20 achieved a high kill percentage in PC3 cells, recording 68% with an IC50 value of 1.28 mg/mL. The second most effective compound, EPS74, also demonstrated 68% efficacy with an IC50 of 1.36 mg/mL. Since two of the crude EPSs tested, EPS20 and EPS106, exhibited cytotoxicity in healthy RWPE1 cells (49% and 56%, respectively), the maximum concentration to be tested in experiments with purified samples was established at 500 µg/ml.

The cell viability of RWPE1 cells exposed to purified EPSs in the concentration range of 3.9–500 µg/mL was above 50% at all concentrations (Fig. 7). When raw EPS samples were applied at a concentration of 500 mg/mL, cell viability ranged from 98–120% for RWPE1 cells, whereas this rate was 92–122% for PC3 cells. There is a significant increase in inhibition when purified EPSs are tested at the same concentration. Viability rates ranged from 54–97% for RWPE1 cells and 32–72% for PC3 cells. EPS111 exhibited the most significant effect on the proliferation of PC3 cells, resulting in a 68% inhibition (IC50 of 0,23 mg/mL) followed by EPS106 in a 55% (IC50 of 0,45 mg/mL).

Publications in the literature regarding EPSs produced by thermophilic bacteria and their biological activities are relatively scarce compared to those from mesophilic producers. EPS production levels of some thermophilic bacteria were given as follows; Geobacillus sp. TS3-9, 87 µg/mL [11], G. thermodenitrifcans ArzA-6, 76 µg/mL; G. toebii ArzA-8, 80 µg/mL [27], Geobacillus sp. strain WSUCF1, 404 µg/mL [10], Bacillus licheniformis BITSL006, 45 µg/mL [28], Aeribacillus pallidus 418, 80 µg/mL [14], Geobacillus sp. 4004, 60 µg/mL [5], Parageobacillus thermantarcticus 400 µg/mL [29]. When we examined the EPS production levels (269,7 to 650,9 µg/mL) of the isolates used in our study, we found that they were comparable to those of other thermophilic Gram-positive producers. Considering the total protein content of EPSs, it is calculated to be 0.3%, 1.4%, 1.5%, 0.3%, and 1.2% for EPS20, EPS74, EPS106, EPS111, and EPS206, respectively. Similarly, the EPS produced by G. tepidamans V264 contains 1.8% protein, whereas the EPS produced by thermophilic B. thermoantarcticus has 2% protein [30, 31]. According to Genc et al. the amount of total protein in EPS solution obtained from Anoxybacillus pushchinoensis G11 was determined as 1.08% [32]. Among the EPSs, only EPS111 was thoroughly characterized in our previous publication, which revealed that it consists of hexose (glucose, fructose, galactose, and mannose) and pentose sugars [8]. Comprehensive chemical analyses of the remaining four EPSs are scheduled for future studies. Again, based on the findings of our previous study [8] it was demonstrated that none of the EPSs exhibited cytotoxic effects on blood cells, prompting us to explore the larvicidal and anticancer activities of these metabolites.

Novel strategies to reduce mosquito populations are an urgent priority. Applying EPS to mosquito breeding sites could help mitigate the risks of mosquito-borne diseases. To our knowledge, there are currently no reports regarding the larvicidal activity of EPS derived from members of the Parageobacillus, or Anoxybacillus genera. In a study conducted by Siddharthan et al. the larvicidal bioactivity of Geobacillus thermodenitrificans PS41 was assessed, revealing insecticidal activity against third-instar larvae of Spodoptera litura, which resulted in a mortality rate of 60.26 ± 1.5%. However, the trials utilized extracted secondary metabolites rather than EPS [33].

Our study revealed the larvicidal effects of thermophilic EPSs against Aedes albopictus, a major vector of arboviral diseases such as Chikungunya, dengue fever, and yellow fever. Among the tested bacterial EPSs, 111 and EPS261 were the most effective with LC₅₀ values after 24 hours were 38 µg/mL and 95.5 µg/mL, respectively. The larvicidal activity of EPS obtained from Bacillus licheniformis was tested against the larvae of Anopheles stephensi, a vector for Plasmodium sp. parasites, and Ae. aegypti, which, like Ae. albopictus transmits arboviruses [34]. The LC₅₀ and LC₉₀ values of Bl-EPS against An. stephensi were 61.31 and 127.45 µg/mL, and against Ae. aegypti were 79.28 and 150.68 µg/mL, respectively. These EPS exhibited maximum mortality against both vectors at 150 µg/mL. Similarly, EPS produced by Pseudomonas aeruginosa B01 exhibited potent larvicidal activity against an unspecified mosquito species at a concentration of 10 mg/dL [35].

When evaluating the antitumor effects of the thermophilic EPSs used in this study, purified samples demonstrate a greater inhibitory effect on both healthy and cancerous cells compared to raw EPSs (Table 1). Several in vitro studies have demonstrated that purified polysaccharides are more functional than crude polysaccharides [36, 37]. The anticancer properties of EPSs may result from other unwanted components or impurities present in the samples examined. Therefore, to assert that the activity in the studied material is attributable to EPS, the metabolite must be obtained and utilized in high purity. In our study, the reason pure samples exhibited superior results regarding anticancer activity may stem from the release of specific functional groups, such as sulfate and uronic acid, which can be found in EPSs through purification. Additionally, the varying rates at which thermophilic EPSs exhibit anticancer effects may be attributed to differences in their molecular weights and monosaccharide structures. However, more detailed chemical and structural analyses are required to validate these assertions.

Table 1

Viability percentages of RWPE1 and PC3 cell lines treated with crude and purified EPS at a concentration of 500 mg/mL, along with IC₅₀ values of EPS.
	CRUDE SAMPLES				PURIFIED SAMPLES
	RWPE1 cell line		PC3 cell line		RWPE1 cell line		PC3 cell line
	Cell viability (%)	IC₅₀ value (mg/mL)	Cell viability (%)	IC₅₀ value (mg/mL)	Cell viability (%)	IC₅₀ value (mg/mL)	Cell viability (%)	IC₅₀ value (mg/mL)
EPS20	120	2,04	92	1,28	54	0,53	60	0,60
EPS74	106	2,44	106	1,36	67	0,67	68	0,68
EPS106	116	1,76	110	2,80	72	0,72	45	0,45
EPS111	111	3,16	112	3,24	91	0,90	32	0,23
EPS261	98	3,08	122	2,56	97	0,97	72	0,72

In our study, the sample exhibiting the most effective anticancer activity against PC3 while maintaining the highest viability of RWPE1 cells is the purified form of EPS111, with an IC50 value of 0.23 mg/mL. It is followed by pure EPS106, which has an LC₅₀ value of 0.45 mg/mL and inhibited PC3 cells by 55%.

It was reported that EPS purified from Bacillus licheniformis AG-06, exhibited significant cytotoxic activity (IC50 value of 37.5 ± 0.87 µg/mL) against human lung carcinoma cells (A549) and thus demonstrates strong anticancer potential [38]. In another study, it was found that crude EPS derived from Anoxybacillus pushchinoensis G11 showed a cytotoxic effect on A-549 and colon cancer (Caco-2 and HT-29) cell lines at a concentration of 2.5 mg/mL [32]. Wang et al. evaluated the antitumor activity of purified samples of EPS from Geobacillus sp. TS3-9 strain on hepatoma carcinoma cells (HepG2) using MTT analysis. The inhibitory effect of EPS on the proliferation of HepG2 was dose-dependent at concentrations ranging from 4 µg/mL to 100 µg/mL, with the inhibition rate varying from 2.2–16.9%, respectively [11]. Two exopolysaccharides (EPS-1 and EPS-2) purified from the Geobacillus sp. WSUCF1 strain exhibited no cytotoxicity against human embryonic kidney-293 (HEK-293) cells at concentrations of 2 and 3 mg/mL, respectively [10]. Arena et al. revealed that exopolysaccharide (EPS-2) produced by G. thermodenitrificans B3-72 strain inhibited HSV-2 replication in human peripheral blood mononuclear cells (PBMC), but not in WISH cells [7].

In this study, we aimed to isolate and purify novel exopolysaccharides from thermophilic isolates and investigate their biological activities. Among the five EPSs we tested, EPS111 and EPS261 distinguished themselves with their larvicidal activities, while EPS106 and EPS111 exhibited a notable inhibitory effect on the proliferation of PC3 cells. Considering all this, among the EPSs we studied, especially EPS111, EPS106, and EPS261 stand out for their unique and valuable biological activities. However, in future studies, these natural and potential exopolysaccharides must be analyzed in detail and tested in vivo and clinically to guarantee the efficacy of the EPSs.

COI description

The authors have no conflicts of interest to declare.

Author Contribution

G.B. was responsible for the organization and coordination of the study. D.A.U., planned and performed EPS extraction and purification steps. M.A., D.Y.B and M.T. were the investigators and responsible for the data collection and analysis. All authors contributed to the writing of the final manuscript and reviewed the manuscript.

Acknowledgement

Mehmet AYTAR and Demet YALÇIN BİNGÜL received funding from the Higher Education Council of Türkiye (YOK) as a participant of the 100/2000 PhD scholarship.

Data Availability

Sequence data were deposited in GenBank, under accession numbers OR896930, OR896946, OR896962, OR896966, OR897006 for HBB-20, HBB-74, HBB-106, HBB-111 and HBB-261, respectively.

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No competing interests reported.

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Larvicidal and Cytotoxic Activities of Exopolysaccharides Produced by Thermophilic Bacteria

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Abstract

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INTRODUCTION

MATERIAL AND METHODS

Taxonomic analysis of bacterial isolates

Extraction and purification of exopolysaccharides

Determination of protein and sugar content

Larvicidal (Insecticidal) activities of exopolysaccharides

Cell culture

Cytotoxicity test

Statistical analysis

RESULTS

Taxonomic analysis of thermophilic isolates

Isolation and purification of EPSs

Larvicidal activities of exopolysaccharides

Cytotoxicity of EPSs

DISCUSSION

CONCLUSION

Declarations

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Status:

Version 1