Molecular detection of the ability of Biosynthesized Titanium dioxide nanoparticles to curing some genes of virulence factors of Entamoeba histolytica

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Introduction
Entamoeba histolytica is one of the protozoan intestinal parasites that cause amoebiasis, whether symptoms appear or not, and it ranks third among the parasites that cause death in the world after

Abstract
The present study included the microscopic and molecular identification of Entamoeba histolytica by using specific primers to detect four virulence factors possessed by Entamoeba histolytica.Virulence factors included Active Cysteine proteinase, Galactose/N-acetyl-D-galactose-lectin, Amoeba pore C and Phospholipase.Titanium dioxide nanoparticles (TiO2NPs) were synthesized from Pseudomonas aeruginosa which producing Pyocyanin pigment as a reducing agent to form it.After that we studied the ability ofTiO2NPs to inhibit virulence factors production and curing the genes responsible for encoding them by using four different dose 2 ,3, 4, 6 mg/Kg and administered by intraperitoneal injection to laboratory mice Mus musculus.The infection was molecularly confirmed, then the treatment for a period of ten days, as it was given on the third, fifth, seventh, and tenth days.The results of the study showed the inability of dose 2, 3 mg/Kg of TiO2NPs to curing the gene responsible for active cysteine proteinase and Gal/Gal NAC lectin upon treatment on the third to the tenth day, but was able to curing the genes when using dose (4,6) mg/Kg, Statistical analysis was performed , it was found that there were significant differences between the use of different dose and days of treatment.While Amoeba pore C, it was found that the four dose did not affect the inhibition on the third day of treatment.While dose 4,6 mg/Kg were able to curing the gene responsible for its encoding.Statistical analysis showed significant differences between the use of TiO2NPs and the days of treatment.Finally, the results showed that the four doses do not affect on phospholipase.Once, performing the statistical analysis it was found that there were no significant differences.Epidemiological studies indicated that 10% of people infected with E. histolytica show symptoms of disease while approximately 90% of patients are asymptomatic 2 .
Entamoeba histolytica possesses many virulence factors that help to penetrate and invade tissues, which contributes to an increase in pathogenesis, including the ability to secrete enzymes, includes active cysteine proteinase and Phospholipase, as well as the binding factor for Galactose/N-acetyl-Dgalactosamine (Gal/ Gal NAC) lectin, amoebapore and phospholipase 3 .
Nanotechnology is a neoteric technology that transforms large molecules into nano-sized particles 1 -100 nanometers 4 .There are several nanoparticles that differ in their sizes, shapes, surface area, and function, as well as metal nanoparticles and metal oxides are of great importance due to their specialized qualities in fighting microbial communities 5 .
Currently, titanium dioxide (TiO2) has become the focus of researchers because in addition to being approved by the Food and Drug Administration (FDA), it has great biological effects in environmental purification, pharmaceutical applications, solar energy cells as well as its catalytic ability Photosynthesis 2 .
There are various methods for the synthesis of titanium dioxide nanoparticles such as physical methods, chemical methods and biological methods.Recently, biological methods are considered the most suitable methods because they are environmentally friendly and have low cost compared to other methods 7 .
Pseudomonas aeruginosa is an opportunistic, nonlactose fermenter, gram-negative bacterium that is resistant to many antibiotics and produces many pigments, including Pyocyanin, a low molecular weight bluish-green pigment that is one of the most important virulence factors of bacteria and acts as a reducing agent 8 .
The main aim of the present study is to prove the effectiveness of biosynthetic Titanium dioxide nanoparticles on inhibiting the production of some virulence factors and curing the genes responsible for their encoding in mice infected with E. histolytica.

Materials and Methods
Twenty-five fecal samples were collected for children infected with E.histolytica from the patients of Al Alalwia Children's Teaching Hospital and Central Child Hospital in Baghdad-Iraq who were suffering from moderate to severe diarrhea.Shield (Fecal Collection tube) used to preserve parasite genetic material for DNA extraction by using Quick-DNA TM Fecal/ Soil Microbe Miniprep Kit /Zymo/ USA and conducting a polymerase chain reaction (PCR) test using four pairs of primers from (Integrated DNA Technologies company, IDT (Canada) to detect the presence of the virulence factors Active cysteine proteinase, Amoebapore C, Lectin (hg13), Phospholipase of the parasite, as they were molecularly detected to confirm their presence in fecal samples.Thirty Musmusculus mice were selected and divided into six groups that included the first group (negative control group), and the amoebic suspension was administered orally to five groups of laboratory mice, and the infection was confirmed both microscopically and molecularly.Biosynthesis of Titanium dioxide nanoparticles Titanium dioxide nanoparticles was synthesized from P.aeruginosaproducing Pyocyanin dye as a reducing agent to form Titanium dioxide nanoparticles.The biosynthetic titanium dioxide was characterized using several tests including: X-ray diffraction, field emission scanning electron microscope (FE-SEM), Fourier transform infrared spectroscopy (FTIR), UV visible spectroscopy, Atomic force microscope (AFM).The average diameter of the Titanium dioxide particles was 59.69 nanometers.

Specific primers of virulence factors:-
After confirming the presence of infection, 5 mice were taken and their stool samples were collected and stored in DNA/RNA Shield (Fecal Collection tube) and considered as a positive control group and were not treated with any treatment.As for the remaining 20 mice, they were divided into four groups that were treated by intraperitoneal injection.Intraperitoneal injection (IP) with four different concentrations of biosynthetic Titanium dioxide nanoparticles 2, 3, 4, 6 mg/ Kg to determine the effect of titanium dioxide nanoparticles in inhibiting the production of virulence factors 9 .

Results and Discussion
Results of detection of virulence factors of E. histolytica samples.

Active Cysteine proteinase
The results of the detection of the virulence factor Cysteine proteinase showed its presence in 11 samples, or only 44%, as shown in (Fig .1) and (Table 5).Cysteine proteinase, which is the most important virulence factor secreted by the parasite, dissolves host tissues, kills host cells in contact with it, induces apoptosis of target host cells and through this mechanism distinguishes the Trophozoite of pathological species of the genus Amoeba such as E. histolytica differs from other non-pathological species and this is agree with 10 who demonstrated the role of Cysteine proteinase and its essential role in the interactions between parasite and host, including nutrient acquisition, facilitating tissue invasion and defense against body immunity.

Amoebapore C
Virulence factor results of the detection of Amoebapore C showed its presence in only 12 samples, as shown in (Fig. 2 and Table 5).The virulence factor Amoebapore C plays a very important role in bloody diarrhea because it causes tissue breakdown and amoebic colitis.The results of this study showed its presence in 52% of the examined fecal samples, and these results agree with 11 they found that Only 58 samples were positive for Ameobapore C, while they do not agree with 12 , which found that the above virulence factor is present in all samples.Studies have shown that trophozoite that lacks Amoabapore C is less virulent 13 .

Lectin (hg13)
The results of the detection of the virulence factor Lectin (hg13) showed its presence in only 13 samples, as shown in (Fig. 3 and Table 5).The virulence factor is GAL/ NaG Lectin, as the results of this study showed that its presence was 52%, and these results are agreeing with 11 who found that the above virulence factor is present in 55% of the samples, as well as compatible with 14 who demonstrated that the adhesion of trophozoite to the intestinal mucosa is mediated by GAL/ /NaG Lectin, which is the first step in pathogenesis.

Phospholipase
The results of the detection of the virulence factor Phospholipase showed its presence in only 12 samples, as shown in (Fig. 4 and Table 5).
Statistical analysis was carried out using chi-square to find out the percentage of virulence factors of E. histolytica in the fecal samples as shown in Table 6, and it was proved that there were no significant differences in the presence of virulence factors in 25 fecal samples, as the value of Chi-Square was 0.321 and the P-Value 0.956, Significance level P ≤ 0.05.

Active Cysteine proteinase
The results of molecular detection of the effect of TiO2NPs in inhibiting the ability of the parasite to produce the virulence factor Cysteine proteinase by curing the gene responsible for encoding it showed that 2, 3 mg/Kg of TiO2NPs did not affect the production of the virulence factor when treated with the third to the tenth day of infection, as shown (Fig. 5 and Table 7).While, the doses of Titanium dioxide nanoparticles 4, 6 mg/Kg showed their ability to inhibit the parasite's ability to produce Cysteine proteinase and curing the gene responsible for encoding it from the third to the tenth day of treatment, which indicates the effectiveness of these doses and since the first use on the third day of infection, and when Conducting the statistical analysis using Dunkin' polynomial test, it was found that there were significant differences between the doses of Titanium dioxide nanoparticles 2 ,3 and 4,6 mg/Kg in inhibiting the parasite's ability to produce virulence factor and the days of treatment as shown in Table 8.Virulence factor Amoebapore C The results of the molecular detection showed difference in the effect of Titanium dioxide nanoparticles on the ability of the parasite to produce the virulence factor under study, as all dose of TiO2NPs did not affect the ability of the parasite to produce the virulence factor Amoebapore C upon treatment on the third day of infection, as shown in Fig. 6 and Table 9.Also, the concentrations 2,3 mg/Kg on the fifth, seventh and tenth days also did not affect the ability of the parasite to produce the virulence factor Amoebapore C. Whereas, the concentrations of 4 ,6 mg/Kg showed inhibition in the ability of the parasite to produce the virulence factor by curing the gene responsible for encoding it, and when statistical analysis was carried out using Dunkin' polynomial test, significant differences were found between the concentrations of Titanium dioxide nanoparticles 2,3 and 4,6 mg/Kg inhibiting the ability of the parasite to produce virulence factor and the days of treatment, as shown in Table 10.

Virulence factor lectin (hg13)
The results of the molecular detection showed difference in the effect of ofTiO2NPs on the ability of the parasite to produce the virulence factor under study, that the dose 2, 3 mg/Kg of TiO2NPs did not affect the ability of the parasite to produce the virulence factor lectin (hg13).During treatment from the third to the tenth day of infection, as shown in Fig. 7 and Table 11.
For instance for the dose 2,3 mg/Kg ofTiO2NPs, they were characterized by their ability to inhibit the virulence factor Lectin (hg13) and curing the gene responsible for its encoding from the third to the tenth day of treatment, which indicates the effectiveness of these concentrations and since the first use on the third day , When conducting statistical analysis using Dunkin's multinomial test, it was found that there were significant differences between the inhibitory effect of 2,3 and 4,6 mg/Kg of TiO2NPs on the parasite's ability to produce virulence factor and treatment days, as shown in Table 12.The number of virulence factor produced according to the number of days after treatment Titanium dioxide dose mg/Kg Days of treatment

Virulence factor Phospholipase
The results of treatment of infection with E. histolytica parasite with nanoparticles of titanium dioxide showed that the four concentrations during the different treatment days did not affect the ability of the parasite to produce the virulence factor Phospholipase of E .histolytica as shown in Fig. 8 and Table 13.Also, when performing the statistical analysis, no significant differences appeared using the four concentrations of biosynthetic Titanium Dioxide Nanoparticles during the different treatment days as shown in Table 14.This study is new of its kind in the synthesis of nanoparticles and their use in inhibiting the genes which are responsible of encoding virulence factors of E. histolytica, and there are studies similar to it to a certain extent, as 9 used commercially manufactured silver nanoparticles to determine their toxicity and invitro effect on the trophozoite of E. hisolytica, as it noticed a significant difference in the decrease in the number of trophozoites after incubation with silver nanoparticles, as dose 50,75 and 100 µg /ml were used, but the study was in vitro.
The results of this study are similar to 15 as the results of their use of copper oxide nanoparticles and silver nanoparticles, respectively, showed a significant effect of reduction the cysts vitality in E. histolytica and Cryptosporidium parvum.These nanoparticles were considered as safe and effective therapeutic alternatives against Parasites above.The studies also demonstrated the effectiveness of gold and silver nanoparticles, as well as metal oxides, that have an inhibitory ability and were considered antiparasitic against different types of parasites Giardia, Plasmodium, and Toxoplasma 16 .Likewise, the results of this study in the use of nanoparticles as antiparasitic agents agreed with the results of the study reached by 17 which showed that Selenium and copper oxide nanoparticles were used to determine the extent of their impact on the number of cysts of the Giardia duodenalis.
The study was conducted by in vitro and nanoparticles have their unique physical and chemical properties, and because they are small in size, they have a large surface area, electric charge, and a distinctive shape 18 Its surface area interferes with various biological molecules and microorganisms and destroys the negative activity of parasites and can enter cells more than other molecules 19 .For instance the nanoparticles interfere with the surface of the parasite and cause damage and destruction to the molecules of Lipophosphoglygan and Glygoprotein that are present on the surface of the parasite and responsible for infection, and that these molecules are affected by the Reactive Oxygen Species (ROS) generated by the nanoparticles and this leads to the failure of infection with the parasite 20 .

Conclusion
The results of the study showed the ability of TiO2NPs as antiparasitic of TiO2NPs to inhibit virulence factors production of parasite and curing the genes which are responsible for coding them when using dose (4, 6) mg/Kg of TiO2NPs thus reducing the pathogenicity of E. histolytica and statistical analysis showed significant differences between the use of TiO2NPs and the days of treatment.

Figure 4 .
Figure 4. PCR product of Phospholipase for E.histolytica, band size 420 bp, the product was electrophoresis on agarose gel 2%, 70 Vol, 60 amp, 1x TBE or buffer for 1.5 h, ladder 1000 bp.Through the detection of parasite virulence factors genes in excrement samples, it was found that the four virulence factors genes are present in samples
/dx.doi.org/10.21123/bsj.2023.7205P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal Table 10.Effect of Titanium dioxide nanoparticles with different concentrations on the ability of E. histolytica to produce the virulence factor Amoebapore C.The number of virulence factor produced according to the number of days after treatment

Table 8 . Effect of Titanium dioxide nanoparticles with different concentrations on the ability of E. histolytica to produce the virulence factor cysteine proteinase.
*P.C refers to the positive control group (infected mice untreated with Titanium dioxide nanoparticles)