Effect of monosaccharides on the interaction between Acanthamoeba culbertsoni and Shigella sonnei and the cytotoxicity and protease activity of A. culbertsoni

Introduction and Aim: In this study, the interaction between Acanthamoeba culbertsoni and Shigella sonnei was analyzed. In addition, the effect of monosaccharides on the cytotoxicity of A. culbertsoni was analyzed through interaction with macrophages. In particular, the effect of monosaccharides on the activity of A. culbertsoni protease was analyzed. Materials and Methods: Association tests were performed for the interaction of A. culbertsoni trophozoites with Shigella sonnei and Escherichia coli DH5-alpha, and the cytotoxicity and proteolytic enzyme activity of A. culbertsoni trophozoites were analyzed. In all these tests, monosaccharides, e.g., mannose, galactose, glucose, and xylose, were pretreated with A. culbertsoni trophozoites. Results: Except for 50 mM and 100 mM mannose, pathogenic S. sonnei by other monosaccharides showed almost 3 times more association than E. coli DH5-alpha. The association of A. culbertsoni trophozoites and S. sonnei by treatment with 100 mM galactose was inhibited by about 102%. The cytotoxicity of A. culbertsoni trophozoites by galactose was inhibited by about 13% and 25% at 50 and 100 mM, respectively. In particular, 50 and 100 mM mannose inhibited A.culbertsoni trophozoites cytotoxicity by 53% and 58%, respectively. A. culbertsoni proteolytic enzymes were strongly induced by 50 mM and 100 mM mannose. Conclusion: The results of this study confirmed that mannose and galactose influenced the interaction of bacteria to A. culbertsoni trophozites.


INTRODUCTION
canthamoeba culbertsoni mainly causes granulomatous amoebic encephalitis (GAE), which is typically fatal in immunocompromised individuals (1,2). A. culbertsoni induced GAE in vitro by inducing cytotoxicity such as contraction of target cells, vesiculation, and nuclear condensation (3). In addition, A. culbertsoni could phagocytose neurons through the cylindrical structures, termed digipodia, binding target cells by a contact-dependent mechanism (4). The function of monosaccharides on the cell membrane surface involved in this contact-dependent mechanism has not been well reported in A. culbertsoni. On the other hand, there is a report that when mannose was treated with A. castellanii causing amoebic keratitis (AK), adhesion to collagen and laminin could be inhibited (5), and protective immunity against AK could be induced by immunization with mannosebinding protein in experimental animals (6). A review reported the role of lectins in pathogenic protozoa as factors related to pathogenicity (7). It has been observed that monosaccharides are in the transport of pathogens into tissues as well as interactions with host cells. Lectins from Plasmodium falciparum, Trypanosoma cruzi, Entamoeba histolytica and Giardia lamblia could mediate interactions with host cells (8)(9)(10)(11). Therefore, lectins could be candidates to study pathogenicity and it was important to understand the role of lectins to induce disease (12). Studies have shown that mannose plays an important role in A. castellanii while galactose plays a role in T. cruzi contact with target cells. For pathogen-host cell interactions, it seemed that the interaction could not be perfect with one monosaccharide. It was thought that not only there was main monosaccharide, but also some monosaccharides played a complementary role for their interactions. In this study, the interaction between A. culbertsoni and the host cell was analyzed and the differences between the interaction of A. culbertsoni with pathogenic and non-pathogenic bacteria by various monosaccharides were analyzed. In addition, the effect of monosaccharides on the cytotoxicity of A. culbertsoni was analyzed through interaction with macrophages. Often, proteolytic enzymes are secreted from the pathogen following pathogen-host contact. Therefore, the effect of monosaccharides on the activity of A. culbertsoni protease was observed through zymography.

Preparation of monosaccharides and association test of bacteria to A. culbertsoni trophozoites
The monosaccharides used in this study were mannose, galactose, glucose, and xylose. Each monosaccharide was dissolved in distilled water (DW), adjusted to 10, 50, and 100 mM concentrations, and used for association, cytotoxicity, and proteolytic enzyme analysis. Unlike previous studies, monosaccharides were treated with A. culbertsoni trophozoites for 2 hr for higher binding of monosaccharides to A. culbertsoni trophozoites. In addition, bacteria were also treated in A. culbertsoni trophozoites for 2 hr. Briefly, each of the concentrations mentioned above was pretreated with A. culbertsoni trophozoites, then added to a 24-well cell culture plate and incubated at 37°C for 24 hr. After washing three times with phosphatebuffered saline (PBS), bacteria were added to the plate for 2 hr. After washing the unbound bacteria with PBS, 0.5% sodium dodecyl sulfate (SDS) was added to destroy A. culbertsoni trophozoites. Bacterial colonies were calculated after 20 ul was added to the Luria-Bertani (LB) agar plate and cultured at 37°C for one day (17). The bacterial association was calculated as follows: grown bacterial colony (cfu)/total bacteria (cfu) × 100 = % bacterial associated with A. culbertsoni trophozoites.

Lactate dehydrogenase release assay for cytotoxicity of A. culbertsoni trophozoites
For cytotoxicity test, macrophages were cultured in a 24-well cell culture plate at 37°C for 24 hr, washed three times with PBS (phosphate-buffered saline), and mixed with A. culbertsoni trophozoites. 10, 50 and 100 mM monosaccharides were pretreated with A. culbertsoni trophozoites for 2 h at 37°C. After mixing A. culbertsoni trophozoites and macrophages at 37°C for 24 hr, cytotoxicity was measured by LDH (cytotoxicity detection kit, Promega) of the destroyed macrophages. The amount of LDH was [LDH activity of the experimental group (measured as optical density at 492 nm) -LDH activity of the negative control group / total LDH activity -LDH activity of the negative control group × 100 = % cytotoxicity). For total LDH activity, the amount of LDH of macrophages treated with 1% Triton X-100 for 30 min at 37°C was used.

Zymography assay to study the effect of monosaccharides on A. culbertsoni trophozoite enzymes
A zymography assay was performed to analyze the activity of A. culbertsoni trophozoites proteolytic enzymes induced by a contact-dependent mechanism by monosaccharides (18). As mentioned above, monosaccharides were added to A. culbertsoni trophozoites at 37°C for 2 hr. Briefly, monosaccharidetreated A. culbertsoni trophozoites was destroyed by sonication, and whole lysate was produced and added to 12% sodium dodecyl-polyacrylamide (SDS-PAGE) gel with gelatin. 5% Triton-X100 was added to the gel at room temperature for 1 hr and the gel was stained with Coomassie brilliant blue.

Statistical analysis
To analyze the statistical difference of the resulting values, Student two-sample t test was performed and P value calculated. A value P <0.05, was considered statistically significant.

Influence of monosaccharides on the interactions between A. culbertsoni trophozoites and pathogenic S. sonnei and nonpathogenic E. coli DH5α
To analyze the interaction between A. culbertsoni trophozoites and bacteria such as S. sonnei, E. coli DH5α by monosaccharides, an association test between A. culbertsoni trophozoites and bacteria treated with monosaccharides was performed (Fig. 1).
Pathogenic S. sonnei, which was not treated with monosaccharides, showed about 150% higher association with A. culbertsoni trophozoites than nonpathogenic E. coli DH5α (Fig. 1A and B). Except for 50 mM and 100 mM mannose, pathogenic S. sonnei by other monosaccharides showed almost 3 times more association than non-pathogenic E. coli DH5α. However, there was little effect of monosaccharides in non-pathogenic E. coli DH5α (Fig. 1B). These findings showed that pathogenic S. sonnei interacted with A. culbertsoni trophozoites much better than non-pathogenic E. coli DH5α. It also suggested that even if non-pathogenic E. coli DH5α associates with A.culbertsoni trophozoites, pathways other than contact-dependent pathways by monosaccharides may be involved. Among mannose, galactose, glucose, and xylose applied in this study, mannose had the greatest effect on the interaction. In particular, when compared with the control group not treated with monosaccharides, association was inhibited by about 128% and 164.5% at 50 and 100 mM, respectively (Fig.  1A). Interestingly, the association of A. culbertsoni trophozoites and S. sonnei by treatment with 100 mM galactose was inhibited by about 102% compared to the control group without monosaccharide treatment, suggesting that mannose and galactose are important factors involved in the interaction between A. culbertsoni trophozoites and S. sonnei.

Cytotoxicity of A. culbertsoni trophozoites against macrophages by monosaccharides
To analyze the cytotoxicity of A. culbertsoni trophozoites by monosaccharides, LDH release assay was performed. Macrophage was used as a target cell, and the amount of LDH was measured from macrophages destroyed by cytotoxicity of A. culbertsoni trophozoites (Fig. 2). mM mannose, galactose, glucose or xylose was treated with A. culbertsoni trophozoites for 2 hr and association test was performed. A and B showed the association between A. culbertsoni trophozoites and S. sonnei and E. coli DH5α, respectively. None was a negative control group without monosaccharides, and 10, 50, and 100 were monosaccharides-treated groups. The experiment was performed in triplicate, and the results are expressed as mean ± standard deviation (SD). Asterisks indicated statistical significance (P<0.05). culbertsoni trophozoites was a percentage value with respect to the amount of LDH induced from destruction of macrophages. "None" was a negative control group that was not treated with monosaccharides and 10, 50, and 100 were experimental groups treated with monosaccharides in mM concentration. This experiment was performed in triplicate, and the results are expressed as mean ± standard deviation (SD). Asterisks indicated statistical significance (P<0.05).
The cytotoxicity of A. culbertsoni trophozoites was about 88% to macrophages, that is, in the negative control group, mixed with A. culbertsoni trophozoites and untreated with monosaccharides. There was little cytotoxic effect by glucose and xylose. However, the cytotoxicity of A. culbertsoni trophozoites by galactose was inhibited by about 13% and 25% at 50 and 100 mM, respectively, compared to the negative control group. Interestingly, 50 and 100 mM mannose inhibited A. culbertsoni trophozoites cytotoxicity by 53% and 58%, respectively. These results suggested that cytotoxicity was the most inhibited by mannose among the four monosaccharides used in this study, and mannose played the largest role in the contact-dependent mechanism of A. culbertsoni trophozoites as shown in Figure 1 above.

Proteolytic enzymes profiles for A. culbertsoni trophozoites induced by monosaccharides
To analyze changes in A. culbertsoni trophozoites proteolytic enzymes by monosaccharides, zymography was performed (Fig. 3).

Fig. 3:
Proteolytic enzyme activities of A. culbertsoni trophozoites by monosaccharides. The proteolytic assay was analyzed by gelatin gel SDS-PAGE. None was a group untreated with monosaccharides, and 10, 50, and 100 were experimental groups treated with monosaccharides in mM concentration.
For sufficient saturation of monosaccharides, A. culbertsoni trophozoites was treated with monosaccharides at 37°C for 2 hr. Induction of A. culbertsoni trophozoites proteolytic enzymes by glucose and xylose was hardly observed. 50 and 100 mM galactose activated proteolytic enzymes 2 -3 times more than 10 mM galactose and the negative group (None). On the other hand, 50 and 100 mM mannoseinduced proteolytic enzymes were most clearly increased. Therefore, 100 mM mannose showed the highest activity of protease among the four monosaccharides used in this study.

DISCUSSION
To understand contact-dependent pathogenic induction, it is necessary to understand the functions of proteins in pathogen cell membranes and their binding factors.
The study of factors related to the cell membrane proteins of the host cell is also important. In this study, among the factors related to the cell membrane of A. culbertsoni trophozoites as the pathogen, the effects of monosaccharides were analyzed. In this study, the interaction between A. culbertsoni trophozoites and pathogenic S. sonnei was confirmed to have the greatest effect by mannose. In addition, although the effect was not as great as that of mannose, it was confirmed that there was an effect on the interaction by 100 mM galactose. However, monosaccharides had little effect on the interaction of A. culbertsoni trophozoites with non-pathogenic E. coli DH5α. Taken together, this suggested a very important fact, that the interaction could be simultaneously inhibited by mannose and galactose. The connection was inhibited by 100 mM mannose in another free-living amoeba, Naegleria fowleri, and the pathogenic bacterium methicillin-resistant Staphylococcus aureus (MRSA) (19). The cytotoxicity of A. culbertsoni trophozoites could also be inhibited by mannose and galactose. It was also observed that mannose had the greatest effect on the activity of proteolytic enzymes secreted from A. culbertsoni trophozoites. There are still no reports on bacterial binding factors that bind to the A. culbertsoni mannose receptor. However, it was confirmed that a factor binding to the mannose domain of A. castellanii was α1-3D-mannobiose in Legionella (20). On the other hand, the mannose receptor, that is, the mannose binding protein (MBP), was extracted from A. culbertsoni by affinity chromatography (21). A. culbertsoni MBP was confirmed to have a molecular weight of about 83 kDa. A. castellanii MBP constitutes multiple 130 kDa subunits (22). Although the difference in MBP between species was evident in A. culbertsoni and A. castellanii, it was assumed that there was a difference in molecular weight depending on the subunits.

CONCLUSION
The understanding of the interaction between A. culbertsoni trophozoites and host cells or bacteria is still lacking. Based on earlier studies on A. castellanii and the results of this study, it could be concluded that mannose (50 mM and 100 mM) and galactose, mannose have the greatest effect on the interaction. In near future, if monosaccharides of different concentrations and types are combined and applied to the study, better results can be predicted in understanding the pathogenicity of A. culbertsoni and the process of penetration into tissues. In addition, if the genes involved in the signaling events occurring in A. culbertsoni trophozoites by monosaccharides are secured through molecular biological methods, GAE by A. culbertsoni and AK infection by A. castellanii may be able to be controlled more effectively through antibody or cell-mediated immunity to recombinant proteins.