A Macroporous Magnesium Oxide-Templated Carbon Adsorbs Shiga Toxins and Type III Secretory Proteins in Enterohemorrhagic Escherichia coli, Which Attenuates Virulence

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is one of the most common foodborne pathogens. However, no drug that prevents the severe complications caused by this bacterium has been approved yet. This study showed that a macroporous magnesium oxide (MgO)-templated carbon material (MgOC₁₅₀) adsorbs Shiga toxins, and Type III secretory EspA/EspB proteins responsible for EHEC pathogenesis, and decreases the extracellular levels of these proteins. On the other hand, this material did not affect the growth of EHEC. Citrobacter rodentium traditionally used to estimate Type III secretion system-associated virulence in mice is highly virulent. The survival period of infected mice was prolonged when MgOC₁₅₀ was administered. This adsorbent disturbed neither mammalian cells nor normal intestinal bacteria, such as Enterococcus hirae, Lactobacillus acidophilus, and Lactobacillus casei. In contrast, MgOC₁₅₀ adsorbed antimicrobial agents, including β-lactams, quinolones, tetracyclines, and trimethoprim/sulfamethoxazole. However, fosfomycin and amikacin were not adsorbed. Thus, MgOC₁₅₀ can be used with fosfomycin and amikacin to treat infections. MgOC₁₅₀ is used for industrial purposes, such as an electrode catalyst, a bioelectrode, and enzyme immobilization. The study proposed another potential application of MgOC₁₅₀, assisting anti-EHEC chemotherapy.

Keywords: Shiga toxin; Type III secretion system; antimicrobial chemotherapy; antimicrobial resistance; bacterial pathogenesis; enterohemorrhagic Escherichia coli; porous carbon; virulence.

Figure 1

Adsorption of Shiga toxin (A) and lysozyme (B) by MgOC₁₅₀. The Shiga toxin standard (0.4 μg) or lysozyme (50 μg) was incubated with and without MgOC₁₅₀, and non-adsorbed proteins were measured in protein assay described in Materials and Methods. Protein levels are presented as the percentage of the value for samples after incubation with MgOC₁₅₀ relative to that after incubation without MgOC₁₅₀. Data plotted are the means from three independent experiments; error bars indicate the standard deviations. Asterisks denote significance for values (p < 0.05) of protein level after incubation with MgOC₁₅₀ relative to that after incubation without MgOC₁₅₀.

Figure 2

Survival of HTB-44 and Vero cells after incubation with and without EHEC supernatant cultured with and without MgOC₁₅₀. Survival rates are presented as the percentages of the RLU value for cells after incubation with each supernatant relative to that after incubation without supernatant. Data are the means of two biological replicates. Error bars indicate the ranges. Experiments were repeated twice, and similar results were obtained.

Figure 3

Determination of EspB levels. (A) EspB levels in the EHEC supernatant cultured with and without MgOC₁₅₀. (B) EspB levels in EHEC whole-cell extracts cultured with and without MgOC₁₅₀. (C) The EHEC supernatant was incubated with and without MgOC₁₅₀ for 2 h, and EspB levels were estimated after MgOC₁₅₀ removal. Proteins including EspB were separated by SDS-PAGE. EspB was visualized by Western blotting with EspB antiserum. For loading control (LC), BSA was visualized by CBB stain. Locations of molecular mass standards (in kilodaltons) are shown on the left.

Figure 4

Determination of EspA levels. (A) EspA levels in the EHEC supernatant cultured with and without MgOC₁₅₀. (B) The EHEC supernatant was incubated with and without MgOC₁₅₀ for 2 h, and EspA levels were estimated after MgOC₁₅₀ removal. Proteins including EspA were separated by SDS-PAGE. EspA was visualized by Western blotting with EspA antiserum. For loading control (LC), BSA was visualized by CBB stain. Locations of molecular mass standards (in kilodaltons) are shown on the left.

Figure 5

Virulence of C. rodentium in C3H/HeJ mice after MgOC₁₅₀ administration. (A) Change in the body weight of C3H/HeJ mice infected with the DBS100 strain. The connecting lines denote the means, and the error bars denote the standard deviations. (B) Survival of C3H/HeJ mice infected with the DBS100 strain. (C) Change in the body weight of C3H/HeJ control mice. The connecting lines denote the means, and the error bars denote the standard deviations. Mice (N = 5 mice bled without MgOC₁₅₀ for infection, N = 6 mice bled with MgOC₁₅₀ for infection, N = 5 control mice bled without MgOC₁₅₀ for non-infection, and N = 5 control mice bled with MgOC₁₅₀ for non-infection) were monitored daily. Asterisks denote the significances (p < 0.05) of survival rate and body weight of mice administrated with MgOC₁₅₀ relative to those of mice administrated without MgOC₁₅₀.

Figure 6

Toxicity of MgOC₁₅₀ in host cells and normal intestinal flora. (A) Survival of Caco-2 cells after incubation with without MgOC₁₅₀. MgOC₁₅₀ was added into 150 μl Caco-2 cell cultures. Amounts (mg) of MgOC₁₅₀ on x-axis were represented as those corresponding to 5 ml cultures. The survival rates are presented as the percentage of the RLU value for the cells after incubation with MgOC₁₅₀ relative to that after incubation without MgOC₁₅₀. Growth of several normal intestinal flora, E. hirae (B), L. acidophilus (C), and L. casei (D) cultured with and without MgOC_150. All strains were anaerobically cultured with and without 30 mg MgOC₁₅₀. Bacterial growth was estimated by measuring the CFUs.

Figure 7

Antimicrobial activities of indicated drugs in the presence of by MgOC₁₅₀. (A) Drug adsorption by MgOC₁₅₀. Each drug was incubated in an aqueous solution with and without 30 mg MgOC₁₅₀ for 2 h. The y axis shows the percent of drug amount (mg) after incubation with MgOC₁₅₀ relative to the drug amount (mg) after incubation without MgOC₁₅₀. (B) Growth of the EHEC strain when cultured with or without indicated drugs in the presence and absence of 30 mg MgOC₁₅₀. Data plotted are the means from three independent experiments; error bars indicate the standard deviations. Asterisks denote significance for values (p < 0.05) of drug amount and CFU (colony-forming unit) after incubation with MgOC₁₅₀ relative to that after incubation without MgOC₁₅₀.