Effects of Melanized Bacteria and Soluble Melanin on the Intestinal Homeostasis and Microbiome In Vivo

Abstract

Radiation damage is associated with inflammation and immunity in the intestinal mucosa, including gut microbiota. Melanin has a unique capacity to coordinate a biological reaction in response to environmental stimuli, such as radiation exposure. Thus, melanin and melanized microbes have potential to be used for mitigation of injury induced by radiation. The purpose of the current study is to examine the safety of these agents for future targeting gut microbiome to prevent radiation-induced injury. We administered mice with soluble allomelanin and observed its effect on the intestinal physiology and body weight. We then established a melanized bacterial strain in probiotic E. coli Nissle. We measured the body weight of the mice treated with melanized E. coli Nissle. We showed the enhanced bacterial abundance and colonization of the melanized bacteria E. coli Nissle in the intestine. Melanized E. coli Nissle colonized the colon in less than 3 h and showed consistent colonization over 24 h post one oral gavage. We did not find significant changes of bodyweight in the mice treated with melanized bacteria. We did not observe any inflammation in the intestine. These results demonstrate the safety of soluble melanin and melanin-producing bacteria and will support the future studies to treat radiation-induced injuries and restore dysbiosis.

Keywords: E. coli Nissle; intestine; melanin; melanized bacteria; microbiome; probiotics.

Figure 1

Gut microbes in the soluble melanin-treated CD-1 mice. The compositions of Escherichia coli and Lactobacillus did not change post 12 and 24 h after CD-1 mice treated with melanin (2.5 mg/mouse), compared to the untreated mice group. Data are expressed as mean ± SD. N = 6, two-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons.

Figure 2

CD-1 mice were one-gavaged with probiotic E. coli Nissle or melanized E. coli Nissle. (A) Relative body weight did not change in CD-1 mice gavaged with probiotic E. coli Nissle or melanized E. coli Nissle. Data are expressed as mean ± SD. N = 6, two-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons. (B) The spleen and liver weight did not change significantly in CD-1 mice gavaged with probiotic E. coli Nissle or melanized E. coli Nissle post 48 h. Data are expressed as mean ± SD. N = 6, Welch’s t test. (C) The length of small intestine, cecum and colon did not change in CD-1 mice gavaged with probiotic E. coli Nissle or melanized E. coli Nissle post 48 h. Data are expressed as mean ± SD. N = 6, Welch’s t test. (D) The melanized E. coli Nissle in feces post one-gavage was tested by culture and PCR. The melanized E. coli Nissle (LB cm agar plates, 10 µg/mL) was cultured using one-gavaged mice fecal samples. The melanized bacteria could be detected at 3, 12, and 24 h post treatment. The number of melanized bacteria increased at 3, 12, and 24 h post treatment compared to the before treatment in CFU count. The E. coli 16S rRNA RT-PCR amplification indicates the increased E. coli and melanized E. coli Nissle in fecal samples. Data are expressed as mean ± SD, N = 6, one-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons. All p values are shown in this figure. (E) The compositions of Salmonella, Lactobacillus and Bacteroides Fragilis did not change in the melanized bacteria-treated mice. Data are expressed as mean ± SD. N = 6, one-way ANOVA test and the p-values were adjusted with Tukey method for multiple comparisons.

Figure 3

The morphology of colon and ileum did not change in the CD-1 mice one-gavaged with probiotic E. coli Nissle or melanized E. coli Nissle in H&E staining. (A,B) Representative H&E images were from a single experiment and were representative of 6 mice per group.