Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

Abstract

Lactic acid bacteria (LAB) are natural inhabitants of the gastrointestinal tract (GIT) of humans and animals, and some LAB species receive considerable attention due to their health benefits. Although many papers have been published on probiotic LAB, only a few reports have been published on the migration and colonization of the cells in the GIT. This is due mostly to the lack of efficient reporter systems. In this study, we report on the application of the fluorescent mCherry protein in the in vivo tagging of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423. The mCherry gene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome of L. plantarum 423 by homologous recombination. In the case of E. mundtii ST4SA, the mCherry gene was cloned into the pGKV223D LAB/Escherichia coli expression vector. Expression of the mCherry gene did not alter the growth rate of the two strains and had no effect on bacteriocin production. Both strains colonized the cecum and colon of mice. Lactic acid bacteria (LAB) are common inhabitants of a healthy human gastrointestinal tract (GIT). Lactobacillus spp. are used as starter cultures in many fermented foods and are well known for their probiotic properties and the exclusion of pathogens from the GIT (1-6). Some strains have been closely associated with the treatment of gastrointestinal disorders, lactose intolerance, and stimulation of the immune system (7,8).Despite the increasing consumer interest in probiotic LAB, the mechanisms whereby these bacteria exert their beneficial effects in the GIT are not well understood (9). Although simulated gastrointestinal models (10-14) produced valuable data on the colonization of probiotic LAB and the exclusion of pathogens, the method remains an in vitro approach and is not a true reflection of in situ conditions. Labeling of probiotic bacteria with genes expressing fluorophores allows studying colonization and competition between gut microorganisms in vivo (15)(16)(17)(18)(19).Since the discovery of the green fluorescent protein (GFP) by Shimomura et al. (20), a number of genetic variants that emit light at longer wavelengths, and that are more suited for in vivo animal studies, have been described (21-23). The mCherry red fluorescence protein (RFP), a variant of the Discosoma red (DsRed) protein (24,25), is excited at wavelengths longer than 600 nm and is photostable (26-28). Studies conducted with Bacillus subtilis (29) and Escherichia coli (30) showed that expression of the mCherry protein, even at high levels, had no effect on the cell's physiology. Tauer and coworkers (31) used the mCherry gene to study recombinant protein expression in Lactobacillus plantarum. Mohedano and coworkers (32) adapted the broad-host-range vector pNZ8048 to study gene expression in Lactobacillus and showed that the mCherry construct could be used to study complex promoter induction mechanisms, such as bacteriocin production by Lactobacillus acidophilus. The role that lactococcin 972 (...

Adhesion Properties of Lactic Acid Bacteria on Intestinal Mucin

“…However, various anatomical differences in the gut structures exist between animal species, and these differences create a host-specific GI environment [ 1 ]. Because murine species (particularly mice) are commonly used as an in vivo experimental colonization model [ 22 , 26 , 27 , 28 ], in this section, we describe the structure of the GI tract mucosa from a histochemical perspective with comparison of the human and murine intestinal systems.…”

Tracer technology of probiotics based on optical imaging

“…GFP蛋白无毒, 在生 表 2 部分红色荧光蛋白特性 [39] Table 2 Partial red fluorescent protein characteristics [39] 荧光蛋白 物体中易表达且不影响生物体的正常功能, 作为标 记蛋白, 其发光蛋白对目标蛋白的正常活性也没有 影响, 因此广泛应用于科学研究 [49] . Yu等人 [50] 以GFP标记德氏乳杆菌(Lactobacillus 灌胃植物乳杆菌 423-pGKV223D的小鼠荧光成像(a), 灌胃植物乳杆 菌 423-cat-mCherry 的 小 鼠 荧 光 成 像 (b), 灌 胃 蒙 氏 肠 球 菌 ST4SA-pGKV223D 的 小 鼠 荧 光 成 像 (c), 灌 胃 蒙 氏 肠 球 菌 ST4SA-pCat-mCherry的小鼠荧光成像(d) [53] :cat-mCherry (pCat-mCherry) (d) [53] 物稳定性较差, 成像效果易受体内生物分子的影响.…”

“…Sunbul等人 [82] [24,25,27] , 但对于长时间的示踪监测, 该技术仍有一定的局限性, 挖掘发光波长更长, 发光 稳定的生物发光基因仍是研究的重点. 传统荧光标记 示踪益生菌, 发光强且可持续发光, 目前已有较多报 道以绿色荧光蛋白或红色荧光蛋白标记益生菌进行体 内示踪监测 [53,87] , 但该技术监测背景光较强, 成像效 The difference between them is that bioluminescence refers to self-luminescence. It catalyzes the substrate reaction and emits visible light by expressing and secreting different sources of luciferase.…”

Fluorescent Lactic Acid Bacteria and Bifidobacteria as Vehicles of DNA Microbial Biosensors

“…The use of fluorescent proteins as a visible marker and as a transcriptional reporter to monitor bacterial gene expression in real-time in LAB and Bifidobacterium spp. in living cells has been addressed through different strategies [ 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ].…”