Expression of Fluorescent Proteins in Bifidobacteria for Analysis of Host-Microbe Interactions

Abstract

Bifidobacteria are an important component of the human gastrointestinal microbiota and are frequently used as probiotics. The genetic inaccessibility and lack of molecular tools commonly used in other bacteria have hampered a detailed analysis of the genetic determinants of bifidobacteria involved in their adaptation to, colonization of, and interaction with the host. In the present study, a range of molecular tools were developed that will allow the closing of some of the gaps in functional analysis of bifidobacteria. A number of promoters were tested for transcriptional activity in Bifidobacterium bifidum S17 using pMDY23, a previously published promoter probe vector. The promoter of the gap gene (P gap ) of B. bifidum S17 yielded the highest promoter activity among the promoters tested. Thus, this promoter and the pMDY23 backbone were used to construct a range of vectors for expression of different fluorescent proteins (FPs). Successful expression of cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry could be shown for three strains representing three different Bifidobacterium spp. The red fluorescent B. bifidum S17/pVG-mCherry was further used to demonstrate application of fluorescent bifidobacteria for adhesion assays and detection in primary human macrophages cultured in vitro. Furthermore, pMGCmCherry was cloned by combining a chloramphenicol resistance marker and expression of the FP mCherry under the control of P gap . The chloramphenicol resistance marker of pMGC-mCherry was successfully used to determine gastrointestinal transit time of B. bifidum S17. Moreover, B. bifidum S17/pMGC-mCherry could be detected in fecal samples of mice after oral administration.

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

“…The adhesion of L. plantarum and Lactobacillus fermentum strains to the intestinal tract of zebrafish was studied by transferring plasmid pRCR12, containing the mCherry gene, into the bacterial cells (34). Grimm and coworkers (35) used the expression of the mCherry gene to study interactions of bifidobacteria with mammalian host cells.…”

Experimental determination and characterization of thegappromoter ofBifidobacterium bifidumS17

“…This is in line with a previous study by our group showing higher P gap -driven reporter activity in B. bifidum S17 compared with other promoters in all growth phases tested. 20 Furthermore, several other groups also reported high transcriptional activity of bifidobacterial gap promoters and these promoters have been used successfully for expression in bifidobacteria. 20,21,23 The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) encoded by the gap gene catalyzes the conversion of glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate.…”

“…20 Furthermore, several other groups also reported high transcriptional activity of bifidobacterial gap promoters and these promoters have been used successfully for expression in bifidobacteria. 20,21,23 The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) encoded by the gap gene catalyzes the conversion of glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate. Although GAPDH is primarily known for its role in glycolysis, it is also required for formation of lactate from glyceraldehyde 3-phosphate and, in consequence, efficient energy conservation from substrates that are fermented via the bifidus shunt.…”

“…20 Briefly, crude extract containing 1-5 mg total protein as determined using 2-D Quant kit (GE Healthcare Life Science) was brought to a volume of 100 ml reaction with GUS assay buffer (50 mM Na 2 HPO 4 pH 7, 1 mM EDTA, 0.1% Triton X-100, 5 mM dithiothreitol) and transferred to the wells of a C. Then 10 ml of the reaction mix were transferred into a fresh 96-well plate containing 190 ml stop buffer (0.2 M Na 2 CO 3 in H 2 O, pH 9.5). The concentration of 4-methylumbelliferone produced from 4-methylumbelliferyl-b-D-glucuronide by GusA was quantified by measuring fluorescence using a Tecan infinite M200 plate reader with excitation at 388 nm and emission at 480 nm.…”

“…These include the sequences up stream of the 16S rRNA, gap, and hup genes of different Bifidobacterium sp. 8,20 However, none of these promoters were determined experimentally or analyzed in more detail.…”