Identification of Streptococcus thermophilus Genes Specifically Expressed under Simulated Human Digestive Conditions Using R-IVET Technology

Abstract

Despite promising health effects, the probiotic status of Streptococcus thermophilus, a lactic acid bacterium widely used in dairy industry, requires further documentation of its physiological status during human gastrointestinal passage. This study aimed to apply recombinant-based in vivo technology (R-IVET) to identify genes triggered in a S. thermophilus LMD-9 reference strain under simulated digestive conditions. First, the R-IVET chromosomal cassette and plasmid genomic library were designed to positively select activated genes. Second, recombinant clones were introduced into complementary models mimicking the human gut, the Netherlands Organization for Applied Scientific Research (TNO) gastrointestinal model imitating the human stomach and small intestine, the Caco-2 TC7 cell line as a model of intestinal epithelium, and anaerobic batch cultures of human feces as a colon model. All inserts of activated clones displayed a promoter activity that differed from one digestive condition to another. Our results also showed that S. thermophilus adapted its metabolism to stressful conditions found in the gastric and colonic competitive environment and modified its surface proteins during adhesion to Caco-2 TC7 cells. Activated genes were investigated in a collection of S. thermophilus strains showing various resistance levels to gastrointestinal stresses, a first stage in the identification of gut resistance markers and a key step in probiotic selection.

Keywords: R-IVET; S. thermophilus; TIM-1 system; adhesion; intestinal microbiota.

Figure 1

Construction of the chromosomal R-IVET cassette. The R-IVET chromosomal construction was cloned into the locus STER 0891 (t0891) of S. thermophilus LMD-9 resulting in strain STUL5003. This strain was constructed in two steps. The first step (1A to 1C) corresponds to the construction of STUL5002 mutant: PCR amplification of individual fragments (A), overlapping PCR (B), and resulting construction on the chromosome of STUL5002 mutant (C). The second step (1D to 1F) illustrates the construction of STUL5003 mutant: PCR amplification of individual fragments (D), overlapping PCR (E), and resulting chromosomal construction of STUL5003 mutant (F). Numerical values indicated in italics in genes (F) correspond to locus tags as described in the annotated sequence of LMD-9 chromosome (NC_008532_1). Oligonucleoti0des used as amplification primers are indicated as black arrows and are labeled with their # numbers (see Table S1, Supplementary Materials). UP and DOWN regions correspond to upstream and downstream sequences flanking the fragments to be cloned, respectively. The specR, kanR, and prom fragments correspond to genes encoding a spectinomycin adenyltransferase conferring resistance to spectinomycin and streptomycin, a 3′5″-aminoglycoside phosphotransferase of type III conferring resistance to kanamycin, and the promoter region of kanR gene, respectively. The gray triangle corresponds to the loxP sequence that is recognized by the specific site recombinase Cre.

Figure 2

Overview of R-IVET experiments in the three complementary human gut models.

Figure 3

Schematic representation of positive-selection R-IVET screen in S. thermophilus STUL5003. The R-IVET technology consists of two elements. The first one is the chromosomal cassette (A) with the spectinomycin resistance gene (specR) flanked by two loxP sites followed by the promoterless kanamycin resistance gene (kanR). The second element is the pULNcreB plasmid (B, Table 1) which possesses the promoterless recombinase gene (cre). Cre recombinase recognizes the loxP sites of the cassette. (C) When the DNA fragment cloned upstream of the cre gene is without promoter activity, only the specR gene is expressed and the two terminators, i.e., specR gene and Tlas downstream of the specR gene, prevent expression of the kanR gene. The corresponding clone is spectinomycin-resistant and kanamycin-sensitive (Spec^RKan^S). (D) When the DNA fragment possesses promoter activity, Cre recombinase is produced and leads to excision of the specR gene; the kanR gene can, therefore, be expressed from its own promoter. Hence, the clone is spectinomycin-sensitive and kanamycin-resistant (Spec^SKan^R).

Figure 4

Bacterial survival and number of activated R-IVET clones obtained and analyzed in each TIM-1 compartment (A) and in fecal microbiota batch cultures (B). (A) In each TIM-1 compartment, data points obtained for the R-IVET library and the theoretical transit marker are represented by gray bars and black circle curves, respectively. In the stomach compartment, the black diamond curve gives the gastric pH evolution over time. Tf represents the cumulated ileal effluents plus gastrointestinal residue collected at the end of TIM-1 experiment. Values are given as means of log₁₀ CFU ± SEM (n = 3). At each time, results for the R-IVET library were compared to those of the transit marker. Significant differences are noted by asterisks (ANOVA and Bonferroni test, p < 0.05). Numbers in bars correspond to the total number of activated R-IVET clones obtained and analyzed at each time point. (B) For batch cultures, dark-gray bars and light-gray bars represent data from volunteer 1 (man) and volunteer 2 (woman), respectively. Values are given as means of log₁₀ CFU ± SEM (four technical replicates). Values at each time point were compared to those obtained at T₀ (^■ p < 0.05) and between each volunteer (* p < 0.05). Numbers in bars correspond to the total number of activated R-IVET clones analyzed at each time point.

Figure 5

Location, orientation, and denomination of inserts of activated R-IVET clones compared to S. thermophilus LMD-9 genes. CDS: coding DNA sequence.

Figure 6

Schematic overview of allele variability of R-IVET activated genes identified in TIM-1 and fecal batch cultures among strains displaying different properties of resistance against GI stress and deduced protein signature. Colors/numbers correspond to different alleles of R-IVET activated genes that were identified. Different proteins signatures are given by different colors/letters. The full name of each gene is detailed in Table S3 (Supplementary Materials). ^Ø No PCR amplicon; ^a locus annotation from 28 January 2014; ^b locus annotation from 30 July 2015 from NCBI; ^c according to Junjua et al. [15]; ^d LMD-9 used as a reference strain for comparison; ^e types of proteins found in strains exhibiting a high level of resistance against GI stress; ^f types of proteins found in strains exhibiting a low level of resistance against GI stress.

Figure 7

Synthetic view of main genes from S. thermophilus LMD-9 specifically induced in each GI models and their possible involvement in cell functions [43,46,47,48,50,52,53,58,59,60,61].