Analyzing global gene expression of Lactobacillus plantarum in the human gastrointestinal tract

The human gastrointestinal (GI)-tract represents a dynamic ecosystem comprising various habitats each with niche-specific microbial communites, collectively called microbiota. Lactic acid bacteria (LAB) are considered to be a large group of the microbiota in the upper GI-tract that is involved in health-stimulating processes within the host. The lactic acid bacterium Lactobacillus plantarum is one of the most versatile and flexible LAB that is encountered in a range of environmental niches, has a proven ability to survive gastric transit, and can colonize the GI-tract of human and other mammals. Several studies describe the effects of L. plantarum consumption on the human physiology and health. The complete genome sequence of L. plantarum WCFS1 makes it a suitable model to study with molecular techniques like fluorescent in situ hybridization (FISH), quantitative reverse transcriptase PCR (qRT-PCR), and DNA microarrays to contribute to unraveling the mechanisms underlying the GI targeted properties of L. plantarum (Chapter 1).</span>Methods for the detection, classification, and to estimate general activity of microbes in complex ecosystems generally use ribosomal RNA (rRNA) and their coding genes as<span class=GramE>target</span>molecules due to their universal distribution and high nucleotide sequence conservation. In <strong>Chapter 2</strong> , the complete genome sequences of the LAB, L. plantarum, L. johnsonii<span style='mso-bidi-font-style:italic'>, and</span>Lactococcus lactis were used to compare location, sequence, organisation, and regulation of the rRNA operons. All operons demonstrate a common organization with the order 5'-16S-23S-5S-3', but differ in the number, location and specificity of the tRNA genes. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. However, in the rRNA operon promoter regions of L. plantarum and Lactococcus lactis marked differences were observed, while the promoter regions of L. johnsonii showed a similar promoter structure in all operons. Although all five L. plantarum rRNA promoters are significantly different, this study demonstrates that their activities were very similar under the conditions tested.</span>The feasibility of using the rRNA-based FISH method to estimate the overall in situ activity of L. plantarum in the human GI-tract was assessed ( <strong>Chapter 3</strong> ). FISH could be used to estimate the in situ activity of L. plantarum WCFS1 in exponentially growing cells. However, L. plantarum is capable of growth to very high cell densities and the properties of the L. plantarum cell-envelope prevented effective entry of the fluorescent oligonucleotide probe into the cells at later stages of growth when high cell densities are reached. Hence, FISH is not appropriate for use in the GI-tract as the growth phase of L. plantarum may vary considerably and differ between cells, as influenced by way of ingestion, and location in the GI-tract.</span>The focus of this research shifted to analyzing specific gene expression of L. plantarum in the human GI-tract using DNA microarrays and qRT-PCR. Gene expression of L. plantarum associated with human mucosa was determined using DNA microarrays in three colonic biopsies and in the ileal and colonic biopsy within a single person<span style='mso-bidi-font-style:italic'>( <strong>Chapter 4</strong> )</span>. The ingested L. plantarum cells were metabolically active in all screens and in all subjects, as demonstrated by the detection of about 10% expressed genes by the DNA microarrays. Genes were detected for all functional classes. The differences in L. plantarum gene expression between the colons of three individuals were larger than the differences between the ileum and colon of a single individual. This is the first report of global gene expression analysis of an ingested microbe in the human intestinal mucosal.</span>To evaluate the effect of passage of the proximal small intestine on gene expression of L. plantarum , the perfusion technique was conducted in healthy human volunteers ( <strong>Chapter 5</strong> ). Subsequently, RNA isolated from the perfusion suspension containing L. plantarum was hybridized to DNA microarrays. Passage of the small intestine left L. plantarum metabolically active as can be seen from up-regulation of genes involved in cell division, and protein synthesis. However, an osmotic shock response was observed, most probably induced by bile salts and including up-regulation of genes involved in ion transport, and glycine, betaine, carnitine, or choline uptake. In addition, expression of many genes involved in the cell envelope was altered, indicating a strengthening of the cell envelope and pointing to specific interactions with the host and further microbiota.<span style='color:blue'><o:p></o:p></span></span>In addition, qRT-PCR established gene expression of L. plantarum from the low amount of RNA recovered from three subjects having an ileostoma who consumed the bacteria in a milk drink ( <strong>Chapter 6</strong> ). In the ileostomy samples the L. plantarum cells were metabolically active as indicated by induced expression of genes predicted to be involved in protein synthesis, an active amino acid biosynthesis machinery, and sugars uptake and degradation for energy to eventually lactate and ethanol production. Genes encoding a potential adherence protein, and α-L-rhamnosidase, which is an enzyme for hydrolysis of rhamnosides and releasing L-rhamnose from various substrates,<span style='color:blue'></span>and a bacteriocin precursor were clearly up regulated.</span>All screens were compared to demonstrate subject-specific expression, differences between gene expression of L. plantarum in the ileum and colon and between luminal cells and cells associated with mucosal cells or mucus ( <strong>Chapter 7</strong> ). In addition, a comparison between bacterial gene expression in humans and animals (mice and rabbits) indicated a large similarity between the gene<span class=GramE>expression</span>in those different hosts.</span>In combination with clinical studies, the approaches for gene expression described in this thesis are powerful and high-throughput tools to provide insight and new perspective on in vivo host-microbe interactions. This is expected to lead to new generations of probiotics with a scientifically proven basis for their health effects