In Situ Profiling Of Transcription In The Gi Tract

As soon as sequence data is available for a few genes in a bacterium of interest, one could think of several sophisticated tools that allow investigation of the in situ expression levels of specific genes. One example of such an approach is the implementation of quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in the gram-negative bacterium Helicobacter pylori (66). This study describes the assessment of gene expression in this pathogen within the mouse and human gastric mucosae. Three genes, encoding urease, catalase, and a putative adhesin specific for adherence to human gastric mucosa, were selected for analysis, as their role during host residence was already established. Using minute quantities of mRNA isolated from human and mouse infected mucosae, the in situ expression of these three genes could be established. Moreover, the results of this study indicate that the relative abundance of transcripts was the same in the human and mouse model system. Hence, this study demonstrates that qRT-PCR is a powerful tool for the detection and quantification of bacterial gene expression in the GI tract (66). Similar experiments were performed in L. plantarum. An in vitro screen and a R-IVET screen were already performed in this LAB to identify genes of which the expression is induced in vitro by bile or in situ in the GI tract of a mouse model system, respectively (25,45). Matching of the results obtained in these two screening procedures revealed two genes, encoding an integral membrane protein and an argininosuccinate synthase that appeared to be induced by bile in vitro as well as in vivo in the GI tract of a mouse model system. Therefore, the expression of these two genes was assessed using qRT-PCR followed by SYBR green fluorescence detection. As the duodenum is the site of bile release, expression in this specific region of the host's GI tract was investigated. The results confirmed that the expression levels of these two genes were significantly higher in L. plantarum cells isolated from the mouse duodenum relative to cells grown in standard laboratory media (25). Current studies aim at the confirmation of gene-induction of several other L. plantarum genes initially identified with the R-IVET screen (Marco et al. unpublished data). Moreover, transcription profiling under a variety of in vitro conditions could identify more matches with the R-IVET screen and these genes could subsequently be analyzed with qRT-PCR. These experiments might reveal the specific environmental cue involved in in situ induction in the GI tract and could eventually elucidate the regulatory mechanism(s) involved. These approaches could help to unravel the geographical differentiation of L. plantarum gene expression along the GI tract, i.e., specific induction in the stomach, small intestine or colon.

Another promising development is the optimization of bacterial RNA isolation protocols from fecal samples (67), and GI-tract samples from conventional mice fed with L. plantarum (Marco et al. unpublished data) or human cancer patients who volunteered to consume an oatmeal-based drink containing high numbers of L. plantarum prior to surgery (de Vries et al. unpublished data). Although it is technically difficult to isolate high quality bacterial mRNAs from these samples, such RNA samples originating from an in vivo animal tissue, could prove extremely valuable, as they should allow analysis using DNA micro-array technology, providing direct information on the in situ expression levels of thousands of bacterial genes. Moreover, comparison of bacterial responses in samples from the GI tract from animal models and of human origin could provide an indication of the overlap in the response of L. plantarum during residence in the GI tract of different hosts.

Studies in gnotobiotic mice have indicated that there is specific signaling between the commensal bacterium B. thetaiotaomicron and its host. Synthesis of host epithelial glycans is elicited by a B. thetaiotaomicron signal of which the expression is regulated by a fucose-binding bacterial transcription factor. This factor senses environmental levels of fucose and coordinates the decision to generate a signal for production of host fucosylated glycans when environmental fucose is limited or to induce expression of the bacteria's fucose utilization operon when fucose is abundant (68). Additional studies have evaluated the global intestinal response to colonization of gnotobiotic mice with B. thetaiotaomicron. This colonization dramatically affected the host's gene expression, including several important intestinal functions such as nutrient absorption, mucosal barrier fortification, and postnatal intestinal maturation (9). From the in situ global transcription profiles mentioned above and follow-up experiments it could be established that the production of a previously uncharacterized angiogenin is induced when gnotobiotic mice are colonized with B. thetaiotaomicron, revealing a mechanism whereby intestinal commensal bacteria influence GI-tract bacterial ecology and shape innate immunity (69). In addition, the cellular origin of the angiogenin response was investigated when different intestinal cell types were separated by laser-capture microdissection and analyzed by qRT-PCR, revealing that angiogenin-3 mRNA is specifically induced only in crypt epithelial cells. Hence, these experiments strongly suggest an intestinal tissue specific response of the host during colonization (9). Interestingly, comparison of the changes in global host gene expression in mice after colonization with B. thetaiotaomicron, Bifidobacterium infantis or E. coli led to the observation that part of this host response was only induced in mice by colonization with B. thetaiotaomicron (9). However, analysis of a broader range of members of the intestinal microbiota will reveal what the level of bacterial response specificity within the host's tissues actually is. One such study is currently performed for L. plantarum (Peters et al. unpublished data). Overall, the aforementioned studies on B. thetaiotaomicron colonization of gnotobiotic mice provided valuable information on the influence of one particular member of the microbiota on the host. However, the host response during colonization by more complex mixtures of microbes and/or the host response in other animal systems remained to be investigated at that time. Recently, it was found that conventionalization of adult gnotobiotic mice with normal microbiota harvested from the distal intestine of conventionally raised mice produced a 60% increase in body fat content and insulin resistance despite reduced food intake. Studies of gnotobiotic and conventionalized mice revealed that the microbiota promotes absorption of monosac-charides from the gut lumen, which results in induction of de novo hepatic lipogenesis. Fastin-induced adipocyte factor (Fiaf), a member of the angiopoietin-like family of proteins, is selectively suppressed in the intestinal epithelium of normal mice by conventionalization. Analysis of gnotobiotic and conventionalized, normal and Fiaf knockout mice established that Fiaf is a circulating lipoprotein lipase inhibitor and that its suppression is essential for the microbiota-induced deposition of triglycerides in adipocytes. These results suggest that the gut microbiota have a major impact on food-derived energy harvest and storage in the host (70). Another recent study investigated the host response during colonization of a different animal model. DNA micro-array comparison of gene expression in the digestive tracts of six days post-fertilization gnotobiotic, conventionalized, and conventionally raised zebrafish (Danio Rerio) revealed 212 genes regulated by the microbiota. Notably, 59 of these genes were also found to be regulated in the mouse intestine during colonization, including genes that encode functions involved in stimulation of epithelial proliferation, promotion of nutrient metabolism, and innate immune response, indicating a substantial overlap in the genetic response of mice and zebrafish towards intestinal colonization (71). Despite these recent developments, an important future challenge lies within the translation of these animal host response analyses to the human system.

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