Concluding Remarks And Future Perspectives

Historically, research on the bacterial flora of the GI tract has concentrated on the inhabitants that have negative effects on their hosts. More recently, research has expanded from these pathogenic to non-pathogenic bacteria, including symbionts and commensals. One obvious reason for this is the accumulating evidence that certain bacteria, especially strains from the genus Lactobacillus and Bifidobacterium, may have probiotic effects in man and animals (72). At present there is a detailed understanding on the distribution of specific microbes along the human colon and the variations that can occur between different individuals (73-75). Moreover, knowledge on the activity and response of specific species to the conditions encountered when they transit through this complex niche is starting to accumulate. Several in vitro studies mimicking specific conditions in the GI tract have been performed, which allowed the identification of the repertoire of genes and their corresponding proteins that respond to the condition applied. More recently, in vivo approaches aiming at the identification of bacterial genes that are induced during passage of the GI tract have been performed in various microbes, including food-grade species. The current knowledge on promoter elements regulating gene expression of food-grade bacteria in the GI tract could have application possibilities, as these bacteria have been shown to possess great potential to serve as delivery vehicles of health-promoting or therapeutic compounds to the human GI tract (76-84). (R-)IVET approaches have provided the required promoters that will allow the construction of LAB-based dedicated GI-tract delivery vehicles that only express certain desired functions in situ. Moreover, geographically more detailed insight in the exact site of in situ gene activation in the GI tract derived from qRT-PCR using specific tissue samples might allow the construction of highly site-specific delivery vehicles. Combination of these promoters with certain genes, e.g., bacteriophage-derived or other lytic cassettes, might generate LAB strains that release their cellular content at a specific location in the GI tract.

At present, a large part of the consortium of bacteria residing in the GI tract has not been cultured in vitro. Since most genetic approaches require the culturability of the microbe under investigation, the expansion of our knowledge of this group of bacteria is highly challenging and very limiting at this stage. Metagenomic approaches might shed light on the genetic complexity of the collective genomic material of the intestinal microbiota (85). Moreover, such studies could reveal previously unknown, critical genes for intestinal microbiota functioning. However, effective exploration of metagenomic functionality will depend on high throughput screening systems that allow function identification. Moreover, the development of effective and robust methods to assess microbiota activity in situ in a culture independent manner will be critical for our functional understanding of the large number of unculturable bacteria in the GI tract.

A promising prospect from the increasing availability of complete genome sequences is the construction of DNA micro-arrays in several laboratories working on food-associated microbes. As a consequence, the first publications presenting data from these DNA micro-arrays appeared recently (86-88). These genomics-based, global investigations of gene expression in food-grade microbes under various conditions will further detail our understanding of their behavior. However, the application of these transcriptome profiling techniques on microbe-containing GI tract samples will still have to overcome some technical hurdles (RNA extraction procedures, response validation, etc.), but will eventually lead to a more complete view of the activity of these bacteria in this complex niche. Besides the application of DNA micro-array technology to reveal the bacterial side of host-microbe interactions in the GI tract, this technology has already been used by Hooper and co-workers in several elegant studies aiming at identification of the response of gnotobiotic mice to colonization with the commensal B. thetaiotaomicron (9,68,69). In addition, more recent gnotobiote studies have shed light on the differences in mouse gene expression upon colonization by a more complex mixture of bacteria (70), and have provided the first steps towards the comparative analysis of host responses in different animal models (71). Nevertheless, an important question that still remains to be answered is to what extent the data obtained on host and bacterial gene expression using animal models can be extrapolated to the situation in humans.

In conclusion, the genome-wide transcript profiling approaches that have been performed to date have provided us with clues of the possible role of individual host and bacterial genes during host-microbe interactions. Combination of bacterium and host transcriptomes should allow the construction of molecular models that describe host-microbe interactions, allowing more pinpointed experiments in the future, designed on the basis of a molecular interaction hypothesis. As Gl-tract bacteria like L. plantarum and B. thetaiotaomicron are genetically accessible, gene deletion and overexpression mutants can be constructed and employed to study the effect of a single bacterial gene and its corresponding function on host gene expression. After profiling of these host genes, knockout mice and/or antisense RNA approaches might allow gene silencing on the host side of the spectrum, thereby enabling us to study the effect of single host gene mutations on the colonization of microbes. Ultimately, such studies may provide a molecular knowledge base to understand Gl-tract colonization of commensals or symbionts, and could lead to the molecular explanation of probiotic effects associated with LAB and related species.

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Pregnancy And Childbirth

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