Overcoming Physiological Challenges in LBP Discovery

Genomics

Genomics revolves around the examination of genomes in an organism or a collection of creatures. The analysis offers assistance in identifying Live biotherapeutic product (LBP) platforms housing colonization-facilitating gene functions and establishing relationships between the LBP and gut symbiotes. The in-silico scrutiny of the Bifidobacterium bifidum genome divulged genes that validate for glycosyl hydrolases and ABC-type transporters intrinsic to bacteria that colonize mucus. These genes facilitate the breakdown and conveyance of host glycans derived from mucin, thereby guaranteeing nutrient abundance in the gut for the LBP framework. To convert these inferences into functional attributes (e.g., colonization) in aggressive gut environments, in vivo studies can authenticate these in silico suppositions. Techniques like shotgun metagenomics effectively outline the shifts in the gut's microbiota composition in vivo and identify microbe-microbe interactions as well.

Transcriptomics

Transcriptomics centers on the functional study of the transcriptome of given entities or organism communities under diverse conditions. Focusing on microbial transcriptional analyses under conditions relatable to human gut physiology helps decode gene regulation pathways crucial to overcoming physiological challenges. For example, the transcriptomic scrutiny of Akkermansia muciniphila revealed several systems involved in bile acid resistance. Furthermore, a similar study featuring a minor community consisting of Faecalibacterium prausnitzii, Blautia hydrogenotrophica, and Roseburia intestinalis clarified that each species showcased different metabolic activities when cultured alone or collectively. These transcriptomic investigations can help comprehend how various platforms adapt and utilize structures of gut mucin.

Proteomics

Proteomics involves the comprehensive examination of proteins expressed by an organism or a collection thereof, under different conditions and at unique points in time. The focus is on protein expression studies that evaluate the protein profiles of single microbial strains, particularly those dealing with physiological challenges typical of the human gut. Previously, the proteomic responses of LBPs have been analyzed in the presence of bile, variable nutrient availability, and oxygen gradients. A study conducted on the surface proteins of different Lactobacillus strains revealed strain-dependent adaptations to physiological challenges such as bile, immune responses, and other microbes. Proteomics allows extensive analysis of proteins involved in overcoming unique physiological challenges related to the gut environment, facilitating the selection of a chassis for LBP design or the incorporation of engineered elements utilizing these proteins.

Metabolomics

LBPs must overcome physiological challenges in the gut and their metabolic functions play a crucial role in conquering these challenges. A careful application of metabolomic analysis, using both untargeted approaches that evaluate all molecules and targeted approaches that investigate specific molecules under varying conditions, can bring these metabolic functions to light. For example, a metabolomic study of L. plantarum strains unveiled the extent of bile acid deconjugation, not only helping survive bile stress but also reducing competition for nutrients by restricting the expansion of certain microbial species. A study using the resistant-starch-degrader Ruminococcus bromii found that this species competes for malto-oligosaccharides with a mucin-degrading R. gnavus strain. Also, metabolomic profiling of L. acidophilus and L. gasseri found that their metabolic capability aids in the breakdown of dietary oxalate, which can cause kidney disorders, confirming LBPs' ability to benefit the host as well as acquire energy for itself in the gut. In other words, metabolomic analysis of LBPs helps comprehension of their molecular-scale mechanisms to overcome gut challenges.

Functional genomics

Functional genomics is another compelling tool to assess LBPs, using genetic engineering for in-depth study of gene functions, regulatory parts, and transformability. On one hand, metagenomic alteration of gut microbiome by in situ conjugation (MAGIC) has showcased the possibility of genetic engineering of gut bacteria through horizontal gene transfer and metagenomic sequencing. On the other hand, techniques such as transcript barcoding can improve our predictions of engineered function in vivo to support regulatory part selection. Thereby, functional genomic screens can identify colonization factors in LBPs. In parallel, certain advancements are being made in the likes of Bacteroides. A high-throughput screening of Bacteroides vulgatus clones unveiled a new class of polysaccharide utilization loci that could better utilize gut mucins.

In conclusion, overcoming physiological challenges in the discovery of live biotherapeutics can be achieved by leveraging the power of genomics, transcriptomics, proteomics, metabolomics, and functional genomics. By unraveling how microbes interact and respond to the human gut at multiple biological levels, we can enhance LBP design strategies to optimize their viability and efficacy.

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