Characterization of Small Non-coding RNAs in the Gut Microbiome

The human body is home to a vast array of microbes whose function in various human ecosystems remain unclear (1-3). It has been recently documented from data generated by the NIH sponsored Human Microbiome Project (HMP) that individuals in the same status of health exhibit considerable microbiome taxonomic diversity and operational taxonomic unit (OTU) abundance variation; however the microbiome associated functional pathways are more uniformed, suggesting that these functional pathways may be better markers of health status. In bacteria, small RNAs (sRNAs) have been shown to play a role in regulating multiple pathways involved in adaptation processes during environmental changes. sRNAs exist as short independent transcripts ranging in size between 50 and 300 nucleotides that function by base-pairing with target RNAs. sRNAs are categorized into two groups, trans-encoded and cis-encoded (or antisense), based on the location of the sRNA gene relative to the target gene. Both cis- and trans-encoded sRNAs regulate gene expression by direct base-pairings, resulting in degradation of the mRNA, or by occlusion of the ribosome binding site (RBS) and preventing translation of the mRNA. In Escherichia coli sRNAs are involved in modulation of tolerance to acid stress, expression of virulence factors, colonization of a host, cell motility and antibiotic tolerance (4, 5). In Salmonella typhimurium sRNAs have been demonstrated to support virulence regulation, replication in macrophages, secretion of effector proteins and cell adhesion (4, 5). In plant cells sRNAs have been demonstrated to support the immune response to pathogenic viruses (6). In mammals sRNAs (microRNAs) are involved in a wide array of physiological and pathological mechanisms, including host response to viral and bacterial infections (7). Pathogenic bacteria in particular dwell in numerous ecological niches and have to adapt to rapidly changing conditions when they enter the gut (8). Many proteins are known to regulate genes at a transcriptional level, but sRNAs have increasingly been recognized as major regulators during these rapid stress-adaptive responses (5, 9, 10).

Commensal bacteria are important for gastrointestinal (GI) homeostasis, are essential for immune system development, and for preventing infections by bacterial pathogens that cause disease. However, it is not fully understood how commensal bacteria deal with invasive pathogens in the GI tract (11-15). Recent reports have demonstrated the expression of a subset sRNAs in healthy human gut microflora using metatranscriptomic analysis (16-18); however there is a significant gap on understanding the role of sRNAs in the context of the microbiome and response to invasive pathogens.

The NIH sponsored HMP has generated a tremendous resource that empowers the community to study the dynamic composition of the microbiome; however, little to nothing is known about sRNA expression by the microbiome. We propose a pilot study to create a sRNA resource for the mouse gut microbiome in the context of infection as a model for humans. This proposed sRNA pilot survey will provide an additional useful community resource to facilitate study of the dynamic changes in the microbiome. We will survey sRNAs during GI homeostasis and during infection with S. typhimurium strain 14028 in mice. We have fecal samples collected from a pool of five mice on -5, -3 and -1 days prior to S. typhimurium infection representing a healthy microbiome; and samples from days +1, +3, +6, +10 and +14 days post-infection with S. typhimurium representing an infection-confronted microbiome. These samples will provide a survey of sRNA expression in healthy state and during S. typhimurium infection. We have already obtained 16S data from the same samples that will permit us to correlate dynamic changes in the microbiome and sRNA expression prior to and following S. typhimurium infection and allow us to interrogate a potential role during the infection process. To aid with analysis we propose to perform metagenomic DNA sequence characterization from time-points -1 and +6 from existing samples. Metagenomic analysis of these samples will support read mapping of identified sRNAs, as well as support the understanding of sRNA differential expression prior to and during S. typhimurium infection. Based on the pre-existing 16S data, it is known that following S. typhimurium infection, dynamic changes occur within the gut microbiome. The proposed pilot project will result in a list of microbiome-expressed sRNAs in the healthy state and after infection that will enable the community to better understand the role of sRNAs during infection and support the development of hypothesis driven studies to further refine the details of the microbiome-pathogen-host interactions in health and disease. 

Funding