RNA recoding in cephalopods tailors microtubule motor protein function.

RNA editing is a widespread epigenetic process that can alter the amino acid sequence of proteins, termed "recoding." In cephalopods, most transcripts are recoded, and recoding is hypothesized to be an adaptive strategy to generate phenotypic plasticity. However, how animals use RNA recoding dynamically is largely unexplored. We investigated the function of cephalopod RNA recoding in the microtubule motor proteins kinesin and dynein. We found that squid rapidly employ RNA recoding in response to changes in ocean temperature, and kinesin variants generated in cold seawater displayed enhanced motile properties in single-molecule experiments conducted in the cold. We also identified tissue-specific recoded squid kinesin variants that displayed distinct motile properties. Finally, we showed that cephalopod recoding sites can guide the discovery of functional substitutions in non-cephalopod kinesin and dynein. Thus, RNA recoding is a dynamic mechanism that generates phenotypic plasticity in cephalopods and can inform the characterization of conserved non-cephalopod proteins.

Spatially regulated editing of genetic information within a neuron.

In eukaryotic cells, with the exception of the specialized genomes of mitochondria and plastids, all genetic information is sequestered within the nucleus. This arrangement imposes constraints on how the information can be tailored for different cellular regions, particularly in cells with complex morphologies like neurons. Although messenger RNAs (mRNAs), and the proteins that they encode, can be differentially sorted between cellular regions, the information itself does not change. RNA editing by adenosine deamination can alter the genome's blueprint by recoding mRNAs; however, this process too is thought to be restricted to the nucleus. In this work, we show that ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. Finally, a transcriptome-wide analysis of RNA editing reveals that tens of thousands of editing sites (>70% of all sites) are edited more extensively in the squid giant axon than in its cell bodies. These results indicate that within a neuron RNA editing can recode genetic information in a region-specific manner.

Enterococcus faecium secreted antigen A generates muropeptides to enhance host immunity and limit bacterial pathogenesis.

We discovered that Enterococcus faecium (E. faecium), a ubiquitous commensal bacterium, and its secreted peptidoglycan hydrolase (SagA) were sufficient to enhance intestinal barrier function and pathogen tolerance, but the precise biochemical mechanism was unknown. Here we show E. faecium has unique peptidoglycan composition and remodeling activity through SagA, which generates smaller muropeptides that more effectively activates nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mammalian cells. Our structural and biochemical studies show that SagA is a NlpC/p60-endopeptidase that preferentially hydrolyzes crosslinked Lys-type peptidoglycan fragments. SagA secretion and NlpC/p60-endopeptidase activity was required for enhancing probiotic bacteria activity against Clostridium difficile pathogenesis in vivo. Our results demonstrate that the peptidoglycan composition and hydrolase activity of specific microbiota species can activate host immune pathways and enhance tolerance to pathogens.

Biochemical Mechanisms of Pathogen Restriction by Intestinal Bacteria.

The intestine is a highly complex ecosystem where many bacterial species interact with each other and host cells to influence animal physiology and susceptibility to pathogens. Genomic methods have provided a broad framework for understanding how alterations in microbial communities are associated with host physiology and infection, but the biochemical mechanisms of specific intestinal bacterial species are only emerging. In this review, we focus on recent studies that have characterized the biochemical mechanisms by which intestinal bacteria interact with other bacteria and host pathways to restrict pathogen infection. Understanding the biochemical mechanisms of intestinal microbiota function should provide new opportunities for therapeutic development towards a variety of infectious diseases.

A secreted bacterial peptidoglycan hydrolase enhances tolerance to enteric pathogens.

The intestinal microbiome modulates host susceptibility to enteric pathogens, but the specific protective factors and mechanisms of individual bacterial species are not fully characterized. We show that secreted antigen A (SagA) from Enterococcus faecium is sufficient to protect Caenorhabditis elegans against Salmonella pathogenesis by promoting pathogen tolerance. The NlpC/p60 peptidoglycan hydrolase activity of SagA is required and generates muramyl-peptide fragments that are sufficient to protect C. elegans against Salmonella pathogenesis in a tol-1-dependent manner. SagA can also be heterologously expressed and secreted to improve the protective activity of probiotics against Salmonella pathogenesis in C. elegans and mice. Our study highlights how protective intestinal bacteria can modify microbial-associated molecular patterns to enhance pathogen tolerance.

Exploiting a host-commensal interaction to promote intestinal barrier function and enteric pathogen tolerance.

Commensal intestinal bacteria can prevent pathogenic infection; however, limited knowledge of the mechanisms by which individual bacterial species contribute to pathogen resistance has restricted their potential for therapeutic application. Here, we examined how colonization of mice with a human commensal Enterococcus faecium protects against enteric infections. We show that E. faecium improves host intestinal epithelial defense programs to limit Salmonella enterica serotype Typhimurium pathogenesis in vivo in multiple models of susceptibility. E. faecium protection is mediated by a unique peptidoglycan hydrolase, SagA, and requires epithelial expression of pattern recognition receptor components and antimicrobial peptides. Ectopic expression of SagA in non-protective and probiotic bacteria is sufficient to enhance intestinal barrier function and confer resistance against S. Typhimurium and Clostridium difficile pathogenesis. These studies demonstrate that specific factors from commensal bacteria can be used to improve host barrier function and limit the pathogenesis of distinct enteric infections.

Rapid visualization and large-scale profiling of bacterial lipoproteins with chemical reporters.

Lipoproteins are a largely uncharacterized class of proteins in bacteria. In this study, metabolic labeling of bacteria with fatty acid chemical reporters allowed rapid profiling of lipid-modified proteins. We identified many candidate lipoproteins in Escherichia coli and detected a novel modification on YjgF. This chemical approach should facilitate future characterization of lipoproteins.