Söll, D. | Search bioRxiv

bioRxiv2022

An Aedes aegypti seryl-tRNA synthetase paralog controls bacteroidetes growth in the midgut

Insect gut microbiota plays important roles in host physiology, such as nutrition, digestion, development, fertility, and immunity. We have found that in the intestine of Aedes aegypti, SLIMP (seryl-tRNA synthetase like insect mitochondrial protein) knockdown followed by a blood meal promotes dysbiosis, characterized by the overgrowth of a specific bacterial phylum, Bacteroidetes. In turn, the latter decreased both infection rates and Zika virus prevalence in the mosquitoes. Previous work in Drosophila melanogaster showed that SLIMP is involved in protein synthesis and mitochondrial respiration in a network directly coupled to mtDNA levels. There are no other reports on this enzyme and its function in other insect species. Our work expands the knowledge of the role of these SerRS paralogs. We show that A. aegypti SLIMP (AaeSLIMP) clusters with SLIMPs of the Nematocera sub-order, which have lost both the tRNA binding domain and active site residues, rendering them unable to activate amino acids and aminoacylate tRNAs. Knockdown of AaeSLIMP did not significantly influence the mosquitoes survival, oviposition, or eclosion. It also neither affected midgut cell respiration nor mitochondrial ROS production. However, it caused dysbiosis, which led to the activation of Dual oxidase and resulted in increased midgut ROS levels. Our data indicate that the intestinal microbiota can be controlled in a blood-feeding vector by a novel, unprecedent mechanism, impacting also mosquito vectorial competence towards zika virus and possibly other pathogens as well. Author SummaryAminoacyl-tRNA synthetases (aaRS) are a family of ubiquitous enzymes responsible for the attachment of specific amino acids to their cognate tRNAs. During evolution some aaRS acquired new domains and/or suffered gene duplications, resulting in the improvement and expansion of their functions some of them being specific to a group of organisms. A paralog of seryl-tRNA synthetase restricted to the class Insecta (SLIMP) is found in Arthropoda. Our goal was to explore the role of SLIMP in the female mosquito Aedes aegypti using RNA interference. We showed that A. aegypti SLIMP (AaeSLIMP) gene expression is up-regulated upon blood feeding through a heme-dependent signaling. Although AaeSLIMP knockdown neither impacted the mosquito survival nor oviposition, it provoked ROS levels augmentation in the midgut via Dual Oxidase activity in order to control the increase in the intestinal native microbiota, specifically bacteria of the Bacteroidetes phylum. Although dysbiosis can result from mitochondrial impairment, this is the first time that the absence of a mitochondrial enzyme is linked to intestinal microbiota without any visible effects in mitochondrial respiration and mitochondrial ROS production. Furthermore, Zika Virus infection of AaeSLIMP silenced mosquitoes is decreased when comparing to control, meaning that Bacteroidetes overgrowth may be protecting the female mosquito. Our data indicate that the intestinal microbiota can be controlled in a blood-feeding vector by a novel, unprecedent mechanism, impacting also mosquito vectorial competence towards zika virus and possibly other pathogens as well.

biochemistry↗

bioRxiv2020

Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

We recently described an orthogonal initiator tRNA (itRNATy2) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here we report that a mutant of itRNATy2 (itRNATy2AUA) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing itRNATy2AUA, UAU can function as a dual-use codon that selectively encodes ncAAs at the initiating position and tyrosine at elongating positions. Using itRNATy2AUA, in conjunction with its cognate tyrosyl-tRNA synthetase and two mutually orthogonal pyrrolysyl-tRNA synthetases, we demonstrate that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. Furthermore, by engineering the substrate specificity of one of the pyrrolysyl-tRNA synthetases, we developed a triply orthogonal system that enables simultaneous reassignment of UAU, UAG, and UAA to produce proteins containing three distinct ncAAs at precisely defined sites. To showcase the utility of this system, we produced proteins containing two or three ncAAs, with unique bioorthogonal functional groups, and demonstrate that these proteins can be separately modified with multiple fluorescent probes. TOC Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/415521v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@10e7ebdorg.highwire.dtl.DTLVardef@a2c26dorg.highwire.dtl.DTLVardef@18ab3aborg.highwire.dtl.DTLVardef@119cf87_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2017

Engineering post-translational proofreading to discriminate non-standard amino acids

Progress in genetic code expansion requires accurate, selective, and high-throughput detection of non-standard amino acid (NSAA) incorporation into proteins. Here, we discover how the N-end rule pathway of protein degradation applies to commonly used NSAAs. We show that several NSAAs are N-end stabilizing and demonstrate that other NSAAs can be made stabilizing by rationally engineering the N-end rule adaptor protein ClpS. We use these insights to engineer a synthetic quality control method, termed \"Post-Translational Proofreading\" (PTP). By implementing PTP, false positive proteins resulting from misincorporation of structurally similar standard amino acids or undesired NSAAs rapidly degrade, enabling high-accuracy discrimination of desired NSAA incorporation. We illustrate the utility of PTP during evolution of the biphenylalanine orthogonal translation system used for synthetic biocontainment. Our new OTS is more selective and confers lower escape frequencies and greater fitness in all tested biocontained strains. Our approach presents a new paradigm for molecular recognition of amino acids in target proteins.

synthetic biology↗