A Synonymous Mutation in Yersinia enterocolitica yopE Affects the Function of the YopE Type III Secretion Signal

Abstract

Yersinia spp. inject virulence proteins called Yops into the cytosol of target eukaryotic cells in an effort to evade phagocytic killing via a dedicated protein-sorting pathway termed type III secretion. Previous studies have proposed that, unlike other protein translocation mechanisms, Yops are not recognized as substrates for secretion via a solely proteinaceous signal. Rather, at least some of this information may be encoded within yop mRNA. Herein, we report that the first seven codons of yopE, when fused to the reporter protein neomycin phosphotransferase (Npt), are sufficient for the secretion of YopE 1-7 -Npt when type III secretion is induced in vitro. Systematic mutagenesis of yopE codons 1 to 7 reveals that, like yopQ, codons 2, 3, 5, and 7 are sensitive to mutagenesis, thereby defining the first empirical similarity between the secretion signals of two type III secreted substrates. Like that of yopQ, the secretion signal of yopE exhibits a bipartite nature. This is manifested by the ability of codons 8 to 15 to suppress point mutations in the minimal secretion signal that change the amino acid specificities of particular codons or that induce alterations in the reading frame. Further, we have identified a single nucleotide position in codon 3 that, when mutated, conserves the predicted amino acid sequence of the YopE 1-7 -Npt but abrogates secretion of the reporter protein. When introduced into the context of the full-length yopE gene, the single-nucleotide mutation reduces the type III injection of YopE into HeLa cells, even though the predicted amino acid sequence remains the same. Thus, yopE mRNA appears to encode a property that mediates the type III injection of YopE.

Substrate-Activated Conformational Switch on Chaperones Encodes a Targeting Signal in Type III Secretion

“…The results have suggested in certain cases that specific regions in the N-terminal region of effectors and translocators constitute the secretion signal (Sory et al, 1995; Lloyd et al, 2001; Amer et al, 2011), whereas in other cases it has been suggested that the secretion signal is encoded in the mRNA sequence (Anderson and Schneewind, 1997; Ramamurthi and Schneewind, 2005). However, secretion is a complex process and targeting is just the first step of it.…”

Substrate recognition of type III secretion machines -testing the RNA signal hypothesis

“…Remarkably, two synonymous substitutions, each at isoleucine codon 3, abrogated the function of yopE and yopQ minimal secretion signals. When introduced into full-length yopE , such single nucleotide substitutions significantly reduce YopE injection into tissue culture cells, although substrate recognition was not completely blocked (Ramamurthi and Schneewind, 2005). In yopE , the AUA and AUU nucleotide triplets of Ile codon 3 promote secretion signalling, whereas AUC does not (Ramamurthi and Schneewind, 2005).…”

“…Even the tolerance of secretion signals to frameshift mutations has limits. Mapping the minimal secretion signal of yopE and yopQ identified the first seven or 10 codons as functional elements (Ramamurthi and Schneewind, 2005). However, frameshift mutations of these minimal secretion signals abrogated function, although that initiation of the encoded hybrids into the type III pathway could be restored by fusion of downstream yopE or yopQ codons (7-15 codons).…”

RNA Localization in Bacteria

“…For example, many mRNAs encoding inner membrane proteins are preferentially localized at the membrane and this localization can be mediated in a translation-dependent manner during synthesis and secretion of N-terminal signal peptides. However, for certain mRNAs encoding inner membrane proteins or secreted effectors, it has been reported that signals within the mRNA itself are instead required for localization/secretion or that the mRNAs have multiple localization signals, including RNA sequence-based and those encoded in the N-terminal peptide amino acid sequence (10, 72, 74, 147). It remains to be seen how many bacterial mRNAs have a eukaryotic-like RNA zip-code, and what the sequence- and/or structural features of these elements are.…”