Reciprocal secretion of proteins by the bacterial type III machines of plant and animal pathogens suggests universal recognition of mRNA targeting signals

Abstract

Bacterial pathogens of both animals and plants use type III secretion machines to inject virulence proteins into host cells. Although many components of the secretion machinery are conserved among different bacterial species, the substrates for their type III pathways are not. The Yersinia type III machinery recognizes some secretion substrates via a signal that is encoded within the first 15 codons of yop mRNA. These signals can be altered by frameshift mutations without affecting secretion of the encoded polypeptides, suggesting a mechanism whereby translation of yop mRNA is coupled to the translocation of newly synthesized polypeptide. We report that the type III machinery of Erwinia chrysanthemi cloned in Escherichia coli recognizes the secretion signals of yopE and yopQ. Pseudomonas syringae AvrB and AvrPto, two proteins exported by the recombinant Erwinia machine, can also be secreted by the Yersinia type III pathway. Mapping AvrPto sequences sufficient for the secretion of reporter fusions in Yersinia revealed the presence of an mRNA secretion signal. We propose that 11 conserved components of type III secretion machines may recognize signals that couple mRNA translation to polypeptide secretion.

Figure 1

The Erwinia type III machine secretes Yersinia Yops. E. coli DH5α (pCPP2156) expressing type III genes of E. chrysanthemi was transformed with plasmids that expressed either the yopE or the yopQ gene under control of the IPTG-inducible tac promoter. Cultures were induced for expression of Yops and centrifuged. Bacteria in the sedimented pellet (P) were separated from the culture medium in the supernatant (S). Supernatant samples were concentrated 60-fold to facilitate quantitation by SDS/PAGE. Proteins were analyzed by immunoblotting by using peptide antibody against the Flag epitope that had been appended to C-terminal yopE and yopQ sequences. The cytoplasmic protein, chloramphenicol acetyl transferase, supplied on an additional plasmid, was not secreted in these strains. As a control for type III secretion, E. coli DH5α (pCPP2368) carrying a transposon insertion in the Erwinia hrc gene cluster did not secrete YopE–Flag andYopQ–Flag. Yersinia YopQ and YopE harbor an mRNA signal within the first 15 codons that functions to couple mRNA translation and type III secretion of these polypeptides.

Figure 2

Yersinia type III machines secrete Pseudomonas secretion substrates. Y. enterocolitica W22703 was transformed with plasmids carrying the avrB and avrPto genes under control of the constitutively expressed npt promoter. Yersinia cultures were induced for type III secretion by temperature shift to 37°C and chelation of calcium from the culture medium. Proteins were detected by immunoblotting with antibody against the Flag epitope, which had been appended to C-terminal avrB and avrPto sequences. Yersinia efficiently recognized the secretion signals of the Pseudomonas proteins, as 43% of AvrB and 78% of AvrPto were secreted, whereas the Yersinia cytoplasmic protein, LcrH, was not secreted. As a control for type III secretion, Yersinia carrying a null mutation in lcrD were unable to secrete AvrB or AvrPto. Deletion of the first 15 or 10 codons of AvrB or AvrPto, respectively, abolished secretion. The secretion of AvrB and AvrPto by the Erwinia type III machine cloned in E. coli was analyzed by concentrating culture supernatants 7.5 (AvrPto) and 80 fold (AvrB).

Figure 3

Pseudomonas AvrPto is secreted by the type III machine via an mRNA secretion signal. (A) The secretion signal encoded by the first 15 codons is not only necessary but also sufficient for the secretion of reporter proteins. The first 15 codons of avrB and avrPto were fused to neomycin phosphotransferase (Npt) and secretion was measured in Y. enterocolitica W22703 with specific antibody to Npt. Both the first 15 codons of avrB and avrPto are sufficient to cause 25% and 47% secretion, respectively, of reporter fusions into the extracellular medium of Yersinia cultures. A fusion of the first 44 codons of avrB to npt caused an increase in secretion of the hybrid protein to 42%. (B) The secretion signal encoded within the first 15 codons of avrPto is recognized at the level of mRNA sequence, because it can tolerate frameshift mutations. Frameshift mutations were generated by nucleotide insertions (+1, +2) or deletions (−1, −2) immediately after the AUG translational start. Frameshifts were suppressed by reciprocal deletions and insertions at the fusion site with npt. The +1, +2, and −1 frameshift mutations did not interfere with Yersinia type III secretion because 49, 28 and 25% of the polypeptides were found in the culture supernatant. The −2 frameshift mutation abolished mRNA expression of the npt reporter.

Figure 4

Predicted RNA structures of the AvrPto and AvrB secretion signals. RNA sequences were subjected to folding analysis by using the Zuker program (24). The displayed structures show areas encompassing the ribosome-binding sites (bold), including the Shine–Dalgarno sequence (−13 to −8), start codon (+1 to +4), and downstream sequence of avrB and avrPto secretion signals. ΔG value (Gibbs energy) for avrB is −19.9 kCal (1 Cal = 4.18 J) and that of avrPto is −17.0 kCal.