Regulation of chaperone/effector complex synthesis in a bacterial type III secretion system

Abstract

Type III protein secretion systems (T3SSs), which have evolved to deliver bacterial proteins into nucleated cells, are found in many species of Gram-negative bacteria that live in close association with eukaryotic hosts. Proteins destined to travel this secretion pathway are targeted to the secretion machine by customized chaperones, with which they form highly structured complexes. Here, we have identified a mechanism that co-ordinates the expression of the Salmonella Typhimurium T3SS chaperone SicP and its cognate effector SptP. Translation of the effector is coupled to that of its chaperone, and in the absence of translational coupling, an inhibitory RNA structure prevents translation of sptP. The data presented here show how the genomic organization of functionally related proteins can have a significant impact on the co-ordination of their expression.

Fig. 1

Identification of the sptP translation start codon. (A) Alignment of sptP nucleotide sequence from the following sequenced Salmonella enterica strains: 1) S. Typhimurium LT2; 2) S. Typhi Ty2 and CT18; 3) S. Paratyphi A strain AKU_12601 and ATCC 9150; 4) S. Salamae Sofia; and 5) S. Arizonae serovar 62:z4,z23. (B) Whole cell lysates of strains carrying the indicated changes to the putative sptP start sites in the context of a strain encoding a sptP::phoA translational reporter were analyzed by western immunoblot with an anti-PhoA antibody.

Fig. 2

Expression of the chaperone SicP is required for wild type expression of the effector SptP. (A) Schematic representation of the sptP::phoA translational reporter constructs. (B) Whole cell lysates of the indicated strains were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Results represent the average of three independent experiments. Student's t test was used to determine the p values. (C) Strains carrying constructs equivalent to those described in (A) in which sptP::phoA was replaced with sptP::lacZ, were analyzed by Miller assays. LacZ activity was quantified and normalized to wild type. Results represent the average of three independent experiments. Student's t test was used to determine the p values.

Fig. 3

A nucleotide region directly upstream of sptP inhibits expression of sptP in the absence of sicP expression. (A) Schematic representation of the sicP deletion mutants. The nucleotides deleted within the sicP coding sequence are indicated. The starting point of all deletions was 14 nucleotides before the sicP start codon (−14) so as to prevent the initiation of sicP translation in these mutants. (B) Whole cell lysates were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Results represent the average of at least three independent experiments. Student's t test was used to determine p values. When compared to sicPSTOP, p<0.01 only for mutants ΔsicP^Δ14-372 and ΔsicP^Δ14-354 (indicated by *).

Fig. 4

An mRNA structure inhibits translation of sptP. (A) The mutations indicated in the schematic representation (in grey boxes) of secondary mRNA structure surrounding the sptP start codon (as predicted by mFOLD) were introduced into a strain encoding a sptP::phoA reporter and whole cell lysates were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Additionally, the same mutations were introduced into a wild type strain and whole cell lysates were analyzed by western immunoblot with an anti-SptP antibody. (B) The mutations indicated in the schematic representation (in grey boxes) predicted to disrupt (A -> U) or restore (A -> U/U-> A) base complementarity in the predicted stem surrounding the sptP start codon were introduced into a background strain encoding a sptP::phoA reporter. Whole cell lysates of these strains were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Student's t test was used to determine p values. (C) The indicated mutations to lengthen the predicted stem surrounding the sptP start codon were introduced into a background strain encoding a sptP::phoA reporter. Whole cell lysates of these strains were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Student's t test was used to determine p values.

Fig. 5

sicP is the only T3SS-specific factor necessary for sicP-expression-dependent regulation of sptP translation. (A) Whole cell lysates of S. Typhimurium bearing plasmids expressing wild type sicP sptP::phoA, sicPSTOP sptP::phoA, and ΔsicP sptP::phoA were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Student's t test was used to determine p values. (B) Whole cell lysates of E. coli bearing the same plasmids were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Student's t test was used to determine p values.

Fig. 6

Providing SicP in trans is not sufficient to promote wild type levels of SptP expression. (A) Whole cell lysates of the indicated strains with or without a plasmid expressing SicP were analyzed by western immunoblot with an anti-PhoA antibody. (B) Whole cell lysates of strains with sicP placed either upstream or downstream of the sptP::phoA reporter (as indicated in the diagram) were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Student's t test was used to determine p values.

Fig. 7

Expression of the effector SptP is translationally coupled to expression of the chaperone SicP. (A) Whole cell lysates of strains carrying the indicated chromosomally encoded sicP deletions in the context of the sptP::phoA background were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Results represent the average of at least three independent experiments. The difference in reporter expression between the mutants and wild type was significant (p<0.05, Student's t test) only for the mutants sicPSTOP, ΔsicP, and ΔSicPΔ 2-31. (B) Whole cell lysates of strains carrying the indicated chromosomally-encoded frame-shifted sicP mutants in the context of the sptP::phoA background were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Results represent the average of at least three independent experiments, and p values were determined by the Student's t test. (C) Strains were constructed in which the ribosome binding site (RBS) of sicP was changed to RBSs of defined strength (“weak” and “strong”). Mutations were introduced in the context of a strain that expresses 3xFLAG epitope tagged SicP and carries a sptP::phoA reporter. Whole cell lysates were analyzed by western immunoblot with an anti-FLAG or an anti-PhoA antibody and the signals were quantified. Results represent the average of at least three independent experiments. Student's t test was used to determine p values. (D) Stop codons were introduced at codons 121, 118, 112, or 104 of sicP (corresponding to 2, 11, 29, and 53 nt upstream of the predicted stem) in the context of a strain carrying a sptP::phoA reporter fusion. Black highlighting indicates nucleotides base-paired in the stem. Whole cell lysates were analyzed by western immunoblot with an anti-PhoA antibody. SptP::phoA reporter signal was quantified and normalized to wild type. Results represent the average of at least three independent experiments. Only the mutants sicPSTOP and SicPY104* are significantly different from wild type (Wt). Student's t test was used to determine p values.

Fig. 8

Translational coupling of sicP and sptP is required for optimal secretion of SptP. Whole cell lysates and culture supernatants of the indicated strains were analyzed by Western immunoblot using an antibody against SptP. SptP signal was quantified and normalized to wild type. Results represent the average of at least three independent experiments. Student's t test was used to determine p values.