A conserved amino acid sequence directing intracellular type III secretion by Salmonella typhimurium

Abstract

Type III secretion systems (TTSS) are important virulence factors that Gram-negative bacteria use to translocate proteins into the cytoplasm of eukaryotic host cells. Salmonellae encode two virulence-associated TTSS. The Salmonella pathogenicity island 1 (SPI1)-encoded TTSS is active on contact with host cells, whereas the Salmonella pathogenicity island 2 (SPI2)-encoded TTSS is expressed after phagocytosis of bacteria by host cells. Previously, no consensus signal sequence for translocation has been identified among TTSS effector proteins. In this work, seven proteins, termed Salmonella-translocated effectors (STE), are described that contain conserved amino acid sequences that direct translocation by TTSS in Salmonella typhimurium. STE that are coordinately regulated with SPI2 gene expression contain translocation signals that are recognized by the SPI2 but not by the SPI1 TTSS. STE that are constitutively expressed contain signals that direct translocation through both SPI1 and SPI2 TTSS. Of the seven STE examined, SspH1 and SspH2 have been previously shown to be translocated and involved in virulence; SlrP and SifA were identified as virulence factors, but were not previously known to be associated with TTSS; and SseI, SseJ, and SifB were previously unidentified. Three STE genes (sspH1, sspH2, and sseI) are located within temperate bacteriophages, suggesting a common mechanism for the dissemination of more recently evolved STE.

Figure 1

Alignment of STE secretion/translocation signals. Residues in bold are high consensus; at least five of seven STE contain these residues. Residues in gray are low consensus; three or four of seven STE contain these residues. Residues that are not shaded have two or fewer of seven STE containing the residue. Alignments were performed with the blosum62 method (28). Residues that have similar properties are grouped as following: !, any one of IV; $, any one of LM; %, any one of FY; and #, any one of NDQE. The consensus sequence is shown under the STE alignment. The percent identity to SspH2 varies between proteins. SspH2₁₄₂ is 89% identical to SseI₁₄₂. SseJ₁₁₁ is 38% identical to SspH2₁₀₉. SifB₁₁₅ is 28% identical to SspH2₁₁₂. SifA₁₁₃ is 32% identical to SspH2₁₁₁. SspH2₁₄₂ is 45% identical to SspH1₁₃₉. SspH2₁₂₈ is 37% identical to SlrP₁₂₅.

Figure 2

Regulation of STE expression. S. typhimurium wild-type or regulatory mutants expressing transcriptional fusions of STE genes to firefly luciferase in the pGPLFR03 expression vector were grown overnight in LB and back diluted in SPI2-inducing conditions (A) or SPI1-inducing conditions (B), and firefly luciferase activity was determined and normalized for cell number by dividing by the activity of the constitutively expressed Renilla luciferase. sspC∷Tn5-lacZY expression is presented as Miller units (as a control for SPI1-regulated gene expression).

Figure 3

Analysis of residues required for translocation. Wild-type S. typhimurium or strains carrying mutations in SPI2 TTSS (ssaT) or SPI1 TTSS (ΔprgH-K) expressing various CyaA fusions were used to infect RAW264.7 cells at a multiplicity of infection (MOI) of 10. Macrophages were infected for 1 h with bacteria in late logarithmic growth for SPI1 TTSS expression (C) or for 1 h with late stationary phase bacteria plus 5 or 6 h of gentamycin treatment for SPI2 TTSS expression (A and B). Infected macrophages were lysed in 0.1 M HCl; cellular cAMP levels were determined by enzyme immunoassay and were normalized for protein content determined by the Bradford assay and are presented as pmol of cAMP per μg of protein. SspH1₁₄₀ and SspH1_{1–31,36–140}CyaA (Δ32–35) were used in B and C. Note that cAMP production may be variable between assays, and infection of bacteria expressing SspH1₁₄₀CyaA results in cAMP increases that are similar to those seen with SspH1₂₀₈CyaA in a direct comparison (data not shown).

Figure 4

Translocation of STE-CyaA fusion proteins. Wild-type S. typhimurium or strains carrying mutations in SPI2 TTSS (ssaT) or SPI1 TTSS (ΔsspC) expressing various CyaA fusions were used to infect RAW264.7 cells as described in Fig. 3, assaying translocation by SPI2 (A) or SPI1 (B) TTSS. SspH1₂₀₈, SspH2₂₁₄, SlrP₁₉₁, SseI₂₀₆, SseJ₁₄₇ and SspA₁₅₈ and LacZ₂₁CyaA fusions were analyzed.