The inner rod protein controls substrate switching and needle length in a Salmonella type III secretion system

Abstract

Type III secretion machines are essential for the biology of many bacteria that are pathogenic or symbiotic for animals, plants, or insects. They exert their function by delivering bacterial effector proteins into target eukaryotic cells. The core component of these machines is the needle complex, a multiprotein structure that spans the bacterial envelope and serves as a conduit for proteins that transit this secretion pathway. The needle complex is composed of a multiring base embedded in the bacterial envelope and a filament-like structure, the needle, that projects from the bacterial surface and is linked to the base by the inner rod. Assembly of the needle complex proceeds in a step-wise fashion that is initiated by the assembly of the base and is followed by the export of the building subunits for the needle and inner rod substructures. Once assembled, the needle complex reprograms its specificity and becomes competent for the secretion of effector proteins. Here through genetic, biochemical, and electron microscopy analyses of the Salmonella inner rod protein subunit PrgJ we present evidence that the assembly of the inner rod dictates the timing of substrate switching and needle length. Furthermore, the identification of mutations in PrgJ that specifically alter the hierarchy of protein secretion provides additional support for a complex role of the inner rod substructure in type III secretion.

Keywords: Salmonella pathogenesis; bacterial pathogenesis; organelle assembly.

Fig. 1.

The inner rod does not assemble in the absence of InvJ. (A) Structural modeling of PrgJ. The monomeric and multimeric modeled structures are shown and the position of the M83 residue selected for cross-linking experiments is indicated (white spheres). (B) S. Typhimurium-expressing PrgJ^M83pBpa yields intact and functional needle complex structures. Shown are electron micrographs of negatively stained needle complexes isolated from wild-type and prgJ^M83pBpa strains (Upper) and Western blot analysis (Lower) of whole-cell lysates and culture supernatants of the indicated strains for the presence of the type III secreted proteins SipB, SptP, and InvJ. (C) Cross-linking of PrgJ^M83pBpa occurs in needle complexes obtained from wild-type but not from ∆invJ S. Typhimurium mutant strains. Needle complexes were isolated from the indicated S. Typhimurium strains, exposed to UV, or left untreated as indicated, and analyzed by Western immunoblot with antibodies directed to PrgJ (Upper) or components of the needle complex (Lower).

Fig. 2.

Mutations in the inner rod protein PrgJ affect TTSS needle length. (A) Culture sedimentation analysis of the indicated PrgJ alanine mutant strains during growth under TTSS inducing conditions. Bacteria that express long needles sediment in the culture tubes due to needle-mediated clumping. Sedimentation was calculated by comparing the OD₆₀₀ values pre- and postvortexing of standing bacterial cultures and are expressed as arbitrary units after standardization relative to wild type, which is given the arbitrary value of 100. Error bars represent the standard deviation of three independent samples. (B) Electron micrographs of negatively stained bacterial cells reveal long TTSS needle filaments. Indicated are the different PrgJ mutants examined as well as wild type and the ∆invJ mutant control.

Fig. 3.

Analysis of PrgJ mutants exhibiting abnormal TTSS needle length. (A) Electron micrographs of isolated needle complexes from the indicated S. Typhimurium strains. (B) Western blot analysis of whole-cell lysates and culture supernatants of the indicated S. Typhimurium mutant strains.

Fig. 4.

PrgJ mutant strains with aberrant needle length assemble a functional TTSS that undergoes substrate switching. (A). Western blot analysis of whole-cell lysates and culture supernatants of the indicated strains examining the presence of the protein translocase SipB and the effector protein SptP. (B) Bacterial invasion of cultured Henle-407 cells by the indicated PrgJ mutant and control strains was determined using the gentamicin protection assay. Values represent the percentage of bacteria that survive gentamicin treatment, are normalized relative to the values of wild-type bacteria, and are the average of three determinations. Error bars represent the standard deviation of three independent experiments.

Fig. 5.

Mutations in PrgJ lead to altered hierarchy in type III secretion. (A) Western blot analysis of whole-cell lysates and culture supernatants of the indicated strains examining the presence of early (InvJ), middle (SipB), and late (SptP) substrates. (B) Electron micrographs of isolated needle complexes from the indicated S. Typhimurium strains. (C) Bacterial invasion of cultured Henle-407 cells by the indicated PrgJ mutant and control strains was determined using the gentamicin protection assay. Values represent the percentage of bacteria that survive gentamicin treatment, are normalized relative to the values of wild-type bacteria, and are the average of three determinations. **P < 0.001 relative to the values of wild type. Error bars represent standard deviation of three independent experiments.