The Salmonella type III secretion system inner rod protein PrgJ is partially folded

Abstract

The type III secretion system (T3SS) is essential in the pathogenesis of many bacteria. The inner rod is important in the assembly of the T3SS needle complex. However, the atomic structure of the inner rod protein is currently unknown. Based on computational methods, others have suggested that the Salmonella inner rod protein PrgJ is highly helical, forming a folded 3 helix structure. Here we show by CD and NMR spectroscopy that the monomeric form of PrgJ lacks a tertiary structure, and the only well-structured part of PrgJ is a short α-helix at the C-terminal region from residues 65-82. Disruption of this helix by glycine or proline mutation resulted in defective assembly of the needle complex, rendering bacteria incapable of secreting effector proteins. Likewise, CD and NMR data for the Shigella inner rod protein MxiI indicate this protein lacks a tertiary structure as well. Our results reveal that the monomeric forms of the T3SS inner rod proteins are partially folded.

FIGURE 1.

Sequence alignment of the T3SS inner rod proteins. Computer programs (39) predicted (Pred.) that PrgJ is mostly helical (h). NMR, however, reveals that only residues 65–82 form a regular α-helix (shaded gray) in monomeric PrgJ. Miao et al. (15) showed that the seven C-terminal residues of PrgJ are important in activating the inflammatory cytokine interleukin 1β (IL-1β). Also denoted are the YscI residues (84, 87, 94–96), that were mutated by Wood et al. (13) and shown to be defective in needle assembly. The proteins used in the alignment are PrgJ S. typhimurium; MxiI, S. flexneri; BsaK, B. pseudomallei; EprJ, E. coli 0157:H7; SctI. Photorhabdus luminescens; LscI. Photorhabdus luminescens; PscI, P. aeruginosa; AscI, Aeromonas salmonicida; and YscI, Y. pestis.

FIGURE 2.

Purified recombinant PrgJ and MxiI were transfected into mouse bone marrow-derived macrophages and probed for IL-1β by ELISA 24 h post-transfection. Control (ϕ) was transfection buffer.

FIGURE 3.

CD spectra of recombinant PrgJ with increasing amount of trifluoroethanol (TFE). A, CD spectra of full-length MxiI (B) and MxiI^CΔ5 a construct where the C-terminal five residues were truncated (C).

FIGURE 4.

Two-dimensional ¹H-¹⁵N HSQC spectra of PrgJ (A), shown with backbone assignments; overlay of the ¹H-¹⁵N HSQC spectra of full-length MxiI (black peaks) and the construct lacking the C-terminal five residues, MxiI^CΔ5 (red peaks) (B); and the ¹H-¹⁵N HSQC spectra of MxiI^CΔ5 (C).

FIGURE 5.

Secondary chemical shifts (Cα, Cβ, and Hα) suggest two potential α-helical regions in PrgJ spanning residues 30–35 and 65–82 (A). PrgJ contains mostly sequential interproton NOEs at the N-terminal region, however, residues 65–82 show α-helical NOEs (B). Heteronuclear {¹H}-¹⁵N NOE values between 0.4–0.6 (shaded) indicate PrgJ residues have backbone flexibility between that of random coil and well-defined secondary structures (C).

FIGURE 6.

Functional analysis of PrgJ I74 and Y77 glycine and proline mutations. A S. typhimurium PrgIJ deletion strain was transformed with a PrgJ plasmid and grown to late logarithmic phase. Culture pellet (P) and supernatant (S) fractions were probed with antibodies for T3SS proteins SipB, SptP, InvJ and PrgJ (A). Purified needle complexes were probed with antibodies for proteins that form the T3SS basal structure, InvG, PrgH, PrgK, and PrgJ (B). Electron micrographs of purified needle complexes (C).