Contribution of Salmonella typhimurium type III secretion components to needle complex formation

Abstract

The prgHIJK operon encodes components of the Salmonella typhimurium pathogenicity island 1 type III secretion system (TTSS). Previously, prgH and prgK were shown to be required for formation of the supramolecular type III secretion needle complex (NC) [Kubori, T., et al. (1998) Science 280, 602-605]. This work indicates that all prg operon genes are required for NC formation. PrgH multimerizes into a distinct tetrameric-shaped structure that may be an early intermediate of NC assembly and may provide the structural foundation required for PrgK oligomerization. PrgH and PrgK, in the absence of other TTSS components, oligomerize into ring-shaped structures identical in appearance and size to the base of the NC, indicating that they are likely the major inner membrane structural components required for secretion. PrgI and PrgJ cofractionate with the NC and are secreted into the culture supernatant. NC from prgI and prgJ mutants have an identical morphology to the envelope-spanning (basal body) NC components, but are missing the external needle, indicating that PrgI and PrgJ are required for full NC assembly and are likely components of the external needle. Therefore, PrgI and PrgJ are secreted through the NC basal body, composed in part of PrgH/K and InvG/H rings, to participate in assembly of the more distal components of the NC.

Figure 1

Each of the prgs is required for type III secretion. SDS/PAGE analysis of secreted proteins from WT S. typhimurium (CS401), isogenic strains harboring nonpolar deletions of each of the prg genes; ΔH (TK164), ΔI (TK25), ΔJ (TK26), ΔK (TK93) and each of the corresponding complemented strains; ΔH + pWprgH (pTK30), ΔI + pWprgI (pTK31), ΔJ + pWprgJ (pTK32), and ΔK + pWprgK (pTK33). The samples were prepared from overnight cultures grown with mild aeration. The identity of proteins secreted by the SPI1 TTSS and flagellar system are indicated on the left.

Figure 2

PrgI and PrgJ are found in the culture supernatant. (A) Western blot analysis of whole cell lysates (C) and culture supernatants (S) from WT S. typhimurium (CS401), the isogenic prg deletion strains: ΔH (TK164), ΔI (TK25), ΔJ (TK26), ΔK (TK93), and each of the corresponding complemented strains: ΔH + pWprgH (pTK30), ΔI + pWprgI (pTK31), ΔJ + pWprgJ (pTK32), ΔK + pWprgK (pTK33). Cells were grown to late log phase with mild aeration. The amounts of whole cell lysate (C) and supernatant (S) fractions loaded per lane represent equivalent amounts of total culture volume. Blots were probed with polyclonal antibodies directed against SipA, InvJ, PrgH, PrgI, PrgJ, and PrgK as indicated. (B) Western blot analysis of whole cell lysates, and culture supernatants from WT (CS401) and prg-deletion strains: ΔH (TK164), ΔI (TK25), ΔJ (TK26), ΔK (TK93) harboring plasmid pBADprgJ (pTK32). Equivalent amounts of whole cell lysate and supernatant fractions were loaded per lane. Blots were probed with anti-PrgJ and anti-SipA polyclonal antiserum as indicated.

Figure 3

prgI and prgJ are required for needle structure formation. (A) Western-blot analysis of whole cell lysate (C), culture supernatant (S), and postCsCl needle complex fractions (NC) isolated from WT Salmonella (TK328). The amount of C and S fractions loaded represents equivalent amounts of total culture volume (0.2 ml), the NC represents 25 ml of culture. The blot was probed with polyclonal antiserum against PrgH, PrgI, PrgJ, PrgK, and InvJ as indicated. (B) Western blot analysis of PreCsCl NC fractions isolated from WT and prg deletion strains: WT (TK328), ΔH (TK334), ΔI (TK335), ΔJ (TK336), ΔK (TK337), ΔH-K (TK338). The blot was probed with polyclonal antiserum against PrgH, PrgI, PrgJ, and PrgK as indicated. (C) Electron micrographs of NC isolated from WT (TK328). Complexes came from same fraction (NC) analyzed in A. Samples were negatively stained with 2% PTA (pH 7.2) and observed under a JEM-1200EXII transmission electron microscope. Approximate sizes of the NC are listed in the schematic (D) (n = 30). (Scale bar, 50 nm.) (D) Electron micrographs of partial NC isolated from WT (TK328), ΔprgI (TK335), and ΔprgJ (TK336) deletion mutants. A schematic comparing the size and morphology of these structures to WT structures is also shown. Approximate sizes are given in schematic (n = 30). (Scale bars, 50 nm and 25 nm.)

Figure 4

Prg complex isolation from E. coli. (A) SDS/PAGE of whole cell lysates (WC) and needle complex fractions (NC) prepared from recombinant E. coli (DH5α) expressing prgH (pTK43), prgK (pTK44), or prgH/prgK (pTK53). (B) Western blot analysis of PrgH isolated from E. coli (from A). The sample was resuspended in SDS loading buffer and boiled (+) or directly loaded (−) onto the gel. Blot was probed with polyclonal antiserum to PrgH. (C and D) Electron micrographs of (C) PrgH isolated from E. coli, and (D) NC isolated from WT S. typhimurium. In both C and D, numerous tetrameric-shaped structures can be seen. (Scale bar, 50 nm.) (E) Enlarged image of one of the tetramers from C with a schematic showing an approximate size of 10 nm × 10 nm (n = 25). (Scale bar, 50 nm.)

Figure 5

PrgH and PrgK form the inner membrane rings of the NC. (A and B) Representative electron micrograph images of PrgH/PrgK complexes from recombinant E. coli (DH5α) expressing prgH/prgK (pTK42) or prgHIJK (pTK53). (B) Electron micrographs of individual ring structures (top), rectangular structures (side), and oblique view of same structures. (Scale bar, 50 nm.) (C) Comparison of two different side views of the PrgH/K complex with NC isolated from WT Salmonella. Note that the differences seen in these structures are reflected in the differences seen in the base structure of the NC. Scale bar, 50 nm. (D) Schematic comparing the approximate size and shape of the isolated Prg components: tetramer, 10 nm (n = 25); rectangular (side), width 21 nm, height 9 nm (n = 27); ring (top), outer diameter 21 nm, inner diameter 12 nm (n = 30).