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Comparative Study
. 2000 Aug 29;97(18):10225-30.
doi: 10.1073/pnas.170128997.

Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system

T Kubori  1 , A Sukhan, S I Aizawa, J E Galán
Affiliations
Comparative Study

Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system

T Kubori et al. Proc Natl Acad Sci U S A. .

Abstract

Many bacterial pathogens of plants and animals have evolved a specialized protein-secretion system termed type III to deliver bacterial proteins into host cells. These proteins stimulate or interfere with host cellular functions for the pathogen's benefit. The Salmonella typhimurium pathogenicity island 1 encodes one of these systems that mediates this bacterium's ability to enter nonphagocytic cells. Several components of this type III secretion system are organized in a supramolecular structure termed the needle complex. This structure is made of discrete substructures including a base that spans both membranes and a needle-like projection that extends outward from the bacterial surface. We demonstrate here that the type III secretion export apparatus is required for the assembly of the needle substructure but is dispensable for the assembly of the base. We show that the length of the needle segment is determined by the type III secretion associated protein InvJ. We report that InvG, PrgH, and PrgK constitute the base and that PrgI is the main component of the needle of the type III secretion complex. PrgI homologs are present in type III secretion systems from bacteria pathogenic for animals but are absent from bacteria pathogenic for plants. We hypothesize that the needle component may establish the specificity of type III secretion systems in delivering proteins into either plant or animal cells.

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Figures

Figure 1
Figure 1
Role of the type III secretion protein-export apparatus in the assembly of the S. typhimurium needle complex. Electron micrographs of negatively stained, osmotically shocked wild-type S. typhimurium and the isogenic invA and invC mutant derivatives. The positions of needle complexes and bases are indicated by arrows. (Bar = 100 nm.)
Figure 2
Figure 2
Identification of the components of the base structure of the needle complex. Type III secretion components were isolated from a S. typhimurium invA mutant strain, purified on a CsCl density gradient, and visualized by silver staining (A) or by Western immunoblotting by using an antibody specific to InvG, PrgH, and PrgK (B). The identity of the components is indicated. (C) Electron micrographs of negatively stained purified complexes from the S. typhimurium invA mutant showing the absence of the needle substructure. (Bar = 50 nm.)
Figure 3
Figure 3
InvJ controls the length of the needle substructure of the type III secretion complex. Electron micrographs of negatively stained, osmotically shocked wild-type S. typhimurium (A) and its isogenic invJ mutant derivative (B), showing the lengthening of the needle substructure in the absence of the InvJ protein (compare A with B). After vortexing, long needle substructures separated from the bacterial cell but still attached to the type III secretion complex base substructure were seen occasionally (C). Introduction into the invJ mutant strain of a plasmid carrying a wild-type copy of invJ restored the proper needle length (D). Overexpression of hilA, which encodes a transcriptional regulator of the type III secretion system, resulted in the accumulation of large amounts of exceedingly long needle substructures observed on electron micrographs of a negatively stained S. typhimurium invJ mutant (E). Introduction into this strain of a plasmid carrying a wild-type copy of invJ resulted in the disappearance of the unusually elongated needle substructures (F). Likewise, introduction of an invA mutation, which abrogates type III secretion, resulted in the absence of needle filaments (G). Arrows indicate the position of the needle complexes in the bacterial envelope. [Bar = 100 nm (Upper) and 1 μm (Lower).]
Figure 4
Figure 4
Isolation and identification of the main subunit of the needle substructure. (A) Needle structures were isolated from a S. typhimurium invJ mutant strain and purified on a CsCl density gradient, and the different fractions were loaded on a SDS/polyacrylamide gel and visualized by silver staining. An electron micrograph of negatively stained purified needle structures from fraction 5 of the CsCl gradient is shown. (Bar = 100 nm.) (B) Purified needle complexes from wild-type S. typhimurium and the isogenic invA or invJ mutants were separated on a SDS/polyacrylamide gel and visualized by silver staining or transferred to nitrocellulose membranes and probed with an antibody directed against PrgI. The identities of the different proteins are indicated.
Figure 5
Figure 5
Amino acid sequence alignment of S. typhimurium PrgI and its homologs in type III secretion systems from other animal pathogenic bacteria. Included in the alignment are PrgI from S. typhimurium, MxiH from S. flexneri, YscF from Yersinia enterocolitica, PscF from P. aeruginosa, and EscF from enteropathogenic E. coli.
Figure 6
Figure 6
Electron micrographs of negatively stained, osmotically shocked S. typhimurium prgI mutant strain (A and B) and the same strain carrying a complementing plasmid encoding wild-type prgI (C). (Bar = 100 nm.)
Figure 7
Figure 7
S. typhimurium prgI mutants are defective for type III secretion. (A) Total culture supernatant protein profile of wild-type S. typhimurium, the secretion-defective invA mutant, the prgI mutant strain, and the complemented prgI mutant strain. (B) Western immunoblot analysis of culture supernatant proteins from the same strains probed with mAbs directed to the type III secreted proteins SipB and SipC.
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