Supramolecular structure of the Salmonella typhimurium type III protein secretion system.

The type III secretion system of Salmonella typhimurium directs the translocation of proteins into host cells. Evolutionarily related to the flagellar assembly machinery, this system is also present in other pathogenic bacteria, but its organization is unknown. Electron microscopy revealed supramolecular structures spanning the inner and outer membranes of flagellated and nonflagellated strains; such structures were not detected in strains carrying null mutations in components of the type III apparatus. Isolated structures were found to contain at least three proteins of this secretion system. Thus, the type III apparatus of S. typhimurium, and presumably other bacteria, exists as a supramolecular structure in the bacterial envelope.

Stimulation by gonococci of chloride ion uptake in human leucocytes in relation to other properties of phagocytosis.

Human polymorphonuclear leucocytes (PMNL) underwent several changes in response to challenge with Neisseria gonorrhoeae, namely (1) an increase in oxygen uptake, (2) changes in membrane electrical properties, and (3) increased transport of chloride ions (Cl-) across the membrane. Mean oxygen consumption and Cl- uptake by PMNL were stimulated by both pilate (P+) and non-pilate (P-) gonococci, although the levels were much reduced in the presence of P+ organisms. P+ gonococci also initiated low levels of polarisation or depolarisation in contrast with P- cells, which caused hyperpolarisation followed by depolarisation in the PMNL. Most of the strains showed these patterns. High performance liquid chromatography of extracts of unstimulated PMNL and of PMNL challenged with gonococci confirmed production of hypochlorous acid (HOCl) in the leucocyte. Furthermore, addition of radiolabelled Cl- to the PMNLs showed that some of the Cl- taken up by the cells in response to gonococcal challenge was incorporated into the HOCl, suggesting a direct relationship between stimulation of Cl- uptake and production of active chlorine compounds in the leucocyte.

The invasion-associated type III secretion system of Salmonella typhimurium: common and unique features.

Several bacterial pathogens make use of a specialized protein secretion system to inject effector proteins into host cells. This system, commonly referred to as type III secretion, is always associated with phenotypes related to intimate interactions between the pathogen and its respective host cells. The enteric pathogen Salmonella typhimurium utilizes a type III secretion system to invade nonphagocytic intestinal epithelial cells. Whereas the invasion-associated type III system of S. typhimurium has evolved to perform a specific function, many of the components of this system are conserved among the type III systems of other bacterial pathogens. This review will discuss the common and unique features of the S. typhimurium system in relation to the type III systems of other human pathogens. Topics discussed include the phenotypes associated with various type III systems, the genetic loci encoding these systems, the components of the type III secretion apparatus, the effector proteins and the mechanisms by which they enter host cells as well as the mechanisms used to regulate the expression of type III systems.

Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP.

The gene encoding the Pseudomonas aeruginosa phosphate-specific porin OprP was subjected to both linker and epitope insertion mutageneses. Nine of the 13 linker mutant genes expressed protein at levels comparable to those obtained with the wild-type gene. These mutant proteins were shown, by indirect immunofluorescence with an OprP-specific antiserum, to be properly exposed at the cell surface. Four of the linker mutant genes expressed protein at reduced levels which were not detectable at the cell surface. A foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum was cloned into the linker sites of 12 of the 13 mutant genes. Seven of the resultant epitope insertion mutant genes expressed surface-exposed protein. Two of these mutant genes presented the foreign epitope at surface-accessible regions as assessed by indirect immunofluorescence with a malarial epitope-specific monoclonal antibody. The data from these experiments were used to create a topological model of the OprP monomer.

The role of specific lysine residues in the passage of anions through the Pseudomonas aeruginosa porin OprP.

When grown under phosphate-limiting conditions Pseudomonas aeruginosa expresses the phosphate-specific porin OprP. In order to determine whether any of the lysine residues located in the amino-terminal half of the protein play a role in the transport of anions through the channels, the first nine amino-terminal lysine residues of OprP were substituted with glutamates. The mutant proteins were purified and the channels they formed were characterized by reconstituting the purified porins in planar lipid membranes. In comparison to the wild-type protein, the Lys74, Lys121, and Lys126 mutants all displayed reduced levels of conductance at KCl concentrations below 1 M, and the Lys74 and Lys121 mutants no longer exhibited a saturation of conductance at high anion concentrations. In addition, the ability of phosphate ions to inhibit the conductance of Cl- ions through the channels formed by the Lys121 mutant was greatly reduced, while their ability to inhibit the Cl- conductance of the Lys74 mutant was reduced by approximately 2-fold. To clarify the roles that Lys74, Lys121, and Lys126 play in regulating the channel characteristics of OprP, these amino acids were replaced with either glycine or glutamine residues. Analysis of these mutants suggested that both Lys74 and Lys126 may serve to funnel anions toward the binding site, but only the presence of Lys121 is required for the formation of the inorganic phosphate-specific binding site of OprP.

Genetic analysis of assembly of the Salmonella enterica serovar Typhimurium type III secretion-associated needle complex.

Several pathogenic bacteria have evolved a specialized protein secretion system termed type III to secrete and deliver effector proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium uses one such system to mediate entry into nonphagocytic cells. This system is composed of more than 20 proteins which are encoded within a pathogenicity island (SPI-1) located at centisome 63 of its chromosome. A subset of these components form a supramolecular structure, termed the needle complex, that resembles the flagellar hook-basal body complex. The needle complex is composed of a multiple-ring cylindrical base that spans the bacterial envelope and a needle-like extension that protrudes from the bacterial outer surface. Although the components of this structure have been identified, little is known about its assembly. In this study we examined the effect of loss-of-function mutations in each of the type III secretion-associated genes encoded within SPI-1 on the assembly of the needle complex. This analysis indicates that the assembly of this organelle occurs in discrete, genetically separable steps. A model for the assembly pathway of this important organelle is proposed that involves a sec-dependent step leading to the assembly of the base substructure followed by a sec-independent process resulting in the assembly of the needle portion.

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

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.