RESUMO
The Pseudomonas aeruginosa type III secretion system (TTSS) protein PscJ belongs to the PrgK family of TTSS proteins. These proteins are predicted to form one of the inner membrane localized ring substructures of the TTSS needle complex. To determine which amino acid residues of PscJ are important for its function, the pscJ gene was subjected to site-directed mutagenesis. Fifteen individual PscJ amino acid residues that are located in conserved regions of the PrgK family were targeted for mutagenesis. Eight of these residues could be subjected to non-conservative substitution mutagenesis without affecting the function of the resultant mutant protein. Substitution of the other 7 residues (E26, K52, E105, A107, G126, H133, and V189) resulted in either a non-functional protein or the loss of detectable protein. When the essential residues were mapped on to the crystal structure of the E. coli PrgK homolog EscJ, the majority appeared to localize to surface-exposed regions of the protein suggesting a role for these regions in the assembly of the PscJ ring structure.
Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Membrana/química , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Transporte Proteico , Pseudomonas aeruginosa/metabolismoRESUMO
Type III secretion systems (TTSSs) mediate translocation of virulence factors into host cells. We report the 17-angstrom resolution structures of a central component of Salmonella typhimurium TTSS, the needle complex, and its assembly precursor, the bacterial envelope-anchored base. Both the base and the fully assembled needle complex adopted multiple oligomeric states in vivo, and needle assembly was accompanied by recruitment of the protein PrgJ as a structural component of the base. Moreover, conformational changes during needle assembly created scaffolds for anchoring both PrgJ and the needle substructure and may provide the basis for substrate-specificity switching during type III secretion.
Assuntos
Proteínas de Bactérias/ultraestrutura , Salmonella typhimurium/ultraestrutura , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Substâncias Macromoleculares , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/ultraestrutura , Salmonella typhimurium/químicaRESUMO
An essential component of type III secretion systems (TTSS) is a supramolecular structure termed the needle complex. In Salmonella enterica, at least four proteins make up this structure: InvG, PrgH, PrgK, and PrgI. Another protein, PrgJ, is thought to play a role in the assembly of this structure, but its function is poorly understood. We have analyzed the expression and localization of PrgJ and the needle protein PrgI in different S. enterica serovar Typhimurium mutant strains. We found that the levels of PrgI and PrgJ were significantly reduced in a TTSS-deficient invA mutant strain and that the decreased levels were due to protein instability. In addition, we found that PrgJ, although associated with the needle complex in wild-type S. enterica serovar Typhimurium, was absent from needle complexes obtained from an invJ mutant strain, which exhibits very long needle substructures. We suggest that PrgJ is involved in capping the needle substructure of the needle complex.
