InvF is required for expression of genes encoding proteins secreted by the SPI1 type III secretion apparatus in Salmonella typhimurium

Abstract

The expression of genes encoding proteins secreted by the SPI1 (Salmonella pathogenicity island) type III secretion apparatus is known to require the transcriptional activators SirA and HilA. However, neither SirA nor HilA is believed to directly activate the promoters of these genes. invF, the first gene of the inv-spa gene cluster, is predicted to encode an AraC-type transcriptional activator and is required for invasion into cultured epithelial cells. However, the genes which are regulated by InvF have not been identified. In this work, an in-frame deletion in invF was constructed and tested for the expression of Phi(sigD-lacZYA), sipC::Tn5lacZY, and a plasmid-encoded Phi(sicA-lacZYA). SigD (Salmonella invasion gene) is a secreted protein required for the efficient invasion of Salmonella typhimurium into cultured eucaryotic cells. sicA (Salmonella invasion chaperone) is the first gene of a putative operon encoding the Sip/Ssp (Salmonella invasion/Salmonella secreted proteins) invasion proteins secreted by the SPI1 type III export apparatus. invF was required for the expression of the sigD, sicA, and sipC fusions. This is the first demonstration that there is a functional promoter in the intergenic sequence between spaS and sicA. In addition, several proteins were either absent from or found in reduced amounts in the culture supernatants of the invF mutant. Therefore, invF is required for the optimal expression of several genes encoding SPI1-secreted proteins. Genetic evidence is also presented suggesting there is HilA-dependent readthrough transcription from the invF promoter at least through sipC.

FIG. 1

Supernatant proteins from wild-type and ΔinvF strains. Lane 1, supernatant proteins from the secretion defective spaS mutant SVM514; lanes 2 to 7, S. typhimurium SL1344 containing pWSK130, pHD9 (invF⁺), and philA (lanes 2 to 4) and the invF mutant SVM579 containing the same plasmids in the same order (lanes 5 to 7); lanes 8 and 9, supernatant proteins from SVM579 strains containing the vector pVLT33 (lane 8) and the IPTG-inducible sigDE clone pHH37 (lane 9). Positions of molecular weight standards are indicated in kilodaltons on the left; previously identified secreted proteins are indicated on the right. Question marks denote proteins that have not been confirmed by immunoblot analysis. Proteins were prepared and analyzed as described in Materials and Methods.

FIG. 2

Model for the regulation of invasion/virulence gene expression in S. typhimurium. The direction of transcription for each gene cluster is indicated by closed arrows; open arrows represent putative transcripts of the inv-spa and sip/ssp genes. Question marks indicate either unidentified regulatory factors or unclear relationships between the designated regulator and the noted promoter.