SsaV Interacts with SsaL to Control the Translocon-to-Effector Switch in the Salmonella SPI-2 Type Three Secretion System

Salmonella Typhimurium is an intracellular pathogen that uses the SPI-2 type III secretion system to deliver virulence proteins across the vacuole membrane surrounding intracellular bacteria. This involves a tightly regulated hierarchy of protein secretion controlled by two molecular switches. We found that SPI-2-encoded proteins SsaP and SsaU are involved in the first but not the second secretion switch. We identify key amino acids of the inner membrane protein SsaV that are required to interact with the so-called gatekeeper protein SsaL and show that the dissociation of SsaV-SsaL causes the second switch, leading to delivery of effector proteins. Our results provide insights into the molecular events controlling virulence-associated type III secretion and suggest a broader model describing how the process is regulated. ABSTRACT Nonflagellar type III secretion systems (nf T3SSs) form a cell surface needle-like structure and an associated translocon that deliver bacterial effector proteins into eukaryotic host cells. This involves a tightly regulated hierarchy of protein secretion. A switch involving SctP and SctU stops secretion of the needle protein. The gatekeeper protein SctW is required for secretion of translocon proteins and controls a second switch to start effector secretion. Salmonella enterica serovar Typhimurium encodes two T3SSs in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. The acidic vacuole containing intracellular bacteria stimulates assembly of the SPI-2 T3SS and its translocon. Sensing the nearly neutral host cytosolic pH is required for effector translocation. Here, we investigated the involvement of SPI-2-encoded proteins SsaP (SctP), SsaU (SctU), SsaV (SctV), and SsaL (SctW) in regulation of secretion. We found that SsaP and SsaU are involved in the first but not the second secretion switch. A random-mutagenesis screen identified amino acids of SsaV that regulate translocon and effector secretion. Single substitutions in subdomain 4 of SsaV or InvA (SPI-1-encoded SctV) phenocopied mutations of their corresponding gatekeepers with respect to translocon and effector protein secretion and host cell interactions. SsaL interacted with SsaV in bacteria exposed to low ambient pH but not after the pH was raised to 7.2. We propose that SsaP and SsaU enable the apparatus to become competent for a secretion switch and facilitate the SsaL-SsaV interaction. This mediates secretion of translocon proteins until neutral pH is sensed, which causes their dissociation, resulting in arrest of translocon secretion and derepression of effector translocation. IMPORTANCE Salmonella Typhimurium is an intracellular pathogen that uses the SPI-2 type III secretion system to deliver virulence proteins across the vacuole membrane surrounding intracellular bacteria. This involves a tightly regulated hierarchy of protein secretion controlled by two molecular switches. We found that SPI-2-encoded proteins SsaP and SsaU are involved in the first but not the second secretion switch. We identify key amino acids of the inner membrane protein SsaV that are required to interact with the so-called gatekeeper protein SsaL and show that the dissociation of SsaV-SsaL causes the second switch, leading to delivery of effector proteins. Our results provide insights into the molecular events controlling virulence-associated type III secretion and suggest a broader model describing how the process is regulated.