Complementary activities of SseJ and SifA regulate dynamics of the Salmonella typhimurium vacuolar membrane

Abstract

SummaryThe Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) of Salmonella typhimurium is required for bacterial replication within host cells. It acts by translocating effector proteins across the membrane of the Salmonella-containing vacuole (SCV). The SifA effector is required to maintain the integrity of the SCV membrane, and for the formation in epithelial cells of Salmonella-induced filaments (Sifs), which are tubular extensions of SCVs. We have investigated the role in S. typhimurium virulence of the putative SPI-2 effector genes sifB, srfJ, sseJ and sseI. An S. typhimurium strain carrying a mutation in sseJ was mildly attenuated for systemic virulence in mice, but strains carrying mutations in either srfJ, sseI or sifB had very little or no detectable virulence defect after intraperitoneal inoculation. Expression of SseJ in HeLa cells resulted in the formation of globular membranous compartments (GMCs), the composition of which appears to be similar to that of SCV membranes and Sifs. The formation of GMCs was dependent on the serine residue of the predicted acyltransferase/lipase active site of SseJ. Transiently expressed SseJ also inhibited Sif formation by wildtype bacteria, and was found to associate with Sifs, SCV membranes and simultaneously expressed SifA. Intracellular vacuoles containing sseJ mutant bacteria appeared normal but, in contrast to a sifA mutant, a sifA sseJ double mutant strain did not lose its vacuolar membrane, indicating that loss of vacuolar membrane around sifA mutant bacteria requires the

Growth and killing of a Salmonella enterica serovar Typhimurium sifA mutant strain in the cytosol of different host cell lines

“…To estimate bacterial death in the absence of bacterial replication, we used an auxotrophic double mutant strain (aroC purD), which is unable to grow in cultured macrophages (Fields et al, 1986). When assayed in time-course replication assays, the aroC purD mutant strain, which remains within a vacuole throughout the course of the infection (Ruiz-Albert et al, 2002), displayed a fivefold decrease in numbers between 2 and 16 h, in close agreement with the results reported by Buchmeier & Libby (1997) (Fig. 7a).…”

“…n et al, 2000). Plasmid pFVP25.1, carrying gfpmut3A under the control of a constitutive promoter, was introduced into bacterial strains Ruiz-Albert et al (2002) for fluorescence visualization where indicated (Valdivia & Falkow, 1997 The infection was allowed to proceed for 15 min at 37 mC in 5% CO # . The monolayers were washed once with DMEM containing FCS and 100 µg gentamicin ml − " and incubated in this medium for 1 h, after which the gentamicin concentration was decreased to 16 µg ml − ".…”

“…A strain carrying a mutation in ssaV, a gene essential for SPI-2 TTSS-mediated protein secretion (Beuzo! n et al, 1999), and required for replication in HeLa cells (Cirillo et al, 1998 ;Ruiz-Albert et al, 2002), was included as a control in the assays. All strains examined carried a plasmid expressing GFP constitutively, for microscopic visualization of bacteria.…”

The Salmonella-containing vacuole—Moving with the times

“…The T3SS2 effector PipB2 enhances this process likely via direct interaction with kinesin light chain [48,49]. Sif formation involves at least one other T3SS2 effector, the deacylase SseJ, that seems to act as a counter balance to SifA since it down-regulates Sif tubule formation and, unlike the sifA deletion mutant, the double sifA sseJ deletion mutant does not escape from the SCV [50][51][52].…”

Maintenance of vacuole integrity by bacterial pathogens

“…The activity of SseJ has been shown to destabilize the SCV membrane in the absence of correct interactions with cytoskeletal motors (described above). Additional deletion of sseJ inhibits the vacuole rupture phenotype of a sifA mutant [34] and the absence of sseJ reduces the vacuole destabilizating affect of myosin II inhibition [6]. Cholesterol affects the fluidity of membranes and it has been suggested that SseJ-dependent cholesterol depletion may contribute to tubulation of the SCV by increasing membrane fluidity while also increasing susceptibility to cytoskeleton motor-dependent forces that cause membrane rupture [32].…”