Aggregation of host endosomes by Salmonella requires SPI2 translocation of SseFG and involves SpvR and the fms–aroE intragenic region

Abstract

SummarySalmonella-induced aggregation of host endosomal compartments into tubules, termed lgp-tubules, requires sifA and ompR. Lgp-tubules result from Salmonella-directed alteration of the endocytic system and typify the unique intracellular locale where Salmonella replicate. A high-throughput method devised to screen 11 520 MudJ mutants for loss of lgp-tubule formation identified one auxotrophic and nine prototrophic mutants. Molecular characterization identified four new loci required to alter epithelial endocytic structure. Salmonella pathogenicity island 2 (SPI2) is the locus central to the phenotype. A subset of SPI2 effectors is essential: SpiC and SseFG are required, but not SseE. A subset of apparatus proteins is also implicated: SsaJ, L, M, V and P are required. SPI2 was implicated further, as SifA shows similarity with known SPI2 translocation targets, and OmpR regulates SPI2. Another locus lies within the smf±aroE intragenic region. Lgp-tubule formation also involves a locus on the virulence plasmid pSLT. The pSLT-encoded SpvR negatively regulates an unknown repressor of the phenotype located on pSLT. Finally, disruption of carB leads to multiple auxotrophy that prevents lgp-tubule formation. This study demonstrates that lgp-tubule formation is a virulence mechanism that underlies the selective disruption of host endocytic trafficking and is associated with the formation of a replicationpermissive locale.

SseA is a chaperone for the SseB and SseD translocon components of the Salmonella pathogenicity-island-2-encoded type III secretion system

“…Although we cannot rule out this possibility, it seems unlikely that this is the case since the phenotypes of strains lacking other proteins encoded in the sseA-G operon are different from those observed for the sseA psseBD strain. Strains defective in SseF and SseG are only mildly attenuated in virulence and partially affected in Sif formation (Guy et al, 2000;Hansen-Wester et al, 2002;Hensel et al, 1998), whereas a strain defective in SseE is not attenuated in virulence and has no other reported phenotype (Hensel et al, 1998). SscA is predicted to be a chaperone for SseC on the basis of sequence similarity (Cirillo et al, 1998;Hensel et al, 1998;Neyt & Cornelis, 1999) and SscB is also predicted to be a chaperone (Cirillo et al, 1998;Hensel et al, 1998).…”

“…Mutation of sseE does not lead to a noticeable attenuation of virulence (Hensel et al, 1998); SseF and SseG have been shown to be translocated into the host cell (Hansen-Wester et al, 2002;Kuhle & Hensel, 2002). Strains carrying mutations in sseF or sseG are mildly attenuated and partially defective in the formation of tubular membranous structures known as Salmonella-induced filaments (Sifs), which are induced upon SPI-2-mediated translocation of the effector protein SifA into HeLa cells (Beuzó n et al, 2000;Brumell et al, 2002;Garcia-del Portillo et al, 1993;Guy et al, 2000;Kuhle & Hensel, 2002;Stein et al, 1996). The first gene of the operon encodes SseA, a small protein that contains a domain with potential to form a coiled-coil structure, but which has no significant sequence similarity to other proteins (Hensel et al, 1998).…”

The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

“…HeLa cells were grown in Earle's minimal essential medium (ATCC 30-2003) containing 2 mM glutamine and supplemented with 10 % fetal calf serum. HeLa cells were invaded using a standard gentamicin protection assay (Guy et al, 2000). Six hours after invasion, cells were fixed in 2?5 % formaldehyde in PBS.…”

“…5b) to restore SPI2 effector translocation via inducible intracellular complementation (Stein et al, 1996;Guy et al, 2000). The indicator of ex vivo effector translocation was the Sif phenotype, which requires translocation of at least three effectors (sifA, sseF and sseG) by the SPI2 TTS system (Stein et al, 1996;Guy et al, 2000).…”

“…Loss of SseA prevents Sif formation, but the phenotype is nearly fully restored when SseA is provided in trans (see below). HeLa cells were infected with DsseA/pSA or DsseA/pSA Q17D,Q31D and induced by addition of arabinose to the extracellular media (Guy et al, 2000). The negative control, DsseA, failed to generate Sif.…”