SseK1 and SseK2 are novel translocated proteins of Salmonella enterica serovar typhimurium

Abstract

Salmonella enterica is a gram-negative, facultative intracellular pathogen that causes disease symptoms ranging from gastroenteritis to typhoid fever. A key virulence strategy is the translocation of bacterial effector proteins into the host cell, mediated by the type III secretion systems (TTSSs) encoded in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. In S. enterica serovar Typhimurium LT2, we identified the protein products of STM4157 and STM2137 as novel candidate secreted proteins by comparison to known secreted proteins from enterohemorrhagic Escherichia coli and Citrobacter rodentium. The STM4157 and STM2137 proteins, which we have designated SseK1 and SseK2, respectively, are 61% identical at the amino acid level and differ mainly in their N termini. Western analysis showed that in vitro accumulation and secretion of these proteins in serovar Typhimurium were affected by mutations in the two-component systems SsrA/B and PhoP/Q, which are key mediators of intracellular growth and survival. SPI-2 TTSS-dependent translocation of recombinant SseK1::Cya was evident at 9 h postinfection of epithelial cells, while translocation of SseK2::Cya was not detected until 21 h. Remarkably, the translocation signal for SseK1 was contained within the N-terminal 32 amino acids. Fractionation of infected epithelial cells revealed that following translocation SseK1 localizes to the host cytosol, which is unusual among the currently known Salmonella effectors. Phenotypic analysis of DeltasseK1, DeltasseK2, and DeltasseK1/DeltasseK2 mutants provided evidence for a role that was not critical during systemic infection. In summary, this work demonstrates that SseK1 and SseK2 are novel translocated proteins of serovar Typhimurium.

FIG. 1.

STM4157 (SseK1) and STM2137 (SseK2) are homologous to secreted proteins from A/E pathogens and are encoded in pathogenicity islets in serovar Typhimurium. (A) Amino acid sequence alignment of NleB, Z4328, STM2137, and STM4157 was performed by using ClustalW (http://www.ebi.ac.uk/clustalw) with default parameters for all settings and formatted using GeneDoc (http://www.psc.edu/biomed/genedoc). The amino acid sequence of NleB was predicted from the unfinished genome sequence of C. rodentium (www.sanger.ac.uk/projects/microbes). The EHEC homologue is Z4328. The amino acid sequences of STM2137 and STM4157 were predicted from the published genome sequence of serovar Typhimurium LT2 (43). (B) The STM2137 and STM4157 genes are present within low-G+C pathogenicity islets in the serovar Typhimurium LT2 chromosome. The upper and lower regions are not contiguous with each other. Arrows indicate the direction of transcription (5′ to 3′) of known and predicted ORFs. Percent G+C is indicated below each region delimited by the vertical dashed lines.

FIG. 2.

SseK1 and SseK2 protein levels are influenced by mutations affecting the SPI-2 TTSS in serovar Typhimurium SL1344. (A) Detection of SseK1::2HA in bacterial cell pellets obtained from wt and isogenic regulatory mutants. (B) Detection of SseK1::2HA in bacterial cell pellets obtained from TTSS apparatus mutants. (C) Detection of SseK1::2HA in bacterial supernatants (secreted proteins) obtained from TTSS apparatus mutants. (D) Detection of SseK2::2HA in bacterial cell pellets obtained from wt and regulatory mutants. The indicated serovar Typhimurium SL1344 strains lacking (*) plasmid or carrying either pACsseK1::2HA or pACsseK2::2HA were grown under SPI-1- or SPI-2-inducing conditions (indicated by 1 or 2, respectively) as described in Materials and Methods. Samples were analyzed by Western blotting. DnaK was included as an internal control for equal loading in each lane. No DnaK signal was detected in the supernatant (data not shown). SigD was included as a positive control for SPI-1-induced proteins (29).

FIG. 3.

Translocation of SseK1 and SseK2 into host cells. (A) Translocation of SseK1::Cya was assayed at 9 h p.i. of HeLa cells. (B) Translocation of SseK2::Cya was assayed at 21 h p.i. of HeLa cells. As indicated on the x axes, wt serovar Typhimurium SL1344 and derivative strains lacking (−) or carrying the plasmids pACsseK1::cya (sseK1), pPipBN180-Cya (PipB), or pACsseK2::cya (sseK2) were used to infect HeLa cells. The cells were lysed, and intracellular cAMP levels were determined as described in Materials and Methods. Each black bar represents the average of three independent samples analyzed in duplicate, with the standard deviations indicated by the error bars.

FIG. 4.

The amino-terminal 32 amino acids of SseK1 are necessary and sufficient to mediate its translocation. HeLa cells were infected for 9 h with wt serovar Typhimurium SL1344 lacking plasmid (wt control) or with the ΔsseK1 strain expressing the N- and C-terminally truncated SseK1::Cya recombinant proteins diagrammed on the left. The intracellular cAMP levels, determined using the Cya assay, that correspond to each construct are indicated in the graph on the right. Each black bar represents the average of three independent samples analyzed in duplicate, with standard deviations indicated by error bars. Expression and enzymatic activity of the recombinant proteins were confirmed in sonicated cellular lysates of serovar Typhimurium (data not shown).

FIG. 5.

Translocated SseK1 localizes to the host cytosol. (A) HeLa cells were infected with the indicated serovar Typhimurium SL1344 strains for 15 h prior to mechanical lysis and fractionation by differential centrifugation. Uninfected HeLa cells and HeLa cells infected with wt Salmonella lacking plasmid were included as negative controls. The resultant pellet (P), membrane (M), and cytosol (C) fractions were separated by SDS-PAGE and subjected to Western analysis as described in Materials and Methods. DnaK was included as a marker for intact bacteria, calnexin was included as a marker for host membranes, and β-tubulin was included as a marker for the host cytosol. (B) Ectopic expression of SseK1-EGFP recombinant protein in HeLa cells confirms a cytosolic localization. The left panel shows HeLa cells transfected with the pEGFP-C1 control, while the right panel shows HeLa cells transfected with pEGFP-C1(sseK1) for 40 h.