InvB is a type III secretion chaperone specific for SspA

Abstract

A wide variety of gram-negative bacteria utilize a specialized apparatus called the type III secretion system (TTSS) to translocate virulence factors directly into the cytoplasm of eukaryotic cells. These translocated effectors contribute to the pathogen's ability to infect and replicate within plant and animal hosts. The amino terminus of effector proteins contains sequences that are necessary and sufficient for both secretion and translocation by TTSS. Portions of these sequences contain binding sites for type III chaperones, which facilitate efficient secretion and translocation of specific effectors through TTSS. In this study, we have utilized the yeast two-hybrid assay to identify protein-protein interactions between effector and chaperone proteins encoded within Salmonella pathogenicity island 1 (SPI-1). Several interactions were identified including a novel interaction between the effector protein, SspA (SipA), and a putative chaperone, InvB. InvB was demonstrated to bind to the amino terminus of SspA in the bacterial cytoplasm. Furthermore, InvB acts as a type III chaperone for the efficient secretion and translocation of SspA by SPI-1. InvB also permitted translocation of SspA through the SPI-2 TTSS, indicating that it is an important regulator in the recognition of SspA as a target of TTSS. Finally, it was determined that InvB does not alter the transcription of sspA but that its absence results in reduced SspA protein levels in Salmonella enterica serovar Typhimurium.

FIG. 1

Yeast two-hybrid interactions. A subset of genes encoding SPI-1-secreted proteins and potential chaperones were cloned into pGBT9 and pGAD424 to create fusion proteins with the GAL4 binding and activation domains, respectively. These fusion proteins were expressed in yeast, and positive interactions (+) were identified by growth on histidine-deficient medium and β-galactosidase activity. When fused to the GAL4 binding domain, some proteins caused nonspecific activation of Gal1-regulated genes (NA). All results were confirmed by testing interactions in fresh background strains.

FIG. 2

Immunoprecipitation of InvB from Salmonella serovar Typhimurium. Either wild-type Salmonella serovar Typhimurium (A) (CS401) or wild-type bacteria expressing either SspA-CyaA (pB500) or SspH1-CyaA (pEM25) (B) were grown to logarithmic phase, and cytoplasmic fractions were isolated after sonication. The resulting lysate was precleared with protein A-Sepharose, and proteins were immunoprecipitated by the addition of various sera and protein A-Sepharose. Proteins associated with the Sepharose beads were collected by centrifugation and solubilized by boiling in SDS-PAGE sample buffer. All protein samples were resolved by SDS-PAGE and analyzed with Western blotting with sera specific for InvB. Results shown are representative of multiple experiments.

FIG. 3

Secretion defects of a nonpolar chromosomal deletion of invB in Salmonella serovar Typhimurium. Culture supernatants were collected from either the wild-type strain (CS401), the ΔinvB strain (PB502), or the ΔinvB strain with a plasmid-borne copy of invB (pB502). Bacterial cultures were grown overnight, and supernatants were collected after centrifugation. Secreted proteins were precipitated with trichloroacetic acid and separated by SDS-PAGE. Proteins were visualized either by Coomassie blue staining (A) or by Western blotting with antibodies specific to SspA (B). Culture supernatants were also collected from wild-type (CS401) and ΔinvB (PB502) strains expressing the SPI-1-secreted fusion protein SspH1-CyaA. Supernatant proteins were separated by SDS-PAGE and analyzed by Western blotting with monoclonal antibodies specific for CyaA (C). Results presented are representative of multiple supernatant preparations.

FIG. 4

SPI-1 TTSS translocation phenotype of ΔinvB strain. Intracellular cAMP levels were determined in RAW264.7 macrophages after a 1-h infection with the wild-type strain (CS401), the ΔsspC strain (CAS108), or the ΔinvB strain (PB502) expressing SspA-CyaA (pB500) or SspH1-CyaA (pEM25). cAMP levels were determined by enzyme immunoassay and normalized for protein content. Standard deviations of triplicate wells are shown for an assay representative of three performed.

FIG. 5

Effects of InvB expression on SPI-2 TTSS translocation. Intracellular cAMP levels were determined in RAW264.7 macrophages after a 6-h infection with the wild-type strain (CS401), the ΔprgH-K strain (TK91), the ssaT strain (EM323), or the ΔinvB strain (PB502) expressing SspA-CyaA (pB500), SspA-CyaA and InvB (pEM120), or SspH1-CyaA (pEM25). cAMP levels were determined as for Fig. 4. Results shown are representative of three assays.

FIG. 6

Effect of invB deletion on transcription of sspA. Transcription of sspA was measured in wild-type strains (PB525), ΔinvB strains (PB523), and ΔinvB strains containing a plasmid expressing invB (pB502). All strains contain sspA::Tn5lac chromosomal fusions. Stationary-phase bacteria were diluted in LB medium and grown for 3 h. Samples were assayed for β-galactosidase activity every 30 min from an OD₆₀₀ of ∼0.3 to ∼2.0. Standard deviations of triplicate samples are shown. These experiments were performed three times with similar results.

FIG. 7

Western blot analysis of cytoplasmic levels of SspA. Bacterial strains deficient for secretion were grown overnight, back diluted, and grown to exponential growth phase. Samples from ΔprgH (TK164) and ΔprgH ΔinvB (PB509) strains were collected, resolved using SDS-PAGE, and analyzed by Western blotting using antibodies specific for SspA. The amount of SspA present in these strains was quantitated using a STORM840 Phosphoimaging System and by analyzing the data with Imagequant V1.2. Percent SspA levels are presented with standard deviations of triplicate samples. These experiments were repeated multiple times.