Quantitative mass spectrometry catalogues Salmonella pathogenicity island-2 effectors and identifies their cognate host binding partners

Abstract

Gram-negative bacterial pathogens have developed specialized secretion systems to transfer bacterial proteins directly into host cells. These bacterial effectors are central to virulence and reprogram host cell processes to favor bacterial survival, colonization, and proliferation. Knowing the complete set of effectors encoded by a particular pathogen is the key to understanding bacterial disease. In addition, the identification of the molecular assemblies that these effectors engage once inside the host cell is critical to determining the mechanism of action of each effector. In this work we used stable isotope labeling of amino acids in cell culture (SILAC), a powerful quantitative proteomics technique, to identify the proteins secreted by the Salmonella pathogenicity island-2 type three secretion system (SPI-2 T3SS) and to characterize the host interaction partners of SPI-2 effectors. We confirmed many of the known SPI-2 effectors and were able to identify several novel substrate candidates of this secretion system. We verified previously published host protein-effector binding pairs and obtained 11 novel interactions, three of which were investigated further and confirmed by reciprocal co-immunoprecipitation. The host cell interaction partners identified here suggest that Salmonella SPI-2 effectors target, in a concerted fashion, cellular processes such as cell attachment and cell cycle control that are underappreciated in the context of infection. The technology outlined in this study is specific and sensitive and serves as a robust tool for the identification of effectors and their host targets that is readily amenable to the study of other bacterial pathogens.

FIGURE 1.

Schematic overview of the secretome analysis. A, S. typhimurium SL1344 ssaR were grown in defined minimal medium (LPM) containing regular abundance arginine (R) and lysine (K); SL1344 wild type were grown in the same medium containing Arg and Lys labeled with heavy isotopes. (Both bacterial strains were also deleted for their argH, lysA, and fliF genes; see text under “Results.”) B, bacterial cells were pelleted and culture supernatants filtered, pooled, and concentrated. C, proteins from concentrated supernatants were precipitated and subjected to in-gel digestion. D, tryptic peptides were analyzed by LC-MS/MS. Proteins secreted by the SPI-2 T3SS are characterized by enrichment of peptides containing heavy isotopes.

FIGURE 2.

SILAC ratios of proteins detected in culture supernatants. Ratios of MS peak intensities under SPI-2 secretion versus nonspecific conditions (SPI-2/nSp) are plotted for each protein. A total of 758 proteins were identified in the analysis (inset). Most hits with a ratio of 3 or higher corresponded to known SPI-2 T3SS effectors (black bars) or translocon components (gray bars).

FIGURE 3.

Secretion of PtpS (STM2949). Shown is a Western blot analysis of bacterial pellets and culture supernatants of wild type SL1344 and ssaR transformed with either ptpS-2xHA or a control plasmid, grown in minimal medium. Western blots (IB) were probed with antibodies directed against HA (for detection of PtpS), DnaK (pellet control), and SseB (secretion control). *, nonspecific band.

FIGURE 4.

Schematic overview of the interaction partner screen. For strategy A, effector genes were inserted into a mammalian expression vector as tandem HA epitope fusions (A); HA-tagged effectors were expressed in HEK293T grown in regular medium (B); lysates from these cells, as well as untransfected control cells labeled with heavy isotopes, were subjected to immunoprecipitation with an anti-HA antibody (C); bound protein was eluted, samples were pooled, proteins were digested with trypsin (D); and tryptic peptides were analyzed by LC-MS/MS (E). For strategy B, effector genes were inserted into a bacterial expression vector as GST tandem HA epitope fusions, and effectors were expressed in E. coli and purified (F); purified effectors were added to lysates from HEK293T cells grown in the presence of heavy isotopes, and these lysates, as well as control lysates, were subjected to immunoprecipitation with an anti-HA antibody (C); the following steps were common to strategy A (D and E). G, Western blot stained with anti-HA showing expression of effectors in HEK293T. In this example, expression levels of SseI and SseL were classified as “high,” SseG as “intermediate,” and SseD and SsaB as “low” (see Table 2). *, nonspecific band. H, SDS-PAGE of effectors purified from E. coli.

FIGURE 5.

Representative mass spectra for immunoprecipitations (IP) with SseJ (A–C) and SspH1 (D and E). Mass/charge (m/z) spectra of individual peptides are shown for GAPDH (A), RhoA (B), RhoC (C), eEF1a (D), and serine/threonine PKN1 (E). Peptide sequences and SILAC ratios (light/heavy) for each peptide are shown.

FIGURE 6.

Confirmation of binding of SspH2 to Sgt1 and Bub3 and SseL to OSBP. A, HEK293T lysates transfected with either SspH2 or a vector control were subjected to immunoprecipitation using antibodies directed against SspH2 or Sgt1 or with normal mouse IgG as an antibody control. Samples were analyzed by Western blot (IB) probing for Sgt1 and SspH2. B, HEK293T lysates transfected with either OSBP or a vector control were incubated with recombinant SseL and subjected to immunoprecipitation using antibodies against OSBP or SseL. Samples were analyzed by Western blot probing for SseL and OBSP. C, HEK293T lysates transfected with either SspH2 or a vector control were subjected to immunoprecipitation using antibodies directed against SspH2 or Bub3 or with normal mouse IgG as an antibody control. Samples were analyzed by Western blot probing for Bub3 and SspH2.

FIGURE 7.

Biological processes affected by effectors analyzed in this study. S. typhimurium effectors binding to host cell targets suggest a set of biological contexts to be influenced by infection. SseG (G) and SspH2 (H2) bind, respectively, to Caprin-1 and Bub3/AH receptor-interacting protein (AIP)/Sgt-1, host proteins influencing cell cycle control. SseF (F), SseG, SseL (L), and SseJ (J) target proteins located at desmosomes or focal adhesions, suggesting a role in the regulation of cell attachment. SspH1 (H1), SspH2, and SseL target PKN1, Sgt1, and OSBP, respectively, arguing for a possible role in the regulation of innate immunity.