Discovery of Novel Secreted Virulence Factors from Salmonella enterica Serovar Typhimurium by Proteomic Analysis of Culture Supernatants

Journal Article · Sat Jan 01 04:00:00 EST 2011 · Infection and Immunity, 79(1):33-43

DOI:https://doi.org/10.1128/IAI.00771-10· OSTI ID:1004798

Niemann, George; Brown, Roslyn N.; Gustin, Jean K.; Stufkens, Afke; Shaikh-Kidwai, Afshan S.; Li, Jie; McDermott, Jason E.; Brewer, Heather M.; Schepmoes, Athena A.; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

The intracellular pathogen Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis in the world. This pathogen has two type-III secretion systems (TTSS) necessary for virulence that are encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) and are expressed during extracellular or intracellular infectious states, respectively, to deliver virulence factors (effectors) to the host cell cytoplasm. While many have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this mass spectrometry-based proteomics study, we identified effector proteins secreted under minimal acidic medium growth conditions that induced the SPI-2 TTSS and its effectors, and compared the secretome from the parent strain to the secretome from strains missing either essential (SsaK) or regulatory components (SsaL) of the SPI-2 secretion apparatus. We identified 75% of the known TTSS effector repertoire. Excluding translocon components, 95% of the known effectors were biased for identification in the ssaL mutant background, which demonstrated that SsaL regulates SPI-2 type III secretion. To confirm secretion to animal cells, we made translational fusions of several of the best candidates to the calmodulin-dependent adenylate cyclase of Bordetella pertussis and assayed cAMP levels of infected J774 macrophage-like cells. From these infected cells we identified six new TTSS effectors and two others that are secreted independent of TTSS. Our results substantiate reports of additional secretion systems encoded by Salmonella other than TTSS.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 1004798
• Report Number(s): PNNL-SA-71385; 33210; 400412000
• Journal Information: Infection and Immunity, 79(1):33-43, Journal Name: Infection and Immunity, 79(1):33-43 Journal Issue: 1 Vol. 79; ISSN 0019-9567; ISSN INFIBR
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS
ACCURATE MASS
ANIMAL CELLS
CONTAINING VACUOLES
CYCLASES
CYTOPLASM
ESCHERICHIA-COLI
Environmental Molecular Sciences Laboratory
IDENTIFICATION
III SECRETION
MASS-SPECTROMETRY
MEMBRANE-VESICLES
MUTANTS
PATHOGENICITY ISLAND 2
PATHOGENS
PROTEINS
SALMONELLA
SECRETION
STRAINS
TYPHOID-FEVER
VIRULENCE
YEAST PROTEOME