Published Ahead of Print 25 October 2010. 2011, 79(1):33. DOI: 10.1128/IAI.00771-10. Infect. Immun. 

Richard D. Smith, Joshua N. Adkins and Fred HeffronMcDermott, Heather M. Brewer, Athena Schepmoes,Stufkens, Afshan S. Shaikh-Kidwai, Jie Li, Jason E. George S. Niemann, Roslyn N. Brown, Jean K. Gustin, Afke Culture Supernatants Typhimurium by Proteomic Analysis of

SerovarSalmonella entericaFactors from Discovery of Novel Secreted Virulence

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INFECTION AND IMMUNITY, Jan. 2011, p. 33–43 Vol. 79, No. 10019-9567/11/$12.00 doi:10.1128/IAI.00771-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Discovery of Novel Secreted Virulence Factors from Salmonella entericaSerovar Typhimurium by Proteomic Analysis of

Culture Supernatants�#George S. Niemann,1† Roslyn N. Brown,2† Jean K. Gustin,3 Afke Stufkens,1 Afshan S. Shaikh-Kidwai,1

Jie Li,1 Jason E. McDermott,4 Heather M. Brewer,2 Athena Schepmoes,2 Richard D. Smith,2Joshua N. Adkins,2 and Fred Heffron1*

Department of Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon1; Biological Sciences Division,Pacific Northwest National Laboratory, Richland, Washington2; Vaccine and Gene Therapy Institute, Oregon Health and

Science University, Portland, Oregon3; and Computational Biology and Bioinformatics,Pacific Northwest National Laboratory, Richland, Washington4

Received 18 July 2010/Returned for modification 16 September 2010/Accepted 12 October 2010

Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout theworld. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicityislands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm andare required for virulence. While many effectors have been identified and at least partially characterized,the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effectorproteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and itseffectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat)or regulatory (�ssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excludingthe translocon components SseBCD, all SPI-2 effectors were biased for identification in the �ssaL mutant,substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled oursecretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation ofvirulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA� reporterfusions, we identified six novel type III effectors and two additional proteins that were secreted into J774macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolardeletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Sixmutants were significantly attenuated for spleen colonization. Our results also suggest that non-type IIIsecretion mechanisms are required for full Salmonella virulence.

Salmonella enterica serovars are intracellular pathogens thatcan cause gastroenteritis and typhoid fever. In the developingworld, they are a leading cause of morbidity and mortalityresulting from dehydration and untreated sepsis (21–22, 43).Salmonella actively secretes effector proteins into the host cellcytoplasm to create a replicative niche and inhibit the im-mune system. Many of these effectors are delivered by oneof two type III secretion systems (TTSS), which are encodedon Salmonella pathogenicity islands 1 and 2 (SPI-1 andSPI-2, respectively) (24). The SPI-1 TTSS facilitates hostcell entry and inflammation, whereas SPI-2 mediates intra-cellular survival (19, 51). Both SPI-1 and SPI-2 are requiredin a mouse model of persistent infection (35). While over 30TTSS effectors have been identified to date (5, 25, 42, 58–59), the list is thought to be an underestimate of the trueeffector repertoire because several virulence phenotypes are

dependent on TTS but are not linked to any known effectors(34, 57).

A proteomic study of Escherichia coli O157:H7 identifiedover 31 new type III effectors. This analysis took advantage ofa sepL mutant that secreted effector proteins into culture me-dium in vitro (60). E. coli SepL interacts with Tir (63), a type IIIeffector that inserts into the host plasma membrane and func-tions as a receptor for the bacterial protein intimin (31). TheSepL-Tir complex is thought to occlude secretion until thetranslocon (host membrane pore) is assembled, thereby ensur-ing that Tir is translocated before any other effectors. SepL isa homolog of Salmonella SsaL, and a recent study found thatan ssaL mutant secreted proteins into medium (68). However,the proposed mechanism regulating Salmonella TTS is differ-ent from that in the E. coli SepL model. SsaL is believed toform a complex with two other proteins, SpiC and SsaM. Thecurrent model suggests that this heterotrimeric complex re-sponds to intracellular pH and permits secretion of transloconproteins at acidic pH (intravacuolar pH) while inhibiting se-cretion of effector proteins. When the SPI-2 TTSS gains accessto the neutral pH of the eukaryotic cytoplasm, the SsaL-SpiC-SsaM complex dissociates, and effector translocation ensues(68). Because an ssaL mutation relieved the pH-dependentinhibition of effector secretion, we hypothesized that culture

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Oregon Health and Science University, 3181S.W. Sam Jackson Park Road, Portland, OR 97201. Phone: (503)494-6841. Fax: (503) 494-6862. E-mail: heffronf@ohsu.edu.

† These authors contributed equally to this work.# Supplemental material for this article may be found at http://iai

.asm.org/.� Published ahead of print on 25 October 2010.

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supernatant from a Salmonella ssaL mutant could be analyzedby proteomics to discover novel effector proteins.

In this study, we used proteomics to identify effector pro-teins secreted by S. enterica serovar Typhimurium undergrowth conditions that induced expression of the SPI-2 TTSSand its many effectors (9–10, 68). A global evaluation of SPI-2effector secretion has not been previously reported. To supportour conclusions, we compared the secretome of the wild-type(WT) parent strain to the secretomes of strains missing essen-tial (ssaK::cat) or regulatory (�ssaL) components of the SPI-2secretion apparatus. Proteins were enriched by reverse-phaseresins and identified by liquid chromatography-tandem massspectrometry (LC-MS/MS). Interpretation of the results wasaided by a machine learning algorithm (SIEVE, SVM-basedidentification and evaluation of virulence effectors) that scoredeach protein for its probability of being a type III effector (50).This approach proved to be an excellent strategy for the iden-tification and discovery of secreted effector proteins. We iden-tified eight novel effectors and approximately 80% of the pre-viously reported repertoire encoded by S. Typhimurium ATCC14028. Excluding translocon components, all of the type IIIeffectors were secreted exclusively or in greater abundance bythe ssaL mutant, suggesting that ssaL and sepL are functionalorthologs.

MATERIALS AND METHODS

Culture conditions, strains, and plasmids. The strains and plasmids used inthis study are listed in Table S1 in the supplemental material. Salmonella entericaserovar Typhimurium (ATCC 14028) was used as the wild-type strain. All bac-teria were grown in Luria-Bertani (LB) broth or mLPM (see below for contents)at 37°C on a shaker set to 300 rpm. Carbenicillin, kanamycin, and chloramphen-icol were used at 100, 60, and 30 �g/ml, respectively. J774 cells were cultured at37°C in 5% CO2 using Dulbecco’s modified Eagle medium supplemented with10% fetal bovine serum, sodium pyruvate, sodium bicarbonate, and nonessentialamino acids. Lambda red recombination (12) was used to construct the genedeletions and sseJ::hemagglutinin (HA) strains. Deletions were constructed usingPCR products derived from primers listed in Table S2 in the supplementalmaterial and the pKD4 or pKD13mod templates. All constructs were transducedwith bacteriophage P22 and resolved to create in-frame, nonpolar deletions.sseJ::HA was derived from a PCR product using primers 3 and 4 with thepNFB15 template (received from Lionello Bossi), which generated a C-terminal,doubly HA-tagged SseJ followed by a nonresolvable kanamycin marker. Thisconstruct was P22 transduced into the wild-type, �ssaL, and ssaK::cat (18) back-grounds so that they each expressed a chromosomal copy of sseJ::HA under thecontrol of its native promoter. The sseJ::HA, �ssaL sseJ::HA, and ssaK::catsseJ::HA strains were transformed with the pBADssrB plasmid (65) to generatethe strains used for proteomic analysis. For CyaA� secretion assays, open readingframes were PCR amplified from S. Typhimurium using primer sets described inTable S2 in the supplemental material. Each forward primer was designed toanneal �20 bp upstream of its start codon to encode the putative Shine-Dal-garno sequence. Flanking 5� XbaI and 3� PvuII or EcoRV restriction sitesenabled directional cloning into pMJW1753 (18) cut with XbaI and SmaI. Theresulting C-terminal CyaA� fusions were verified by automated sequencing andtransformed into the wild-type, ssaK::cat, and invA::cat (18) backgrounds. SssAand SssB::CyaA� fusions were transformed into two additional backgrounds:�invA ssaK::cat and �invA ssaK::cat �flgB. Transcription was driven by theconstitutive lac promoter in Salmonella, and expression was confirmed by West-ern blotting against CyaA� (Santa Cruz; 1:1,000).

mLPM. mLPM contained 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4,0.3% (vol/vol) glycerol, 0.00001% thiamine, 0.5 �M ferric citrate, 8 �M MgCl2,337 �M PO43�, and 80 mM MES [2-(N-morpholino)ethanesulfonic acid]-freeacid adjusted to pH 5.8 with NaOH.

Sample preparation for LC-MS/MS. Bacteria were grown overnight in 50 mlLB broth. The following day, bacteria were washed three times in mLPM andthen diluted 1:10 to a final volume of 500 ml. Carbenicillin (100 �g/ml) wasadded to select for inoculated bacteria, and one tablet of protease inhibitorcocktail without EDTA (Roche) was added to inhibit protein degradation. At the

4-, 8-, and 16-h time points, the medium was centrifuged for 10 min at 5,000 �g to pellet the bacteria. Spent medium was filtered through a 0.45-�m Duraporefilter assembly (Millipore) and pumped at �1 ml/min overnight at 4°C throughserial 6-ml columns containing 50 mg of C4, C8, and C18 solid-phase extraction(SPE) resins (Strata and Supelco). Prior to loading of the samples, SPE columnswere conditioned with 100% methanol followed by 0.1% trifluoroacetic acid(TFA). After sample processing, SPE columns were washed with 95:5 H2O-acetonitrile (ACN), 0.1% TFA and stored at �20°C until they were elutedwith 80:20 ACN-H2O, 0.1% TFA. Eluted samples were concentrated with aSpeedVac, quick-frozen in liquid nitrogen, and stored at �80°C until needed.Protein concentration was assessed by a bicinchoninic acid (BCA) proteinassay (Pierce). When required, tryptic digests were performed as previouslydescribed (1, 3).

Capillary LC-MS/MS analysis. The high-performance liquid chromatography(HPLC) system and method used for nanocapillary liquid chromatography havebeen described in detail elsewhere (1, 52). Analysis was performed using an LTQOrbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) withelectrospray ionization. The HPLC column was coupled to the mass spectrom-eter using an in-house-manufactured interface. The heated capillary temperatureand spray voltage were 200°C and 2.2 kV, respectively. Data acquisition began 20min after the sample was injected and continued for 100 min over an m/z rangeof 400 to 2,000. For each cycle, the six most abundant ions from MS analysis wereselected for MS/MS analysis, using a collision energy setting of 35 eV. A dynamicexclusion time of 60 s was used to discriminate against previously analyzed ions.Each sample was analyzed in duplicate.

Data analysis. Peptides were identified by using SEQUEST to search the massspectra from LC-MS/MS analyses. These searches were performed using theannotated S. Typhimurium LT2 FASTA data file, containing 4,550 protein se-quences provided by the J. Craig Venter Institute, a standard parameter file withno modifications to amino acid residues. The searches were allowed for allpossible peptide termini, i.e., not limited by tryptic terminus state. Results werefiltered using a modification of criteria established by Washburn et al. (64) anda statistical approach to estimate the accuracy of peptide identifications (30),with a score of at least 0.9 used to increase confidence in identified peptides. Anestimate of the false-positive rate was obtained by searching against a reversedFASTA database, as described elsewhere (48). An estimated false-discovery rateof 2% was determined by using the combined filtering rules. The number ofpeptide observations from each protein (spectral count) was used as a roughmeasure of relative abundance. Multiple charge states of a single peptide wereconsidered individual observations, as were the same peptides detected in dif-ferent mass spectral analyses. Similar approaches for quantitation have beendescribed previously (1–3). Spectral counts for peptide identifications weresummed across technical replicates and were analyzed separately for the eluentsof the C4, C8, and C18 SPE columns. The results of the C4 and C8 columns weresimilar, and C18 column yields were sparse to null. Therefore, results from the C4

and C8 column eluents were combined, and C18 eluents were not used in sub-sequent analyses.

The accurate mass and elution time (AMT) tag approach (4, 54–55) was usedas a complementary approach to increase the sensitivity of peptide detection andas a secondary method of relative peptide quantification. For this method, areference database containing accurate peptide masses and normalized LC elu-tion times was generated from 72 LC-MS/MS analyses. C18 SPE column datawere excluded from the AMT database due to the paucity of peptides observed.The corresponding peptide sequences were determined using SEQUEST. Pep-tides were identified in subsequent high-throughput LC-MS analyses by match-ing masses and elution times observed in the high-resolution MS spectra to thosecontained in the reference database of peptides. Quantitation was based onmeasuring the mass spectral peak intensity of each peptide. This approach wasenabled by published and unpublished in-group tools which can be downloadedfrom http://omics.pnl.gov. The software program DAnTE (49) was used to per-form the abundance roll-up procedure to convert peptide information into pro-tein information, allowing relative protein abundances to be inferred. Statisticalanalyses were performed using Microsoft Excel. Hierarchical clustering andconstruction of heat maps were carried out using OmniViz 6.0.

Western blotting of secretome samples. To evaluate cellular protein expres-sion, pelleted bacteria were suspended in phosphate-buffered saline (PBS), nor-malized to an optical density at 600 nm (OD600), and lysed in Laemmli samplebuffer, and a volume corresponding to �1 � 105 bacteria was resolved bySDS-PAGE. To evaluate secretion, a 20-ml aliquot of spent, filter-sterilizedmedium was taken from each of the samples. Protein was precipitated overnightat 4°C in 20% trichloroacetic acid (TCA) and centrifuged at 12,000 � g at 4°C for30 min the following day. Precipitated protein pellets were washed twice inice-cold acetone, placed on a 95°C heat block to evaporate solvent, and then

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suspended in Laemmli sample buffer. If a sample turned yellow, acidity wasneutralized by adding 1 M Tris (pH 9) until the sample turned blue. Mediumsamples were normalized to the number of bacteria in the pellet and resolved bySDS-PAGE. Western blots using antibodies to DnaK (Assay Design; 1:10,000)and the C-terminal hemagglutinin (HA) tag of SseJ (Covance; 1:1,000) were usedto detect intracellular and secreted proteins, respectively.

CyaA� secretion assays. CyaA� secretion assays were performed as previouslydescribed (18). Secretion was evaluated under SPI-1 or SPI-2 gene-inducingconditions using the wild-type, invA::cat, and ssaK::cat genetic backgrounds.InvA and SsaK are structural components of the SPI-1 and SPI-2 TTSS, respec-tively. To induce SPI-1 expression, overnight LB cultures were diluted 1:33 in LBbroth and incubated at 37°C with shaking until the cultures reached late logphase. J774 cells were infected at a multiplicity of infection (MOI) of 50 for 1 h,and cyclic AMP (cAMP) levels were measured by enzyme-linked immunosorbentassay (ELISA) (Assay Designs). An effector was considered to be secreted by theSPI-1 TTSS if the cAMP responses between the wild-type (WT) and invA::catbackgrounds were �10-fold different. To induce SPI-2 expression, bacteria weregrown to late stationary phase overnight in LB broth. J774 cells were infected atan MOI of 250 for 6 h. An effector was deemed secreted by the SPI-2 TTSS if thecAMP responses between the WT and ssaK::cat backgrounds were �10-folddifferent. Three biological replicates were performed for each CyaA� fusion, anderror bars were calculated by determining standard errors of the means.

CI infections. We performed a mixed-infection competitive-index (CI) exper-iment using 6- to 8-week-old female 129/SvJ mice (Jackson Laboratories, BarHarbor, ME). This protocol is based upon the work of H. Yoon, C. Ansong, J. N.Adkins, and F. Heffron (submitted for publication) and is summarized here.Using lambda red recombination, we designed each Salmonella mutant and awild-type control with a unique scar sequence so that relative quantities could beanalyzed in a mixed infection. Bacteria were grown overnight in LB broth andwere washed three times in PBS the following day. Equal numbers of bacteriawere mixed together, and 15 129/SvJ mice were infected intraperitoneally (i.p.)with a total of 104 CFU. Spleens were harvested on days 1, 4, and 7 postinfectionand plated on LB agar for overnight growth at 37°C, where they yielded isolatedcolonies of approximately the same size. The following day, bacteria were har-vested and normalized to an OD600, and the unique scar sequence encoded byeach Salmonella strain was amplified by nested PCR using primers ScarF andScarR. The resulting PCR products containing unique scar sequences werepurified (Qiagen), normalized to concentration, and analyzed by quantitativereal-time PCR (qRT-PCR) to determine the quantity of each mutant relative tothat of the wild-type reference strain. qRT-PCR was performed using primerScarR with primers 67 to 74 (see Table S2 in the supplemental material), SYBRgreen reagent (Applied Biosystems), and a StepOnePlus real-time PCR system(Applied Biosystems). The CI is equal to �Emutant�

�Ct mutant/�Ewild type��Ct wild type, the

�Ct (change in threshold cycle) is equal to �Ctoutput � Ctinput, and E is equalto e1/�slope, where E is the amplification efficiency for each primer pair derivedfrom the slope of a calibration curve. E values ranged between 1.7 and 1.9.Statistical significance was calculated using Student’s t test.

RESULTS

Rationale for medium and strains used in this study. Theoverall objective of this study was to identify effectors secretedinto medium designed to induce SPI-2 expression. mLPM is adefined, acidic, low-phosphate, low-magnesium minimal growthmedium that induces SPI-2 expression (8–10, 14, 44).

Three different isogenic S. Typhimurium ATCC 14028strains were used for proteomic analysis: the wild-type (WT),ssaK::cat, and �ssaL strains. To each of these strains we addeda C-terminal, HA-tagged copy of SseJ to monitor secretion ofa known SPI-2 effector by Western blotting. SsaK is an essen-tial component of the SPI-2 secretion apparatus, and thessaK::cat mutant is deficient for SPI-2 TTSS. As mentionedpreviously, ssaL deletion is thought to disrupt the SpiC-SsaM-SsaL complex and enable secretion of type III effector proteinsat acidic pH in vitro (68). Secretion of SseJ into mLPM at pH5.8 was confirmed (see Fig. S1 in the supplemental material).

SsrB is a positive, global regulator of SPI-2 and of virulencefactors spread throughout the chromosome (36, 65). Despite

the use of medium that induced SPI-2 gene expression (seeFig. S2 in the supplemental material), we hypothesized thatSsrB overexpression might increase the probability of identi-fying novel type III effectors. SsrB overexpression likely causesself-activation via spontaneous dimerization and self-phosphor-ylation, a phenotype that has been previously demonstrated forthe PhoP two-component regulator (39). We therefore over-expressed SsrB in a parallel set of samples to drive effectorsynthesis. To accomplish this, all strains were transformed withthe pBADssrB plasmid, which encodes an arabinose-induciblecopy of ssrB (65).

Assessing secretion by Western blotting. Samples were pre-pared by growing cultures for 4, 8, or 16 h in mLPM with andwithout 0.2% arabinose. Bacteria were pelleted by centrif-ugation, and the supernatant was filter sterilized using alow-protein-affinity membrane (Fig. 1). At each time point,we evaluated the cell pellet and supernatant for intracellularand secreted levels of the tagged secreted effector, SseJ::HA,by Western blotting. We also probed for the cytoplasmic pro-tein DnaK to verify retention of this protein in the cell pellets.

DnaK was not observed in any of the secreted fractions (Fig.2), suggesting that the bacteria maintained cell integritythroughout the course of the experiment. In samples grownwithout arabinose, SseJ was detected in the supernatant at 8and 16 h but not at 4 h. Secretion of SseJ was observed only inthe ssaL mutant, not the parental strain nor the ssaK mutant(Fig. 2). Intracellular levels of SseJ decreased over time in thewild type and ssaK mutant but not in the ssaL mutant (Fig. 2).Given the lack of DnaK in the secreted fractions, these resultsargue against cell lysis and suggest that SsaL can regulate sseJtranscription and/or translation. Further experiments are neededto determine how this repression takes place and whether itaffects other type III effectors. When pBADssrB was inducedwith arabinose, SseJ secretion was still dependent upon thessaL deletion. However, SseJ secretion was observed only at4 h, not at 8 or 16 h (Fig. 2). Collectively, these results suggesta complex, poorly understood process of regulation and feed-back. Nonetheless, the results shown in Fig. 2 supported ourexperimental design because SseJ secretion was restricted tothe ssaL mutant and because two novel effectors were exclusiveto SsrB overexpression (see below).

Characterization of the secretome. Filtered culture superna-tants from each strain were applied to serial C4, C8, and C18

solid-phase extraction (SPE) columns to collect proteins forLC-MS/MS analysis (Fig. 1). Data from LC-MS/MS were an-alyzed with SEQUEST, which matches mass spectra to peptidesequences. Protein abundance was estimated by summing thenumbers of peptide mass spectra corresponding to a particularprotein (16). This approach, referred to as spectral counting, iswidely used in the proteomics community (1, 3, 26, 40).SEQUEST analysis with our study conditions (72 LC-MS/MSdata sets) yielded �1,400 unique secreted peptides thatmapped to �434 proteins. To increase confidence in the iden-tifications, we limited our analysis to those proteins identifiedby two or more peptides (n 300) (see Table S3 in thesupplemental material).

As an alternative to spectral counting, we also employed theaccurate mass and elution time (AMT) tag approach (69),which reduces under-sampling and provides mass spectral peakarea/height calculations for use as relative peptide abundance

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measurements. When the results of the analyses were com-pared to each other, the relative protein expression patternsbetween the AMT tag and spectral counting methods were insurprisingly close agreement (raw and processed results may beobtained from http://www.sysbep.org). The low level of com-plexity of the secretome compared to that of a typical cellularproteome may account for this observation. As a result, under-sampling of the MS/MS data was not a significant factor in thisstudy and improved confidence in the peptide identifications.For these reasons, and because of its broader use within theproteomics community, only results derived from spectralcounting are presented here.

Protein secretion patterns. To normalize the LC-MS/MSdata for each protein, summed spectral counts observed for asingle strain were divided by the total numbers of peptidesobserved for all strains to give relative abundance values rang-ing from 0 to 1. A secreted protein was considered stronglybiased for a particular strain if its relative abundance was �0.7.The remaining proteins were considered nonbiased, withroughly equal numbers observed for all strains. Identified pro-teins were also assessed with the PSORTb algorithm (66) topredict subcellular localization and by a computational methodthat predicts TTS (SIEVE [http://www.sysbep.org/sieve/]) (50).A SIEVE score of �1 indicated that a protein has a reasonableprobability of being a type III effector. These and other notablefeatures are highlighted in Fig. 3 and Table 1 and are discussednext.

(i) Identification of known type III effectors. We observed15 known effectors secreted exclusively by the SPI-2 TTSS(SopD2, SrfH, PipB, SifB, SseK2, SseJ, SifA, SseG, SseL, SteC,SspH2, PipB2, SseC, SseB, and SseD) and five additional ef-fectors secreted by both the SPI-1 and SPI-2 TTSS (SteB,SteA, SpvC, AvrA, and SlrP) (Fig. 4). Based on statisticalanalyses, these effectors fell into two distinct strain-specificcategories, ssaL dependent and WT dependent. All effectorswere ssaL dependent except the translocon proteins SseBCD,which fell into the WT-dependent category (Fig. 4). Takentogether, these results support and extend the model of Yu etal. (68) where SsaL inhibits the secretion of TTSS effectors, butnot translocon proteins, at acidic pH.

(ii) ssaL bias. Sixty-five proteins were biased for identifica-tion in the ssaL mutant (Fig. 3 and Table S4 in the supple-mental material), 17 of which were known SPI-2 effectors. Ofthe 65 ssaL-biased proteins, 89% had zero peptides or onepeptide detected in the ssaK mutant. Furthermore, ssaL-biasedproteins had significantly higher SIEVE scores than the rest ofthe secretome (mean, 0.9 versus 0.06; P 7 � 10�7) and thegenome (mean, 0.9 versus 0; P 5 � 10�14), suggesting that asignificant proportion of these proteins may be novel secretedeffectors (Table 1). Overall, these proteins were not signifi-cantly different from the rest of the secretome or genome interms of molecular weight. In terms of subcellular localization,22% of ssaL-biased proteins were predicted to be cytoplasmic,compared to 34% for the whole secretome (P 0.05, 2) and

FIG. 1. Sample preparation of extracellular proteins for LC-MS analysis. Five-hundred-milliliter mLPM culture volumes were grown for 4, 8,or 16 h. The bacterial pellet and an aliquot of filter-sterilized medium were analyzed by Western blotting as described in Materials and Methods.The remaining medium was then pumped through serial C4, C8, and C18 solid-phase extraction (SPE) resins. Samples were eluted and thenanalyzed by LC-MS/MS.

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40% for the genome (P 0.002, 2). The ssaL-biased subsetwas also enriched in predicted periplasmic proteins comparedto the ssaL-biased subsets of the secretome and genome. Onlythree proteins in the ssaL subset were among the 200 mostabundantly expressed in Salmonella under similar growth con-ditions (1), further suggesting that cell lysis was minimizedduring growth and sample preparation (Table 1).

(iii) WT bias. In the WT subset, 68 proteins were observedat higher levels than were observed in mutant samples (Fig. 3and Table S4 in the supplemental material). Eighty four per-cent of these proteins in the WT subset had zero (42 proteins)or one (15 proteins) peptide identification in the ssaK mutant.The WT-biased subset had a mean SIEVE score of �0.46,which indicates, overall, a low probability of TTS. However,this group also included the SPI-2 translocon componentsSseB, SseC, and SseD, confirming the Yu et al. model aspreviously described (68). Unlike the ssaL-biased subset, whichhad a molecular mass distribution similar to that of the ge-nome, the WT-biased proteins had a higher proportion(63%) of small cytoplasmic proteins with molecular masses

of �20 kDa. The proportion of proteins that are �20 kDa aspredicted from the genome sequence is 28%. Notably, 21 ofthe WT-biased proteins were among the top 200 most highlyexpressed Salmonella proteins observed under similargrowth conditions (Table 1) (1). Thus, the WT-biased groupwas enriched for small, abundant, intracellular proteins. It isintriguing and unclear why small, cytosolic proteins predom-inated in the WT-biased group.

(iv) ssaK bias. Nine proteins fell into the ssaK-biased cate-gory (Fig. 3 and Table S4 in the supplemental material). Thisgroup was enriched for outer membrane and extracellular pro-teins and had a SIEVE score of 0.41, which indicates a low-to-moderate probability of TTS (Table 1).

(v) Proteins with no strain differences observed. One hun-dred fifty-eight proteins were observed at nearly equal levelsin all strain backgrounds, including components of the SPI-2TTS apparatus, SsaC, SsaG, and SsaI (Fig. 3 and Table S4 inthe supplemental material). However, 76% of this grouphad two or more peptide identifications in the ssaK mutant,and the mean SIEVE score was �0.09, indicating that themajority of identified proteins were unlikely to be type IIIeffectors. Note that the secretome was prepared from strainsgrown in acidic minimal medium, i.e., inducing conditionsfor SPI-2. Compared to the genome, this subset was en-riched for proteins that are highly expressed under thesegrowth conditions (n 38), and a significant fraction wasperiplasmic or outer membrane localized (24% versus 6%

FIG. 2. Western blots of secretome samples illustrating secretionfrom the ssaL mutant. At each time point (4, 8, and 16 h), culturesupernatants were filter sterilized and TCA precipitated to concen-trate secreted proteins. The bacterial pellet (�1 � 105 CFU) andTCA precipitate were analyzed by Western blotting to evaluate SseJsecretion and retention of the intracellular protein DnaK in the cellpellet.

FIG. 3. Heat map representation of peptide identifications display-ing strain bias. Columns indicate the strain and normalized spectralcounts for each observed protein. To normalize the data, spectralcounts from each strain were summed across all time points with andwithout SsrB overexpression and then divided by the sum of valuesacross that protein row, resulting in a scale ranging from 0 (leastabundance) to 1 (highest abundance). A protein was considered biasedfor observation in a strain if its relative abundance value was �0.7.About 50% of proteins were observed at similar levels in all strains.Notable features of the strain-biased groups are indicated to the rightof each grouping.

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for the genome; P 2 � 10�20, 2) (Table 1). Flagellins(FliC and FljB) were also abundant in this subset, confirm-ing a previous Salmonella study that analyzed the content ofLB culture supernatants (32). Collectively, these observa-tions suggest that abundant outer membrane and periplas-mic proteins belong to this group. This group was also no-

table for the presence of PagC, PagD, PagK, and SrfN, ofwhich PagC, PagD, and SrfN have been reported to berequired for virulence (7, 23, 46–47). We determined thatthese proteins are secreted to host cells in outer membranevesicles (OMV) (Yoon et al., submitted). Thus, OMV mayhave contributed to the identification of other proteins.

TABLE 1. Properties of proteins identified in the secretome

Property WT bias ssaL bias ssaK bias No bias Secretome Genomea SPI-2effectorsb

Mean molecular mass in kDa 24 32 35 26 27 35 39Mean SIEVE score �0.46 0.90 0.41 �0.09 0.06 0.00 2.18No. of proteins among 200 most

abundantly expressedproteins (% of total)c

21 (31) 3 (5) 1 (11) 38 (24) 63 (21) NA 0 (0)

Mean peptide count 3 5 1 5 4 NA 9Mean spectral count 10 31 3 90 57 NA 69Total no. of proteins 68 65 9 158 300 4,525 20

No. of proteins by subcellularlocation(% of total)d

Cytoplasm 36 (53) 14 (22) 2 (22) 51 (32) 103 (34) 1819 (40) 8 (40)Inner membrane 11 (16) 9 (14) 0 (0) 18 (11) 37 (12) 1103 (24) 2 (10)Periplasm 0 (0) 13 (20) 1 (11) 21 (13) 35 (12) 158 (3) 0 (0)Outer membrane 1 (1) 1 (2) 2 (22) 17 (11) 21 (7) 102 (2) 0 (0)Extracellular 1 (1) 7 (11) 4 (44) 4 (3) 16 (5) 68 (2) 6 (30)Unknown location 19 (28) 21 (32) 0 (0) 47 (30) 87 (29) 1275 (28) 4 (20)

a NA, not applicable.b Known SPI-2 TTSS effectors and translocon proteins.c Previously observed in Salmonella proteomic studies.d Predicted using the PSORTbv3.0 algorithm.

FIG. 4. Analysis of known type III effector identifications. Spectral-count heat map of known type III effectors identified in the secretome.Columns indicate the strain and spectral counts for each observed protein across all time points with and without SsrB overexpression. Proteinswere binned by their association with the SPI-1/SPI-2 or SPI-2 TTSS. Known TTSS effectors, but not translocon components, were observedprimarily from the ssaL mutant. MAPKs, mitogen-activated protein kinases; SIF, Salmonella-induced filament; SCV, Salmonella-containingvacuole; GEF, guanine nucleotide exchange factor; SKIP, Sifa kinesin interacting protein; VAP, vacuolar actin polymerization.

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Selection of candidate secreted proteins. Based on the se-cretion patterns exhibited by known SPI-2 type III effectors(presence in the �ssaL and/or WT background, low/no detec-tion in the ssaK mutant, and SIEVE scores of �1) (Fig. 4), 12candidate proteins were tested for secretion into host cells.The candidate proteins were PSLT037 (SpvD), STM0359,STM1026 (GtgA), STM1055 (GtgE), STM1087 (PipA),STM1478 (YdgH), STM1599 (PdgL), STM1809, STM2139,STM2585, STM3762 (CigR), and STM4082 (YiiQ).

Confirmation of novel secreted proteins. To determine ifcandidate effectors were secreted into host cells, we con-structed a cyaA� fusion to each open reading frame (18, 56).Expression was verified by Western blotting (data not shown),J774 macrophage-like cells were infected, and cAMP levelswere measured. We identified three novel proteins secreted bythe SPI-2 TTSS (GtgA, CigR, and STM2139) and three se-creted by both the SPI-1 and SPI-2 TTSS (SpvD, GtgE, andSTM2585) (Fig. 5 and 6). In accordance with an existing nam-ing scheme for Salmonella type III effectors (18), we desig-nated STM2139 SteD (Salmonella translocated effector D) andSTM2585 SteE. Notably, each of these novel effectors was ssaLbiased, an observation that further supports the role of SsaL inregulating SPI-2 TTS. In addition, two proteins were secretedindependently of TTS: STM0359, which we designated SssA(Salmonella secreted substrate A), and YdgH, a conservedunknown protein that we designated SssB (Salmonella secretedsubstrate B) (Fig. 5 and 6). Of the remaining four candidates,we observed no secretion into host cells. There are severalreports that suggest that secretion may be cell or tissue specific(17, 20), but due to the general success of the screen, we didnot exhaustively try alternative methods. As a result, some ofthe unconfirmed candidates may in fact be secreted but notunder the conditions described here. Nevertheless, 8 out of 12candidates were confirmed as novel secreted substrates, dem-onstrating an unprecedented level of accuracy in our approach.

Effects of time and SsrB overexpression on secreted proteinidentifications. When the secretome was analyzed as a whole,time point and SsrB-related abundance patterns of secretionvaried among the wild-type, ssaL mutant, and ssaK mutantbackgrounds. Most proteins had greater abundances at 8 hthan at 4 h (see Fig. S3 in the supplemental material). Aminority of proteins further increased in abundance by 16 h.Among the known SPI-2 effectors, there was a trend of in-

creased secretion with time in the samples lacking arabinose.With SsrB overexpression from the pBADssrB plasmid, secre-tion of known effectors increased from 4 to 8 h, followed by adecline at 16 h (Fig. S3). Optimal effector identification cor-related with stationary-phase growth in mLPM (Fig. S4), butthe distinct secretion profiles at 8 and 16 h (Fig. S3) argue foradditional, uncharacterized developments between timepoints.

Most type III effectors were detected in culture supernatantsregardless of SsrB overexpression. However, there were sev-eral exceptions. SteB and SseK2 were exclusively identified inthe samples lacking arabinose (see Table S3 in the supplemen-tal material). Conversely, the novel effectors CigR and SssBwere restricted to the samples where SsrB was overexpressed(Table S3). Thus, SsrB overexpression had a modest impactupon effector identification, but it nevertheless permitted dis-covery of two additional secreted proteins.

Chromosomal location and virulence phenotypes of novelsecreted proteins. Most of the novel effector proteins are en-coded within pathogenicity islands such as Gifsy-2 (gtgA andgtgE), Gifsy-3 (steE), and SPI-3 (cigR) or the Salmonella viru-lence plasmid (spvD), suggesting that they were horizontallyacquired. On the other hand, steD, sssA, and sssB do notappear to be located in a pathogenicity island. Since SssA andSssB were secreted independently of TTS (Fig. 6), these pro-teins could belong to a more ancient secretion mechanism thanthe type III mechanism. SssA is unique in that it is only 33amino acids in length and is the smallest effector reported todate. SssA has no enzymatic activity likely because of its smallsize, has no primary sequence homology, and is unstructured,based on nuclear magnetic resonance (NMR) analysis (G.Buchko, unpublished result). Conversely, SssB possesses a do-main of unknown function (DUF1471) that is conservedamong numerous members of the Enterobacteriaceae. We the-orize that these two proteins may be secreted by an alternative,evolutionarily conserved system(s).

We next assessed the virulence properties of our novel se-creted proteins. SpvD is encoded by most pathogenic Salmo-nella strains but is not required for virulence in BALB/c mice(6, 41). Conversely, a gtgE mutant was attenuated 7-fold forspleen colonization of i.p. infected BALB/c mice (28). Viru-lence phenotypes have not been reported for the other sixnovel secreted proteins. We constructed nonpolar deletions of

FIG. 5. Discovery of novel secreted effectors. Spectral-count heat map for 8 novel secreted effectors. Columns indicate the strain and spectralcounts for each observed protein across all time points with and without SsrB overexpression. Proteins were binned by their secretion via the SPI-2TTSS or both the SPI-1 and SPI-2 TTSS. NA, not applicable (secreted independently of SPI-1, SPI-2, and flagellar TTSS).

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each novel candidate effector except gtgA via lambda red-me-diated allelic replacement and performed a competitive-indexexperiment with 129/SvJ mice infected i.p. These inbred miceexpress natural-resistance-associated macrophage protein 1(Nramp 1), a myeloid-cell-specific transporter of divalent cat-ions that affects susceptibility to Salmonella and other intra-cellular pathogens. In contrast to BALB/c mice, which areacutely sensitive to Salmonella infection, 129/SvJ mice presenta persistent infection that can last for weeks (11, 45). Deletionof spvD, steE, gtgE, steD, and sssA significantly attenuated col-onization of mouse spleens on days 1, 4, and 7 postinfection.The sssB mutant was attenuated on day 1 but appeared torecover by day 4 (Table 2). Our data agree with the previouslyreported virulence phenotype for GtgE but not for SpvD. The

FIG. 6. CyaA� secretion assays demonstrating protein translocation into the host cell cytoplasm. (A) SpvD, GtgA, GtgE, SteD, SteE, and CigRare novel type III effector proteins. To determine if an effector was translocated by SPI-2 or both the SPI-1 and SPI-2 TTSS, bacteria expressingadenylate cyclase (CyaA�) fused to the indicated proteins were induced for SPI-1 or SPI-2 gene expression. Following infection of J774 cells, cAMPlevels were measured. CyaA� fusions were tested in the WT (black bars), SPI-1 mutant (invA::cat) (light-gray bars), and SPI-2 mutant (ssaK::cat)(open bars) backgrounds. Left, SPI-1 infection conditions; right, SPI-2 infection conditions. (B) SssA and SssB are secreted into J774 macrophagesindependently of TTS. In addition to in the WT, invA::cat, and ssaK::cat backgrounds, SssA and SssB::CyaA� fusions were tested in an SPI-1, SPI-2double mutant (�invA ssaK::cat) (striped bars) and an SPI-1, SPI-2, flagellum triple mutant (�invA ssaK::cat �flgB) (dark-gray bars). cAMP levelsand error bars were obtained from three independent experiments but were not normalized to an internal control.

TABLE 2. Competitive indexes derived from the spleens of i.p.infected 129/SvJ mice

TTSS Mutantgenotype

Relative quantity on:

Day 1 Day 4 Day 7

SPI-1/SPI-2 �spvD 0.50 � 0.31a 0.43 � 0.07a 0.28 � 0.05a

�gtgE 0.22 � 0.05a 0.25 � 0.07a 0.18 � 0.04a

�steE 0.46 � 0.03a 0.27 � 0.09a 0.14 � 0.03a

SPI-2 �steD 0.22 � 0.07a 0.32 � 0.07a 0.21 � 0.06a

�cigR 0.68 � 0.26 0.96 � 0.17 1.04 � 0.39

NAb �sssA 0.52 � 0.10a 0.66 � 0.16a 0.50 � 0.08a

�sssB 0.35 � 0.04a 0.91 � 0.14 1.02 � 0.35

a P � 0.05.b NA, not applicable.

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disparity may be attributable to the different mouse strainsused in each study.

DISCUSSION

We used mass spectrometry to investigate the secretomes ofS. Typhimurium ATCC 14028, �ssaL mutant, and ssaK mutantbacteria grown under SPI-2-inducing conditions. An ssaL mu-tant secreted 17 known SPI-2 type III effectors into mediumbut not the translocon proteins SseBCD. Coupling our pro-teomic data with the SIEVE algorithm proved to be an efficientway to select novel proteins for characterization. We reporteight novel effectors that are translocated into host macro-phages, six of which are required for full virulence in thespleens of i.p. infected 129/SvJ mice.

Additional phenotypes have been reported for an ssaL mu-tant of S. Typhimurium SL1344 (8, 10). Coombes and cowork-ers observed that SsaL was required for in vitro secretion ofeffectors encoded within SPI-2 (SseG and SseL) but was dis-pensable for secretion of effectors encoded outside SPI-2(PipB and SopD2) (8). In contrast, we detected all four ofthese proteins secreted into �ssaL culture supernatants (Fig.4). Indeed, our data support a more general mechanism whereSsaL inhibits SPI-2 effector secretion at acidic pH (68). Ourdata also suggest that Salmonella SsaL is a functional orthologof SepL. SepL is found in enterohemorrhagic E. coli (EHEC),enteropathogenic E. coli (EPEC), and Citrobacter rodentium.When sepL was mutated in these bacteria, they all secretedtype III effector proteins into culture media and, thus, havebeen similarly exploited for proteomic analysis (15, 33, 60).

Both SepL and SsaL contain an HrpJ-like domain. HrpJ is acomponent of the TTSS from the plant pathogen Pseudomonassyringae and is required for its virulence (27). Since HrpJ-likedomains are also present in the SPI-1 protein InvE and in typeIII apparatus proteins from Vibrio, Yersinia, and Shigella spp.,deletion of these HrpJ-like genes may permit secretion intomedia. Therefore, proteomic approaches similar to ours maybe extendable to numerous Gram-negative pathogens.

The secretome data suggest that Salmonella secretes numer-ous effector proteins into host cells independently of TTS.SssA and SssB fell into this category, and the secretion mech-anism is under investigation in our laboratory. Of particularinterest to us is secretion via OMV. We likely copurified OMVbecause we identified known Salmonella OMV cargo (OmpA,OmpC, OmpF, OmpX, and Pal) (13, 62), as well as cytoplas-mic, periplasmic, and outer membrane proteins consistent withOMV content (13, 38). We also used filtered culture superna-tants, which are the starting material for OMV purificationstrategies (13, 29, 62). Furthermore, our proteomic analysisidentified SrfN, PagC, PagD, and PagK, which are secretedinto host cells via OMV and are required for virulence in mice(Yoon et al., submitted). Since other virulence factors arelikely to be translocated into cells by OMV, this presents anunexplored aspect of Salmonella pathogenesis for study.

In conclusion, we took advantage of an ssaL mutant andhigh-resolution LC-MS/MS to identify proteins secreted by S.Typhimurium in vitro. This has been the most comprehensivescreen for Salmonella-secreted effectors to date. Most impor-tantly, this study uncovered novel secreted effectors and im-

plies the existence of novel secretory pathways for Salmonellavirulence factors.

ACKNOWLEDGMENTS

We are grateful to Jennifer Niemann, Karl Weitz, Therese Clauss,Angela Norbeck, Meagan Burnet, and Penny Colton for contributionsto this work.

Support for this work was provided by the National Institute ofAllergy and Infectious Diseases, NIH/DHHS, through interagencyagreement Y1-A1-8401-01 and by grant NIH/NIAID A1022933-22A1to F.H. We used instrumentation and capabilities developed with sup-port from the National Center for Research Resources (grant RR018522 to R.D.S.) and the DOE/BER.

Proteomic analyses were performed in the Environmental Molecu-lar Sciences Laboratory, a U.S. Department of Energy Office of Bio-logical and Environmental Research (DOE/BER) national scientificuser facility on the Pacific Northwest National Laboratory (PNNL)campus in Richland, WA. PNNL is a multiprogram national labo-ratory operated by Battelle for the DOE under contract DE-AC05-76RL01830. Mass spectrometry results are available at SysBEP.organd Omics.pnl.gov.

REFERENCES

1. Adkins, J. N., H. M. Mottaz, A. D. Norbeck, J. K. Gustin, J. Rue, T. R.Clauss, S. O. Purvine, K. D. Rodland, F. Heffron, and R. D. Smith. 2006.Analysis of the Salmonella typhimurium proteome through environmentalresponse toward infectious conditions. Mol. Cell. Proteomics 5:1450–1461.

2. Adkins, J. N., S. M. Varnum, K. J. Auberry, R. J. Moore, N. H. Angell, R. D.Smith, D. L. Springer, and J. G. Pounds. 2002. Toward a human bloodserum proteome: analysis by multidimensional separation coupled with massspectrometry. Mol. Cell. Proteomics 1:947–955.

3. Ansong, C., H. Yoon, A. D. Norbeck, J. K. Gustin, J. E. McDermott, H. M.Mottaz, J. Rue, J. N. Adkins, F. Heffron, and R. D. Smith. 2008. Proteomicsanalysis of the causative agent of typhoid fever. J. Proteome Res. 7:546–557.

4. Ansong, C., H. Yoon, S. Porwollik, H. Mottaz-Brewer, B. O. Petritis, N.Jaitly, J. N. Adkins, M. McClelland, F. Heffron, and R. D. Smith. 2009.Global systems-level analysis of Hfq and SmpB deletion mutants in Salmo-nella: implications for virulence and global protein translation. PLoS One4:e4809.

5. Bakowski, M. A., V. Braun, and J. H. Brumell. 2008. Salmonella-containingvacuoles: directing traffic and nesting to grow. Traffic 9:2022–2031.

6. Bauerfeind, R., S. Barth, R. Weiss, and G. Baljer. 2001. Prevalence of theSalmonella plasmid virulence gene “spvD” in Salmonella strains from ani-mals. Dtsch. Tierarztl. Wochenschr. 108:243–245.

7. Belden, W. J., and S. I. Miller. 1994. Further characterization of the PhoPregulon: identification of new PhoP-activated virulence loci. Infect. Immun.62:5095–5101.

8. Coombes, B., N. Brown, Y. Valdez, J. Brumell, and B. Finlay. 2004. Expres-sion and secretion of Salmonella pathogenicity island-2 virulence genes inresponse to acidification exhibit differential requirements of a functionaltype III secretion apparatus and SsaL. J. Biol. Chem. 279:49804–49815.

9. Coombes, B., M. Wickham, N. Brown, S. Lemire, L. Bossi, W. Hsiao, F.Brinkman, and B. Finlay. 2005. Genetic and molecular analysis of GogB, aphage-encoded type III-secreted substrate in Salmonella enterica serovartyphimurium with autonomous expression from its associated phage. J. Mol.Biol. 348:817–830.

10. Coombes, B. K., M. J. Lowden, J. L. Bishop, M. E. Wickham, N. F. Brown,N. Duong, S. Osborne, O. Gal-Mor, and B. B. Finlay. 2007. SseL is aSalmonella-specific translocated effector integrated into the SsrB-controlledSalmonella pathogenicity island 2 type III secretion system. Infect. Immun.75:574–580.

11. Cuellar-Mata, P., N. Jabado, J. Liu, W. Furuya, B. B. Finlay, P. Gros, andS. Grinstein. 2002. Nramp1 modifies the fusion of Salmonella typhimurium-containing vacuoles with cellular endomembranes in macrophages. J. Biol.Chem. 277:2258–2265.

12. Datsenko, K., and B. Wanner. 2000. One-step inactivation of chromosomalgenes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci.U. S. A. 97:6640–6645.

13. Deatherage, B. L., J. C. Lara, T. Bergsbaken, S. L. Rassoulian Barrett, S.Lara, and B. T. Cookson. 2009. Biogenesis of bacterial membrane vesicles.Mol. Microbiol. 72:1395–1407.

14. Deiwick, J., T. Nikolaus, S. Erdogan, and M. Hensel. 1999. Environmentalregulation of Salmonella pathogenicity island 2 gene expression. Mol. Mi-crobiol. 31:1759–1773.

15. Deng, W., J. Puente, S. Gruenheid, Y. Li, B. Vallance, A. Vazquez, J. Barba,J. Ibarra, P. O’Donnell, P. Metalnikov, K. Ashman, S. Lee, D. Goode, T.Pawson, and B. Finlay. 2004. Dissecting virulence: systematic and functional

VOL. 79, 2011 SALMONELLA SECRETOME ANALYSIS 41

on March 21, 2012 by O

regon Health &

Science U

niversityhttp://iai.asm

.org/D

ownloaded from

analyses of a pathogenicity island. Proc. Natl. Acad. Sci. U. S. A. 101:3597–3602.

16. Eng, J. K., A. L. McCormack, and J. R. Yates III. 1994. An approach tocorrelate tandem mass spectral data of peptides with amino acid sequencesin a protein database. J. Am. Soc. Mass Spectrom. 5:976–989.

17. Geddes, K., F. Cruz, and F. Heffron. 2007. Analysis of cells targeted bySalmonella type III secretion in vivo. PLoS Pathog. 3:e196.

18. Geddes, K., M. Worley, G. Niemann, and F. Heffron. 2005. Identification ofnew secreted effectors in Salmonella enterica serovar Typhimurium. Infect.Immun. 73:6260–6271.

19. Ginocchio, C. C., S. B. Olmsted, C. L. Wells, and J. E. Galan. 1994. Contactwith epithelial cells induces the formation of surface appendages on Salmo-nella typhimurium. Cell 76:717–724.

20. Gong, H., G. Vu, Y. Bai, E. Yang, F. Liu, and S. Lu. 2010. Differentialexpression of Salmonella type III secretion system factors InvJ, PrgJ, SipC,SipD, SopA, and SopB in cultures and in mice. Microbiology. 156(Pt. 1):116–127.

21. Graham, S. 2002. Salmonellosis in children in developing and developedcountries and populations. Curr. Opin. Infect. Dis. 15:507–512.

22. Guerrant, R. L., J. M. Hughes, N. L. Lima, and J. Crane. 1990. Diarrhea indeveloped and developing countries: magnitude, special settings, and etiol-ogies. Rev. Infect. Dis. 12(Suppl. 1):S41–S50.

23. Gunn, J. S., C. M. Alpuche-Aranda, W. P. Loomis, W. J. Belden, and S. I.Miller. 1995. Characterization of the Salmonella typhimurium pagC/pagDchromosomal region. J. Bacteriol. 177:5040–5047.

24. Hansen-Wester, I., and M. Hensel. 2001. Salmonella pathogenicity islandsencoding type III secretion systems. Microbes Infect. 3:549–559.

25. Haraga, A., M. B. Ohlson, and S. I. Miller. 2008. Salmonellae interplay withhost cells. Nat. Rev. Microbiol. 6:53–66.

26. Hendrickson, E. L., Q. Xia, T. Wang, J. A. Leigh, and M. Hackett. 2006.Comparison of spectral counting and metabolic stable isotope labeling foruse with quantitative microbial proteomics. Analyst 131:1335–1341.

27. Hirano, S. S., A. O. Charkowski, A. Collmer, D. K. Willis, and C. D. Upper.1999. Role of the Hrp type III protein secretion system in growth of Pseudo-monas syringae pv. syringae B728a on host plants in the field. Proc. Natl.Acad. Sci. U. S. A. 96:9851–9856.

28. Ho, T. D., N. Figueroa-Bossi, M. Wang, S. Uzzau, L. Bossi, and J. M. Slauch.2002. Identification of GtgE, a novel virulence factor encoded on the Gifsy-2bacteriophage of Salmonella enterica serovar Typhimurium. J. Bacteriol.184:5234–5239.

29. Kadurugamuwa, J. L., and T. J. Beveridge. 1995. Virulence factors arereleased from Pseudomonas aeruginosa in association with membrane vesi-cles during normal growth and exposure to gentamicin: a novel mechanismof enzyme secretion. J. Bacteriol. 177:3998–4008.

30. Keller, A., A. I. Nesvizhskii, E. Kolker, and R. Aebersold. 2002. Empiricalstatistical model to estimate the accuracy of peptide identifications made byMS/MS and database search. Anal. Chem. 74:5383–5392.

31. Kenny, B., R. DeVinney, M. Stein, D. J. Reinscheid, E. A. Frey, and B. B.Finlay. 1997. Enteropathogenic E. coli (EPEC) transfers its receptor forintimate adherence into mammalian cells. Cell 91:511–520.

32. Komoriya, K., N. Shibano, T. Higano, N. Azuma, S. Yamaguchi, and S. I.Aizawa. 1999. Flagellar proteins and type III-exported virulence factors arethe predominant proteins secreted into the culture media of Salmonellatyphimurium. Mol. Microbiol. 34:767–779.

33. Kresse, A. U., F. Beltrametti, A. Muller, F. Ebel, and C. A. Guzman. 2000.Characterization of SepL of enterohemorrhagic Escherichia coli. J. Bacteriol.182:6490–6498.

34. Lapaque, N., J. L. Hutchinson, D. C. Jones, S. Meresse, D. W. Holden, J.Trowsdale, and A. P. Kelly. 2009. Salmonella regulates polyubiquitinationand surface expression of MHC class II antigens. Proc. Natl. Acad. Sci.U. S. A. 106:14052–14057.

35. Lawley, T. D., K. Chan, L. J. Thompson, C. C. Kim, G. R. Govoni, and D. M.Monack. 2006. Genome-wide screen for Salmonella genes required for long-term systemic infection of the mouse. PLoS Pathog. 2:e11.

36. Lee, A. K., C. S. Detweiler, and S. Falkow. 2000. OmpR regulates thetwo-component system SsrA-SsrB in Salmonella pathogenicity island 2. J.Bacteriol. 182:771–781.

37. Reference deleted.38. Lee, E. Y., J. Y. Bang, G. W. Park, D. S. Choi, J. S. Kang, H. J. Kim, K. S.

Park, J. O. Lee, Y. K. Kim, K. H. Kwon, K. P. Kim, and Y. S. Gho. 2007.Global proteomic profiling of native outer membrane vesicles derived fromEscherichia coli. Proteomics 7:3143–3153.

39. Lejona, S., M. E. Castelli, M. L. Cabeza, L. J. Kenney, E. Garcia Vescovi,and F. C. Soncini. 2004. PhoP can activate its target genes in a PhoQ-independent manner. J. Bacteriol. 186:2476–2480.

40. Liu, H., R. G. Sadygov, and J. R. Yates III. 2004. A model for randomsampling and estimation of relative protein abundance in shotgun proteom-ics. Anal. Chem. 76:4193–4201.

41. Matsui, H., C. M. Bacot, W. A. Garlington, T. J. Doyle, S. Roberts, and P. A.Gulig. 2001. Virulence plasmid-borne spvB and spvC genes can replace the90-kilobase plasmid in conferring virulence to Salmonella enterica serovar

Typhimurium in subcutaneously inoculated mice. J. Bacteriol. 183:4652–4658.

42. McGhie, E. J., L. C. Brawn, P. J. Hume, D. Humphreys, and V. Koronakis.2009. Salmonella takes control: effector-driven manipulation of the host.Curr. Opin. Microbiol. 12:117–124.

43. Mermin, J., R. Villar, J. Carpenter, L. Roberts, A. Samaridden, L.Gasanova, S. Lomakina, C. Bopp, L. Hutwagner, P. Mead, B. Ross, and E.Mintz. 1999. A massive epidemic of multidrug-resistant typhoid fever inTajikistan associated with consumption of municipal water. J. Infect. Dis.179:1416–1422.

44. Miao, E. A., J. A. Freeman, and S. I. Miller. 2002. Transcription of the SsrABregulon is repressed by alkaline pH and is independent of PhoPQ andmagnesium concentration. J. Bacteriol. 184:1493–1497.

45. Monack, D. M., D. M. Bouley, and S. Falkow. 2004. Salmonella typhimuriumpersists within macrophages in the mesenteric lymph nodes of chronicallyinfected Nramp1 / mice and can be reactivated by IFNgamma neutral-ization. J. Exp. Med. 199:231–241.

46. Nishio, M., N. Okada, T. Miki, T. Haneda, and H. Danbara. 2005. Identifi-cation of the outer-membrane protein PagC required for the serum resis-tance phenotype in Salmonella enterica serovar Choleraesuis. Microbiology151:863–873.

47. Osborne, S. E., D. Walthers, A. M. Tomljenovic, D. T. Mulder, U. Silphadu-ang, N. Duong, M. J. Lowden, M. E. Wickham, R. F. Waller, L. J. Kenney,and B. K. Coombes. 2009. Pathogenic adaptation of intracellular bacteria byrewiring a cis-regulatory input function. Proc. Natl. Acad. Sci. U. S. A.106:3982–3987.

48. Peng, J., J. E. Elias, C. C. Thoreen, L. J. Licklider, and S. P. Gygi. 2003.Evaluation of multidimensional chromatography coupled with tandem massspectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeastproteome. J. Proteome Res. 2:43–50.

49. Polpitiya, A. D., W. J. Qian, N. Jaitly, V. A. Petyuk, J. N. Adkins, D. G. CampII, G. A. Anderson, and R. D. Smith. 2008. DAnTE: a statistical tool forquantitative analysis of -omics data. Bioinformatics 24:1556–1558.

50. Samudrala, R., F. Heffron, and J. E. McDermott. 2009. Accurate predictionof secreted substrates and identification of a conserved putative secretionsignal for type III secretion systems. PLoS Pathog. 5:e1000375.

51. Shea, J. E., M. Hensel, C. Gleeson, and D. W. Holden. 1996. Identification ofa virulence locus encoding a second type III secretion system in Salmonellatyphimurium. Proc. Natl. Acad. Sci. U. S. A. 93:2593–2597.

52. Shen, Y., N. Tolic, R. Zhao, L. Pasa-Tolic, L. Li, S. J. Berger, R. Harkewicz,G. A. Anderson, M. E. Belov, and R. D. Smith. 2001. High-throughputproteomics using high-efficiency multiple-capillary liquid chromatographywith on-line high-performance ESI FTICR mass spectrometry. Anal. Chem.73:3011–3021.

53. Reference deleted.54. Smith, R. D., G. A. Anderson, M. S. Lipton, C. Masselon, L. Pasa-Tolic, Y.

Shen, and H. R. Udseth. 2002. The use of accurate mass tags for high-throughput microbial proteomics. OMICS 6:61–90.

55. Smith, R. D., G. A. Anderson, M. S. Lipton, L. Pasa-Tolic, Y. Shen, T. P.Conrads, T. D. Veenstra, and H. R. Udseth. 2002. An accurate mass tagstrategy for quantitative and high-throughput proteome measurements. Pro-teomics 2:513–523.

56. Sory, M. P., A. Boland, I. Lambermont, and G. R. Cornelis. 1995. Identifi-cation of the YopE and YopH domains required for secretion and internal-ization into the cytosol of macrophages, using the cyaA gene fusion ap-proach. Proc. Natl. Acad. Sci. U. S. A. 92:11998–12002.

57. Srinivasan, A., M. Nanton, A. Griffin, and S. J. McSorley. 2009. Culling ofactivated CD4 T cells during typhoid is driven by Salmonella virulence genes.J. Immunol. 182:7838–7845.

58. Steele-Mortimer, O. 2008. The Salmonella-containing vacuole: moving withthe times. Curr. Opin. Microbiol. 11:38–45.

59. Thomas, M., and D. W. Holden. 2009. Ubiquitination—a bacterial effector’sticket to ride. Cell Host Microbe 5:309–311.

60. Tobe, T., S. A. Beatson, H. Taniguchi, H. Abe, C. M. Bailey, A. Fivian, R.Younis, S. Matthews, O. Marches, G. Frankel, T. Hayashi, and M. J. Pallen.2006. An extensive repertoire of type III secretion effectors in Escherichiacoli O157 and the role of lambdoid phages in their dissemination. Proc. Natl.Acad. Sci. U. S. A. 103:14941–14946.

61. Reference deleted.62. Wai, S., B. Lindmark, T. Soderblom, A. Takade, M. Westermark, J. Oscars-

son, J. Jass, A. Richter-Dahlfors, Y. Mizunoe, and B. Uhlin. 2003. Vesicle-mediated export and assembly of pore-forming oligomers of the enterobac-terial ClyA cytotoxin. Cell 115:25–35.

63. Wang, D., A. Roe, S. McAteer, M. Shipston, and D. Gally. 2008. Hierarchaltype III secretion of translocators and effectors from Escherichia coliO157:H7 requires the carboxy terminus of SepL that binds to Tir. Mol.Microbiol. 69:1499–1512.

64. Washburn, M. P., D. Wolters, and J. R. Yates III. 2001. Large-scale analysisof the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 19:242–247.

65. Worley, M. J., K. H. Ching, and F. Heffron. 2000. Salmonella SsrB activatesa global regulon of horizontally acquired genes. Mol. Microbiol. 36:749–761.

42 NIEMANN ET AL. INFECT. IMMUN.

on March 21, 2012 by O

regon Health &

Science U

niversityhttp://iai.asm

.org/D

ownloaded from

66. Yu, N. Y., J. R. Wagner, M. R. Laird, G. Melli, S. Rey, R. Lo, P. Dao, S. C.Sahinalp, M. Ester, L. J. Foster, and F. S. Brinkman. 2010. PSORTb 3.0:improved protein subcellular localization prediction with refined localizationsubcategories and predictive capabilities for all prokaryotes. Bioinformatics26:1608–1615.

67. Reference deleted.

68. Yu, X. J., K. McGourty, M. Liu, K. E. Unsworth, and D. W. Holden. 2010. pHsensing by intracellular Salmonella induces effector translocation. Science328:1040–1043.

69. Zimmer, J. S., M. E. Monroe, W. J. Qian, and R. D. Smith. 2006. Advancesin proteomics data analysis and display using an accurate mass and time tagapproach. Mass Spectrom. Rev. 25:450–482.

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