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Proteomics-based Expression Library Screening (PELS)

A Novel Method for Rapidly Defining Microbial Immunoproteomes*S
  • Indira T. Kudva
    Footnotes
    Affiliations
    From the Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

    Departments of Medicine, Harvard Medical School, Boston, Massachusetts 02114
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  • Bryan Krastins
    Footnotes
    Affiliations
    Harvard Partners Center For Genetics and Genomics, Cambridge, Massachusetts 02139
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  • Haiqing Sheng
    Affiliations
    Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, Idaho 83844
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  • Robert W. Griffin
    Affiliations
    From the Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
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  • David A. Sarracino
    Affiliations
    Harvard Partners Center For Genetics and Genomics, Cambridge, Massachusetts 02139
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  • Phillip I. Tarr
    Affiliations
    Departments of Pediatrics and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Carolyn J. Hovde
    Footnotes
    Affiliations
    Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, Idaho 83844
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  • Stephen B. Calderwood
    Affiliations
    From the Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

    Departments of Medicine, Harvard Medical School, Boston, Massachusetts 02114

    Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02114
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  • Manohar John
    Correspondence
    To whom correspondence should be addressed. Tel.: 617-724-7528; Fax: 617-726-7416;
    Affiliations
    From the Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

    Departments of Medicine, Harvard Medical School, Boston, Massachusetts 02114
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants R21 AI055963 (to I. T. K.) and R01 DK52081 (to P. I. T.).
    S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
    ¶ Both authors contributed equally to this work.
    §§ Supported in part by the National Research Initiative of the United States Department of Agriculture Cooperative State Research, Education, and Extension Service Grants 99-35201-8539 and 04-04562 and National Institutes of Health Grants NO1-HD-0-3309, U54-AI-57141, P20-RR16454, and P20-RR15587.
Open AccessPublished:May 31, 2006DOI:https://doi.org/10.1074/mcp.T600013-MCP200
      Current methodologies for global identification of microbial proteins that elicit host humoral immune responses have several limitations and are not ideally suited for use in the postgenomic era. Here we describe a novel application of proteomics, proteomics-based expression library screening, to rapidly define microbial immunoproteomes. Proteomics-based expression library screening is broadly applicable to any cultivable, sequenced pathogen eliciting host antibody responses and hence is ideal for rapidly mining microbial proteomes for targets with diagnostic, prophylactic, and therapeutic potential. In this report, we demonstrate “proof-of-principle” by identifying 207 proteins of the Escherichia coli O157:H7 immunome in bovine reservoirs in only 3 weeks.
      Proteins constituting microbial immunomes (the subset of microbial antigens that elicit host immune responses) have excellent diagnostic, prophylactic, and therapeutic potential because a subset of such immunogenic proteins is part of the repertoire of microbial factors that function to help pathogens counter host defenses, facilitate niche adaptation, and survive and replicate in these hosts. Methodologies to rapidly identify such protein targets would facilitate exploitation of microbial genome sequence data and expedite the development of novel management strategies against infectious diseases.
      Traditional methodologies for proteome wide identification of immunogenic microbial proteins (IMPs)
      The abbreviations used are: IMP, immunogenic microbial protein; PELS, proteomics-based expression library screening; IVIAT, in vivo induced antigen technology; pAb, polyclonal antibody; 1D, one-dimensional; GeLC-MS/MS, 1D SDS-PAGE ESI nano-LC-MS/MS; NHS, N-hydroxysuccinimide; IPTG, isopropyl β-d-thiogalactopyranoside; NOG, n-octyl β-d-glucopyranoside; HP, high performance.
      1The abbreviations used are: IMP, immunogenic microbial protein; PELS, proteomics-based expression library screening; IVIAT, in vivo induced antigen technology; pAb, polyclonal antibody; 1D, one-dimensional; GeLC-MS/MS, 1D SDS-PAGE ESI nano-LC-MS/MS; NHS, N-hydroxysuccinimide; IPTG, isopropyl β-d-thiogalactopyranoside; NOG, n-octyl β-d-glucopyranoside; HP, high performance.
      involve screening microbial recombinant genomic expression libraries in plasmid/phage expression vectors and laboratory host strains with sera from colonized or infected hosts. However, colony immunoscreening and in vivo induced antigen technology (IVIAT) (
      • Rollins S.M.
      • Peppercorn A.
      • Hang L.
      • Hillman J.D.
      • Calderwood S.B.
      • Handfield M.
      • Ryan E.T.
      In vivo induced antigen technology (IVIAT).
      ), a variation of colony immunoscreening that defines only partial immunoproteomes, and bacterial surface display coupled with magnetic cell sorting (
      • Etz H.
      • Minh D.B.
      • Henics T.
      • Dryla A.
      • Winkler B.
      • Triska C.
      • Boyd A.P.
      • Sollner J.
      • Schmidt W.
      • von Ahsen U.
      • Buschle M.
      • Gill S.R.
      • Kolonay J.
      • Khalak H.
      • Fraser C.M.
      • von Gabain A.
      • Nagy E.
      • Meinke A.
      Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus.
      ) are laborious and require several months or more for definitive IMP identification. Immunoproteomics of pathogens cultured in vitro under either standard laboratory conditions or those that attempt to mimic the host environment are also popular; however, pathogens cultured in vitro might not express the entire spectrum of virulence proteins. In view of the challenging task of accurately reproducing the host environment, such approaches might overlook those immunogenic virulence proteins that are expressed exclusively in response to host environmental cues and contribute significantly to pathogenicity (
      • Mahan M.J.
      • Heithoff D.M.
      • Sinsheimer R.L.
      • Low D.A.
      Assessment of bacterial pathogenesis by analysis of gene expression in the host.
      ). Although these limitations may be circumvented by immunoproteomics of pathogens isolated directly from either biological specimens or host anatomical sites of infection, consistent recovery of sufficient numbers of suitable organisms for analysis presents a significant challenge (
      • Xu Q.
      • Dziejman M.
      • Mekalanos J.J.
      Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro.
      ). Protein microarray/chip technology has tremendous potential for rapid, global definition of IMPs but is constrained by bottlenecks in proteome scale purification of microbial proteins and currently permits immunological characterization of only a partial proteome (
      • Li B.
      • Jiang L.
      • Song Q.
      • Yang J.
      • Chen Z.
      • Guo Z.
      • Zhou D.
      • Du Z.
      • Song Y.
      • Wang J.
      • Wang H.
      • Yu S.
      • Wang J.
      • Yang R.
      Protein microarray for profiling antibody responses to Yersinia pestis live vaccine.
      ). Newer formats such as nucleic acid programmable protein arrays (
      • Ramachandran N.
      • Hainsworth E.
      • Bhullar B.
      • Eisenstein S.
      • Rosen B.
      • Lau A.Y.
      • Walter J.C.
      • LaBaer J.
      Self-assembling protein microarrays.
      ) are still experimental, and neither nucleic acid programmable protein arrays nor antibody arrays/protein chips utilizing SELDI-TOF mass spectrometry for antigen identification (
      • Hess J.L.
      • Blazer L.
      • Romer T.
      • Faber L.
      • Buller R.M.
      • Boyle M.D.
      Immunoproteomics.
      ) have been demonstrated to rapidly define microbial immunoproteomes.
      To rapidly identify proteins comprising microbial immunomes, we have developed a novel technique, proteomics-based expression library screening (PELS), that couples standard recombinant DNA and immunochemistry techniques with proteomics. The principle of PELS is outlined in Fig. 1 and involves capture of recombinant proteins expressed from an inducible, microbial genomic DNA expression library using polyclonal antibodies (pAbs) affinity-purified from acute/convalescent sera of infected hosts or sera from reservoirs colonized by the cognate pathogen (“bait” pAbs) coupled to a solid support. Proteins captured by the bait pAbs are subjected to one-dimensional (1D) SDS-PAGE ESI nano-LC-MS/MS (GeLC-MS/MS) and identified via SEQUEST database searching (
      • Steen H.
      • Mann M.
      The ABC’s (and XYZ’s) of peptide sequencing.
      ) (Fig. 1). The entire process, from recombinant genomic expression library construction to definitive protein identification, is accomplished in only 3 weeks without biases inherent to manual screening. To our knowledge, this is the first application of proteomics for rapid, global identification of IMPs from among proteins expressed from genes on inserts within recombinant clones comprising microbial genomic expression DNA libraries.

      RESULTS AND DISCUSSION

      To validate PELS (Fig. 1), we sought to rapidly define a protein subset constituting the O157 immunome in bovine reservoirs for future evaluation as candidates for a vaccine for elimination of this pathogen from the gastrointestinal tracts of cattle, a principal source of human infection. For this, we constructed and induced recombinant protein expression from genes on inserts within clones comprising an optimized O157 expression library using a range of IPTG concentrations followed by growth at different incubation temperatures for varying time intervals. To capture recombinant proteins contained in cell lysate and pellet fractions of library clones, we affinity-purified bait pAbs from pooled hyperimmune cattle sera previously generated against diverse O157 strains following confirmation of reactivity of this hyperimmune cattle serum pool against previously identified O157 antigens (Fig. 2, a and b). We then charged HiTrap NHS-activated columns by coupling to bait pAbs after which pooled cell lysate and pellet fractions from above were applied separately to charged columns. Specifically captured O157 recombinant proteins (Fig. 3, a–d) were identified by subjecting pooled elution fractions to GeLC-MS/MS and SEQUEST database searching (Table I and Supplemental Table 1).
      Table IPreviously reported adhesins of O157 and other pathogens identified by PELS
      ProteinLocus ID in O157 EDL 933
      Ref. 11.
      /Sakai
      Ref. 12.
      strains
      MassNo. peptide hitsProtein coverageContributes to adherence inRef.
      kDa%
      OmpA; outer membrane protein 3a (II*;G;d); adhesinZ1307/Ecs1041384243O157
      • Torres A.G.
      • Kaper J.B.
      Multiple elements controlling adherence of enterohemorrhagic Escherichia coli O157:H7 to HeLa cells.
      Iha; adhesin; exogenous ferric siderophore receptor R4Z1178/Ecs13607748O157, uropathogenic E. coli
      • Tarr P.I.
      • Bilge S.S.
      • Vary Jr., J.C.
      • Jelacic S.
      • Habeeb R.L.
      • Ward T.R.
      • Baylor M.R.
      • Besser T.E.
      Iha: a novel Escherichia coli O157:H7 adherence-conferring molecule encoded on a recently acquired chromosomal island of conserved structure.
      ,
      • Johnson J.R.
      • Jelacic S.
      • Schoening L.M.
      • Clabots C.
      • Shaikh N.
      • Mobley H.L.T.
      • Tarr P.I.
      The IrgA homologue adhesin Iha is an Escherichia coli virulence factor in murine urinary tract infection.
      a Ref.
      • Perna N.T.
      • Plunkett G.
      • Burland V.
      • Mau B.
      • Glasner J.D.
      • Rose D.J.
      • Mayhew G.F.
      • Evans P.S.
      • Gregor J.
      • Kirkpatrick H.A.
      • Posfai G.
      • Hackett J.
      • Klink S.
      • Boutin A.
      • Shao Y.
      • Miller L.
      • Grotbeck E.J.
      • Davis N.W.
      • Lim A.
      • Dimalanta E.T.
      • Potamousis K.D.
      • Apodaca J.
      • Anantharaman T.S.
      • Lin J.
      • Yen G.
      • Schwartz D.C.
      • Welch R.A.
      • Blattner F.R.
      Genome sequence of enterohaemorrhagic Escherichia coli O157:H7.
      .
      b Ref.
      • Hayashi T.
      • Makino K.
      • Ohnishi M.
      • Kurokawa K.
      • Ishii K.
      • Yokoyama K.
      • Han C.G.
      • Ohtsubo E.
      • Nakayama K.
      • Murata T.
      • Tanaka M.
      • Tobe T.
      • Iida T.
      • Takami H.
      • Honda T.
      • Sasakawa C.
      • Ogasawara N.
      • Yasunaga T.
      • Kuhara S.
      • Shiba T.
      • Hattori M.
      • Shinagawa H.
      Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12.
      .
      PELS identified 207 proteins, comprising 3.8% of the proteome of the sequenced O157 strain EDL933 (
      • Perna N.T.
      • Plunkett G.
      • Burland V.
      • Mau B.
      • Glasner J.D.
      • Rose D.J.
      • Mayhew G.F.
      • Evans P.S.
      • Gregor J.
      • Kirkpatrick H.A.
      • Posfai G.
      • Hackett J.
      • Klink S.
      • Boutin A.
      • Shao Y.
      • Miller L.
      • Grotbeck E.J.
      • Davis N.W.
      • Lim A.
      • Dimalanta E.T.
      • Potamousis K.D.
      • Apodaca J.
      • Anantharaman T.S.
      • Lin J.
      • Yen G.
      • Schwartz D.C.
      • Welch R.A.
      • Blattner F.R.
      Genome sequence of enterohaemorrhagic Escherichia coli O157:H7.
      ), as components of the immunome of this organism in bovine reservoirs (Table I and Supplemental Table 1). PELS was strongly validated by the fact that 35 of 207 (17%) proteins were also part of the O157 immunome in humans convalescing from extraintestinal O157 disease (
      • John M.
      • Kudva I.T.
      • Griffin R.W.
      • Dodson A.W.
      • McManus B.
      • Krastins B.
      • Sarracino D.
      • Progulske-Fox A.
      • Hillman J.D.
      • Handfield M.
      • Tarr P.I.
      • Calderwood S.B.
      Use of in vivo-Induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection.
      ). Here we wish to emphasize that further experimentation is required to ascertain whether the rest of the PELS-identified proteins are unique to the O157 immunome in cattle. This is because the immunoproteome defined by IVIAT is a partial one in that it includes only proteins that are expressed either uniquely during human infection or at significantly higher levels in vivo than during in vitro growth because of a series of adsorptions of human convalescent sera against O157 grown in standard laboratory media (
      • John M.
      • Kudva I.T.
      • Griffin R.W.
      • Dodson A.W.
      • McManus B.
      • Krastins B.
      • Sarracino D.
      • Progulske-Fox A.
      • Hillman J.D.
      • Handfield M.
      • Tarr P.I.
      • Calderwood S.B.
      Use of in vivo-Induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection.
      ). In contrast, the unadsorbed cattle hyperimmune sera used in PELS facilitated the definition of a more complete immunoproteome. Consequently, immunogenic O157 proteins expressed either equally well both in vitro and in vivo or at higher levels during laboratory growth than during human infection and are part of the PELS-identified protein repertoire remain excluded from the immunome identified by IVIAT. Further validation of PELS came from identification of previously identified adhesins of O157 (
      • Torres A.G.
      • Kaper J.B.
      Multiple elements controlling adherence of enterohemorrhagic Escherichia coli O157:H7 to HeLa cells.
      ,
      • Tarr P.I.
      • Bilge S.S.
      • Vary Jr., J.C.
      • Jelacic S.
      • Habeeb R.L.
      • Ward T.R.
      • Baylor M.R.
      • Besser T.E.
      Iha: a novel Escherichia coli O157:H7 adherence-conferring molecule encoded on a recently acquired chromosomal island of conserved structure.
      ) and of other pathogens (
      • Johnson J.R.
      • Jelacic S.
      • Schoening L.M.
      • Clabots C.
      • Shaikh N.
      • Mobley H.L.T.
      • Tarr P.I.
      The IrgA homologue adhesin Iha is an Escherichia coli virulence factor in murine urinary tract infection.
      ) (Table I) as well as several bovine colonization factors (
      • Dziva F.
      • van Diemen P.M.
      • Stevens M.P.
      • Smith A.J.
      • Wallis T.S.
      Identification of Escherichia coli O157:H7 genes influencing colonization of the bovine gastrointestinal tract using signature-tagged mutagenesis.
      ) (Supplemental Table 1); this is consistent with the fact that this human pathogen only colonizes the gastrointestinal tracts of cattle but does not cause disease in these reservoirs. Also validating PELS was the identification of two (EspB and EspP) of four secreted O157 proteins (EspA, EspB, Tir, and EspP) that are strongly immunogenic in cattle as constituents of an experimental vaccine (
      • Potter A.A.
      • Klashinsky S.
      • Li Y.
      • Frey E.
      • Townsend H.
      • Rogan D.
      • Erickson G.
      • Hinkley S.
      • Klopfenstein T.
      • Moxley R.A.
      • Smith D.R.
      • Finlay B.B.
      Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins.
      ). Parenteral administration of this vaccine formulated with an adjuvant reportedly results in a reduction in fecal shedding of O157 following oral challenge of bovine reservoirs (
      • Potter A.A.
      • Klashinsky S.
      • Li Y.
      • Frey E.
      • Townsend H.
      • Rogan D.
      • Erickson G.
      • Hinkley S.
      • Klopfenstein T.
      • Moxley R.A.
      • Smith D.R.
      • Finlay B.B.
      Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins.
      ). The reasons why EspA and Tir were not identified in the current study are unclear. Plausible explanations include the fact that such proteins might be (i) poorly expressed by wild-type O157 strains following oral inoculation and therefore engender suboptimal antibody responses in the bovine host and/or (ii) expressed at very low levels following induction by the E. coli host strain used in this study. As in the earlier report on the O157 immunome in humans identified by IVIAT (
      • John M.
      • Kudva I.T.
      • Griffin R.W.
      • Dodson A.W.
      • McManus B.
      • Krastins B.
      • Sarracino D.
      • Progulske-Fox A.
      • Hillman J.D.
      • Handfield M.
      • Tarr P.I.
      • Calderwood S.B.
      Use of in vivo-Induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection.
      ), PELS identified outer membrane components of high affinity iron transport, bacteriophage proteins, biosynthetic and metabolic enzymes, and proteins involved in energy generation and anaerobic respiration in accordance with requirements for adaptation to the host environment, in vivo growth, and survival. PELS also identified several hypothetical and unknown proteins, including those unique to this study (Supplemental Table 1).
      In conclusion, this report details a new application of proteomics and highlights the power of proteomics-based methodologies. A striking feature is the rapidity of proteome wide IMP identification, which renders PELS an ideal alternative/complement to emerging protein chip/array technologies. Other attractive features include broad applicability, robustness, and an elimination of subjective bias. Also because proteins are expressed from genes on inserts within clones of a genomic DNA expression library, a more comprehensive determination of the immunoproteome of the cognate pathogen is possible. Limitations relate to constraints on expression of heterologous proteins in E. coli hosts (
      • Rollins S.M.
      • Peppercorn A.
      • Hang L.
      • Hillman J.D.
      • Calderwood S.B.
      • Handfield M.
      • Ryan E.T.
      In vivo induced antigen technology (IVIAT).
      ); however, improved prokaryotic cell-free in vitro translation systems coupled with the advent of technologies for facile generation of expression ORFeomes of microbes (
      • Brasch M.A.
      • Hartley J.L.
      • Vidal M.
      ORFeome cloning and systems biology: standardized mass production of the parts from the parts-list.
      ) should further enhance the power of this methodology.

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