Background Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in developing countries, where it accounts for millions of infections and hundreds of thousands of deaths annually. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive effort is needed to identify conserved antigenic targets. Pathogenic Escherichia coli, including ETEC, use the autotransporter (AT) secretion mechanism to export virulence factors. AT proteins are comprised of a highly conserved carboxy terminal outer membrane beta barrel and a surface-exposed amino terminal passenger domain. Recent immunoproteomic studies suggesting that multiple autotransporter passenger domains are recognized during ETEC infection prompted the present studies. Methodology Available ETEC genomes were examined to identify AT coding sequences present in pathogenic isolates, but not in the commensal E. coli HS strain. Passenger domains of the corresponding autotransporters were cloned and expressed as recombinant antigens, and the immune response to these proteins was then examined using convalescent sera from patients and experimentally infected mice. Principal Findings Potential AT genes shared by ETEC strains, but absent in the E. coli commensal HS strain were identified. Recombinant passenger domains derived from autotransporters, including Ag43 and an AT designated pAT, were recognized by antibodies from mice following intestinal challenge with H10407, and both Ag43 and pAT were identified on the surface of ETEC by flow cytometry. Likewise, convalescent sera from patients with ETEC diarrhea recognized Ag43 and pAT, suggesting that these proteins are expressed during both experimental and naturally occurring ETEC infections and that they are immunogenic. Vaccination of mice with recombinant passenger domains from either pAT or Ag43 afforded protection against intestinal colonization with ETEC. Conclusions Passenger domains of conserved autotransporter proteins could contribute to protective immune responses that develop following infection with ETEC, and these antigens consequently represent potential targets to explore in vaccine development.
References
[1]
WHO (2006) Future directions for research on enterotoxigenic Escherichia coli vaccines for developing countries. Wkly Epidemiol Rec 81: 97–104.
(2008) Enterotoxigenic Escherichia coli: advances in technical and laboratory aspects of research and development of vaccines. Wkly Epidemiol Rec 83: 92–95.
[4]
Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, et al. (2010) Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes Infect 12: 89–98. doi: 10.1016/j.micinf.2009.10.002
Sjoling A, Wiklund G, Savarino SJ, Cohen DI, Svennerholm AM (2007) Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors. Journal of clinical microbiology 45: 3295–3301. doi: 10.1128/JCM.00471-07
[7]
Walker RI, Steele D, Aguado T (2007) Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E. coli (ETEC) disease. Vaccine 25: 2545–2566. doi: 10.1016/j.vaccine.2006.12.028
[8]
Oyofo BA, Subekti DS, Svennerholm AM, Machpud NN, Tjaniadi P, et al. (2001) Toxins and colonization factor antigens of enterotoxigenic Escherichia coli among residents of Jakarta, Indonesia. Am J Trop Med Hyg 65: 120–124.
[9]
Peruski LF Jr, Kay BA, El-Yazeed RA, El-Etr SH, Cravioto A, et al. (1999) Phenotypic Diversity of Enterotoxigenic Escherichia coli Strains from a Community-Based Study of Pediatric Diarrhea in Periurban Egypt. J Clin Microbiol 37: 2974–2978.
[10]
Clemens JD, Sack DA, Harris JR, Chakraborty J, Neogy PK, et al. (1988) Cross-protection by B subunit-whole cell cholera vaccine against diarrhea associated with heat-labile toxin-producing enterotoxigenic Escherichia coli: results of a large-scale field trial. J Infect Dis 158: 372–377. doi: 10.1093/infdis/158.2.372
Fleckenstein JM, Roy K, Fischer JF, Burkitt M (2006) Identification of a two-partner secretion locus of enterotoxigenic Escherichia coli. Infect Immun 74: 2245–2258. doi: 10.1128/IAI.74.4.2245-2258.2006
[13]
Roy K, Hilliard GM, Hamilton DJ, Luo J, Ostmann MM, et al. (2009) Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells. Nature 457: 594–598. doi: 10.1038/nature07568
[14]
Patel SK, Dotson J, Allen KP, Fleckenstein JM (2004) Identification and molecular characterization of EatA, an autotransporter protein of enterotoxigenic Escherichia coli. Infect Immun 72: 1786–1794. doi: 10.1128/IAI.72.3.1786-1794.2004
[15]
Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, Ala'Aldeen D (2004) Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68: 692–744. doi: 10.1128/MMBR.68.4.692-744.2004
[16]
Wells TJ, Tree JJ, Ulett GC, Schembri MA (2007) Autotransporter proteins: novel targets at the bacterial cell surface. FEMS Microbiol Lett 274: 163–172. doi: 10.1111/j.1574-6968.2007.00833.x
[17]
Gustafsson L, Hallander HO, Olin P, Reizenstein E, Storsaeter J (1996) A controlled trial of a two-component acellular, a five-component acellular, and a whole-cell pertussis vaccine. N Engl J Med 334: 349–355. doi: 10.1056/NEJM199602083340602
[18]
Greco D, Salmaso S, Mastrantonio P, Giuliano M, Tozzi AE, et al. (1996) A controlled trial of two acellular vaccines and one whole-cell vaccine against pertussis. Progetto Pertosse Working Group. N Engl J Med 334: 341–348. doi: 10.1056/NEJM199602083340601
[19]
Ward JI, Cherry JD, Chang SJ, Partridge S, Lee H, et al. (2005) Efficacy of an acellular pertussis vaccine among adolescents and adults. N Engl J Med 353: 1555–1563. doi: 10.1056/NEJMoa050824
[20]
Relman DA, Domenighini M, Tuomanen E, Rappuoli R, Falkow S (1989) Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci U S A 86: 2637–2641. doi: 10.1073/pnas.86.8.2637
[21]
Alonso S, Reveneau N, Pethe K, Locht C (2002) Eighty-kilodalton N-terminal moiety of Bordetella pertussis filamentous hemagglutinin: adherence, immunogenicity, and protective role. Infect Immun 70: 4142–4147. doi: 10.1128/IAI.70.8.4142-4147.2002
[22]
Charles IG, Dougan G, Pickard D, Chatfield S, Smith M, et al. (1989) Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis. Proc Natl Acad Sci U S A 86: 3554–3558. doi: 10.1073/pnas.86.10.3554
[23]
Novotny P, Chubb AP, Cownley K, Charles IG (1991) Biologic and protective properties of the 69-kDa outer membrane protein of Bordetella pertussis: a novel formulation for an acellular pertussis vaccine. J Infect Dis 164: 114–122. doi: 10.1093/infdis/164.1.114
[24]
Roy K, Hamilton D, Ostmann MM, Fleckenstein JM (2009) Vaccination with EtpA glycoprotein or flagellin protects against colonization with enterotoxigenic Escherichia coli in a murine model. Vaccine 27: 4601–4608. doi: 10.1016/j.vaccine.2009.05.076
[25]
Roy K, Hamilton D, Allen KP, Randolph MP, Fleckenstein JM (2008) The EtpA exoprotein of enterotoxigenic Escherichia coli promotes intestinal colonization and is a protective antigen in an experimental model of murine infection. Infect Immun 76: 2106–2112. doi: 10.1128/IAI.01304-07
[26]
Roy K, Bartels S, Qadri F, Fleckenstein JM (2010) Enterotoxigenic Escherichia coli elicits immune responses to multiple surface proteins. Infect Immun 78: 3027–3035. doi: 10.1128/IAI.00264-10
[27]
Cravioto A, Reyes RE, Trujillo F, Uribe F, Navarro A, et al. (1990) Risk of diarrhea during the first year of life associated with initial and subsequent colonization by specific enteropathogens. Am J Epidemiol 131: 886–904.
[28]
Rao MR, Wierzba TF, Savarino SJ, Abu-Elyazeed R, El-Ghoreb N, et al. (2005) Serologic correlates of protection against enterotoxigenic Escherichia coli diarrhea. J Infect Dis 191: 562–570. doi: 10.1086/427662
[29]
Steinsland H, Valentiner-Branth P, Gjessing HK, Aaby P, Molbak K, et al. (2003) Protection from natural infections with enterotoxigenic Escherichia coli: longitudinal study. Lancet 362: 286–291. doi: 10.1016/S0140-6736(03)13971-2
[30]
McNeil LK, Reich C, Aziz RK, Bartels D, Cohoon M, et al. (2007) The National Microbial Pathogen Database Resource (NMPDR): a genomics platform based on subsystem annotation. Nucleic Acids Res 35: D347–353. doi: 10.1093/nar/gkl947
[31]
Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, et al. (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9: 75. doi: 10.1186/1471-2164-9-75
[32]
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948. doi: 10.1093/bioinformatics/btm404
[33]
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797. doi: 10.1093/nar/gkh340
[34]
Harlow E, Lane D, Harlow E (1999) Using antibodies : a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.
[35]
Goodrich W (2006) The Kinetic ELISA Advantage in Quantitative Assays. Winooski, Vermont.
[36]
Tsang VC, Wilson BC, Maddison SE (1980) Kinetic studies of a quantitative single-tube enzyme-linked immunosorbent assay. Clin Chem 26: 1255–1260.
[37]
Roy K, Hamilton D, Ostmann MM, Fleckenstein JM (2009) Vaccination with EtpA glycoprotein or flagellin protects against colonization with enterotoxigenic Escherichia coli in a murine model. Vaccine. doi: 10.1016/j.vaccine.2009.05.076
[38]
Allen KP, Randolph MM, Fleckenstein JM (2006) Importance of heat-labile enterotoxin in colonization of the adult mouse small intestine by human enterotoxigenic Escherichia coli strains. Infect Immun 74: 869–875. doi: 10.1128/IAI.74.2.869-875.2006
[39]
Chabot S, Brewer A, Lowell G, Plante M, Cyr S, et al. (2005) A novel intranasal Protollin-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice. Vaccine 23: 1374–1383. doi: 10.1016/j.vaccine.2004.09.010
[40]
Dorsey FC, Fischer JF, Fleckenstein JM (2006) Directed delivery of heat-labile enterotoxin by enterotoxigenic Escherichia coli. Cellular Microbiology 8: 1516–1527. doi: 10.1111/j.1462-5822.2006.00736.x
[41]
Rasko DA, Rosovitz MJ, Myers GS, Mongodin EF, Fricke WF, et al. (2008) The pan-genome structure of Escherichia coli: comparative genomic analysis of E. coli commensal and pathogenic isolates. J Bacteriol. doi: 10.1128/jb.00619-08
[42]
van der Woude MW, Henderson IR (2008) Regulation and function of Ag43 (flu). Annu Rev Microbiol 62: 153–169. doi: 10.1146/annurev.micro.62.081307.162938
[43]
Toh H, Oshima K, Toyoda A, Ogura Y, Ooka T, et al. (2010) Complete genome sequence of the wild-type commensal Escherichia coli strain SE15, belonging to phylogenetic group B2. Journal of bacteriology 192: 1165–1166. doi: 10.1128/JB.01543-09
[44]
Yi H, Cho YJ, Hur HG, Chun J (2011) Genome sequence of Escherichia coli AA86, isolated from cow feces. Journal of bacteriology 193: 3681. doi: 10.1128/JB.05193-11
[45]
Al-Hasani K, Navarro-Garcia F, Huerta J, Sakellaris H, Adler B (2009) The immunogenic SigA enterotoxin of Shigella flexneri 2a binds to HEp-2 cells and induces fodrin redistribution in intoxicated epithelial cells. PLoS One 4: e8223. doi: 10.1371/journal.pone.0008223
[46]
Turner DP, Marietou AG, Johnston L, Ho KK, Rogers AJ, et al. (2006) Characterization of MspA, an immunogenic autotransporter protein that mediates adhesion to epithelial and endothelial cells in Neisseria meningitidis. Infect Immun 74: 2957–2964. doi: 10.1128/IAI.74.5.2957-2964.2006
[47]
Litwin CM, Rawlins ML, Swenson EM (2007) Characterization of an immunogenic outer membrane autotransporter protein, Arp, of Bartonella henselae. Infect Immun 75: 5255–5263. doi: 10.1128/IAI.00533-07
[48]
Fries LF, Montemarano AD, Mallett CP, Taylor DN, Hale TL, et al. (2001) Safety and immunogenicity of a proteosome-Shigella flexneri 2a lipopolysaccharide vaccine administered intranasally to healthy adults. Infect Immun 69: 4545–4553. doi: 10.1128/IAI.69.7.4545-4553.2001
[49]
Jones T, Cyr S, Allard F, Bellerose N, Lowell GH, et al. (2004) Protollin: a novel adjuvant for intranasal vaccines. Vaccine 22: 3691–3697. doi: 10.1016/j.vaccine.2004.03.035
[50]
Boedeker EC (2005) Vaccines for enterotoxigenic Escherichia coli: current status. Curr Opin Gastroenterol 21: 15–19.
[51]
Rasko DA, Rosovitz MJ, Myers GS, Mongodin EF, Fricke WF, et al. (2008) The pangenome structure of Escherichia coli: comparative genomic analysis of E. coli commensal and pathogenic isolates. Journal of bacteriology 190: 6881–6893. doi: 10.1128/JB.00619-08
[52]
Sahl JW, Steinsland H, Redman JC, Angiuoli SV, Nataro JP, et al. (2011) A comparative genomic analysis of diverse clonal types of enterotoxigenic Escherichia coli reveals pathovar-specific conservation. Infection and immunity 79: 950–960. doi: 10.1128/IAI.00932-10
[53]
Qadri F, Das SK, Faruque ASG, Fuchs GJ, Albert MJ, et al. (2000) Prevalence of Toxin Types and Colonization Factors in Enterotoxigenic Escherichia coli Isolated during a 2-Year Period from Diarrheal Patients in Bangladesh. J Clin Microbiol 38: 27–31.
[54]
Chowdhury F, Rahman MA, Begum YA, Khan AI, Faruque AS, et al. (2011) Impact of Rapid Urbanization on the Rates of Infection by Vibrio cholerae O1 and Enterotoxigenic Escherichia coli in Dhaka, Bangladesh. PLoS neglected tropical diseases 5: e999. doi: 10.1371/journal.pntd.0000999
[55]
Elsinghorst EA, Weitz JA (1994) Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein. Infect Immun 62: 3463–3471.
[56]
Lindenthal C, Elsinghorst EA (2001) Enterotoxigenic Escherichia coli TibA glycoprotein adheres to human intestine epithelial cells. Infect Immun 69: 52–57. doi: 10.1128/IAI.69.1.52-57.2001
[57]
Roy K, Kansal R, Bartels SR, Hamilton DJ, Shaaban S, et al. (2011) Adhesin Degradation Accelerates Delivery of Heat-labile Toxin by Enterotoxigenic Escherichia coli. The Journal of biological chemistry 286: 29771–29779. doi: 10.1074/jbc.M111.251546
[58]
Crossman LC, Chaudhuri RR, Beatson SA, Wells TJ, Desvaux M, et al. (2010) A commensal gone bad: complete genome sequence of the prototypical enterotoxigenic Escherichia coli strain H10407. Journal of bacteriology 192: 5822–5831. doi: 10.1128/JB.00710-10
[59]
Del Canto F, Valenzuela P, Cantero L, Bronstein J, Blanco JE, et al. (2011) Distribution of Classical and Nonclassical Virulence Genes in Enterotoxigenic Escherichia coli Isolates from Chilean Children and tRNA Gene Screening for Putative Insertion Sites for Genomic Islands. Journal of clinical microbiology 49: 3198–3203. doi: 10.1128/JCM.02473-10
[60]
Anderson GG, Palermo JJ, Schilling JD, Roth R, Heuser J, et al. (2003) Intracellular bacterial biofilm-like pods in urinary tract infections. Science 301: 105–107. doi: 10.1126/science.1084550
[61]
Ulett GC, Valle J, Beloin C, Sherlock O, Ghigo JM, et al. (2007) Functional analysis of antigen 43 in uropathogenic Escherichia coli reveals a role in long-term persistence in the urinary tract. Infect Immun 75: 3233–3244. doi: 10.1128/IAI.01952-06
[62]
Alteri CJ, Mobley HL (2007) Quantitative profile of the uropathogenic Escherichia coli outer membrane proteome during growth in human urine. Infect Immun 75: 2679–2688. doi: 10.1128/IAI.00076-06
de Luna MG, Scott-Tucker A, Desvaux M, Ferguson P, Morin NP, et al. (2008) The Escherichia coli biofilm-promoting protein Antigen 43 does not contribute to intestinal colonization. FEMS Microbiol Lett 284: 237–246. doi: 10.1111/j.1574-6968.2008.01207.x
[65]
Restieri C, Garriss G, Locas MC, Dozois CM (2007) Autotransporter-encoding sequences are phylogenetically distributed among Escherichia coli clinical isolates and reference strains. Appl Environ Microbiol 73: 1553–1562. doi: 10.1128/AEM.01542-06
[66]
Plotkin SA (2008) Vaccines: correlates of vaccine-induced immunity. Clin Infect Dis 47: 401–409. doi: 10.1086/589862
[67]
Plotkin SA (2010) Correlates of protection induced by vaccination. Clin Vaccine Immunol 17: 1055–1065. doi: 10.1128/CVI.00131-10
[68]
Ouyang-Latimer J, Ajami NJ, Jiang ZD, Okhuysen PC, Paredes M, et al. (2010) Biochemical and genetic diversity of enterotoxigenic Escherichia coli associated with diarrhea in United States students in Cuernavaca and Guadalajara, Mexico, 2004–2007. The Journal of infectious diseases 201: 1831–1838. doi: 10.1086/652797
[69]
Levine MM, Nalin DR, Hoover DL, Bergquist EJ, Hornick RB, et al. (1979) Immunity to enterotoxigenic Escherichia coli. Infect Immun 23: 729–736.
[70]
Dupont H, Formal S, Hornick R, Snyder M, Libonati J, et al. (1971) Pathogenesis of Escherichia coli diarrhea. N Engl J Med 285: 1–9. doi: 10.1056/NEJM197107012850101
[71]
Evans DG, Silver RP, Evans DJ Jr, Chase DG, Gorbach SL (1975) Plasmid-controlled colonization factor associated with virulence in Escherichia coli enterotoxigenic for humans. Infect Immun 12: 656–667.
[72]
Evans DJ Jr, Evans DG (1973) Three characteristics associated with enterotoxigenic Escherichia coli isolated from man. Infect Immun 8: 322–328.
[73]
Levine MM, Ristaino P, Marley G, Smyth C, Knutton S, et al. (1984) Coli surface antigens 1 and 3 of colonization factor antigen II-positive enterotoxigenic Escherichia coli: morphology, purification, and immune responses in humans. Infect Immun 44: 409–420.
[74]
Ogura Y, Ooka T, Iguchi A, Toh H, Asadulghani M, et al. (2009) Comparative genomics reveal the mechanism of the parallel evolution of O157 and non-O157 enterohemorrhagic Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 106: 17939–17944. doi: 10.1073/pnas.0903585106
[75]
Levine MM, Nataro JP, Karch H, Baldini MM, Kaper JB, et al. (1985) The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. The Journal of infectious diseases 152: 550–559. doi: 10.1093/infdis/152.3.550
[76]
Paulozzi LJ, Johnson KE, Kamahele LM, Clausen CR, Riley LW, et al. (1986) Diarrhea associated with adherent enteropathogenic Escherichia coli in an infant and toddler center, Seattle, Washington. Pediatrics 77: 296–300.
[77]
De Rycke J, Comtet E, Chalareng C, Boury M, Tasca C, et al. (1997) Enteropathogenic Escherichia coli O103 from rabbit elicits actin stress fibers and focal adhesions in HeLa epithelial cells, cytopathic effects that are linked to an analog of the locus of enterocyte effacement. Infection and immunity 65: 2555–2563.
[78]
Roos V, Ulett GC, Schembri MA, Klemm P (2006) The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E. coli strains in human urine. Infection and immunity 74: 615–624. doi: 10.1128/IAI.74.1.615-624.2006
[79]
Stapleton A, Moseley S, Stamm WE (1991) Urovirulence determinants in Escherichia coli isolates causing first-episode and recurrent cystitis in women. The Journal of infectious diseases 163: 773–779. doi: 10.1093/infdis/163.4.773
[80]
Chen SL, Hung CS, Xu J, Reigstad CS, Magrini V, et al. (2006) Identification of genes subject to positive selection in uropathogenic strains of Escherichia coli: a comparative genomics approach. Proceedings of the National Academy of Sciences of the United States of America 103: 5977–5982. doi: 10.1073/pnas.0600938103
[81]
Korhonen TK, Valtonen MV, Parkkinen J, Vaisanen-Rhen V, Finne J, et al. (1985) Serotypes, hemolysin production, and receptor recognition of Escherichia coli strains associated with neonatal sepsis and meningitis. Infection and immunity 48: 486–491.
