• View in gallery

    Secretion of serine protease autotransporter toxin (SPATE) proteins from enteroaggregative Escherichia coli (EAEC) strains. Supernatants from five representative EAEC strains are shown. Supernatant proteins were precipitated with 10% trichoroacetic acid and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Coomassie staining. SPATE proteins represented by bands directly identified by mass spectrometry were SepA from strains H194-2 and 239-1, Pic from strains H77-1 and 239-1, and Sat from strain 232-1-1 (indicated by arrows). MW = molecular weight; HB101 = negative control strain.

  • View in gallery

    Distribution of class I and class II serine protease autotransporter toxin (SPATE) proteins among enteroaggregative Escherichia coli (EAEC) strains. The distribution of the SPATE genes among 55 EAEC strains was determined by polymerase chain reaction A, Distribution of class I and class II SPATEs among the EAEC collection. B, Frequency of Pet, Sat, SigA, and EspP.

  • View in gallery

    Identification of serine protease autotransporter toxin (SPATE) sequences from enteroaggregative Escherichia coli (EAEC) and Shigella isolates by multiplex polymerase chain reaction (PCR). The PCR products representing genes for Sat, SepA, Pic, SigA, and Pet were separated by electrophoresis on 1.2% agarose gels. A, PCR products from 11 EAEC strains. B, PCR products from 12 Shigella strains. EAEC strain 042 is the positive control for pet and pic, and S. flexneri 2a strain 2457T is the positive control for sat, sepA, pic, and sigA.

  • 1

    Glandt M, Adachi JA, Mathewson JJ, Jiang ZD, DiCesare D, Ashley D, Ericsson CD, DuPont HL, 1999. Enteroaggregative Escherichia coli as a cause of traveler’s diarrhea: clinical response to ciprofloxacin. Clin Infect Dis 29 :335–338.

    • Search Google Scholar
    • Export Citation
  • 2

    Adachi JA, Jiang ZD, Mathewson JJ, Verenkar MP, Thompson S, Martinez-Sandoval F, Steffen R, Ericsson CD, DuPont HL, 2001. Enteroaggregative Escherichia coli as a major etiologic agent in traveler’s diarrhea in 3 regions of the world. Clin Infect Dis 32 :1706–1709.

    • Search Google Scholar
    • Export Citation
  • 3

    Adachi JA, Ericsson CD, Jiang ZD, DuPont MW, Pallegar SR, DuPont HL, 2002. Natural history of enteroaggregative and enterotoxigenic Escherichia coli infection among US travelers to Guadalajara, Mexico. J Infect Dis 185 :1681–1683.

    • Search Google Scholar
    • Export Citation
  • 4

    Tompkins DS, Hudson MJ, Smith HR, Eglin RP, Wheeler JG, Brett MM, Owen RJ, Brazier JS, Cumberland P, King V, Cook PE, 1999. A study of infectious intestinal disease in England: microbiological findings in cases and controls. Commun Dis Public Health 2 :108–113.

    • Search Google Scholar
    • Export Citation
  • 5

    Okeke IN, Lamikanra A, Czeczulin J, Dubovsky F, Kaper JB, Nataro JP, 2000. Heterogeneous virulence of enteroaggregative Escherichia coli strains isolated from children in southwest Nigeria. J Infect Dis 181 :252–260.

    • Search Google Scholar
    • Export Citation
  • 6

    Wanke CA, Gerrior J, Blais V, Mayer H, Acheson D, 1998. Successful treatment of diarrheal disease associated with enteroaggregative Escherichia coli in adults infected with human immunodeficiency virus. J Infect Dis 178 :1369–1372.

    • Search Google Scholar
    • Export Citation
  • 7

    Wanke CA, Mayer H, Weber R, Zbinden R, Watson DA, Acheson D, 1998. Enteroaggregative Escherichia coli as a potential cause of diarrheal disease in adults infected with human immunodeficiency virus. J Infect Dis 178 :185–190.

    • Search Google Scholar
    • Export Citation
  • 8

    Durrer P, Zbinden R, Fleisch F, Altwegg M, Ledergerber B, Karch H, Weber R, 2000. Intestinal infection due to enteroaggregative Escherichia coli among human immunodeficiency virus-infected persons. J Infect Dis 182 :1540–1544.

    • Search Google Scholar
    • Export Citation
  • 9

    Mossoro C, Glaziou P, Yassibanda S, Lan NT, Bekondi C, Minssart P, Bernier C, Le Bouguenec C, Germani Y, 2002. Chronic diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome associated with HEp-2 adherent Escherichia coli in adults infected with human immunodeficiency virus in Bangui, Central African Republic. J Clin Microbiol 40 :3086–3088.

    • Search Google Scholar
    • Export Citation
  • 10

    Gassama-Sow A, Sow PS, Gueye M, Gueye-N’diaye A, Perret JL, M’Boup S, Aidara-Kane A, 2004. Characterization of pathogenic Escherichia coli in human immunodeficiency virus-related diarrhea in Senegal. J Infect Dis 189 :75–78.

    • Search Google Scholar
    • Export Citation
  • 11

    Menard LP, Dubreuil JD, 2002. Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1): a new toxin with an old twist. Crit Rev Microbiol 28 :43–60.

    • Search Google Scholar
    • Export Citation
  • 12

    Paiva de Sousa C, Dubreuil JD, 2001. Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella. Int J Med Microbiol 291 :15–20.

    • Search Google Scholar
    • Export Citation
  • 13

    Savarino SJ, Fasano A, Robertson DC, Levine MM, 1991. Enteroaggregative Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in vitro rabbit intestinal model. J Clin Invest 87 :1450–1455.

    • Search Google Scholar
    • Export Citation
  • 14

    Eslava C, Navarro-Garcia F, Czeczulin JR, Henderson IR, Cravioto A, Nataro JP, 1998. Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli. Infect Immun 66 :3155–3163.

    • Search Google Scholar
    • Export Citation
  • 15

    Vila J, Vargas M, Henderson IR, Gascon J, Nataro JP, 2000. Enteroaggregative Escherichia coli virulence factors in traveler’s diarrhea strains. J Infect Dis 182 :1780–1783.

    • Search Google Scholar
    • Export Citation
  • 16

    Henderson IR, Nataro JP, 2001. Virulence functions of autotransporter proteins. Infect Immun 69 :1231–1243.

  • 17

    Henderson IR, Hicks S, Navarro-Garcia F, Elias WP, Philips AD, Nataro JP, 1999. Involvement of the enteroaggregative Escherichia coli plasmid-encoded toxin in causing human intestinal damage. Infect Immun 67 :5338–5344.

    • Search Google Scholar
    • Export Citation
  • 18

    Fasano A, Noriega FR, Liao FM, Wang W, Levine MM, 1997. Effect of Shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo. Gut 40 :505–511.

    • Search Google Scholar
    • Export Citation
  • 19

    Huang DB, Okhuysen PC, Jiang ZD, DuPont HL, 2004. Enteroaggregative Escherichia coli: an emerging enteric pathogen. Am J Gastroenterol 99 :383–389.

    • Search Google Scholar
    • Export Citation
  • 20

    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.

    • Search Google Scholar
    • Export Citation
  • 21

    Dutta PR, Cappello R, Navarro-Garcia F, Nataro JP, 2002. Functional comparison of serine protease autotransporters of enterobacteriaceae. Infect Immun 70 :7105–7113.

    • Search Google Scholar
    • Export Citation
  • 22

    Henderson IR, Czeczulin J, Eslava C, Noriega F, Nataro JP, 1999. Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. Infect Immun 67 :5587–5596.

    • Search Google Scholar
    • Export Citation
  • 23

    Benjelloun-Touimi Z, Sansonetti PJ, Parsot C, 1995. SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion. Mol Microbiol 17 :123–135.

    • Search Google Scholar
    • Export Citation
  • 24

    Provence DL, Curtiss R 3rd, 1994. Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain. Infect Immun 62 :1369–1380.

    • Search Google Scholar
    • Export Citation
  • 25

    Czeczulin JR, Whittam TS, Henderson IR, Navarro-Garcia F, Nataro JP, 1999. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. Infect Immun 67 :2692–2699.

    • Search Google Scholar
    • Export Citation
  • 26

    Olesen B, Neimann J, Bottiger B, Ethelberg S, Schiellerup P, Jensen C, Helms M, Scheutz F, Olsen KE, Krogfelt K, Petersen E, Molbak K, Gerner-Smidt P, 2005. Etiology of diarrhea in young children in Denmark: a case-control study. J Clin Microbiol 43 :3636–3641.

    • Search Google Scholar
    • Export Citation
  • 27

    Boisen N, Struve C, Scheutz F, Krogfelt KA, Nataro JP, 2008. New adhesin of enteroaggregative Escherichia coli related to the Afa/Dr/AAF family. Infect Immun 76 :3281–3292.

    • Search Google Scholar
    • Export Citation
  • 28

    Dudley EG, Thomson NR, Parkhill J, Morin NP, Nataro JP, 2006. Proteomic and microarray characterization of the AggR regulon identifies a pheU pathogenicity island in enteroaggregative Escherichia coli. Mol Microbiol 61 :1267–1282.

    • Search Google Scholar
    • Export Citation
  • 29

    Sheikh J, Hicks S, Dall’Agnol M, Phillips AD, Nataro JP, 2001. Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli. Mol Microbiol 41 :983–997.

    • Search Google Scholar
    • Export Citation
  • 30

    Wei J, Goldberg MB, Burland V, Venkatesan MM, Deng W, Fournier G, Mayhew GF, Plunkett G 3rd, Rose DJ, Darling A, Mau B, Perna NT, Payne SM, Runyen-Janecky LJ, Zhou S, Schwartz DC, Blattner FR, 2003. Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T. Infect Immun 71 :2775–2786.

    • Search Google Scholar
    • Export Citation
  • 31

    Orskov F, Orskov I, 1992. Escherichia coli serotyping and disease in man and animals. Can J Microbiol 38 :699–704.

  • 32

    Guyer DM, Henderson IR, Nataro JP, Mobley HL, 2000. Identification of sat, an autotransporter toxin produced by uropathogenic Escherichia coli. Mol Microbiol 38 :53–66.

    • Search Google Scholar
    • Export Citation
  • 33

    Guignot J, Chaplais C, Coconnier-Polter MH, Servin AL, 2007. The secreted autotransporter toxin, Sat, functions as a virulence factor in Afa/Dr diffusely adhering Escherichia coli by promoting lesions in tight junction of polarized epithelial cells. Cell Microbiol 9 :204–221.

    • Search Google Scholar
    • Export Citation
  • 34

    Maroncle NM, Sivick KE, Brady R, Stokes FE, Mobley HL, 2006. Protease activity, secretion, cell entry, cytotoxicity, and cellular targets of secreted autotransporter toxin of uropathogenic Escherichia coli. Infect Immun 74 :6124–6134.

    • Search Google Scholar
    • Export Citation
  • 35

    Canizalez-Roman A, Navarro-Garcia F, 2003. Fodrin CaM-binding domain cleavage by Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption. Mol Microbiol 48 :947–958.

    • Search Google Scholar
    • Export Citation
  • 36

    Taddei CR, Moreno AC, Fernandes Filho A, Montemor LP, Martinez MB, 2003. Prevalence of secreted autotran sporter toxin gene among diffusely adhering Escherichia coli iso lated from stools of children. FEMS Microbiol Lett 227 :249–253.

    • Search Google Scholar
    • Export Citation
  • 37

    Al-Hasani K, Henderson IR, Sakellaris H, Rajakumar K, Grant T, Nataro JP, Robins-Browne R, Adler B, 2000. The sigA gene which is borne on the she pathogenicity island of Shigella flexneri 2a encodes an exported cytopathic protease involved in intestinal fluid accumulation. Infect Immun 68 :2457–2463.

    • Search Google Scholar
    • Export Citation
  • 38

    Roy S, Thanasekaran K, Dutta Roy AR, Sehgal SC, 2006. Distribution of Shigella enterotoxin genes and secreted autotransporter toxin gene among diverse species and serotypes of Shigella isolated from Andaman Islands, India. Trop Med Int Health 11 :1694–1698.

    • Search Google Scholar
    • Export Citation
  • 39

    Parsot C, 2005. Shigella spp. and enteroinvasive Escherichia coli pathogenicity factors. FEMS Microbiol Lett 252 :11–18.

  • 40

    Kaper JB, Nataro JP, Mobley HL, 2004. Pathogenic Escherichia coli. Nat Rev Microbiol 2 :123–140.

  • 41

    Levine MM, Bergquist EJ, Nalin DR, Waterman DH, Hornick RB, Young CR, Sotman S, 1978. Escherichia coli strains that cause diarrhoea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive. Lancet 1 :1119–1122.

    • Search Google Scholar
    • Export Citation
  • 42

    Bilge SS, Clausen CR, Lau W, Moseley SL, 1989. Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells. J Bacteriol 171 :4281–4289.

    • Search Google Scholar
    • Export Citation
  • 43

    RestieriC,GarrissG,LocasMC,DozoisCM, 2007. Autotransporter-encoding sequences are phylogenetically distributed among Escherichia coli clinical isolates and reference strains. Appl Environ Microbiol 73 :1553–1562.

    • Search Google Scholar
    • Export Citation
  • 44

    Bernier C, Gounon P, Le Bouguenec C, 2002. Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a sensitive probe for detecting the AAF-encoding operon family. Infect Immun 70 :4302–4311.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 

 

 

 

 

High Prevalence of Serine Protease Autotransporter Cytotoxins among Strains of Enteroaggregative Escherichia coli

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  • 1 Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Copenhagen, Denmark: Departments of Pediatrics and Medicine, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland

Enteroaggregative Escherichia coli (EAEC) pathogenesis is thought to comprise intestinal colonization followed by the release of enterotoxins and cytotoxins. Here, we use a polymerase chain reaction (PCR) to determine the prevalence of 10 genes encoding serine protease autotransporter toxins (SPATEs) in a collection of clinical EAEC isolates. Eighty-six percent of EAEC strains harbored genes encoding one or more class I cytotoxic SPATE proteins (Pet, Sat, EspP, or SigA). Two Class II, non-cytotoxic, SPATE genes were found among EAEC strains: pic and sepA, each originally described in Shigella flexneri 2a. Using a multiplex PCR for five SPATE genes (pet, sat, sigA, pic, and sepA), we found that most of the Shigella isolates also harbored more than one SPATE, whereas members of most other E. coli pathotypes rarely harbored a cytotoxic SPATE gene. SPATEs may be relevant to the pathogenesis of both EAEC and Shigella spp.

Enteroaggregative Escherichia coli (EAEC) has been associated with several clinical scenarios, including travelers’ diarrhea,13 endemic pediatric diarrhea among children in industrialized4 and developing countries,5 as well as persistent diarrhea among human immunodeficiency virus–infected patients.610 The current pathogenetic paradigm for EAEC includes colonization of the intestinal mucosa followed by the elaboration of one or more cytotoxins and enterotoxins, of which several have been described. 1118 Enterotoxins include the enteroaggregative heat-stable toxin and Shigella enterotoxin-1. The roles of these toxins in EAEC pathogenesis and epidemiology are not yet known.

Infection of human intestinal explants suggests that most EAEC strains elicit frank mucosal damage, accompanied by rounding and exfoliation of colonocytes. Henderson and others have reported that the serine protease autotransporter (SPATE) toxin called Pet (plasmid-encoded toxin) is required for strain 042 to elicit cytotoxic effects on human explants 17; however, only a small number of EAEC strains carry the pet gene, although a much larger number of strains are toxic to explants. This paradox occurs in the context of substantial heterogeneity of EAEC adhesins and other putative virulence factors, presenting a confusing clinical and epidemiologic scenario. 19

The Pet protease is a member of the SPATEs of the Enterobacteriaceae family of secreted proteases. SPATEs comprise a large group of trypsin-like serine proteases, which are secreted by Shigella spp., uropathogenic E. coli, and all of the diarrheagenic E. coli pathotypes.19,20 The toxins are translocated across the outer membrane by the autotransporter pathway, in which translocation requires a dedicated C-terminal beta barrel domain. The N-terminal, mature SPATE toxins are 104–110 kD and feature a typical N-terminal serine protease catalytic domain, followed by a highly conserved beta-helix motif, which is present in nearly all autotransporters. 20 The SPATE family has been organized phylogenetically into two classes. Members of class I SPATEs (which include Pet) are all cytotoxic to epithelial cells. 21 In addition to Pet, class I SPATEs include, prominently, EspP from enterohemorrhagic E. coli (EHEC), EspC from enteropathogenic E. coli (EPEC), SigA from Shigella flexneri, and Sat, from uropathogenic and diffusely adhering E. coli (DAEC).16 Class II SPATEs are more diverse with regard to phenotype, although several are known to cleave mucin. Many EAEC and Shigella strains encode Pic, a mucinase encoded on the bacterial chromosome. 22 Pic may promote intestinal colonization by an unknown mechanism (Harrington S, Nataro JP, unpublished data). Class II includes, besides Pic, SepA from S. flexneri23 and Tsh from avian pathogenic E. coli.24

We sought to resolve the paradox between observed cytotoxic effects attributed to most EAEC strains and the low carriage rate of the Pet cytotoxin. Specifically, we hypothesized that other class I SPATE cytotoxins would be commonly found among EAEC strains. We tested this hypothesis on 55 EAEC and 10 each of ETEC, EHEC, EPEC, enteroinvasive E. coli (EIEC), and DAEC strains, as well as 12 Shigella strains. All strains were isolated in the course of epidemiologic studies 25,26 and were derived from the collections of the Statens Serum Institut in Denmark or the Center for Vaccine Development of the University of Maryland School of Medicine (Tables 1 and 2).25,2729 In addition, we selected 12 non-pathogenic E. coli strains: six strains were isolated from healthy humans in the course of the Danish Integrated Antimicrobial Resistance Monitoring and Research Program, and six strains were isolated from minced meat as part of the Danish food surveillance program. Shigella flexneri 2a strain 2457T was used as a control. 30 Stock cultures were frozen at −80°C in Luria broth or Statens Serum Institut broth containing 10% (v/v) glycerol. All strains were grown at 37°C. Serotyping of the EAEC strains was performed at the Statens Serum Institut by using standard methods. 31

A polymerase chain reaction (PCR) was used to detect the presence of genes corresponding to known SPATE sequences. Primers used are listed in Table 3. DNA template was obtained as previously described. 27 The accuracy of a subset of PCRs was verified by nucleotide sequencing in the Biopolymer Laboratory Core Facility, University of Maryland School of Medicine. A multiplex PCR was designed to detect the most common SPATE genes; the Multiplex PCR kit was used according to manufacturer’s instructions (Qiagen, Valencia, CA). Products were amplified by using the Mastercycler Gradient Thermal Cycler (Eppendorf North America Inc., Westbury, NY). The specific temperatures and primers are listed in Table 3. Monoplex PCRs were performed as described. 27 Multiplex PCR cycles were denaturation at 95°C for 15 minutes, 35 cycles of denaturation (depending on the size of the product, with a 30-second increase for each 500 basepairs) at 94°C for 30 seconds, annealing at 72°C for 1.5 minutes, and extension for 1.5 minutes at 72°C, and a final extension at 72°C for 10 minutes.

Results of monoplex PCR for 10 SPATE-encoding genes (pet, sigA, espC, espP, sat, vat, pic, sepA, tsh, and eatA) are shown in Table 1. To corroborate results of the PCR assays, the supernatants of several strains that were positive for SPATE sequences by PCR were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 17.5% polyacrylamide gel. Strains positive for up to three SPATEs by PCR also showed the expected number of supernatant proteins at the predicted molecular mass (104–130 kD). Several protein bands were excised and subjected to tryptic digestion and mass spectrometry in a Finnigan LCQ Advantage Ion Trap Mass Spectrometer in the Protein Analysis Core of the Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine. These analyses yielded protein identifications that correlated with PCR assays for pic, sat, and sepA (Figure 1).

Of 55 EAEC strains tested, 94.5% harbored genes for one or more of the SPATE proteins, including members of class I (Pet, Sat, SigA, EspP) and/or class II (Pic, SepA, Tsh). No EAEC strain yielded a product corresponding to vat, espC, eatA, or tsh. A total of 18.2% of the strains had only a class I SPATE and no class II SPATE, 9.1% of the strains had only a class II SPATE, and 67.3% had a class I SPATE and a class II SPATE (Figure 2A). Surprisingly, the most common SPATE among the EAEC strains was Sat (74.5% of strains), which was first described in uropathogenic E. coli strains, but which has more recently been reported among DAEC. In contrast, we found pet in only 9.1% of our strains, and one strain (H92-1, Figure 2A , Table 1 ) also harbored the sat gene. The mature Sat and Pet toxins are 52% identical at the amino acid level and thus they may be allelic toxins that fulfill the same roles in pathogenesis. 32 Sat is cytotoxic to urinary epithelial cells in vitro,32 and Guignot and others have suggested that Sat induces cytoskeletal perturbation in intestinal epithelium accompanied by rearrangement of tight junction proteins. 33 Although the fundamental mode of action of Sat is unknown, Maroncle and others have suggested that the protein enters epithelial cells and directly cleaves spectrin, 34 an effect also attributed to its closest homolog, Pet. 35 Further experiments are required to determine whether the two toxins induce identical effects.

Some EAEC strains did not have pet nor sat, but interestingly, most of these strains harbored another class I SPATE (Figure 2B). A total of 7.3% of the EAEC strains harbored sigA, a cytotoxin originally described in S. flexneri, and 3.6% harbored espP, reported as a cytotoxin in Shiga toxin-producing E. coli strains. Overall, nearly 85% of EAEC strains harbored a gene encoding a class I cytotoxic SPATE protein.

The class II SPATEs were also common among the EAEC collections: 63.6% of the strains harbored pic and 38.2% harbored sepA. Pic, originally described in strains of S. flexneri 2a, has been shown to cleave submaxillary mucin, 22 and our data suggest that Pic promotes intestinal colonization in a mouse model (Harrington S, Nataro JP, unpublished data). SepA, also originally reported in S. flexneri 2a, may promote intestinal inflammation induced by Shigella strains, but its mode of action has not been described. 23

We hypothesized that the distribution of SPATE proteases would correlate with that of the aggregative adherence fimbriae (AAF) adhesins and/or with EAEC phylogeny as reported. 25 All five strains positive for the AAF/II pilin also carried the gene encoding Pet; none of these strains carried genes encoding Sat or SigA. Interestingly, these five Pet-encoding strains were distributed into three distantly related clusters on the EAEC phylogram, suggesting either horizontal co- transmission of AAF/II with Pet-encoding genes or functional linkage of these factors. In contrast, strains harboring aggA (encoding the pilin of AAF/I) or agg4A (the pilin of AAF/IV, also called Hda) were commonly found to encode sat or sigA but not pet.

SigA was first reported to be encoded on a large chromosomal pathogenicity island in S. flexneri, which also encoded the Pic protease. In our collection, four EAEC strains harbored sigA; two of the four also carried pic. In contrast, 33 strains carried pic but not sigA. These data suggest that the linkage of pic and sigA as originally described is uncommon. No other correlations among toxins or adhesins were observed, and no correlation of toxin genes with the phylogenetic clusters was apparent.

To facilitate later analysis for the five SPATEs commonly found in EAEC strains (pet, sat, sigA, pic, and sepA), we developed a multiplex PCR. The multiplex assay yielded clear products at the appropriate mass in all relevant controls, with no erroneous bands detected. All EAEC strains positive for these genes by monoplex were re-tested with the multiplex and showed a strong correlation between the multiplex and monoplex PCR assays. A representative image of multiplex PCR products derived from a subset of the EAEC and Shigella strains is shown in Figure 3 . Use of this assay as a tool for prospective epidemiologic analysis to address the potential role of SPATEs in human disease will be reported elsewhere.

We performed our multiplex PCR analysis on sets of ETEC, EHEC, EPEC, EIEC, DAEC, Shigella, and non-pathogenic E. coli strains (Table 2). The prevalence of the genes varied markedly by pathotype. In contrast to EAEC, we did not find Pic, SepA, Sat, SigA, or Pet-encoding genes among any of the 10 ETEC strains (Table 2). Similarly, only two of 12 non-pathogenic E. coli strains were positive for any of these five SPATEs (one for sat and one for sigA; Table 2). One EPEC strain was positive for Pic, two EHEC strains were positive for Sat and either Pic or SigA, three DAEC strains were positive for Sat and one strain was positive for Pic (Table 2). Previous studies have reported the presence of sat in the diarrheagenic E. coli pathotypes DAEC, EHEC, and EPEC. 32,36 Because several of the SPATEs have previously been reported among Shigella strains,22,23,37,38 we subjected 12 clinical Shigella isolates (11 S. flexneri and one S. sonnei) to our multiplex PCR assay. As we observed in EAEC strains, most of the Shigella strains harbored more than one SPATE protein (Figure 3). Whereas none of the Shigella strains harbored the pet gene, all were positive for sat and/or sigA, with four strains harboring both genes. Interestingly 6 of the 10 EIEC strains harbored SigA as the only SPATE protein. EIEC is distinguished from Shigella by biochemical tests, but EIEC strains and Shigella spp. share essential virulence factors. 39,40 Thus, it is plausible that SigA plays an important role in the pathogenesis of both EIEC and Shigella.

We showed that cytotoxic SPATE proteins were found almost exclusively among microbial pathogens that have been shown to induce mucosal damage and inflammation. Clinically, Shigella are more virulent in this regard than EAEC, and they commonly carried more than one cytoxic SPATE toxin. Whether the SPATE toxins exhibit primary or secondary roles in pathogenesis by Shigella and EAEC is under further investigation in our laboratory.

Table 1

Origin, serotype, and monoplex polymerase chain reaction results of 55 enteroaggregative Escherichia coli (EAEC) strains used in this study*

Table 1
Table 2

Shigella, ETEC, and non-pathogenic Escherichia coli strains used in this study*

Table 2
Table 2
Table 3

Primers used for monoplex PCR and multiplex PCR*

Table 3
Figure 1.
Figure 1.

Secretion of serine protease autotransporter toxin (SPATE) proteins from enteroaggregative Escherichia coli (EAEC) strains. Supernatants from five representative EAEC strains are shown. Supernatant proteins were precipitated with 10% trichoroacetic acid and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Coomassie staining. SPATE proteins represented by bands directly identified by mass spectrometry were SepA from strains H194-2 and 239-1, Pic from strains H77-1 and 239-1, and Sat from strain 232-1-1 (indicated by arrows). MW = molecular weight; HB101 = negative control strain.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 2; 10.4269/ajtmh.2009.80.294

Figure 2.
Figure 2.

Distribution of class I and class II serine protease autotransporter toxin (SPATE) proteins among enteroaggregative Escherichia coli (EAEC) strains. The distribution of the SPATE genes among 55 EAEC strains was determined by polymerase chain reaction A, Distribution of class I and class II SPATEs among the EAEC collection. B, Frequency of Pet, Sat, SigA, and EspP.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 2; 10.4269/ajtmh.2009.80.294

Figure 3.
Figure 3.

Identification of serine protease autotransporter toxin (SPATE) sequences from enteroaggregative Escherichia coli (EAEC) and Shigella isolates by multiplex polymerase chain reaction (PCR). The PCR products representing genes for Sat, SepA, Pic, SigA, and Pet were separated by electrophoresis on 1.2% agarose gels. A, PCR products from 11 EAEC strains. B, PCR products from 12 Shigella strains. EAEC strain 042 is the positive control for pet and pic, and S. flexneri 2a strain 2457T is the positive control for sat, sepA, pic, and sigA.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 2; 10.4269/ajtmh.2009.80.294

*

Address correspondence to James P. Nataro, Center for Vaccine Development, 685 West Baltimore Street, Room 480, Baltimore, MD 21201-1509. E-mail: jnataro@medicine.umaryland.edu

Authors’ addresses: Nadia Boisen, Flemming Scheutz, and Karen A. Krogfelt, Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark. Fernando Ruiz-Perez and James P. Nataro, Departments of Pediatrics and Medicine, Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore Street, Room 480, Baltimore, MD 21201-1509.

Acknowledgments: We thank Dr. Søren Persson for helpful advice regarding primer design, Erik Juncker Boll for verifying PCR results, Thomas Lund Sørensen for providing the six strains from the Danish Integrated Antimicrobial Resistance Monitoring and Research Program in Table 2, and Jeppe Boel for providing the six strains from the Danish food surveillance program.

Financial support: This study was supported by U.S. Public Health Service grant AI33096 to James P. Nataro and Danish Council for Strategic Research grant 2101-07-0023 to Karen A. Krogfelt. Nadia Boisen was supported in part by Statens Serum Institut, Copenhagen, Denmark.

REFERENCES

  • 1

    Glandt M, Adachi JA, Mathewson JJ, Jiang ZD, DiCesare D, Ashley D, Ericsson CD, DuPont HL, 1999. Enteroaggregative Escherichia coli as a cause of traveler’s diarrhea: clinical response to ciprofloxacin. Clin Infect Dis 29 :335–338.

    • Search Google Scholar
    • Export Citation
  • 2

    Adachi JA, Jiang ZD, Mathewson JJ, Verenkar MP, Thompson S, Martinez-Sandoval F, Steffen R, Ericsson CD, DuPont HL, 2001. Enteroaggregative Escherichia coli as a major etiologic agent in traveler’s diarrhea in 3 regions of the world. Clin Infect Dis 32 :1706–1709.

    • Search Google Scholar
    • Export Citation
  • 3

    Adachi JA, Ericsson CD, Jiang ZD, DuPont MW, Pallegar SR, DuPont HL, 2002. Natural history of enteroaggregative and enterotoxigenic Escherichia coli infection among US travelers to Guadalajara, Mexico. J Infect Dis 185 :1681–1683.

    • Search Google Scholar
    • Export Citation
  • 4

    Tompkins DS, Hudson MJ, Smith HR, Eglin RP, Wheeler JG, Brett MM, Owen RJ, Brazier JS, Cumberland P, King V, Cook PE, 1999. A study of infectious intestinal disease in England: microbiological findings in cases and controls. Commun Dis Public Health 2 :108–113.

    • Search Google Scholar
    • Export Citation
  • 5

    Okeke IN, Lamikanra A, Czeczulin J, Dubovsky F, Kaper JB, Nataro JP, 2000. Heterogeneous virulence of enteroaggregative Escherichia coli strains isolated from children in southwest Nigeria. J Infect Dis 181 :252–260.

    • Search Google Scholar
    • Export Citation
  • 6

    Wanke CA, Gerrior J, Blais V, Mayer H, Acheson D, 1998. Successful treatment of diarrheal disease associated with enteroaggregative Escherichia coli in adults infected with human immunodeficiency virus. J Infect Dis 178 :1369–1372.

    • Search Google Scholar
    • Export Citation
  • 7

    Wanke CA, Mayer H, Weber R, Zbinden R, Watson DA, Acheson D, 1998. Enteroaggregative Escherichia coli as a potential cause of diarrheal disease in adults infected with human immunodeficiency virus. J Infect Dis 178 :185–190.

    • Search Google Scholar
    • Export Citation
  • 8

    Durrer P, Zbinden R, Fleisch F, Altwegg M, Ledergerber B, Karch H, Weber R, 2000. Intestinal infection due to enteroaggregative Escherichia coli among human immunodeficiency virus-infected persons. J Infect Dis 182 :1540–1544.

    • Search Google Scholar
    • Export Citation
  • 9

    Mossoro C, Glaziou P, Yassibanda S, Lan NT, Bekondi C, Minssart P, Bernier C, Le Bouguenec C, Germani Y, 2002. Chronic diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome associated with HEp-2 adherent Escherichia coli in adults infected with human immunodeficiency virus in Bangui, Central African Republic. J Clin Microbiol 40 :3086–3088.

    • Search Google Scholar
    • Export Citation
  • 10

    Gassama-Sow A, Sow PS, Gueye M, Gueye-N’diaye A, Perret JL, M’Boup S, Aidara-Kane A, 2004. Characterization of pathogenic Escherichia coli in human immunodeficiency virus-related diarrhea in Senegal. J Infect Dis 189 :75–78.

    • Search Google Scholar
    • Export Citation
  • 11

    Menard LP, Dubreuil JD, 2002. Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1): a new toxin with an old twist. Crit Rev Microbiol 28 :43–60.

    • Search Google Scholar
    • Export Citation
  • 12

    Paiva de Sousa C, Dubreuil JD, 2001. Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella. Int J Med Microbiol 291 :15–20.

    • Search Google Scholar
    • Export Citation
  • 13

    Savarino SJ, Fasano A, Robertson DC, Levine MM, 1991. Enteroaggregative Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in vitro rabbit intestinal model. J Clin Invest 87 :1450–1455.

    • Search Google Scholar
    • Export Citation
  • 14

    Eslava C, Navarro-Garcia F, Czeczulin JR, Henderson IR, Cravioto A, Nataro JP, 1998. Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli. Infect Immun 66 :3155–3163.

    • Search Google Scholar
    • Export Citation
  • 15

    Vila J, Vargas M, Henderson IR, Gascon J, Nataro JP, 2000. Enteroaggregative Escherichia coli virulence factors in traveler’s diarrhea strains. J Infect Dis 182 :1780–1783.

    • Search Google Scholar
    • Export Citation
  • 16

    Henderson IR, Nataro JP, 2001. Virulence functions of autotransporter proteins. Infect Immun 69 :1231–1243.

  • 17

    Henderson IR, Hicks S, Navarro-Garcia F, Elias WP, Philips AD, Nataro JP, 1999. Involvement of the enteroaggregative Escherichia coli plasmid-encoded toxin in causing human intestinal damage. Infect Immun 67 :5338–5344.

    • Search Google Scholar
    • Export Citation
  • 18

    Fasano A, Noriega FR, Liao FM, Wang W, Levine MM, 1997. Effect of Shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo. Gut 40 :505–511.

    • Search Google Scholar
    • Export Citation
  • 19

    Huang DB, Okhuysen PC, Jiang ZD, DuPont HL, 2004. Enteroaggregative Escherichia coli: an emerging enteric pathogen. Am J Gastroenterol 99 :383–389.

    • Search Google Scholar
    • Export Citation
  • 20

    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.

    • Search Google Scholar
    • Export Citation
  • 21

    Dutta PR, Cappello R, Navarro-Garcia F, Nataro JP, 2002. Functional comparison of serine protease autotransporters of enterobacteriaceae. Infect Immun 70 :7105–7113.

    • Search Google Scholar
    • Export Citation
  • 22

    Henderson IR, Czeczulin J, Eslava C, Noriega F, Nataro JP, 1999. Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. Infect Immun 67 :5587–5596.

    • Search Google Scholar
    • Export Citation
  • 23

    Benjelloun-Touimi Z, Sansonetti PJ, Parsot C, 1995. SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion. Mol Microbiol 17 :123–135.

    • Search Google Scholar
    • Export Citation
  • 24

    Provence DL, Curtiss R 3rd, 1994. Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain. Infect Immun 62 :1369–1380.

    • Search Google Scholar
    • Export Citation
  • 25

    Czeczulin JR, Whittam TS, Henderson IR, Navarro-Garcia F, Nataro JP, 1999. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. Infect Immun 67 :2692–2699.

    • Search Google Scholar
    • Export Citation
  • 26

    Olesen B, Neimann J, Bottiger B, Ethelberg S, Schiellerup P, Jensen C, Helms M, Scheutz F, Olsen KE, Krogfelt K, Petersen E, Molbak K, Gerner-Smidt P, 2005. Etiology of diarrhea in young children in Denmark: a case-control study. J Clin Microbiol 43 :3636–3641.

    • Search Google Scholar
    • Export Citation
  • 27

    Boisen N, Struve C, Scheutz F, Krogfelt KA, Nataro JP, 2008. New adhesin of enteroaggregative Escherichia coli related to the Afa/Dr/AAF family. Infect Immun 76 :3281–3292.

    • Search Google Scholar
    • Export Citation
  • 28

    Dudley EG, Thomson NR, Parkhill J, Morin NP, Nataro JP, 2006. Proteomic and microarray characterization of the AggR regulon identifies a pheU pathogenicity island in enteroaggregative Escherichia coli. Mol Microbiol 61 :1267–1282.

    • Search Google Scholar
    • Export Citation
  • 29

    Sheikh J, Hicks S, Dall’Agnol M, Phillips AD, Nataro JP, 2001. Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli. Mol Microbiol 41 :983–997.

    • Search Google Scholar
    • Export Citation
  • 30

    Wei J, Goldberg MB, Burland V, Venkatesan MM, Deng W, Fournier G, Mayhew GF, Plunkett G 3rd, Rose DJ, Darling A, Mau B, Perna NT, Payne SM, Runyen-Janecky LJ, Zhou S, Schwartz DC, Blattner FR, 2003. Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T. Infect Immun 71 :2775–2786.

    • Search Google Scholar
    • Export Citation
  • 31

    Orskov F, Orskov I, 1992. Escherichia coli serotyping and disease in man and animals. Can J Microbiol 38 :699–704.

  • 32

    Guyer DM, Henderson IR, Nataro JP, Mobley HL, 2000. Identification of sat, an autotransporter toxin produced by uropathogenic Escherichia coli. Mol Microbiol 38 :53–66.

    • Search Google Scholar
    • Export Citation
  • 33

    Guignot J, Chaplais C, Coconnier-Polter MH, Servin AL, 2007. The secreted autotransporter toxin, Sat, functions as a virulence factor in Afa/Dr diffusely adhering Escherichia coli by promoting lesions in tight junction of polarized epithelial cells. Cell Microbiol 9 :204–221.

    • Search Google Scholar
    • Export Citation
  • 34

    Maroncle NM, Sivick KE, Brady R, Stokes FE, Mobley HL, 2006. Protease activity, secretion, cell entry, cytotoxicity, and cellular targets of secreted autotransporter toxin of uropathogenic Escherichia coli. Infect Immun 74 :6124–6134.

    • Search Google Scholar
    • Export Citation
  • 35

    Canizalez-Roman A, Navarro-Garcia F, 2003. Fodrin CaM-binding domain cleavage by Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption. Mol Microbiol 48 :947–958.

    • Search Google Scholar
    • Export Citation
  • 36

    Taddei CR, Moreno AC, Fernandes Filho A, Montemor LP, Martinez MB, 2003. Prevalence of secreted autotran sporter toxin gene among diffusely adhering Escherichia coli iso lated from stools of children. FEMS Microbiol Lett 227 :249–253.

    • Search Google Scholar
    • Export Citation
  • 37

    Al-Hasani K, Henderson IR, Sakellaris H, Rajakumar K, Grant T, Nataro JP, Robins-Browne R, Adler B, 2000. The sigA gene which is borne on the she pathogenicity island of Shigella flexneri 2a encodes an exported cytopathic protease involved in intestinal fluid accumulation. Infect Immun 68 :2457–2463.

    • Search Google Scholar
    • Export Citation
  • 38

    Roy S, Thanasekaran K, Dutta Roy AR, Sehgal SC, 2006. Distribution of Shigella enterotoxin genes and secreted autotransporter toxin gene among diverse species and serotypes of Shigella isolated from Andaman Islands, India. Trop Med Int Health 11 :1694–1698.

    • Search Google Scholar
    • Export Citation
  • 39

    Parsot C, 2005. Shigella spp. and enteroinvasive Escherichia coli pathogenicity factors. FEMS Microbiol Lett 252 :11–18.

  • 40

    Kaper JB, Nataro JP, Mobley HL, 2004. Pathogenic Escherichia coli. Nat Rev Microbiol 2 :123–140.

  • 41

    Levine MM, Bergquist EJ, Nalin DR, Waterman DH, Hornick RB, Young CR, Sotman S, 1978. Escherichia coli strains that cause diarrhoea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive. Lancet 1 :1119–1122.

    • Search Google Scholar
    • Export Citation
  • 42

    Bilge SS, Clausen CR, Lau W, Moseley SL, 1989. Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells. J Bacteriol 171 :4281–4289.

    • Search Google Scholar
    • Export Citation
  • 43

    RestieriC,GarrissG,LocasMC,DozoisCM, 2007. Autotransporter-encoding sequences are phylogenetically distributed among Escherichia coli clinical isolates and reference strains. Appl Environ Microbiol 73 :1553–1562.

    • Search Google Scholar
    • Export Citation
  • 44

    Bernier C, Gounon P, Le Bouguenec C, 2002. Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a sensitive probe for detecting the AAF-encoding operon family. Infect Immun 70 :4302–4311.

    • Search Google Scholar
    • Export Citation
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