The Presence of the pAA Plasmid in the German O104:H4 Shiga Toxin Type 2a (Stx2a)–Producing Enteroaggregative Escherichia coli Strain Promotes the Translocation of Stx2a Across an Epithelial Cell Monolayer

MAJOR ARTICLE The Presence of the pAA Plasmid in the German O104:H4 Shiga Toxin Type 2a (Stx2a)–Producing Enteroaggregative Escherichia coli Strain Promotes the Translocation of Stx2a Across an Epithelial Cell Monolayer Nadia Boisen,1 Anne-Marie Hansen,2 Angela R. Melton-Celsa,3 Tonia Zangari,3 Ninell Pollas Mortensen,4,5 James B. Kaper,2 Alison D. O’Brien,3 and James P. Nataro1 1 (See the editorial commentary by Steiner on pages 1860–2.) Background. A Shiga toxin type 2a (Stx2a)–producing enteroaggregative Escherichia coli (EAEC) strain of serotype O104:H4 caused a large outbreak in 2011 in northern Europe. Pathogenic mechanisms for this strain are unclear. We hypothesized that EAEC genes encoded on the pAA virulence plasmid promoted the translocation of Stx2a across the intestinal mucosa. Methods. We investigated the potential contribution of pAA by using mutants of Stx-EAEC strain C227-11, either cured of the pAA plasmid or deleted for individual known pAA-encoded virulence genes (ie, aggR, aggA, and sepA). The resulting mutants were tested for their ability to induce interleukin 8 (IL-8) secretion and translocation of Stx2a across a polarized colonic epithelial (T84 cell) monolayer. Results. We found that deletion of aggR or aggA significantly reduced bacterial adherence and (independently) translocation of Stx2a across the T84-cell monolayer. Moreover, deletion of aggR, aggA, sepA, or the Stx2a-encoding phage from C227-11 resulted in reduced secretion of IL-8 from the infected monolayer. Conclusions. Our data suggest that the AggR-regulated aggregative adherence fimbriae I enhance inflammation and enable the outbreak strain to both adhere to epithelial cells and translocate Stx2a across the intestinal epithelium. Keywords. enteroaggregative Escherichia coli (EAEC); O104:H4; Shiga toxin; pAA plasmid; diarrhea. Enteroaggregative Escherichia coli (EAEC) is a pathotype of diarrheagenic Escherichia coli that is a cause of acute and persistent diarrhea in many settings [1–7]. EAEC strains express a heterogeneous array of putative virulence factors [8–12] encoded on the bacterial chromosome or on the EAEC-specific pAA plasmid. EAEC Received 28 January 2014; accepted 11 June 2014; electronically published 18 July 2014. Correspondence: Nadia Boisen, PhD, Department of Pediatrics, University of Virginia School of Medicine, P.O. Box 800326, 409 Lane Road, Charlottesville, VA 22908 (). The Journal of Infectious Diseases® 2014;210:1909–19 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: . DOI: 10.1093/infdis/jiu399 strains often harbor a transcriptional activator of the AraC/XylS class, called “AggR” [13], which controls genes on both the plasmid and the chromosome. Among the genes under AggR control include those that encode the aggregative adherence fimbriae (AAF), of which at least 4 variants exist [14–18]. AAF adhesins have been shown to be essential for EAEC adherence to human intestinal explants and to elicit both cytokine release and opening of epithelial tight junctions in a polarized epithelial model [19, 20]. EAEC strains also often harbor a variable number of serine protease autotransporters of Enterobacteriaceae (SPATEs) that are implicated in immune evasion, mucosal damage, secretogenicity, and colonization [21]. pAA and Stx2a Translocation During EAEC O104:H4 Infection • JID 2014:210 (15 December) • 1909 Department of Pediatrics, University of Virginia School of Medicine, Charlottesville; 2Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, and 3Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland; 4Technology for Industry and the Environment, Discovery–Science–Technology, RTI International, Research Triangle Park, North Carolina; and 5Biological and Nanoscale Systems, BioSciences Division, Oak Ridge National Laboratory, Tennessee In 2011, an outbreak of foodborne hemorrhagic colitis originated in Germany, spreading to other European countries. Over 4000 individuals were affected, including primary and secondary cases [22]. Hemolytic uremic syndrome (HUS) developed in approximately 22% of the cases [22], and 54 people died [23, 24]. The implicated pathogen was an EAEC strain of the rare serotype O104:H4 [23], which was lysogenized with an Stx2a-converting phage. Genomic analysis [25] demonstrated that the outbreak strain contained the genes required to produce the AAF/I variant and 3 SPATEs (Pic, SigA, and SepA). Although much is known about the pathogenesis of serotype O157:H7 Shiga toxin (Stx)–producing E. coli, it is unclear how an EAEC strain would be able to elicit severe hemorrhagic colitis and HUS, even when harboring an Stx-encoding gene. In this study, we tested the hypothesis that the plasmid-borne virulence factors of EAEC contributed to the high pathogenicity of the German outbreak strain by promoting strong adherence to the epithelium and/or by opening epithelial tight junctions. Bacterial Strains, Plasmids, and Growth Conditions The Stx2a-producing O104:H4 strain C227-11 was isolated in Denmark during the outbreak [23]. Bacterial strains and plasmids used in this study are described in Table 1. Strains were grown at 37°C in Luria broth (LB) or Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.45% glucose. Recovery of bacteria was done on MacConkey agar (Mac) or LB agar. When indicated, media were supplemented with kanamycin (Km; 50 µg/mL), carbenicillin (Ca; 100 µg/mL), tetracycline (Tc; 30 µg/mL), streptomycin (Str; 50 µg/mL), penicillin (Pen; 50 µg/mL), chloramphenicol (Cm; 30 µg/mL), gentamicin (Gm; 100 µg/mL), and/or 0.2% arabinose. Plasmid and Strain Constructions Oligonucleotides used are listed in Table 1. Phusion Flash (Fermentas, Waltham, MA) and EasyA (Agilent, Santa Clara, CA) high-fidelity DNA polymerases were used for polymerase chain reaction (PCR) analysis. To obtain a construct expressing aggDCBA from an arabinose-inducible promoter, a Cm cassette was amplified from pACYC184 with primers AH1036/1037 and cloned into the pBAD24 XmnI site (pAMH187); aggDCBA amplified from C227-11 genomic DNA with aggBADF/aggBADR primers was ligated into the pAMH187 NheI/SalI sites. A complementation plasmid with aggR expressed from its native promoter was generated by cloning a DNA fragment amplified from C227-11 genomic DNA, using primers AH1044/ AH1045, into the pACYC184 XmnI site ( pAMH188). An aggR mutant was generated by cloning a Km cassette, amplified from pACYC177 with primers AH1056/AH1057, into an XmnI site in aggR ( pAMH189); the aggR::km-containing DNA fragment was amplified from pAMH189 with primers AH1083/ AH1084 and cloned into the pSRS1 NdeI/SacI sites 1910 • JID 2014:210 (15 December) • Boisen et al Biofilm Assay Biofilm assay was performed as described previously [30]. Epithelial Cell Infections Hu (...truncated)