Identification and characterization of NleA, a non‐LEE‐encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7

Abstract

Summary EnterohaemorrhagicEscherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC. In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified. NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium , and was absent from nonpathogenic strains of E. coli and non-LEE-containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.

Hierarchical Delivery of an Essential Host Colonization Factor in Enteropathogenic Escherichia coli

“…The ligation served as template in a PCR with primers 159 and 162 with the resulting 1.8-kb product being cloned into SacI/KpnI-digested pRE112, generating pNT256. pNT256 was conjugated into EPEC ⌬sepD, followed by the isolation and genotypic verification of ⌬sepD⌬nleA strains, as previously described (37). Similarly, a pRE112-based espZ deletion construct was created using primer pairs EPespZ(1) (GCGGTACCTGCT-TGTCGAGCAACGAGGCG) and ⌬EPespZ(R) (CCGCTAGCG-GATTAGCGATGAAATATGCC) and ⌬EPespZ(F) (GCGCTA-GCTGGTAATACTGCACCAGAAGG) and EPespZ(2) (CCGA-GCTCGAGTATCTTTGTATATTGACTC), followed by the isolation of ⌬espZ mutants.…”

“…Infected HeLa monolayers were washed with phosphate-buffered saline, followed by fixation with paraformaldehyde (2.5%, v/v). The samples were processed for immunofluorescence microscopy as previously described (37). Monoclonal antibodies were used to stain fixed samples: anti-FLAG (1:2000) (Sigma), anti-Tir (1:500), and anti-PY (1:500) clone 4G10 (Upstate Biotechnology, Inc.), followed by extensive washing and staining with secondary antibodies (Alexa 488-or 568-conjugated anti-mouse (Molecular Probes), 1:400).…”

Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission

“…Five translocated effectors are encoded by LEE: Tir/EspE [35,88], Map [87], EspF [109], EspG [45] and EspH [169]. Two effectors are encoded outside the LEE by lambdoid prophages: Cif [104] and NleA/EspI [62,118]. Blast analysis revealed that homologues of NleA/EspI are encoded by an Stx1-converting phage in EHEC of serotype O84:H4 (unpublished results) or by the phage coding for Cif [104].…”

In Vitro and In Vivo Model Systems for Studying Enteropathogenic Escherichia coli Infections

“…Although much of the in vivo work on A/E pathogens has focused on the virulence roles of secreted proteins, others have attempted to determine tissue localization of effectors such as Tir (Deng et al 2003), in an effort to uncover function. Infection of mice with C. rodentium mutants confirms a critical role in colonization and virulence of the LEE-encoded effectors Tir, EspB, and EspZ, as well as the non-LEE-encoded effectors NleA and NleB (Newman et al 1999;Deng et al 2003Deng et al , 2004Gruenheid et al 2004;Mundy et al 2004;Kelly et al 2006), whereas others are thought to contribute collectively (Dean and Kenny 2009). Mice were orally infected with C. rodentium and colonic tissues were harvested and subjected to immunofluorescent staining at 6 days postinfection.…”