An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination

Abstract

Several pathogenic strains of Escherichia coli exploit type III secretion to inject "effector proteins" into human cells, which then subvert eukaryotic cell biology to the bacterium's advantage. We have exploited bioinformatics and experimental approaches to establish that the effector repertoire in the Sakai strain of enterohemorrhagic E. coli (EHEC) O157:H7 is much larger than previously thought. Homology searches led to the identification of >60 putative effector genes. Thirteen of these were judged to be likely pseudogenes, whereas 49 were judged to be potentially functional. In total, 39 proteins were confirmed experimentally as effectors: 31 through proteomics and 28 through translocation assays. At the protein level, the EHEC effector sequences fall into >20 families. The largest family, the NleG family, contains 14 members in the Sakai strain alone. EHEC also harbors functional homologs of effectors from plant pathogens (HopPtoH, HopW, AvrA) and from Shigella (OspD, OspE, OspG), and two additional members of the Map/IpgB family. Genes encoding proven or predicted effectors occur in >20 exchangeable effector loci scattered throughout the chromosome. Crucially, the majority of functional effector genes are encoded by nine exchangeable effector loci that lie within lambdoid prophages. Thus, type III secretion in E. coli is linked to a vast phage "metagenome," acting as a crucible for the evolution of pathogenicity.

Fig. 1.

Experimental flow chart. BLAST searches with a comprehensive database of known and predicted effector sequences were used to identify 62 candidate effectors in 25 loci within the genome of EHEC O157:H7 Sakai. Using a proteomic approach, 31 candidate effectors were found to be secreted by T3S. Subsequently, three methods for measuring T3S-dependent translocation into eukaryote cells were used to identify 28 translocated effectors. In total, 39 candidate effectors were confirmed experimentally either by proteomics or by translocation assays or both methods. Representative translocation data (including controls) are provided for Cya, TEM1 and FLAG translocation assays. Cya: cAMP levels of Caco-2 cell extracts after infection with wildtype (WT) or escC− (T3S negative) E. coli carrying CyaA-effector fusion plasmids (v = vector only); TEM1: HeLa cell fluorescence was observed after infection with WT or escN− (T3S negative) E. coli carrying TEM1-effector fusion plasmids. Blue or green fluorescence indicates translocation or no translocation, respectively. (A) ECs1567 (escN− mutant). (B) ECs1567 (WT); FLAG: Caco-2 cell fluorescence was observed after infection with WT E. coli carrying FLAG-effector fusion plasmids. FLAG-effector fusion proteins, nucleus and F-actin were fluorescently stained green, blue, and red, respectively. Green fluorescence within Caco-2 cells indicates translocation. (C) ECs1567. (D) ECs1814. (E) Vector only. (F) ECs1994.(G) ECs3485. Further details of the experimental approach are described in Results and Discussion.

Fig. 2.

The genome view shows lambdoid prophages in orange and effector loci in blue. The blow-ups of the terminal portions of lambdoid phages shows the clear distinction in GC content between effector genes and the phage backbone. Effector genes highlighted by increased height. For reasons of space, a cluster of insertion sequence remnants has been omitted from the end of Sp12. Sp5 and Sp15, the two Shiga toxin-encoding prophages, do not encode any T3SS effectors. In all cases, phage encoded effector genes fall within the prophage boundaries defined by Hayashi et al. (15).