An inducible lambdoid prophage encoding cytolethal distending toxin (Cdt-I) and a type III effector protein in enteropathogenic Escherichia coli

Abstract

Cytolethal distending toxins (CDTs) are inhibitory cyclomodulins, which block eukaryotic cell proliferation and are produced by a diverse group of Gram-negative bacteria, including Escherichia coli strains associated with intestinal and extraintestinal infections. However, the mode of transmission of the toxin gene clusters among diverse bacterial pathogens is unclear. We found that Cdt-I produced by enteropathogenic E. coli strains associated with diarrhea is encoded by a lambdoid prophage, which is inducible and infectious. The genome of Cdt-I converting phage (CDT-1Phi) comprises 47,021 nucleotides with 60 predicted ORFs organized into six genomic regions encoding the head and tail, virulence, integrase, unknown functions, regulation, and lysis. The genomic organization of CDT-1Phi is similar to those of SfV, a serotype-converting phage of Shigella flexneri, and UTI89, a prophage identified in uropathogenic E. coli. Besides the cdtI gene cluster, the virulence region of CDT-1Phi genome contains sequences homologous to a truncated cycle inhibiting factor and a type 3 effector protein. Mutation analysis of susceptible E. coli strain C600 suggested that the outer membrane protein OmpC is a putative receptor for CDT-1Phi. CDT-1Phi genome was also found to integrate into the host bacterial chromosome forming lysogens, which produced biologically active Cdt-I. Furthermore, phage induction appeared to cause enhanced toxigenicity of the E. coli strains carrying lysogenic CDT-1Phi. Our results suggest that CDT-1Phi is the latest member of a growing family of lambdoid phages encoding bacterial cyclomodulins and that the phage may have a role in horizontal transfer of these virulence genes.

Fig. 1.

Electron micrograph showing the morphology of a CDT-1Φ virion. The hexagonal head (≈44 nm) and the long tail (≈150 nm) are characteristic of the family Siphoviridae.

Fig. 2.

Comparison of the functional structure of the CDT-1Φ genome with those of prophage UTI89, prophage-like element B171, and bacteriophage SfV. The numbers in the bars indicate the homology between CDT-1Φ and each phage (UTI89 prophage, B171-prophage-like element, or bacteriophage SfV). The numbers below the bars indicate the size of each region, and the vertical arrow at the top indicates the attP site.

Fig. 3.

Determination of the region of insertion of CDT-1Φ into the E. coli genome. (A) Schematic diagram showing the region of integration of CDT-1Φ genome in the chromosome of strain C600. The yjjG gene encodes nucleoside 5′-monophosphate phosphohydrolase, and prfC encodes peptide chain release factor RF-3. Arrows indicate the regions where PCR primers P1 and P2, as well as B1 and B2, anneal to the integrated phage and bacterial genome (see Results for details). (B) Ethidium bromide-stained agarose gel showing the amplicons from different combinations of PCR primers and template DNA. Lane 1, HyperLadderI; lanes 2–5, P1 and P2; lanes 6–9, B1 and P1; lanes 10–13, P2 and B2); lanes 14–17, B1 and B2; lanes 2, 6, 10, and 14, E. coli strain NT3363; lanes 3, 7, 11, and 15, E. coli strain C600 with CDT-1Φ; lanes 4, 8, 12, and 16, E. coli strain C600; lanes 5, 9, 13, and 17, CDT-1Φ genome. The combination of primers used and expected sizes of the respective amplicons are indicated below the gel.