Cytolethal Distending Toxin Type I and Type IV Genes Are Framed with Lambdoid Prophage Genes in Extraintestinal Pathogenic Escherichia coli

Abstract

Five types of cytolethal distending toxin (CDT-I to CDT-V) have been identified in Escherichia coli.In the present study we cloned and sequenced the cdt-IV operon and flanking region from a porcine extraintestinal pathogenic E. coli (ExPEC) strain belonging to serogroup O75. We confirmed that similar to other CDTs, CDT-IV induced phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks, and blocked the HeLa cell cycle at the G 2 -M transition. The cdt-IV genes were framed by lambdoid prophage genes. We cloned and sequenced the cdt-I operon and flanking regions from a human ExPEC O18:K1:H7 strain and observed that cdt-I genes were also flanked by lambdoid prophage genes. PCR studies indicated that a gene coding for a putative protease was always associated with the cdtC-IV gene but was not associated with cdtC genes in strains producing CDT-I, CDT-III, and CDT-V. Our results suggest that the cdt-I and cdt-IV genes might have been acquired from a common ancestor by phage transduction and evolved in their bacterial hosts. The lysogenic bacteriophages have the potential to carry nonessential "cargo" genes or "morons" and therefore play a crucial role in the generation of genetic diversity within ExPEC.Cytolethal distending toxins (CDTs) were the first bacterial toxins that block the eukaryotic cell cycle that were described, and they suppress cell proliferation and eventually lead to cell death. CDTs represent an emerging toxin family, which was initially described for Escherichia coli but is actually widely distributed among a variety of pathogenic bacteria, including Shigella dysenteriae, Salmonella enterica serovar Typhi, Campylobacter spp., Helicobacter spp., Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, and Haemophilus ducreyi (31,34).A CDT is a tripartite holotoxin (28) in which CdtB is the active subunit and CdtA and CdtC form a heterodimeric subunit apparatus required for delivery of CdtB into the cell (8,23,24). The toxin of S. enterica serovar Typhi is an exception. This organism does not encode CdtA or CdtC; instead, the enzymatic CdtB is delivered directly into host cells by a bacterial internalization pathway (14). Nuclear entry of CdtB, which relies on an atypical nuclear localization signal, is crucial for the cytotoxic activity (25,27,29). CdtB has DNase I-like activity (10,15,23). Once in the host cell nucleus, CdtB induces DNA double-strand breaks in both proliferating and nonproliferating cells (13,15,26). Although pathogenic roles of CDT have been shown for chronic infection in mouse models (12, 39), CDT has not played a significant role in acute infection models tested to date (25,40,48,49). Nevertheless, CDT appears to be a virulence factor; accordingly, studying its spread is important.So far, five different CDTs have been reported for E. coli, and they were designated in order of publication. CDT-I (44) and CDT-II (38) were identified in enteropathogenic E. coli (EPEC) serotype O86:H34 and O128:NM strains, respectively. CDT-III was cloned from an...

Cytolethal distending toxin A, B and C subunit proteins are necessary for the genotoxic effect of Escherichia coli CDT-V

“…Five types of CDT (CDT-I to CDT-V) have been reported in pathogenic E. coli strains (Tóth et al, 2009a); among them CDT-V is the most recently identified (Janka et al, 2003), and the only type to be detected in Shiga toxin producing (STEC) and enterohaemorrhagic (EHEC) E. coli (Orth et al, 2006;Hinenoya et al, 2009;Hinenoya et al, 2014). Additionally, CDT-V has been detected in several strains of uncertain pathotype (Hinenoya et al, 2009;Tóth et al, 2009b), and in some of these strains it is the only identified toxin so far (Tóth et al, 2009b).…”

The cytolethal distending toxin-IV cdt coding region in an avian pathogenic Escherichia coli (APEC) strain shows instability and irregular excision pattern

“…Accordingly the downstream region of the cdt locus as cdtC was always associated with a putative protease (rorf1) gene [12]. In contrast the upstream region of the cdt locus showed diversity and deletion was detected in some CDT-IV strains including APEC E250 strain.…”

“…The PCR primers used for (i) comparison the genomic organisations of the upstream regions of cdt loci between 28C [16] reference strain and APEC E250 [12]; (ii) the cloning steps of labelling, and (iii) screening for deletions after labelling are compiled in Table II. In vitro labelling the cdt-IV locus Introduction of a chloramphenicol resistance marker, mobilisation genes, an origin of replication, and an origin of transfer into cdt-IV locus of the avian pathogenic E. coli strain E250 was accomplished with the help of plasmid pSG704 [15] as it is depicted in Figure 1, pSG704 is a chloramphenicol-resistant derivative of the conjugative suicide vector pGP704 [15].…”

“…Investigation revealed that cdt-IV operon of the avian pathogenic E. coli (APEC) strains compared to the structure of the reference cdt-IV ExPEC 28C strain contains an altered and still unknown upstream region [12]. That is why in the present study we have carried out a functional analysis with the APEC strain E250 in order to reveal its stability and possible transferability.…”

“…CDTs are quite disseminated in several species and so far in E. coli fi ve genotypes were identifi ed (reviewed by Tóth and Sváb) [7]. In all cases the cdtABC operons were either part of the genome of an inducible converting lambdoid prophage [8], a defective P-2 like prophage [9], a large plasmid [10], as part of a PAI [11] or framed with lambdoid phage genes [12].…”