The ETT2 Gene Cluster, Encoding a Second Type III Secretion System from Escherichia coli , Is Present in the Majority of Strains but Has Undergone Widespread Mutational Attrition

Abstract

ETT2 is a second cryptic type III secretion system in Escherichia coli which was first discovered through the analysis of genome sequences of enterohemorrhagic E. coli O157:H7. Comparative analyses of Escherichia and Shigella genome sequences revealed that the ETT2 gene cluster is larger than was previously thought, encompassing homologues of genes from the Spi-1, Spi-2, and Spi-3 Salmonella pathogenicity islands. ETT2-associated genes, including regulators and chaperones, were found at the same chromosomal location in the majority of genome-sequenced strains, including the laboratory strain K-12. Using a PCR-based approach, we constructed a complete tiling path through the ETT2 gene cluster for 79 strains, including the well-characterized E. coli reference collection supplemented with additional pathotypes. The ETT2 gene cluster was found to be present in whole or in part in the majority of E. coli strains, whether pathogenic or commensal, with patterns of distribution and deletion mirroring the known phylogenetic structure of the species. In almost all strains, including enterohemorrhagic E. coli O157:H7, ETT2 has been subjected to varying degrees of mutational attrition that render it unable to encode a functioning secretion system. A second type III secretion systemassociated locus that likely encodes the ETT2 translocation apparatus was found in some E. coli strains. Intact versions of both ETT2-related clusters are apparently present in enteroaggregative E. coli strain O42.The species Escherichia coli contains a wide range of commensal strains and pathogenic varieties (pathotypes) in addition to the model laboratory organism, E. coli K-12 (16). At least six pathotypes are associated with human intestinal disease: they are enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli, enteroaggregative E. coli (EAEC), and diffusely adherent E. coli. Two pathotypes are associated with extraintestinal disease in humans, namely uropathogenic E. coli (UPEC) and neonatal meningitic E. coli (NMEC). In addition, it is now clear that on phylogenetic grounds, all members of the genus Shigella belong within the species of E. coli (50). Furthermore, this dazzling phenotypic variety is matched by remarkable variations in genome size, with the largest E. coli genomes possessing more than a megabase more DNA than the smallest ones (43).Initial studies of UPEC, and later of other pathotypes, suggested that E. coli strains often acquire new complex pathogenic phenotypes in a single step by the acquisition of pathogenicity islands, which contain virulence genes clustered on the chromosome and which are acquired en bloc by horizontal gene transfer (21). Similar studies with the related bacterium Salmonella enterica have delineated several Salmonella pathogenicity islands (Spi-1, Spi-2, Spi-3, etc.) (2, 22). The horizontal transfer of DNA by mobile elements such as bacteriophages and plasmids is also known to play a role in the evolution of virulence in E. coli and S...

Sequencing and Functional Annotation of Avian Pathogenic Escherichia coli Serogroup O78 Strains Reveal the Evolution of E. coli Lineages Pathogenic for Poultry via Distinct Mechanisms

“…The ETT2 locus in 7122 and IMT2125 is truncated from within eivA to the end of the locus described for E. coli O157:H7 strain Sakai, consistent with the truncation described for another sepsis-associated APEC O78 strain, termed 789 (30). Within ETT2, the predicted spaS and eivJ genes contain distinct frameshift mutations in 7122 and IMT2125, consistent with the varied patterns of mutational attrition of the locus reported among E. coli strains (30,63). The ymcDCBA-yccZ-etp-etk genes, which determine O-antigen capsule biosynthesis (located between appA and yccM) (64), were conserved in both 7122 and IMT2125 but absent in APEC O1.…”

“…S1 and S2 in the supplemental material). Among the virulence-associated loci present in 7122 and IMT2125 but absent from APEC O1 is the degenerate type III secretion locus ETT2, located between yqeG and the glyU tRNA locus in many E. coli strains (63). The ETT2 locus in 7122 and IMT2125 is truncated from within eivA to the end of the locus described for E. coli O157:H7 strain Sakai, consistent with the truncation described for another sepsis-associated APEC O78 strain, termed 789 (30).…”

Characterization of Genomic Island 3 and Genetic Variability of Chilean Field Strains of Brucella abortus

“…A technique called tiling-path PCR scanning (TP-PCR), previously described by Ren et al (29), was used with some modifications. Briefly, the method consists of several interlocking PCRbased approaches able to characterize large genomic regions.…”

“…To construct a complete tiling path through GI-3 and to determine the genetic organization within the island for the 69 strains, we used the TP-PCR method (29), which exploits short-and long-PCR protocols; this method allowed us to construct a complete tiling path through the GI-3 gene cluster for 69 strains and for type strain 544. Initially, we confirmed that reference strain 544 possessed GI-3 gene clusters by short and long PCR, first amplifying ORFs BruAb1_0251, BruAb1_0256, BruAb1_0266, and BruAb1_0274 by short PCR (data not shown) and then amplifying fragments A, B, C, and D by long PCR.…”

Animal models of enteroaggregativeEscherichia coliinfection

“…37 Additionally, the ETT2 gene cluster has been identified in the EAEC O42 genome sequence providing evidence for T3SS mechanism prevalence as well. 38 These secretion systems may play a key role in EAEC virulence due to expulsion of toxic proteins and association with biofilm formation; 39 their roles in pathogenesis remain widely unknown. Heterogeneity among EAEC strains remains an overarching issue that complicates elucidating pathogenic mechanisms underlying infection.…”