The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK.

Enteropathogenic Escherichia coli (EPEC) is a gastrointestinal pathogen that utilizes a type III secretion system (T3SS) to inject an array of virulence effector proteins into host enterocytes to subvert numerous cellular processes for successful colonization and dissemination. The T3SS effector NleD is a 26-kDa zinc metalloprotease that is translocated into host enterocytes, where it directly cleaves and inactivates the mitogen-activated protein kinase signaling proteins JNK and p38. Here a library of 91 random transposon-based, in-frame, linker insertion mutants of NleD were tested for their ability to cleave JNK and p38 during transient transfection of cultured epithelial cells. Immunoblot analysis of p38 and JNK cleavage showed that 7 mutant derivatives of NleD no longer cleaved p38 but maintained the ability to cleave JNK. Site-directed mutation of specific regions surrounding the insertion sites within NleD revealed that a single amino acid, R203, was essential for cleavage of p38 but not JNK in a direct in vitro cleavage assay, in transiently transfected cells, or in EPEC-infected cells. Mass spectrometry analysis narrowed the cleavage region to within residues 187 and 213 of p38. Mutation of residue R203 within NleD to a glutamate residue abolished the cleavage of p38 and impaired the ability of NleD to inhibit AP-1-dependent gene transcription of a luciferase reporter. Furthermore, the R203 mutation abrogated the ability of NleD to dampen interleukin-6 production in EPEC-infected cells. Overall, this work provides greater insight into substrate recognition and specificity by the type III effector NleD.

Mutagenesis and Functional Analysis of the Bacterial Arginine Glycosyltransferase Effector NleB1 from Enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) interferes with host cell signaling by injecting virulence effector proteins into enterocytes via a type III secretion system (T3SS). NleB1 is a novel T3SS glycosyltransferase effector from EPEC that transfers a single N-acetylglucosamine (GlcNAc) moiety in an N-glycosidic linkage to Arg(117) of the Fas-associated death domain protein (FADD). GlcNAcylation of FADD prevents the assembly of the canonical death-inducing signaling complex and inhibits Fas ligand (FasL)-induced cell death. Apart from the DXD catalytic motif of NleB1, little is known about other functional sites in the enzyme. In the present study, members of a library of 22 random transposon-based, in-frame, linker insertion mutants of NleB1 were tested for their ability to block caspase-8 activation in response to FasL during EPEC infection. Immunoblot analysis of caspase-8 cleavage showed that 17 mutant derivatives of NleB1, including the catalytic DXD mutant, did not inhibit caspase-8 activation. Regions of interest around the insertion sites with multiple or single amino acid substitutions were examined further. Coimmunoprecipitation studies of 34 site-directed mutants showed that the NleB1 derivatives with the E253A, Y219A, and PILN(63-66)AAAA (in which the PILN motif from residues 63 to 66 was changed to AAAA) mutations bound to but did not GlcNAcylate FADD. A further mutant derivative, the PDG(236-238)AAA mutant, did not bind to or GlcNAcylate FADD. Infection of mice with the EPEC-like mouse pathogen Citrobacter rodentium expressing NleBE253A and NleBY219A showed that these strains were attenuated, indicating the importance of residues E253 and Y219 in NleB1 virulence in vivo In summary, we identified new amino acid residues critical for NleB1 activity and confirmed that these are required for the virulence function of NleB1.

Evolutionary analysis and distribution of type III effector genes in pathogenic Escherichia coli from human, animal and food sources.

Molecular analysis of Shiga toxin-producing Escherichia coli (STEC) from different sources is considered as a major approach to assess their risk potential. However, only limited data are available about the correlation of evolutionary relationship, the presence of major virulence factor genes and the putative risk of an STEC strain for human infection. In this study, we analysed the evolutionary relationship of 136 pathogenic E. coli strains from human, animal and food sources by multi-locus sequence typing (MLST) and molecular subtyping of their Shiga toxin (stx) and intimin (eae) genes. Moreover, the distribution of three type III effector genes, encoded within the locus of enterocyte effacement (LEE), and 16 effector genes, which are encoded outside the LEE, was analysed. One hundred and five strains from different sources harboured 5-15 of the analysed non-LEE-encoded effector genes. In 101 of these strains, the LEE genes eae, map, espF and espG were present simultaneously. Thirty-one isolates deriving mainly from food and patients suffering from haemolytic uraemic syndrome (HUS) were eae-negative and did not carry any of the analysed effector genes. By combination of MLST and virulence gene data, we defined five genetic clusters. Within these clusters a clear-cut affiliation of particular sequence types and the occurrence of certain effector genes was observed. However, in contrast to other studies, a significant correlation between the amount and type of effector genes and the risk to cause HUS could not be demonstrated.

Genetic background and mobility of variants of the gene nleA in attaching and effacing Escherichia coli.

In this study, we characterized the genetic background of various nleA variants in 106 Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) strains. The flanking regions of eight nleA variants were analyzed by DNA sequencing and compared with the corresponding regions of five previously described NleA-encoding prophages. The analyzed nleA variants were all located downstream of the DNA region responsible for phage morphogenesis. In particular, the type III effector genes avrA, ospB, nleH, and nleG and IS elements were detected in the neighborhood of nleA. The structure of the eight analyzed regions flanking nleA primarily resembled the corresponding region of the NleA4795-encoding prophage BP-4795. Using PCR, the gene order flanking 13 nleA variants in strains of different serogroups was compared to the respective regions in reference strains. The analyses showed that strains which harbor prophages with conserved flanking regions of a particular nleA variant predominantly occurred, and IS elements were additionally detected in these regions. We were able to mobilize nleA by transduction in 20% of strains determined, which comprised in particular EPEC strains harboring an nleA variant, the gene encoding the protein known as "EspI-like." Plaque hybridization was used to identify phages that harbor the genes stx and nleA. However, only two strains harbored variant nleA4795 in the genome of an Stx1 prophage.

Virulence genes and molecular typing of different groups of Escherichia coli O157 strains in cattle.

Characterization of an Escherichia coli O157 strain collection (n = 42) derived from healthy Hungarian cattle revealed the existence of diverse pathotypes. Enteropathogenic E. coli (EPEC; eae positive) appeared to be the most frequent pathotype (n = 22 strains), 11 O157 strains were typical enterohemorrhagic E. coli (EHEC; stx and eae positive), and 9 O157 strains were atypical, with none of the key stx and eae virulence genes detected. EHEC and EPEC O157 strains all carried eae-gamma, tir-gamma, tccP, and paa. Other virulence genes located on the pO157 virulence plasmid and different O islands (O island 43 [OI-43] and OI-122), as well as espJ and espM, also characterized the EPEC and EHEC O157 strains with similar frequencies. However, none of these virulence genes were detected by PCR in atypical O157 strains. Interestingly, five of nine atypical O157 strains produced cytolethal distending toxin V (CDT-V) and carried genes encoding long polar fimbriae. Macro-restriction fragment enzyme analysis (pulsed-field gel electrophoresis) revealed that these E. coli O157 strains belong to four main clusters. Multilocus sequence typing analysis revealed that five housekeeping genes were identical in EHEC and EPEC O157 strains but were different in the atypical O157 strains. These results suggest that the Hungarian bovine E. coli O157 strains represent at least two main clones: EHEC/EPEC O157:H7/NM (nonmotile) and atypical CDT-V-producing O157 strains with H antigens different from H7. The CDT-V-producing O157 strains represent a novel genogroup. The pathogenic potential of these strains remains to be elucidated.

Type III effector genes and other virulence factors of Shiga toxin-encoding Escherichia coli isolated from wastewater.

Pathogenic Shiga toxin-producing Escherichia coli (STEC) strains share the genes encoding Shiga toxins (stx) and many other virulence factors. The classification and evolutionary studies of pathogenic E. coli based on their virulence genes have been conducted with E. coli isolated from human and animal infections or outbreaks. In this study, we used 103 STEC strains isolated from faecally polluted water environments to analyse 23 virulence genes (stx1 , cdt, hlyA, saa, eae, three type III effector genes encoded within the locus of enterocyte effacement (LEE) and 15 non-LEE-encoded type III effector genes). Despite the presence of several stx2 variants, our isolates demonstrated low prevalence of the virulence genes (only 46.6% of the strains were positive for virulence determinants). Among these, the largest repertoire was found in a few O157:H7 isolates (most from cattle wastewater and one from sewage), while other serotypes showed fewer virulence determinants. The occurrence of most virulence genes seemed to be independent from one another. This was clear for hlyA (the most prevalent), cdt and cif (the least prevalent). Other effector genes, could be found or not in combination with others, suggesting that they can be mobilized independently. Our data suggest that E. coli strains can evolve separately by independently acquiring mobile genetic elements.

Shiga toxin-producing Escherichia coli and their bacteriophages as a model for the analysis of virulence and stress response of a food-borne pathogen.

Enterohemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing Escherichia coli (STEC) that are able to cause serious food-borne intestinal diseases which can be followed in 5 to 15% by extraintestinal sequelae such as the hemolytic-uremic syndrome (HUS). One of the major pathogenicity factors of EHEC is the production of one or more Shiga toxins (Stx), which act as inhibitors of protein biosynthesis and have profound effects on the signal transduction and immunological response in eukaryotic cells. The stx genes are located in the genome of heterogeneous, lambdoid, functional or cryptic bacteriophages and are expressed during the phage life cycle. Due to the linkage between the phage life cycle and stx expression, STEC and their bacteriophages are useful as a model for the analysis of stress response and virulence of this food-borne pathogen.

Molecular characterization and distribution of genes encoding members of the type III effector nleA family among pathogenic Escherichia coli strains.

In this study, we investigated the occurrence of the previously described gene nleA(4795) and variants of nleA, putatively encoding non-locus-of-enterocyte-effacement-encoded type III effector proteins with functions that are unknown. nleA variants were detected in 150 out of 170 enteropathogenic Escherichia coli strains and enterohemorrhagic E. coli strains, two of them being eae negative. Besides the known variants nleA(4795), Z6024, and the espI-like gene, 11 novel nleA variants with different lengths and sequence identities at the deduced amino acid level (between 71% and 96%) have been identified. Whereas most of the serogroups associated with more severe disease were quite homogenous with respect to the presence of a particular nleA variant, other serogroups were not. Moreover, Southern blot hybridization revealed that certain strains carry two copies of nleA in their chromosome, frequently encoding different variants. In most cases, the open reading frame of one of the copies was disrupted, usually by an insertion element. Furthermore, transmission of the type III effector-encoding gene could be shown by transduction of nleA-carrying bacteriophages to a laboratory E. coli strain.

The Shiga toxin 1-converting bacteriophage BP-4795 encodes an NleA-like type III effector protein.

In this study, the complete DNA sequence of Shiga toxin 1-converting bacteriophage BP-4795 was determined. The genome of BP-4795 consists of 85 open reading frames, including two complete IS629 elements and three morons at the end of its late regulatory region. One of these morons encodes a type III effector that is translocated by the locus of enterocyte effacement-encoded type III secretion system into HeLa cells, where it localizes with the Golgi apparatus.

Genetic structure and chromosomal integration site of the cryptic prophage CP-1639 encoding Shiga toxin 1.

The sequence of 50 625 bp of chromosomal DNA derived from Shiga-toxin (Stx)-producing Escherichia coli (STEC) O111: H- strain 1639/77 was determined. This DNA fragment contains the cryptic Stx1-encoding prophage CP-1639 and its flanking chromosomal regions. The genome of CP-1639 basically resembles that of lambdoid phages in structure, but contains three IS629 elements, one of which disrupts the gene of a tail fibre component. The prophage genome lacks parts of the recombination region including integrase and excisionase genes. Moreover, a capsid protein gene is absent. CP-1639 is closely associated with an integrase gene of an ancient integrative element. This element consists of three ORFs of unknown origin and a truncated integrase gene homologous to intA of CP4-57. By PCR analysis and sequencing, it was shown that this integrative element is present in a number of non-O157 STEC serotypes and in non-STEC strains, where it is located at the 3'-end of the chromosomal ssrA gene. Whereas in most E. coli O111: H- strains, prophages are inserted in this site, E. coli O26 strains contain the integrative element not connected to a prophage. In E. coli O103 strains, the genetic structure of this region is variable. Comparison of DNA sequences of this particular site in E. coli O157: H7 strain EDL933, E. coli O111: H- strain 1639/77 and E. coli K-12 strain MG1655 showed that the ssrA gene is associated in all cases with the presence of foreign DNA. The results of this study have shown that the cryptic prophage CP-1639 is associated with an integrative element at a particular site in the E. coli chromosome that possesses high genetic variability.