SummaryShiga toxin-producing Escherichia coli (STEC) induce so-called attaching and effacing lesions that enable the tight adherence of these pathogens to the gut epithelium. All of the genes necessary for this process are present in the locus of enterocyte effacement, which encodes a type III secretion system, the secreted Esp proteins and the surface protein intimin. In this study we sequenced the espA gene of STEC, generated and characterized a corresponding deletion mutant and raised EspA-specific monoclonal antibodies to analyse the functional role of this protein during infection. EspA was detected in often filament-like structures decorating all bacteria that had attached to HeLa cells. These appendages were especially prominent on bacteria that had not yet induced the formation of actin pedestals, indicating that they mediate the initial contact of STEC to their target cells. Consistently, a deletion of the espA gene completely abolished the capacity of such STEC mutants to bind to HeLa cells and to induce actin rearrangements. Surface appendages similar to those described in this study are also formed by Pseudomonas syringae and may represent a structural element common to many bacterial pathogens that deliver proteins into their target cells via a type III secretion system.

SummaryPseudomonas aeruginosa chronically colonizing the lungs of cystic fibrosis (CF) patients undergoes fast evolution leading to clonal divergence. More than half of the genotypes of P. aeruginosa clone C isolates exclusively from CF lung infection exhibit large chromosomal inversions (LCIs). To analyse the impact of LCIs, as a novel mechanism of bacterial adaptation, the underlying molecular mechanism was examined. Analysis of inversion breakpoints suggested an IS 6100 -induced coupled insertion-inversion mechanism. A selective advantage was created by insertion of IS 6100 into wbpM , pilB and mutS which leads to common CF phenotypes such as O-antigen and type IV pili deficiency and hypermutability. Speciation in bacteria is accompanied by LCIs. Therefore adaptation by LCIs that allows persistence of P. aeruginosa in the CF lung and species diversification in that new ecological niche can serve as a model for bacterial genome evolution.

The sepL gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up-and downstream of this gene. In this study, the sepL gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sepL is transcribed monocistronically and independently from the esp operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the sepL start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions ؊5 and ؊36. Translational fusions using lacZ as a reporter gene demonstrated that sepL is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37°C, a nutrient-rich environment, high osmolarity, and the presence of Mn 2؉ . Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This sepL mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the sepL gene in trans. The EDL933⌬sepL mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing sepL in trans. These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.Enterohemorrhagic Escherichia coli (EHEC) strains are the major cause of bloody diarrhea and acute renal failure (10, 18). EHEC interacts with the gut mucosa, leading to histopathological changes which are collectively called attaching and effacing (A/E) lesions (18). While the production of Shiga toxins is a distinctive feature of Shigella and EHEC, the capacity to cause A/E lesions of EHEC is shared by many other enteric pathogens like enteropathogenic E. coli (EPEC), diffusely adhering E. coli, Hafnia alvei, and Citrobacter freundii (2,37,44). These bacteria contain a pathogenicity island called the locus of enterocyte effacement (LEE), which codes for bacterial products sufficient for triggering the A/E lesions (33, 34). The LEE sequences of EPEC and EHEC have been published (15,42), and most of the open reading frames (ORFs) are highly conserved, in particular the identified components of the type III secretion apparatus (98 to 100% identity), except for the sepZ gene. The secreted structural and putative effector proteins EspA, EspB, EspD, and Tir are more diverse (84.63, 74.01, 80.36, and 66.48% identity, respectively).Despite the overall sequence similarities of the ORFs within the LEE in EPEC and EHEC, Esp proteins appear to be involved to different...

We have determined that the genes encoding the secreted proteins EspA, EspD, and EspB of enterohemorrhagic Escherichia coli(EHEC) are organized in a single operon. The esp operon is controlled by a promoter located 94 bp upstream from the ATG start codon of the espA gene. The promoter is activated in the early logarithmic growth phase, upon bacterial contact with eukaryotic cells and in response to Ca2+, Mn2+, and HEPES. Transcription of the esp operon seems to be switched off in tightly attached bacteria. The activation process is regulated by osmolarity (induction at high osmolarities), modulated by temperature, and influenced by the degree of DNA supercoiling. Transcription is ςS dependent, and the H-NS protein contributes to its fine tuning. Identification of the factors involved in activation of the esp operon and the signals responsible for modulation may facilitate understanding of the underlying molecular events leading to sequential expression of virulence factors during natural infections caused by EHEC.

Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal disease in humans and livestock, and these bacteria have recently emerged as a leading cause of renal failure in children. In this study, we have examined medium-and temperature-dependent production of secreted proteins from a STEC O26 serotype strain. Growth of bacteria in Luria broth led to the detection of secreted polypeptides of 104, 55, 54, and 37 kDa (p104, p55, p54, and p37, respectively). When grown in serum-free tissue culture medium, only p104, p37 and two additional polypeptides of 25 and 22 kDa (p25 and p22) were present in supernatant fluids. Production of these polypeptides was growth temperature dependent and induced in cultures grown at 37؇C. N-terminal amino acid sequencing revealed that p104 was homologous to the secreted p110 of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a family of secreted proteins in pathogenic bacteria of which the immunoglobulin A protease of Neisseria gonorrhoeae is the prototype. The N-terminal amino acid sequences of p55 and p54 were unique to the STEC strain, while p37 and p25 were found to be highly homologous to the similarly sized EspA and EspB proteins, previously detected in culture supernatants of EPEC. Molecular cloning and sequencing of STEC espB alleles from two different serotypes showed that the encoded polypeptides were about 80% homologous. A monoclonal antibody raised against STEC EspB also cross-reacted with its EPEC analog and allowed us to demonstrate medium-and temperature-dependent production of this important virulence factor in STEC and EPEC strains of differing serotypes.

Enterohemorrhagic Escherichia coli (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological phenotype collectively known as the attaching and effacing lesion. The genes encoding the products responsible for this phenotype are located on a 35-kb pathogenicity island designated the locus of enterocyte effacement, which is also shared by enteropathogenic E. coli. We have identified an open reading frame (ORF) which is located upstream of the espA, espB, andespD genes on the complementary strand and which exhibits high homology to the genes spiB fromSalmonella, yscD from Yersinia, andpscD from Pseudomonas. Localization studies showed that the encoded product is present in the cytoplasmic and inner membrane fractions of EHEC. The construction and characterization of a recombinant clone containing an in-frame deletion of this ORF demonstrated that the encoded product is a putative member of a type III system required for protein secretion. Disruption of this ORF, designated pas (protein associated with secretion), abolished the secretion of Esp proteins. The mutant adhered only poorly and lost its capacities to trigger attaching and effacing activity and to invade HeLa cells. These results demonstrate that Pas is a virulence-associated factor that plays an essential role in EHEC pathogenesis.

The formation of EspA-containing surface appendages in pathogenicEscherichia coli strains, both enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. colistrains, is essential for critical events in the infective process, e.g., localized bacterial adherence to host cells with formation of microcolonies and induction of attaching and effacing lesions. It has been reported that EPEC mutants deficient in the production of EspD, which is encoded by the esp operon, are unable to accumulate actin underneath adherent bacteria but exhibit an attachment similar to that of the wild type. Here, we report the construction and characterization of an in-frame espD deletion mutant of the enterohemorrhagic E. coli (EHEC) strain EDL933. In contrast to what was observed in EPEC mutants, the EDL933 espDmutant not only lacked the capacity to accumulate actin but also exhibited an impaired attachment to HeLa cells. The synthesis of the EspD protein was also essential for the formation of EspA-containing filaments. Finally, localization studies demonstrated that the EspD protein is transferred to the cytoplasm and integrated into the cytoplasmic membranes of infected cells. These results help to elucidate the underlying molecular events in infections caused by EHEC.

Pseudomonas aeruginosa clone C strains, which chronically colonize the lungs of cystic fibrosis patients reorganize their genome structure. In this study, a novel member of the IS3 subfamily of IS elements, ISPa20, was detected which was specific for clone C subclone C13 strains. ISPa20, which was present in high copy number, mediated events of genomic reorganization. ISPa20 was inserted into P. aeruginosa backbone genes leading to adaptation to the cystic fibrosis lung habitat and into DNA acquired through horizontal gene transfer. Further on, large chromosomal inversions were mediated by ISPa20. In contrast to strains of other subclonal linages high rates of genomic rearrangements of subclone C13 strains were observed in vitro. The acquisition of mobile elements by P. aeruginosa clone C strains in the lungs of cystic fibrosis patients supports the chronic colonization by insertional mutagenesis and chromosome restructuring leading to microevolution within clone C that reflects macroevolution observed on the species level.