Estrés postraumático en escolares a 8 meses del 27F / Post-traumatic stress disorder in children eight months after february 27, 2010

Introduction: there are several epidemiological studies regarding the incidence of post-traumatic stress disorder (PTSD) in children exposed to natural disasters. Objective: to describe the prevalence of PTSD in a school-age population in a coastal town from the Maule Region, 8 months after the earthquake/tsunami in february 2010, and to compare differences among PTSD groups of symptom (re-experiencing, avoidance and activation) according to demographic variables such as age, grade, gender and family type. Methodology: the Child PTSD Symptom Scale (CPSS), validated in Chile in 2009, was used in 89 children between 3rd and 6th grade, corresponding to 94.7 percent of the children enrolled in the local school in such classes. Data are analyzed by gender, age, grade and type of family. 89 surveys were applied, 59.6 percent were male and 40.4 percent female aged 8-13. Results: 40.4 percent of children had symptoms consistent with PTSD, with higher incidence in women and younger children; the most significant association among women was age. Both groups (women and young children) presented the highest scores on all group of symptoms. There were no differences by type of family. Conclusions: the incidence of PTSD measured by CPSS scale in the study population was 40.4 percent, considered to be among the highest percentages reported in the international literature.

Introducción: existen diversos datos epidemiológicos respecto a la incidencia de Trastorno por Estrés Pos-traumático (TEPT) en niños expuestos a desastres naturales. Objetivo: describir la prevalencia de TEPT en una población infantil escolarizada de una localidad costera de la Región del Maule después de 8 meses de ocurrido el terremoto/maremoto de febrero/2010, y comparar las diferencias entre grupos de síntomas del TEPT (reexperimentación, evitación y activación) según variables demográficas, como edad, curso, sexo y tipo de familia. Metodología: se aplicó la escala Child PTSD Symptom Scale (CPSS) validada en Chile el año 2009, a 89 niños de 3° a 6° básico lo que corresponde al 94,7 por ciento de los niños matriculados en la escuela de la localidad en dichos cursos. Se analizan los datos según sexo, edad, curso y tipo de familia. Se aplicaron 89 encuestas, 59,6 por ciento eran varones y 40,4 por ciento mujeres de 8 a 13 años de edad. Resultados: el 40,4 por ciento del total de niños tuvo una evaluación compatible con TEPT, con mayor incidencia en mujeres y niños de menor edad, siendo la edad un factor de asociación significativa en las mujeres. Ambos grupos (mujeres y niños más pequeños) presentaron mayores puntajes en todos los grupos sintomáticos. No se encontraron diferencias según el tipo de familia. Conclusiones: la incidencia de TEPT medida a través de la escala CPSS en la población estudiada fue de 40.4 por ciento, encontrándose entre las más altas reportadas en la bibliografía internacional.

Intervención de salud mental en niños expuestos a desastre natural / Mental health intervention in children exposed to natural disasters

Introduction: this study is part of the mental health intervention, conducted by a child psychiatry team for children exposed to the february 2010 earthquake/tsunami in a community of the VII Region that was strongly affected by the natural disaster. Objective: to describe the intervention and evaluate the effectiveness of the strategies implemented for both children and teachers. Methodology: interventions are described and classified in three categories. (1) Case report and child care consulting, referred by their teacher. (2) Psychoeducational workshops for teachers of the intervened school. (3) Self-Care day aimed at professionals of the same school. The evaluation is done through an anonymous survey designed to measure the effectiveness of the intervention. Results: 33 children were evaluated and treated, the most common diagnoses were adaptive disorders (8/33) and ADHD (11/33), and only 3 patients met the criteria for post-traumatic stress disorder (PTSD). Intervention implementation included psychoeducation for parents (100 percent), coordination with schools and local health network (100 percent), counseling (70 percent) and drug prescription (45 percent). Only 45 percent of the cases evaluated had symptoms triggered or exacerbated by the earthquake/tsunami. Regarding teacher evaluation (N: 11), 100 percent described the intervention as "very good". 90 percent considered it appropriate to the needs at the moment and as a contribution to their educational work. Conclusions: after events of this nature, many interventions take place to support the affected population. It is important to have more scientific information about the effectiveness of such interventions to prevent the development of post-traumatic psychopathology.

Introducción: este trabajo forma parte de la intervención de salud mental, realizada por un equipo de psiquiatría infantil para niños expuestos al terremoto/maremoto de febrero de 2010, en una comunidad de la VII Región fuertemente afectada por el desastre natural. Objetivos: describir la intervención realizada y evaluar la efectividad de las estrategias implementadas tanto a niños como a profesores. Metodología: se describe las intervenciones realizadas, clasificadas en 3 categorías: 1) Consultoria de casos clínicos y atención de niños derivados por sus profesores. 2) Talleres psicoeducativos a profesores de la escuela intervenida. 3) Jornada de autocuidado, dirigida a los profesionales de la misma escuela. La evaluación se realiza a través de encuesta anónima a los profesores diseñada para cuantificar la efectividad de la intervención. Resultados: se evaluaron y trataron 33 niños, los diagnósticos más frecuentes fueron Trastornos adaptativos (8/33) y Déficit atencional (11/33); sólo 3 casos cumplían criterios de Trastorno por Estrés Postraumático(TEPT). Las intervenciones utilizadas incluyeron psicoeducación a padres (100 por ciento), coordinación con colegios y red de salud municipal (100 por ciento), apoyo psicológico (70 por ciento) y farmacológico (45 por ciento). Sólo en el 45 por ciento de los casos evaluados la sintomatología se había desencadenado o agravado con el terremoto/maremoto. En relación a la evaluación de profesores (n: 11), 100 por ciento consideró la intervención como "muy buena". Un 90 por ciento la consideró adecuada a las necesidades y constituyó un aporte a su quehacer educativo. Conclusiones: tras eventos como éste, se realizan variadas intervenciones de apoyo a la población afectada. Es importante contar con mayor información científica acerca de la efectividad de dichas intervenciones para prevenir el desarrollo de psicopatología postraumática.

Asp f6, an Aspergillus allergen specifically recognized by IgE from patients with allergic bronchopulmonary aspergillosis, is differentially expressed during germination.

BACKGROUND: Aspergillus fumigatus is a pathogenic mould causing allergic and invasive respiratory diseases. Allergic bronchopulmonary Aspergillosis (ABPA) is a severe pulmonary complication resulting from hypersensitivity to A. fumigatus proteins. Aspergillus allergen Asp f6 is recognized by IgE from ABPA patients, but not from sensitized individuals, a fact that can be used to differentiate between these two groups of allergic patients. METHODS: Proteins from hyphae, resting and germinating conidia of A. fumigatus were compared by SDS-PAGE. Protein identification was performed using MALDI-TOF mass spectrometry. Recombinant A. fumigatus allergens were used to isolate specific monoclonal antibodies (mab) from a hybridoma bank generated against Aspergillus proteins. RESULTS: A hyphae-specific 23 kDa A. fumigatus protein was identified as the allergen Asp f6/manganese-dependent superoxide dismutase (MnSOD). Differential expression of MnSOD was confirmed by immunoblot using a specific mab. In contrast, Asp f8 another intracellular, but not ABPA-specific allergen, was detected in hyphae and conidia. CONCLUSIONS: Aspergillus fumigatus is able to colonize its environment by the formation of hyphae. Hyphae are found in the lung of ABPA patients, but not in patients suffering from atopic asthma. Our finding that Asp f6 is specifically expressed in hyphae might explain why an IgE response to Asp f6 is specific for ABPA patients.

Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages.

Aspergillus fumigatius is a ubiquitous saprophytic fungus that has become the most prevalent airborne fungal pathogen for immunocompromised patients during the last two decades. In this report we have analysed how macrophages recognize this microorganism. Using transfected human HEK 293 cells we demonstrate that NF-kappaB-dependent promoter activation triggered by A. fumigatus is mediated by Toll-like receptors TLR2 and TLR4, whereas no activation was observed in cells overexpressing other distinct TLR proteins (TLR1, TLR3, TLR5-10). Using macrophages derived from mice lacking TLR2 expression, expressing defective TLR4 or both we found that A. fumigatus conidia and hyphae induce NF-kappaB translocation, release of pro-inflammatory molecules, like TNFalpha, and the chemoattractant MIP-2 in a TLR2- and TLR4-dependent manner. Recognition of A. niger and A. fumigatus, was similar in terms of the parameters analysed, suggesting that pathogenic and non-pathogenic aspergilli are sensed by macrophages in a similar fashion. Finally, we found that recruitment of neutrophils is severely impaired in mice lacking both functional TLR2 and TLR4, but is less impaired in single TLR2- or TLR4-deficient mice, providing evidence that both receptors are required for an optimal immune response to Aspergillus in vivo.

Modulation of host cell signalling by enteropathogenic and Shiga toxin-producing Escherichia coli.

The majority of Escherichia coli strains are harmless symbionts in the intestinal tract. However, there are several pathogenic forms, which are responsible for various diseases in humans and live stock. In this review we discuss the interactions between Shiga toxin-producing E. coli and enteropathogenic E. coli and their target host cells, describing their strategies to activate specific cellular signalling pathways which lead to subversion of critical physiological functions. We mainly concentrate on those pathogenic mechanisms that are dependent on a functional type III secretion system, but we also briefly discuss additional factors that contribute to the specific pathogenic profiles of Shiga toxin-producing E. coli and enreropathogenic E. coli.

Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis.

Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

EspA filament-mediated protein translocation into red blood cells.

Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC), three type III secreted proteins, EspA, EspB and EspD, have been shown to be required for translocation of the Tir effector protein into host cells. EspB and EspD have been proposed to form a pore in the host cell membrane, whereas EspA, which forms a large filamentous structure bridging bacterial and host cell surfaces, is thought to provide a conduit for translocation of effector proteins between pores in the bacterial and host cell membranes. Type III secretion has been correlated with an ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro. As EspA filaments link bacteria and the host cell, we predicted that intimate bacteria-RBC contact would not be required for EPEC-induced haemolysis and, therefore, in this study we investigated the interaction of EPEC with monolayers of RBCs attached to polylysine-coated cell culture dishes. EPEC caused total RBC haemolysis in the absence of centrifugation and osmoprotection studies were consistent with the insertion of a hydrophilic pore into the RBC membrane. Cell attachment and haemolysis involved interaction between EspA filaments and the RBC membrane and was dependent upon a functional type III secretion system and on EspD, whereas EPEC lacking EspB still caused some haemolysis. Following haemolysis, only EspD was consistently detected in the RBC membrane. This study shows that intimate bacteria-RBC membrane contact is not a requirement for EPEC-induced haemolysis; it also provides further evidence that EspA filaments are a conduit for protein translocation and that EspD may be the major component of a translocation pore in the host cell membrane.

Intimin from Shiga toxin-producing Escherichia coli and its isolated C-terminal domain exhibit different binding properties for Tir and a eukaryotic surface receptor.

The outer membrane protein intimin plays a crucial role in the attaching and effacing process employed by different enteropathogens to colonize the epithelial surface of their hosts. In this study we have characterized the C-terminal binding domain of intimin from the Shiga toxin-producing Escherichia coli strain 413/89-1, that belongs to the beta-subtype of intimins. We found that a fusion of this domain to the maltose-binding protein binds efficiently to both the translocated intimin receptor (Tir) and the surface of uninfected eukaryotic host cells. In contrast, no such binding was observed with the full-length protein localized on the bacterial surface. As the C-terminal domain of intimin and the full-length protein differ in their binding activity, we suggest that the intimin-binding domain might be controlled by the N-terminal portion of the molecule to prevent unproductive interactions with molecules in the lumen of the gut.

Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton.

Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram-negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the "needle complex" (NC), produced an average image at 17 A resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2-3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N-terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.

Characterization of SepL of enterohemorrhagic Escherichia coli.

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 degrees 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 EDL933DeltasepL 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.

Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE.

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.

The actin-based motility of intracellular Listeria monocytogenes is not controlled by small GTP-binding proteins of the Rho- and Ras-subfamilies.

In this study, we analyzed whether the actin-based motility of intracellular Listeria monocytogenes is controlled by the small GTP-binding proteins of the Rho- and Ras-subfamilies. These signalling proteins are key regulatory elements in the control of actin dynamics and their activity is essential for the maintenance of most cellular microfilament structures. We used the Clostridium difficile toxins TcdB-10463 and TcdB-1470 to specifically inactivate these GTP-binding proteins. Treatment of eukaryotic cells with either of these toxins led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the invasion of epithelial cells by L. monocytogenes and had no effect on the actin-based motility of this bacterial parasite. Our data indicate that intracellular Listeria reorganize the actin cytoskeleton in a way that circumvents the control mechanisms mediated by the members of the Rho- and Ras-subfamilies that can be inactivated by the TcdB-10463 and TcdB-1470 toxins.

Initial binding of Shiga toxin-producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA-containing surface appendages.

Shiga 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.

Pas, a novel protein required for protein secretion and attaching and effacing activities of enterohemorrhagic Escherichia coli.

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, and espD genes on the complementary strand and which exhibits high homology to the genes spiB from Salmonella, yscD from Yersinia, and pscD 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.

Small GTP-binding proteins of the Rho- and Ras-subfamilies are not involved in the actin rearrangements induced by attaching and effacing Escherichia coli.

Attaching and effacing Escherichia coli (AEEC) are extracellular pathogens that induce the formation of actin-rich structures at their sites of attachment to eukaryotic host cells. We analysed whether small GTP-binding proteins of the Rho- and Ras-subfamilies, which control the cellular actin system, are essential for these bacterial-induced microfilament reorganizations. For this purpose we specifically inactivated them using the Clostridium difficile toxins TcdB-10463 and TcdB-1470. Such treatment led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the bacterial-induced actin rearrangements. Our data therefore indicate that the microfilament reorganizations induced by AEEC are independent of those small GTP-binding proteins that under normal conditions control the dynamics and maintenance of the actin cytoskeleton.

EspE, a novel secreted protein of attaching and effacing bacteria, is directly translocated into infected host cells, where it appears as a tyrosine-phosphorylated 90 kDa protein.

Shiga toxin-producing Escherichia coli (STEC), enteropathogenic E. coli (EPEC) and some strains of Hafnia alvei are capable of inducing attaching and effacing (A/E) lesions, characterized by tight apposition of the bacteria to the eukaryotic membrane and formation of actin-based pedestals. In this study, we report on the identification of EspE, a novel secreted 80 kDa protein of A/E bacteria. During infection, EspE is delivered into the cytoplasm of the infected host cell, where it is detected as a higher-molecular-weight form of 90 kDa. We present evidence that translocated EspE becomes tyrosine phosphorylated and that this modified form of EspE may be identical to Hp90, the putative receptor of EPEC intimin. Bacteria of the classic enterohaemorrhagic E. coli (EHEC) serotype O157:H7 fail to induce a tyrosine phosphorylation of EspE and differ in this respect from other A/E bacteria. Translocated EspE, whether tyrosine phosphorylated or not, becomes incorporated into the bacteria-induced cytoskeletal structures, where it normally colocalizes with filamentous actin. EPEC are also able to induce 'pseudopods', elongated pedestals that have recently been implicated in a novel kind of actin-based motility. EspE is enriched at the tip of these structures, suggesting its involvement in the process of actin dynamics, which is triggered during the attaching and effacing process.

A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family.

The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. We identify E/DFPPPPXD/E, a motif repeated 4-fold within the primary sequence of ActA, as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein-protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. Taken together we demonstrate that ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling.

Characterization of an exported protease from Shiga toxin-producing Escherichia coli.

The gene for a novel, high molecular weight protein secreted by Shiga toxin-producing Escherichia coli (STEC) has been cloned, sequenced and characterized with respect to its activity. This gene, designated pssA, is localized on the large plasmid that also harbours the STEC haemolysin operon. Sequencing of a region comprising 10630nt revealed that the sequences flanking the pssA gene are composed of several remnants of different insertion elements. The PssA protein is produced as a 142kDa precursor molecule that, after N- and C-terminal processing, is released into the culture supernatant as a mature polypeptide of approximately 104kDa. The primary sequence of PssA is highly related to a family of autonomously transported putative virulence factors from different Gram-negative pathogens, which includes the Tsh protein of an avian-pathogenic E. coli strain, the SepA protein from Shigella flexneri and the EspC protein from enteropathogenic E. coli. A common motif present in all four proteins is reminiscent of the catalytic centre of certain serine proteases. PssA (protease secreted by STEC) indeed shows serine protease activity in a casein-based assay and is moreover cytotoxic for Vero cells. This activity of PssA and probably of other proteins of the Tsh family may be of functional importance during infection of the mucosal cell layer by the bacterial pathogen.

Temperature- and medium-dependent secretion of proteins by Shiga toxin-producing Escherichia coli.

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 degrees 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.

The enterohemolysin phenotype of bovine Shiga-like toxin-producing Escherichia coli (SLTEC) is encoded by the EHEC-hemolysin gene.

Naturally occurring enterohemolysin negative variants were observed during studies on bovine Shiga-like toxin-producing E. coli (SLTEC). Examination of three strains (413/89-1 and 332, 026:H-, and 570/89, O111:H-) and their isogenic variants (413/89-6, 332-I and 570/89-I, respectively) showed, that in each strain loss of the enterohemolytic phenotype correlated with the loss of a large plasmid ranging from 94 to 104 kb in size. The hemolysin determinant present on the 94 kb plasmid of strain 413/89-1 was cloned and discovered by DNA and N-terminal aminoacid sequence analysis to be highly homologous to the recently published EHEC-hemolysin (HlyEHEC; Schmidt et al., 1994; 1995). When a recombinant plasmid harboring this determinant was reintroduced into the enterohemolysin negative isogenic mutant 413/89-6, the enterohemolytic phenotype was restored. Southern blot hybridization analysis was used to demonstrate that the HlyEHEC is plasmid-borne in SLTEC-strains. Our cumulative data suggest that the enterohemolytic phenotype of SLTEC is encoded by the plasmid-borne HlyEHEC. These results further demonstrate the close similarity between SLTEC-isolates from bovine and human.