Structure and Function Relationship of the Autotransport and Proteolytic Activity of EspP from Shiga Toxin-Producing Escherichia coli

BACKGROUND: The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPalpha, EspPbeta, EspPgamma, and EspPdelta), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown. METHODOLOGY/PRINCIPAL FINDINGS: The structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the beta-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His(127), Asp(156) and Ser(263) in Domain 1 form the catalytic triad of EspP. CONCLUSIONS/SIGNIFICANCE: Our data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPbeta and EspPdelta

Cytolethal Distending Toxin from Shiga Toxin-Producing Escherichia coli O157 Causes Irreversible G(2)/M Arrest, Inhibition of Proliferation, and Death of Human Endothelial Cells

Recently, cytolethal distending toxin V (CDT-V), a new member of the CDT family, was identified in Shiga toxin-producing Escherichia coli (STEC) O157 and particular non-O157 serotypes. Here we investigated the biological effects of CDT-V from STEC O157:H(−) (strain 493/89) on human endothelial cells, which are believed to be major pathogenetic targets in severe STEC-mediated diseases. CDT-V caused dose-dependent G(2)/M cell cycle arrest leading to distension, inhibition of proliferation, and death in primary human umbilical vein endothelial cells (HUVEC) and two endothelial cell lines, EA.hy 926 cells (HUVEC derived) and human brain microvascular endothelial cells (HBMEC). The cell cycle effects of CDT-V were cell type specific. In HUVEC and EA.hy 926 cells, CDT-V caused a slowly developing but persistent G(2)/M block which resulted in delayed nonapoptotic cell death. In contrast, in HBMEC, CDT-V induced a rapidly evolving but transient G(2)/M block which was followed by progressive, mostly apoptotic cell death. In both HBMEC and EA.hy 926 cells, G(2)/M arrest was preceded by the early accumulation of a phosphorylated inactive form of cdc2 kinase. Significant G(2)/M arrest and inhibition of proliferation in both HUVEC and each of the endothelial cell lines were induced by 2 to 15 min of exposure to CDT-V, indicating that the effects of the toxin are irreversible. CDT-V-treated HBMEC and EA.hy 926 cells displayed fragmented nuclei and expressed phosphorylated histone protein H2AX, indicative of DNA damage followed by a DNA repair response. Our data demonstrate that CDT-V causes irreversible damage to human endothelial cells and thus may contribute to the pathogenesis of STEC-mediated diseases

Distinct Expression of Immunoglobulin-Binding Proteins in Shiga Toxin-Producing Escherichia coli Implicates High Protein Stability and a Characteristic Phenotype

Several immunoglobulin-binding proteins of Escherichia coli (Eib) have been isolated from both non-pathogenic and pathogenic E. coli strains. Shiga toxin (Stx)-producing E. coli (STEC) contain eibG either as a single gene or in combination with eibC, while other E. coli strains harbour single or multiple eib genes. The Eib proteins bind human immunoglobulins in a non-immune manner and contribute to bacterial chain-like adherence to human epithelial cells. In this study, the EibG expression in several STEC strains was analysed under different environmental conditions. STEC produced high levels of EibG in complex media and lower levels in low-grade and minimal media under static growth conditions. This characteristic was independent on the Eib subtypes. Microscopically, EibG-expressing STEC exhibited chain formation and aggregation in all employed media, while aggregates were only visible after growth in complex medium. Once expressed, EibG proteins demonstrate high stability during prolonged incubation. Our findings indicate that the regulation of the expression of Eib proteins is highly complex, although the protein levels vary among STEC strains. However, positive upregulation conditions generally result in distinct phenotypes of the isolates

The glycosylation states influenced changes in solubility of mouse prion isoforms as a result of metal-binding.

<p>Pooled mouse brains from C57BL wild-type and transgenic T182N mice were homogenized (10%) and supplemented with 2% N-octyl-β-D-glucopyranoside (OGP) followed by centrifugation to obtain supernatant (S1) and sedimented protein (P1). Highly soluble proteins from the S1 fraction were pre-incubated in the absence or presence of metal ions such as CuCl₂ (Cu), ZnCl₂ (Zn), MgCl₂ (Mg) and CaCl₂ (Ca), 1 mM each. Proteins were once again separated by centrifugation into fractions of high and low solubility represented as supernatants (S2) and pellets (P2), respectively. After immunoblotting, PrP^C proteins were identified using mab SAF70 and signals were visualized by chemiluminescence substrate development. Glyosylated full-length PrP^C proteins of both mouse types were detected in the pellet fraction when bound to zinc ions. A considerably lower effect was observed in the T182N PrP^C interaction with copper ions, whereas other metals played no role in structural changes resulting in sedimentation. When metal ions were bound, mainly full length glycosylated PrP^C changed into the pellet fraction.</p

Reduced solubility due to ZnCl₂ and CuCl₂ is reversed by EDTA and SDS application.

<p>Pooled brain tissues derived from C57BL wild-type mice (A) and bovine (B) homogenized in TBS and 2% N-octyl-β-D-glucopyranoside were supplemented with ZnCl₂ (1 mM) and CuCl₂ (1 mM) followed by incubation in the absence (0) or presence of EDTA in concentrations indicated or 1% SDS. Following centrifugation, proteins were separated into fractions of high solubility in the supernatant and low solubility in the pellet. PrP^C signals were detected using mab SAF34 and visualized by chemiluminescence substrate development.</p