Cytotoxic Effects of Recombinant StxA2-His in the Absence of Its Corresponding B-Subunit.

AB5 protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB5 toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.

[Case report of acute encephalopathy caused by enterohemorrhagic Escherichia coli infection in a 24-year-old woman].

Hemolytic uremic syndrome (HUS) and acute encephalopathy caused by enterohemorrhagic Escherichia coli infection occur commonly in children, whereas adult-onset disease is rare. Here we report the case of a 24-year-old woman who developed acute encephalopathy and recovered without sequelae. She initially developed abdominal pain and diarrhea. On day 6, O-157 Shiga toxin was detected in her stool and she developed HUS. On day 11, acute encephalopathy developed and she required artificial ventilation. She was treated with steroid pulse therapy and plasma exchange (PE) and then discharged on day 53 without any sequelae. Globotriaosylceramide, a Shiga toxin receptor, is more frequently present on the cellular membranes of women than on those of men. Therefore, it is conceivable that adult women are at a higher risk of developing acute encephalopathy than men. Steroid pulse therapy and PE may effectively treat acute encephalopathy by reducing inflammatory cytokine levels in the blood; therefore, these treatments should be proactively considered.

Análisis in silico de la capacidad de dos técnicas de PCR para la detección del gen stx / In silico analysis of the capability of two polimerase chain reaction techniques for stx gene detection.

Escherichia coli productor de toxina Shiga es un patógeno emergente cuyo principal factor de virulencia son las toxinas Shiga (Stx), codificadas por los genes stx. Estas toxinas se clasifican en 6 tipos (1, 2, 2c, 2d, 2e y 2f) que agrupan a 22 variantes. En Argentina se validaron dos técnicas de PCR para la detección de los genes stx, PCR-MK y PCR múltiple. Los objetivos del trabajo fueron analizar mediante el uso de herramientas bioinformáticas la capacidad de dichas técnicas para detectar las variantes del gen stx y demostrar experimentalmente la amplificación de 8 variantes stx. Se recopilaron 25 secuencias nucleotídicas de la base de datos GenBank correspondientes a 21 variantes de stx. Se utilizó el programa BLAST 2 sequences para analizar la complementariedad de las bases nucleotídicas entre las secuencias de las variantes y las secuencias de los cebadores utilizados en las PCR estudiadas. La técnica de PCR-MK permite detectar los tipos stx1, stx2, stx2c, stx2d y stx2f, aunque no permite detectar el tipo stx2e y tres variantes del tipo stx2c. La PCR múltiple permite detectar los tipos stx1, stx2, stx2c, stx2d, pero no los tipos stx2e y stx2f. Se demostró experimentalmente que ambas técnicas de PCR son apropiadas para la detección de las variantes que están asociadas a enfermedad grave en el hombre.

Shiga toxin-producing Escherichia coli is an emergent pathogen, being the Shiga toxin (Stx) the main virulence factor. These toxins are classified into 6 types (1, 2, 2c, 2d, 2e and 2f) and 22 variants. In Argentina, two PCR for stx gene detection, PCR-MK and multiplex-PCR, were validated. The aim of this work was to analyze, by using bioinformatic tools, the stx variants that could be amplified by these PCRs, and to experimentally show the amplification of 8 stx variants. Twentyfive nucleotide sequences were collected from GenBank corresponding to 21 stx variants. The BLAST 2 sequences program was used to analyze the complementarities between the nucleotide sequence of the variants and the primers corresponding to the PCR studied. PCR-MK could detect types stx1, stx2, stx2c, stx2d and stx2f, but not type stx2e and three type stx2c variants. On the other hand, the multiplex-PCR could detect types stx1, stx2, stx2c, stx2d, but not stx2e and stx2f types. It was experimentally determined that both PCRs can detect those variants that cause severe disease in humans.

Synthesis of globo- and isoglobotriosides bearing a cinnamoylphenyl tag as novel electrophilic thiol-specific carbohydrate reagents.

The galactosyl donor, 4,6-di-O-acetyl-2,3-di-O-benzyl-D-galactopyranosyl trichloroacetimidate, was efficiently coupled with regioselectively benzylated lactoside acceptors under standard conditions to stereoselectively afford the corresponding globotrioside and isoglobotrioside derivatives in very good yields. These glycosides were smoothly functionalized with a 6-(p-cinnamoylphenoxy)-hexyl tether tag as novel electrophilic thiol-specific carbohydrate reagents. Immobilization of the globotrioside conjugate to Thiopropyl Sepharose 6B for purification of B-subunit of Shiga toxin (StxB) and coupling of a model cysteine-containing protein (StxB-Z(n)-Cys) to the isoglobotrioside conjugate were both performed with high efficiency.

El rol epidemiológico de las mascotas en el ciclo de transmisión urbana de cepas STEC / Epidemiological role of pets in urban transmission cycle of STEC

Escherichia coli is an important constitutional element of both animal's and men's microbiote. Rumiants in general and bovine in particular have been pointed out as reservoirs of shigatoxigenic (STEC) and enterohemorragic strains (EHEC). These strains are rarely reported in pets isolations and are badly documented. Revisions of the pathogenic role of E. coli in small animals, particulary based on intestinal and urogenital isolations, involve in a very low proportion the STEC strains. In dogs, ocasional isolations are reported, from feces coming from both healthy and diarreic dogs. The STEC intestinal strains prevalence in dogs and cats from urban centers, based on the habit of including regular or ocasional raw meat in their diet, should not be understimated.