Abstract
Shiga toxins (Stx) released by Stx-producing E. coli (STEC) are virulence factors that are most closely associated with hemolytic uremic syndrome (HUS), a life-threatening complication of intestinal infections by STEC. Stx have to enter into the circulatory system before they are delivered to target organs and cause damage. The presence of Stx in sera could be a risk indicator for HUS development. However, the detection of Stx, particularly Stx2, has been difficult due to the presence of Stx2-binding components in human serum. Here, we report new ELISA-based methods for the detection of Stx1 and Stx2 in human serum and the effect of guanidinium chloride on enhancing the sensitivity for the detection of Stx2. The recovery rate for Stx2 was 62% when Stx2-spiked serum samples were treated with guanidinium chloride at a concentration of 200 mM, in contrast to 17% without guanidinium chloride treatment. The effectiveness of guanidinium chloride treatment for the detection of Stx2 in human serum was validated using sera from STEC-infected patients. Coimmunoprecipitation results indicated a specific physical interaction between Stx2 and the human serum amyloid P component (HuSAP) in human serum samples. Our in vitro study demonstrated that the inhibition from HuSAP alone for the detection of Stx2 was only 20%, much less than 69.6% from human serum at Stx2 level 10 ng/mL, suggesting that there may be other factors that bind Stx2 in human serum. This study indicates that treatment of serum samples with guanidinium chloride may be useful for the early and sensitive detection of Stx2 in sera of STEC-infected patients, so preventive measures can be adopted in a timely manner.
Highlights
Shiga toxin-producing E. coli (STEC) has been widely acknowledged as one of the major causative agents of foodborne illness
Shiga toxins (Stx) are the primary virulence factors that lead to hemolytic uremic syndrome (HUS), a clinical composite of thrombocytopenia, hemolytic anemia, and thrombotic microangiopathy that contributes to acute kidney injury, often requiring dialysis, which can progress to acute renal failure and death [1]
Our results indicated that all serum samples collected from these STEC-infected patients were positive for Stx2
Summary
Shiga toxin-producing E. coli (STEC) has been widely acknowledged as one of the major causative agents of foodborne illness. There are two types of Stx produced by STEC, Stx and Stx2 [5] They are distinct genetically and antigenically, but possess similar 3D structures and modes of action. Both Stx and Stx are composed of one A subunit and five B subunits [6]. The A subunit (32 kDa) contains an enzymatically active domain that cleaves a specific adenine base from the 28S rRNA and prevents host cell protein synthesis [7]. The B subunits (7.7 kDa each) bind glycolipids on the surface of host cells and facilitate the internalization of the toxin [8]. Stx is 1000 times more toxic for human renal glomerular endothelia cells than Stx1 [10]
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