Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography.

Julie Heggelund,Gregor Bajc,Vesna Hodnik,Ute Krengel,Gregor Anderluh,Joel Heim

doi:10.3390/ijms20030703

Abstract

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galβ4GlcNAcβ6[Galβ4GlcNAcβ3]Galβ4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.

Highlights

The heat-labile enterotoxin (LT), a homologue of the cholera toxin (CT), is produced by enterotoxigenic Escherichia coli (ETEC)
In order to verify proper folding of the toxin variants, we subjected all proteins to circular dichroism (CD) spectroscopy (Figure S1)
Wild-type pLTB and variant T4N bound with similar affinity to the GM1 pentasaccharide as previously reported, pLTB variant N94H bound more strongly, whereas S95A had a slightly lower affinity, but still in the nanomolar range (Table 1)

Summary

Introduction

The heat-labile enterotoxin (LT), a homologue of the cholera toxin (CT), is produced by enterotoxigenic Escherichia coli (ETEC). ETEC is responsible for millions of diarrheal cases and more than 50,000 deaths every year [1]. The mortality of the disease is declining, but the morbidity is not, despite improvements in sanitation facilities. ETEC infection in children often leads to long-term health problems like stunted growth and reduced cognitive abilities, triggering a vicious cycle of poverty [2]. The disease affects travellers to endemic areas, including medical and military personnel, and has further been linked to chronic diseases like irritable bowel syndrome [3]. The infection spreads through the fecal–oral route, aggravated by the watery diarrhea caused by the enterotoxin

Methods

Results

Discussion

Conclusion