Dr. Rafael Vincent M. Manalo
University of the Philippines Manila

Konzo is a debilitating neurologic disease in Africa caused by a monotonous consumption of cassava, which contains the cyanogenic compound linamarin. Affected patients are commonly women of reproductive age and children beyond 3 years, who acutely present with spastic paraparesis of bilateral lower limbs. It was hypothesized that since linamarin metabolism occurred in the gut, a changing gut microbiota may be important to its pathogenesis. 

The objectives of the study were 1) to determine the occurrence and location of cyanide conversion from linamarin in the mammalian gut; 2) to determine the neurotoxic mechanisms of linamarin metabolites, and 3) to determine the behavioral changes in C57BL/6 mice exposed to the known nutritional risk factors of konzo. 

We developed nickel-based and picric acid-based cyanide detection assays to determine the amount of cyanide that forms when linamarin is digested by gut bacteria as fingerprints to detect linamarase activity in dissected mice gut regions. We similarly performed the assay on cultured stool samples from patients and controls in Kahemba and Masi-Manimba to determine any trends between disease severity and cyanogenic potentials. We correlated these data with in vitro cell culture of neural cells and behavioral assays in C57BL/6 mice to determine the likely pathophysiology underlying the disease. 

We show that cyanogenesis occurred mostly in the small and large intestines of C57BL/6 mice and was affected by a low-protein (6%) diet. Among konzo stool samples, disease severity correlated inversely with cyanogenic potential, with shotgun metagenomics suggesting that Klebsiella pneumoniae may have the highest rate of cyanogenesis. When exposed to KCN, necrotic cell death of neurons (10.76 mM) and microglia (20 mM) and apoptosis of astrocytes (15 mM) were observed at their IC50, with a reduction in their ∆ψm. Interestingly, exposure to NaOCN at much lower concentrations resulted in the apoptosis of both neurons (5.43 mM) and astrocytes (2.85 mM), suggesting that cyanate was more neurotoxic than cyanide and favored the apoptosis of neural cells. Exposure of linamarin to protein-restricted C57BL/6 mice (6%) supplemented with L. plantarum 14917 resulted in non-significant tremors and general weakness without the desired spastic paralysis phenotype.

Cyanogenesis in the gut occurs mostly in the small and large intestines, and may be mediated by highly cyanogenic bacteria such as L. plantarum and K. pneumoniae. A low-protein diet (6%) may decrease gut richness and diversity resulting in the growth of these pathogenic bacteria. However, exposure to linamarin alone in protein-restricted mice supplemented with cyanogenic bacteria fails to demonstrate the spastic paraparesis of konzo, suggesting the emerging role of oxidative stress as a necessary factor for pathogenesis.