Metabolic Rigidity as a Mechanical Barrier to Malaria: Flickering Loss in PKLR-Deficient Erythrocytes.

Pyruvate kinase (PK) deficiency is a rare hereditary enzymopathy caused by mutations in the PKLR gene, leading to reduced glycolytic ATP production in red blood cells (RBCs) and contributing to chronic hemolytic anemia. Here, we use high-speed flickering spectroscopy and non-invasive microrheology to assess how ATP depletion reshapes the nanomechanical properties of RBC membranes. Compared to healthy controls, PKLR-mutant erythrocytes exhibit marked reductions in ATP-dependent flickering amplitude and membrane fluidity, consistent with impaired metabolic elasticity. Strikingly, when infected with Plasmodium yoelii, these metabolically rigidified cells retain mechanical properties that appear to hinder parasite-induced membrane remodeling. By mapping single-cell viscoelastic landscapes across healthy, mutated, infected, and coinfected mouse RBC populations, we uncover a potential biomechanical barrier against malaria imposed by glycolytic insufficiency. These findings highlight a mechanobiological axis of host resistance and position label-free flickering analysis as a powerful tool for diagnosing RBC enzymopathies and probing infection susceptibility at the single-cell level.