Conservation of Bacterial Lipopolysaccharide Binding by SARS-CoV-2 Spike across Major Viral Variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis is shaped not only by viral entry mechanisms but also by interactions with host and microbial factors. The viral spike (S) protein can bind Gram-negative bacterial lipopolysaccharide (LPS), a driver of hyperinflammation in severe COVID-19. How viral evolution over the years alters this interaction remains unclear. Here, we investigated LPS binding across major SARS-CoV-2 variants that emerged over the course of the pandemic (2019-2023) from the ancestral Wuhan-Hu-1 strain to Omicron subvariants BA.1, XBB.1.5, and BA.2.86. Structural mapping revealed multiple mutations near a cryptic lipid-binding pocket in the receptor binding domain (RBD). Using extensive atomic-resolution molecular dynamics (MD) simulations with free energy calculations, validated by biochemical binding assays and fluorescence quenching experiments, we show that these mutations weaken binding to the lipid A component of LPS. However, full-length LPS binds with similar affinity to most variants likely due to increased positive electrostatic potential of the RBD, promoting compensatory interactions with negatively charged LPS inner core sugars. Together, these findings uncover an evolutionary balance that preserves S protein-LPS engagement through distinct molecular mechanisms, suggesting that emerging variants may retain the capacity to potentiate hyperinflammation during infection.