Structural Characterization of Haemophilus influenzae Enolase and Its Interaction with Human Plasminogen by In Silico and In Vitro Assays

Haemophilus influenzae is the causal agent of invasive pediatric diseases, such as meningitis, epiglottitis, pneumonia, septic arthritis, pericarditis, cellulitis, and bacteremia (serotype b). Non-typeable H. influenzae (NTHi) strains are associated with localized infections, such as otitis media, conjunctivitis, sinusitis, bronchitis, and pneumonia, and can cause invasive diseases, such as as meningitis and sepsis in immunocompromised hosts. Enolase is a multifunctional protein and can act as a receptor for plasminogen, promoting its activation to plasmin, which leads to the degradation of components of the extracellular matrix, favoring host tissue invasion. In this study, using molecular docking, three important residues involved in plasminogen interaction through the plasminogen-binding motif ((251)EFYNKENGMYE(262)) were identified in non-typeable H. influenzae enolase (NTHiENO). Interaction with the human plasminogen kringle domains is conformationally stable due to the formation of four hydrogen bonds corresponding to enoTYR(253)-plgGLU(1) (K2), enoTYR(253)-plgGLY(310) (K3), and enoLYS(255)-plgARG(471)/enoGLU(251)-plgLYS(468) (K5). On the other hand, in vitro assays, such as ELISA and far-western blot, showed that NTHiENO is a plasminogen-binding protein. The inhibition of this interaction using polyclonal anti-NTHiENO antibodies was significant. With these results, we can propose that NTHiENO-plasminogen interaction could be one of the mechanisms used by H. influenzae to adhere to and invade host cells.

Automated summary

Haemophilus influenzae causes invasive pediatric diseases, while non-typeable H. influenzae strains are associated with localized infections. Enolase in these bacteria acts as a receptor for plasminogen, promoting tissue invasion. The interaction between NTHi enolase and human plasminogen may be a mechanism used by H. influenzae for host cell adhesion and invasion.