The Enolase of the Haemophilus influenzae Mediates Binding to Collagens: An Extracellular Matrix Component.

Enolase proteins play a significant role as moonlighting proteins. In their role as surface-associated enolase, they have multiple functions as they interact with extracellular matrix proteins. Type I and III collagens are the major constituents of this extracellular matrix, and collagen is one of the targets of interaction with the enolase of many pathogens, thereby helping the colonization process and promoting the subsequent invasion of the host. This work aimed to determine the participation of non-typeable H. influenzae enolase as a collagen-binding protein. In this study, through the use of in vitro tests it was demonstrated that recombinant enolase of non-typeable H. influenzae (rNTHiENO) strongly binds to type I collagen. Using molecular docking, the residues that could take part in the interaction of non-typeable H. influenzae enolase-type I collagen (NTHiENO-Cln I) and non-typeable H. influenzae enolase-type III collagen (NTHiENO-Cln III) were identified. However, in vitro assays show that NTHiENO has a better affinity to interact with Cln I, concerning type Cln III. The interaction of NTHiENO with collagen could play a significant role in the colonization process; this would allow H. influenzae to increase its virulence factors and strengthen its pathogenesis.

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 (251EFYNKENGMYE262) 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 enoTYR253-plgGLU1 (K2), enoTYR253-plgGLY310 (K3), and enoLYS255-plgARG471/enoGLU251-plgLYS468 (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.

Cloning and Characterization of Immunological Properties of Haemophilus influenzae Enolase.

Haemophilus influenzae is a common organism of the human upper respiratory tract; this bacterium is responsible of a wide spectrum for respiratory infections and can generate invasive diseases such as meningitis and septicemia. These infections are associated with H. influenzae encapsulated serotype b. However, the incidence of invasive disease caused by nontypeable H. influenzae (NTHi) has increased in the post-H. influenzae serotype b (Hib) vaccine era. Currently, an effective vaccine against NTHi is not available; due to this, it is important to find an antigen capable to confer protection against NTHi infection. In this study, 10 linear B cell epitopes and 13 CTL epitopes and a putative plasminogen-binding motif (252FYNKENGMY260) and the presence of enolase on the surface of different strains of H. influenzae were identified in the enolase sequence of H. influenzae. Both in silico and experimental results showed that recombinant enolase from H. influenzae is immunogenic that could induce a humoral immune response; this was observed mediating the generation of specific polyclonal antibodies anti-rNTHiENO that recognize typeable and nontypeable H. influenzae strains. The immunogenic properties and the superficial localization of enolase in H. influenzae, important characteristics to be considered as a new candidate for the development of a vaccine, were demonstrated.