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

Yesenia Osorio-Aguilar,Alejandro Carabarin-Lima,Cesar Raul Gonzalez-Bonilla,Rosa Del Carmen Rocha-Gracia,Patricia Lozano-Zarain,Diana Elizabeth Hernandez-Ceron,Ygnacio Martinez-Laguna,Maria Cristina Gonzalez-Vazquez

doi:10.3390/pathogens10121614

Yesenia Osorio-Aguilar, Alejandro Carabarin-Lima + Show 6 more

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https://doi.org/10.3390/pathogens10121614

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Abstract

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.

Highlights

Haemophilus influenzae is a Gram-negative bacterium that normally colonizes the respiratory tract in humans
When conducting experiments to inhibit the interaction between rNTHiENO and human plasminogen by adding different dilutions of anti-rNTHiENO polyclonal antibodies (1/100–1/40,000), the results showed that the addition of increasing concentrations of anti-rNTHiENO antibody led to a dose-dependent decrease in the binding of rNTHiENO to Plg, evidencing a significant difference with the negative control (Figure 6B)
The results obtained in this work, we propose that enoGLU251, enoTYR253, and enoLYS255 constitute the main plasminogen binding sites; there could be amino acids outside of pbmHiENO participating in the interaction with plasminogen or maintaining a protein folding that is favorable to their interaction with plasminogen, but this hypothesis will have to be confirmed in further studies

Summary

Introduction

Haemophilus influenzae is a Gram-negative bacterium that normally colonizes the respiratory tract in humans. H. influenzae type b (Hib) causes many severe infections, including sepsis, epiglottitis, pneumonia, and meningitis [3]; in contrast, NTHi is a major cause of mucosal infections, such as otitis media, sinusitis, conjunctivitis, and exacerbations of chronic obstructive pulmonary disease [4]. The initial step in the pathogenesis of disease due to NTHi involves the establishment of bacteria on the respiratory mucosa, and several studies suggest that NTHi can cross between cells and invade the subepithelial space [5]. This process can be favored by the degradation of components of the extracellular matrix (ECM)

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