Mantova, Giuseppe (2024) Investigating pyroptotic pathways in a mouse model of meningococcal meningitis: insights into the role of HrpA/HrpB Two-Partner Secretion System. [Tesi di dottorato]

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Abstract

Neisseria meningitidis emerges as a prominent causative agent of septicemia and cerebral inflammation worldwide among humans. Although it establishes in the upper respiratory tract of asymptomatic and healthy carriers, it exhibits the remarkable capacity to evade the host's immune defenses and traverse from the bloodstream to the brain, provoking unregulated localized inflammation. The shift between transitory colonization to a devastating illness appears to be the result of a complex interplay of various factors from both the host organism and the bacterial side. Among these factors, the HrpA/HrpB two partner secretion (TPS) system has emerged as a key player in establish meningococcal meningitis. Recent in vitro study demonstrates the involvement of this secretion system in the formation of biofilm, the escaping from internalization vacuoles within host cells and the attachment to the dynein motor, ultimately culminating in the initiation of pyroptosis process. Pyroptosis, a form of pro-inflammatory cell death, has been posited as a one of principal effectors of inflammation associated with invasive meningococcal infection. Based on in vitro evidence about the role of HrpA/HrpB TPS, it was assessed its involvement in establishing meningococcal meningitis and activating pyroptotic pathway in a suitable animal model. It was employed a model of meningococcal meningitis based on intracisternal infection in adult BALB/c mice, using the reference Serogroup C meningococcal strain 93/4286 (ET-37) and an isogenic mutant, 93/4286ΩhrpB, deficient in the synthesis of the HrpB transporter. The virulence of the hrpB-defective strain was assessed in the mouse model of meningococcal meningitis by analyzing the survival of the animals at different time points. Preliminary data showed high survival rates in animals infected with the hrpBdefective mutant compared to those infected with the wild type. Comparative analysis revealed a stark difference in the mutant strain's ability to replicate within the mouse brain compared to the wild type strain. Histological analysis further reinforced the attenuated virulence trait of 93/4286ΩhrpB mutant strain, with the presence of inflammatory infiltrates only in the brains of mice infected with the wild type strain. Furthermore, the study shed light in the role of the pyroptotic pathway in the damage induced by N. meningitidis in mouse brain. Western blot analysis demonstrated that N. meningitidis activate pyroptotic pathway in the mouse brain: the activation of caspase-11, caspase-1 and gasdermin-D (GASDMD) were markedly reduced in the brain of mice infected with hrpb mutant strain. These findings offer valuable insights about the role of pyroptosis process in the context of invasive meningococcal disease and suggest a promising avenue for potential adjunctive therapies based on pyroptosis inhibitors.

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