Final Report Summary - T4P_CROSSING_BARRIER (Strategy of piliated meningococci to cross multicellular defense barriers) Project resultsWe have investigated whether bacterial pathogen, Neisseria meningitidis, could utilise not the transcellular, as currently accepted, but paracellular path to cross tissue defence barriers. We searched for possible mechanisms involved.We utilised two complementary strategies to achieve these goals: the analysis of clinical samples of lethal cases of meningitis and their in vitro reconstitution model. We have elaborated the set of tools to address these tasks. Results of our work on both aspects provided important insights into the possible mechanism of meningitis. Clinical samples of the brain, kidney and heart demonstrated that the dominating form of meningococcus in a human was a cluster, made of piliated bacteria, represented in all the tissues investigated. These clusters, under the conditions of blood flow within vessels were capable of withstanding both shear forces of blood stream and activity in the vascular wall. We have adopted the immunofluorescent approach to analyse the paraffin-embedded sections with confocal microscopy. The immunofluorescence-based 3-D image reconstruction is a non-trivial application in these type of tissue preparation since the process of classical embedding is not suitable for fluorescent labelling; to our knowledge, it was the first time that the antigen-recovery and 3-D reconstruction of the confocal optical sections has been used in the tissue material of lethal cases of meningitis. The results of the clinical material-part of our analysis have not only demonstrated the piliated state of the bacteria around the time of the patient's death. They also proved that optical sectioning and multidimensional reconstruction is a powerful tool to study the disease process in clinical material, complementary to the classical staining of pathologic material.The in vitro part of the project provided significant and multidirectional evidence for the paracellular path taken by meningococci to defend the tissue barriers. We have set the experimental design based on the results of the clinical material-analysis part of the project, mentioned above. Key findings of intense piliation and pili retraction activity in the lethal case's brain and other tissues directed our efforts to improve the existing techniques of meningococcal culture. We achieved dynamics of the pili-mediated clustering matching the size and physical withstanding at the levels deduced from clinical material imaging. Such models of infection provided several findings consistent with the tested hypothesis of paracellular paths of infection. We demonstrated in an electron microscopy approach on in vitro models that the endothelial monolayer does not engulf meningococci frequently enough to ensure the massive transmigration across the cellular bodies (transcellular model, so far the commonly accepted one). The number of engulfing processes was strikingly low. Moreover, the meningococci involved had shown signs of morphological deterioration, speaking against their potential roles in further steps of the barrier penetration. On the other side, the integrity of intercellular connections has been rapidly disrupted throughout the monolayer of endothelial cells, in the form of focal windows formed between the cells (paracellular prototype of the bacterial penetration in vitro). We further demonstrated that actomyosin complexes are involved as systematic response of the endothelium to the infection. The evidence from the Western blotting shows activation of myosin phosphorylation, typical for the activated state of myosin complex. The live imaging showed dramatic retraction of the cell boundaries, both consistent with the active process of cell migration as a key response of host to meningococcal infection. A key involvement of pili and of their contracting activity into the process of penetration of the endothelial monolayers prompted us to systematically study the process at several levels of biological organisation.
