The effects of bacterial inflammagens from Porphyromonas gingivalis on blood clotting

Massimo, Nunes (2020-03)

Thesis (MSc)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Abnormal blood coagulation, systemic inflammation and microbial dysbiosis are shared pathological characteristics of cardiovascular and neurodegenerative diseases. In addition, defective clotting processes and vascular complications are proposed as prominent links between these two disorders as they present simultaneously in respective patients. Another seemingly unrelated pathology that is seen associated with cardiovascular and neurodegenerative disease is periodontitis – a chronic inflammatory condition characterized by oral tissue degradation which is predominantly caused and driven by dysbiotic microbe populations. Out of several species of bacteria that have been identified to contribute to the pathogenesis of periodontal disease, Porphyromonas gingivalis – a gram negative anaerobe –has been deemed as a keystone pathogen capable of causing periodontitis in solitude. Thisbacterium has been implicated in both cardiovascular disease (CVD) and neurodegeneration. In this current study, the aims are to identify an integral virulent product, specifically a protease called gingipain R1 (RgpA), from P. gingivalis in the blood of individuals suffering from neurodegeneration and determine the effects that this protease and lipopolysaccharide (LPS) from the same species impose on normostatic blood coagulation. Research on the effect of these bacterial inflammagens on clot kinetics, rheology and fibrin network formation is and thus emphasizes the novelty of this study. The primary neurodegenerative disease of choice is Parkinson’s disease (PD) as it is also significantly associated with periodontal pathologies and hence likely involves P. gingivalis infection exterior to the oral cavity. We also probed for its protease in the haematological system of patients with Alzheimer’s disease (AD), another neurodegenerative closely correlated to periodontitis. Identifying membrane components of this oral pathogen in the blood of PD patients, and other neurodegenerative diseases such as AD, is of particular importance and may provide further insight into the early pathogenesis and cardiovascular involvement in neurodegenerative disease as a whole. Aims and objectives: This thesis is divided into two main objectives. The first being the identification of a RgpA produced by P. gingivalis in blood samples of PD and AD patients; the second objective is to study the effects of this bacterium’s protease and LPS on coagulation using a healthy blood model and a purified fibrinogen model. To achieve the first objective, the aim is to identify, using polyclonal antibodies against the protease in question, RgpA, in the blood of individuals suffering from PD and AD. To achieve the second objective of this study we characterized the effects of RgpA and LPS on blood coagulation kinetics and terminal fibrin network structure by using a healthy blood model as well as a purified fibrinogen model. The techniques utilized include thromboelastography, rheometry, confocal microscopy and scanning electron microscopy (SEM). Results: Using a polyclonal antibody against RgpA, it is shown that RgpA is present in the haematological system of both a PD population and AD population; this finding was quantitatively significant (p < 0.0001) in both groups as measured via fluorescent intensity. Additionally, the impact of RgpA on the viscoelastic parameters of blood clotting is extensive. RgpA exerts an inhibitory effect on clot kinetics, increases the stiffness of fibrin networks and decreases the total clot load via fibrin(ogen)olytic mechanisms in platelet-poor plasma (PPP) and whole-blood (WB). In purified fibrinogen models, however, this effect was exaggerated as minimal or no fibrin formed after RgpA incubation. This effect was abrogated in the presence of LPS from P. gingivalis whereby a decrease in fibrin load was absent; however, LPS still induced anomalous clot formation characterized by dense matter deposits – a pathological form of fibrin networks. Conclusion: There exists a significant correlation between periodontitis and neurodegeneration, yet findings of P. gingivalis, the chief periodontopathic bacterium, in the blood of PD and AD patients are inexistent. Here, for the first time, it is demonstrated that RgpA is evidently present within the haematological system of individuals suffering from PD and AD. These findings pave way for a new view of neurodegenerative pathology and offers insight into prospective preventative and therapeutic interventions. Furthermore, RgpA may provide useful as a biomarker in these debilitating disorders and therefore requires such attention. In the context of the coagulation system, RgpA is highly active. In contrast to the general prothrombotic state observed in most chronic inflammatory conditions, RgpA seems to shift the normostatic state of coagulation to hypocoagulation. Furthermore, the proteolytic activity of this particular gingipain inhibits the formation of fibrin formed thereby decreasing the clot load, the total amount of fibrin formed. The magnitude of this effect differs in plasma and purified fibrinogen and most likely exits due to the presence of inhibitory and target molecules in plasma such as albumin and other proteins. Another important finding is that the proteolytic capability of RgpA is dampened in the presence of LPS from the same bacterial species which offers insight into realistic physiological functioning whereby LPS and gingipains are co-secreted by P. gingivalis. These data emphasises the degree of influence that bacterial species and inflammagens have on the coagulation system, and highlights their presence in PD and AD.

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