Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

Abstract

We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date.

Leptospira Infection Interferes with the Prothrombinase Complex Assembly during Experimental Leptospirosis

“…Accumulating data indicate that compromised hemostasis might be involved in the manifestation of leptospirosis hemorrhagic syndromes. Evidence point toward activation of coagulation, impaired anticoagulation, and activation of fibrinolysis (Sitprija et al, 1980; Edwards et al, 1986; De Francesco Daher et al, 2002; Chierakul et al, 2008; Wagenaar et al, 2010; Vieira et al, 2013). …”

“…We revealed the important role of tissue factor and tissue factor-bearing microvesicles in the induction of the systemic pro-coagulant state during leptospirosis. We have also described the interaction of Leptospira with the fibrinolytic system by capturing human plasminogen on its surface and stimulating an imbalance of the normal fibrinolysis by enhancing the availability of plasminogen activators and plasmin generation (Vieira et al, 2009, 2011, 2013; Vieira and Nascimento, 2016). …”

Novel Leptospira interrogans protein Lsa32 is expressed during infection and binds laminin and plasminogen

“…with PLG generates PLA on the bacterial surface, leading to laminin and fibronectin degradation, a mechanism that could help bacterial penetration (Vieira et al, 2009. Pathogenic Leptospira endowed with this proteolytic system were capable of penetrating endothelial cells more efficiently than were control bacteria (Vieira et al, 2013). Moreover, several leptospiral proteins have been described to have ECM-and PLG-binding capacities Fernandes et al, 2012;Mendes et al, 2011;Oliveira et al, 2011;Souza et al, 2012;Verma et al, 2010;Vieira et al, 2010b).…”

Avaliação e caracterização de candidatos vacinais voltados para o controle da leptospirose.

“…Para isso, tem sido demonstrado que a interação da bactéria com o sistema fibrinolítico do hospedeiro, um sistema enzimático complexo cuja principal função é dissolver o coágulo sanguíneo, contribui significativamente para a virulência de muitos patógenos (LAHTEENMAKI; KUUSELA; KORHONEN, 2001). Nosso grupo demonstrou pela primeira vez, a capacidade das leptospiras de capturar o plasminogênio (PLG) em sua superfície e, na presença de um ativador, ser convertida em plasmina (PLA), o que confere à bactéria um poder proteolítico capaz de degradar laminina e fibronectina, contribuindo na invasão das leptospiras (VIEIRA et al, 2013;VIEIRA et al, 2009). Além disso, foi demonstrado que a atividade de plasmina pode conferir alguma vantagem relacionada à evasão imune, visto que plasmina gerada na superfície das leptospiras foi capaz de interferir na deposição de IgG e C3b na superfície da bactéria, reduzindo o processo de opsonização (VIEIRA et al, 2011).…”