Pathogenic Leptospira Secreted Proteases Target the Membrane Attack Complex: A Potential Role for Thermolysin in Complement Inhibition

Abstract

Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira. This disease is common in tropical and subtropical areas, constituting a serious public health problem. Pathogenic Leptospira have the ability to escape the human Complement System, being able to survive when in contact with normal human serum. In a previous study, our group demonstrated that supernatants of pathogenic Leptospira (SPL) inhibit the three activation pathways of the Complement System. This inhibition can be directly correlated with the activity of secreted proteases, which cleave the Complement molecules C3, Factor B (Alternative Pathway), C4 and C2 (Classical and Lectin Pathways). In this work, we analyze the activity of the leptospiral proteases on the components of Terminal Pathway of Complement, called the membrane attack complex (MAC). We observed that proteases present in SPL from different Leptospira strains were able to cleave the purified proteins C5, C6, C7, C8, and C9, while culture supernatant from non-pathogenic Leptospira strains (SNPL) had no significant proteolytic activity on these substrates. The cleavages occurred in a time-dependent and specificity manner. No cleavage was observed when we used whole serum as a source of C5–C9 proteins, probably because of the abundant presence of plasma protease inhibitors such as α2-macroglobulin. Complement protein cleavage by SPL was inhibited by 1,10-phenanthroline, indicating the involvement of metalloproteases. Furthermore, 1,10-phenanthroline- treated normal human serum diminished pathogenic leptospira survival. We also analyzed the proteolytic activity of thermolysin (LIC13322) a metalloprotease expressed exclusively by pathogenic Leptospira strains. Recombinant thermolysin was capable of cleaving the component C6, either purified or as part of the SC5b-9 complex. Furthermore, we found that the MAC proteins C6–C9 interact with thermolysin, indicating that this metalloprotease may have an additional inhibitory effect on these molecules by direct interactions. Finally, a functional assay demonstrated that thermolysin was able to inhibit MAC-dependent erythrocytes lysis. We conclude that proteases secreted exclusively by pathogenic Leptospira strains are capable of degrading several Complement effector molecules, representing potential targets for the development of new therapies and prophylactic approaches in leptospirosis.

Role of Murine Complement Component C5 in Acute in Vivo Infection by Pathogenic Leptospira interrogans

“…Nonpathogenic leptospires are rapidly killed in vitro after CS activation while pathogenic species such as Leptospira interrogans serovar Kennewicki strain Fromm (LPF) are resistant. LPF immune evasion mechanisms include: (i) binding to host CS regulatory proteins Factor H (Meri et al, 2005 ), C4b binding protein (Barbosa et al, 2009 , 2010 ; Breda et al, 2015 ) and vitronectin (da Silva et al, 2015 ); (ii) binding to host proteases such as plasminogen (Vieira et al, 2010 , 2011 ; Castiblanco-Valencia et al, 2016 ) which once converted to its active form, plasmin, may cleave CS proteins; and (iii) secretion of leptospiral proteases that cleave Complement proteins (Fraga et al, 2014 ; Amamura et al, 2017 ).…”

Leptospira interrogans Secreted Proteases Degrade Extracellular Matrix and Plasma Proteins From the Host

“…The secretion of proteases that inactivate essential host proteins is an important tool used by diverse microorganisms during the colonization process. Leptospira is no exception to this phenomenon, since pathogenic strains have been shown to secrete proteases capable of degrading several proteins of the complement cascade, contributing to serum resistance (Fraga et al, 2014 ; Amamura et al, 2017 ). Many bacterial proteolytic enzymes can be considered virulence factors.…”

Participação do componente C3 do sistema complemento murino na produção de anticorpos específicos e fagocitose contra <i>Leptospira interrogans</i>.

“…A importância do SC contra as leptospiras é denotada pela eliminação eficiente de algumas estirpes saprofíticas, enquanto outras estirpes que apresentam fatores de virulência não são eliminadas pelos seus mecanismos (Meri et al, 2005;Barbosa et al, 2009 (Fraga et al, 2014;Amamura et al, 2017). Para demonstrar a importância da molécula TLR no reconhecimento das leptospiras em células murinas, nos estudos realizados por Viriyakosol et al (2006) foram utilizados camundongos C3H/HeJ, pertencentes a uma linhagem que apresenta deficiência especificamente na molécula TLR-4 e, como resultado, foi observado que esses animais foram susceptíveis à infecção letal causada pela estirpe patogênica L.…”