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

Thais A Amamura,Sílvio A Vasconcellos,Tatiana R Fraga,Lourdes Isaac,Angela S Barbosa

doi:10.3389/fmicb.2017.00958

Thais A Amamura, Sílvio A Vasconcellos + Show 3 more

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https://doi.org/10.3389/fmicb.2017.00958

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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.

Highlights

Leptospirosis is considered the most important zoonosis worldwide, affecting approximately one million patients every year, being fatal in approximately 5–10% of cases (Costa et al, 2015)
Since C6, C7, C8, and C9 share common protein domains (Hadders et al, 2012; Bubeck, 2014), we decided to explore the proteolytic activity of leptospiral culture supernatants on these proteins
The interactions between thermolysin and C6–C9 components inhibits membrane attack complex (MAC)-mediated hemolysis (Figure 11B) in a dose-dependent manner. These results demonstrated that the proteases secreted by the pathogenic Leptospira may inhibit the formation of MAC by cleaving C6, and/or by direct binding to C6-C9

Summary

Introduction

Leptospirosis is considered the most important zoonosis worldwide, affecting approximately one million patients every year, being fatal in approximately 5–10% of cases (Costa et al, 2015) This disease is considered a global public health problem affecting especially developing countries with tropical or subtropical temperatures and deficient sewage conditions. This bacterial infection is caused by spirochetes from the genus Leptospira which includes pathogenic, intermediate and saprophytic (non-pathogenic) species (Ko et al, 2009; Fouts et al, 2016). The host immune responses against Leptospira are mostly dependent on phagocytosis, production of specific antibodies and activation of the Complement System. Several important biological functions are generated from this system contributing for Leptospira killing, such as production of opsonins, which facilitates the phagocytosis, increase in the production of specific antibodies and formation of the membrane attack complex (MAC) (reviewed in Fraga et al, 2011)

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