immunology of infectious and parasitic diseases - XXXVII Congress ...

Leptospira evades the human complement attack by the secretion of
metalloproteases that directly cleave complement proteins
TATIANA RODRIGUES FRAGA 1 , DANIELLA DOS SANTOS COURROL 1 ,
SÍLVIO ARRUDA VASCONCELLOS 2 , ANGELA SILVA BARBOSA 3 , LOURDES
ISAAC 1
1 Department of Immunology, University of São Paulo; 2 Faculty of Veterinary
Medicine, University of São Paulo; 3 Bacteriology Department, Butantan
Institute, Brazil
Introduction: Leptospirosis is a neglected infectious disease of public health
importance. Complement represents a central immune mechanism in blood
circulation, but the high ability of Leptospira to spread indicates a low efficacy of
complement against this microorganism. Pathogenic Leptospira have
successfully developed strategies to evade the complement system. They
acquire the regulators Factor H (FH) and C4BP, which control complement
activation in pathogen´s surface. However, complement evasion may also occur
in the fluid phase, by the secretion of bacterial proteases. The aim of this work
was to evaluate the Leptospira ability to secrete proteases that directly cleave
complement molecules and also to identify the proteins responsible for the
cleavages.
Methods and Results: The proteolytic cleavages of complement molecules
were analyzed by Western blot. Seven different strains of pathogenic Leptospira
were able to secrete proteases that cleaved C3, C3b, iC3b, C2, C4b, C5 and
FB, but not C1q, C4 and IgG. The cleavages of C3, C4, FB and C2 were also
observed when normal human sera were used as a complement source. In
contrast, non-pathogenic Leptospira did not present significant proteolytic
activity. The protease activity was inhibited by ortho-phenanthroline, a
metalloprotease inhibitor. We cloned, expressed and purified the leptospiral
metalloprotease thermolysin NprT and showed that it was able to cleave C3 and
that its activity was inhibited by ortho-phenantroline. NprT specifically cleaved
C3 in the alfa chain, generating a C3b-like molecule, which was susceptible to
the cleavage by host factor FI. NprT also cleaved C3b, but only in the presence
of FH, indicating a co-factor activity of this molecule. We also performed a
purification of the native proteases from the pathogenic leptospiral supernatant
by gel filtration. A fraction of 15 to 30 kDa was able to cleave the complement
protein C3b. Finally, we showed the alternative pathway activity of normal
human serum was reduced by the treatment with pathogenic leptospiral
proteases.
Conclusions: We describe a novel immune evasion mechanism in Leptospira:
the secretion of proteases that cleave complement proteins. We also identified
the thermolysin NprT, a metalloprotease that cleaves the complement molecule
C3. The leptospiral proteases can be considered as virulence factors, since