Gabriel de Barros Corrêa1, Jazmin Huerta-Cantillo2, Fernando Navarro-Garcia2, Angela Silva Barbosa1, Waldir Pereira Elias1, Claudia Trigo Pedroso de Moraes1
The complement system plays a key role in innate immunity through the activation of classical, lectin, and alternative pathways, leading to pathogen elimination. Through evolution, Gram positive and negative bacteria developed mechanisms to evade host immunity. Proteases produced by pathogenic bacteria can promote immune evasion through complement component cleavage. The plasmid-encoded toxin (Pet), member of the serine protease autotransporters of Enterobacteriaceae (SPATE) family, is a virulence factor secreted by E. coli. Although originally described as a cytotoxin, recent studies showed that Pet can also have immunomodulatory effects. In addition, Pet can also cleave complement components in vitro, like EspP, Pic and Sat, other members of the SPATE family. Considering these factors, we further investigated Pet proteolytic activity against complement components and its importance in promoting bacterial resistance to human serum. Proteolytic assays were performed by incubating commercial components C1q-C9 of the complement system with Pet and Pet S260I proteins, obtained from the supernatants of clones HB101(pCEFN1) and HB101(pCEFN2), respectively. Synergic effects of C3 and C3b cleavage were also analyzed in the presence of complement regulators Factor I and Factor H, in the presence or absence of Pet. Cleavage patterns were analyzed by Immunoblotting using specific antibodies for each complement component tested. Our results showed that, excluding C1q and C2, Pet can cleave the molecules C3, C3b, C4, C4b, C5, C6, C7, C8 and C9, in 1 and 5 hours of incubation. No cleavage products were observed in the presence of Pet S260I, confirming the serine protease motif importance in these degradations, as shown by previous published results. The proteolytic activity of Pet on C3a and the synergic effect of C3 and C3b degradation with factor I and cofactor H are currently being evaluated. Comparing our results with previously published works, there are some similarities between C3 and other elastase or elastase-like proteins cleavage pattern. Since Pet was described as an elastase-like, C3 degradation similarity of elastase proteases could be explained by this protein characteristic. We also observed similarities comparing Pet and Sat cleavage patterns, which could be explained by the amino acid sequence identity observed between these SPATEs (53%). Proteomic studies will be necessary to identify complement molecules cleavage sites of these fragments, obtained after bacterial proteases incubation. Therefore, Pet is an important virulence factor that degrades complement components in vitro and could contribute to the immune system evasion by E. coli, inhibiting all the three activation pathways, besides opsonization, anaphylatoxins and MAC formation. More experiments will be conducted by the group to further investigate Pet contribution to immune evasion.
Agradecimentos: We would like to thank FAPESP (FAPESP 2017/14821-7) and CAPES for the financial support.