Characterization of Caiap and Wdr90 as novel inflammasome components involved in the resistance to Salmonella enterica serovar Typhimurium

Laburpena

Inflammasomes are cytosolic molecular platforms that alert the immune system about the presence of the infection. As multiprotein complexes, they consist of NLRs (NOD-like receptors), adaptor proteins ASC and caspase-1. The assembly of the complexes requires the activation signal and depends on the homotypic interactions. Our laboratory has found that zebrafish guanylate-binding protein 4 (Gbp4), an IFN?-inducible GTPase harbouring a C-terminal CARD domain, is required for the inflammasome-dependent clearance of Salmonella Typhimurium (ST) by neutrophils in vivo. Although several key components of the inflammasome have already been characterized, little is known about other potential components. In this Thesis we describe two new inflammasome components and we developed antibodies against inflammasome components in Alpacas as tools to shed light in to inflammasome assembly and function. On one hand, we report the identification of an evolutionarily conserved protein, that we term Caiap (from CARD- and ANK-containing Inflammasome Adaptor Protein), which has an N-terminal CARD domain and 16 C-terminal ANK domains, and is required for the inflammasome-dependent resistance to Salmonella Typhimurium in zebrafish. Intriguingly, Caiap is highly conserved from cartilaginous fish to marsupials but is absent in placental mammals. Mechanistically, Caiap acts downstream flagellin and interacts with catalytic active Caspa, the functional homolog of mammalian caspase-1, through its ANK domain, while its CARD domain promotes its self-oligomerization. Our results therefore point to ANK domain-containing proteins as key inflammasome adaptors required for the stabilization of active caspase-1 in functionally stable, high molecular weight complexes. On the other hand, we found that zebrafish wdr90 is highly induced by Gbp4 independently of inflammasome activation. This gene encodes a large protein of unknown functions until date that contains several WD40 domains, which are involved in coordinating multi-protein complex assembly. Its overexpression increased caspase-1 activity and the resistance of the larvae to ST infection, acting upstream of Caspa and Asc. In addition, it was able to alter the distribution of NLRC4, but not of NLRP3 or AIM2, when expressed in HEK293T cells. Finally, we have generated WDR90 knockout immortalized bone marrow-derived macrophages and the CRISPR tools to generate KI cell lines to tag endogenous WDR90 with the FLAG epitope to facilitate its detection. In addition, we have produced alpacas antibodies against zebrafish Asc and human WDR90, which will help to shed light into inflammasome assembly, activation and biological functions.