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Experimental Procedures
Author Contributions
Acknowledgments We thank the Mouse Modeling, Integrated Microscopy, and FACS Facilities of IGB-CNR, Naples. Dr. Laura Pisapia is acknowledged for flow cytometry analyses. We are grateful to Prof. Chris Ponting and George A. Calin for insightful scientific discussion. We thank Dr. Monica Autiero for helpful discussion and careful reading of the manuscript. We are indebted to Prof. Malin Parmar and her lab for support with human ESC experiments. Dr Silvia Parisi is acknowledged for providing Lin28 cDNA plasmid and Dr Valeria Tarallo for helpful discussion and experimental support in setting the northern blot conditions. This work is supported by Epigenomics Flagship Project (EPIGEN) MIUR-CNR; TRANSCAN-2 Project BeFIT; the Italian Ministry of Education-University-Research (grant CTN01_00177); AIRC (IG 20736); and Telethon grant GP15209. Emilia Pascale was supported by a PhD fellowship financed by IGB, CNR.
Introduction Adaptor proteins are major contributors to the control of cross-talk between signaling cascades (Flynn, 2001). They contain a variety of protein-binding modules that link protein-binding partners together and facilitate the creation of larger signaling complexes. Non-catalytic region of tyrosine kinase (NCK) and Abelson interactor (ABI) are important adaptor proteins that are involved in many signaling pathways (Cowan and Henkemeyer, 2001). In mammals, NCK protein family has two members, NCK1 and NCK2 (Chen et al., 1998), while ABI protein family has three members, ABI1, ABI2 and ABI3 (Hirao et al., 2006). NCK proteins contain one SRC homology 2 (SH2) domain and three SRC homology 3 (SH3) domains, while ABI proteins contain one SH3 domain and one target SNARE coiled-coil homology (T_SNARE) domain (www.uniprot.org). NCK is known to be adopted by several microbial pathogens to mediate actin polymerization. NCK is essential for the actin-based motility of vaccinia virus (Frischknecht et al., 1999). NCK has also been implicated in the initiation of actin signaling, binding to a tyrosine-phosphorylated 12 amino NS3694 sequences of enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (TIR) in a Y474 phosphorylation-dependent manner (Nieto-Pelegrin et al., 2014). Recently, NCK1 was identified to be a candidate gene for enteric septicemia of catfish (ESC) disease resistance of catfish, and was speculated to play similar roles during ESC and EPEC pathogenicity (Zhou et al., 2017). ABI proteins are key regulators for many molecular signaling pathways, which also play important roles in disease response. The ABI1 was reported as a positional candidate gene for ESC disease resistance of catfish (Zhou et al., 2017), and bacterial cold water disease (BCWD) resistance in rainbow trout (Palti et al., 2015). ABI proteins are components of the ABI/Wave complex which regulates actin polymerization (Hirao et al., 2006). Overexpression of ABI genes negatively regulate cell growth and transformation by specifically targeting the extracellular signal regulated kinases (ERK) pathway (Fan and Goff, 2000). It was postulated that the binding of ABI1 to v-Abl might block activation of critical signal transduction pathways. Signaling molecules activated downstream of v-Abl include RAS (Sawyers et al., 1995), phosphatidylinositol 3-kinase (PI3K) (Varticovski et al., 1991), RAC (Renshaw et al., 1996), c-Jun N-terminal kinase (JNK) (Raitano et al., 1995, Renshaw et al., 1996), and ERKs (Raitano et al., 1995, Renshaw et al., 1996). RAS, RAC, JNK, ERKs signaling molecules are important targets of viral and bacterial virulence factors (Alto and Orth, 2012, Bangi et al., 2012, Krachler et al., 2011). Although adaptor proteins are important regulators of pathogen infection, their involvement in the ESC disease has not been reported. ESC, caused by the bacterium Edwardsiella ictaluri, is one of the most severe bacterial diseases in catfish. Although E. ictaluri pathogenesis is relatively well characterized, the knowledge about E. ictaluri intestinal interaction is limited (Santander et al., 2014). Besides, little is known about the molecular mechanism of pathogenicity of E. ictaluri. Intestinal tract was confirmed as a site of E. ictaluri entry by bacteriological and microscopic methods (Baldwin and Newton, 1993). It has been suggested that E. ictaluri survives in intestinal macrophages (Santander et al., 2013) and causes intestinal barrier disruption and immune suppression (Li et al., 2012). E. ictaluri traverses the epithelial lining with no epithelial cell damage by exploiting normal cellular transport systems (Skirpstunas and Baldwin, 2002). Actin polymerization and receptor-mediated endocytosis are involved in the uptake of E. ictaluri by rat small intestinal epithelial cells (Skirpstunas and Baldwin, 2002). It was corroborated that E. ictaluri lipopolysaccharide oligo-polysaccharide (LPS O-PS) plays a major role during catfish intestinal infection and immune protective stimulation, since they influence the recognition of the LPS by the intestinal mucosal immune system of catfish (Santander et al., 2014). However, the internalization process of E. ictaluri into its natural host, channel catfish (Ictalurus punctatus), together with the involved pathways, genes, and coordinators are still unknown.