Identification of β tubulin IVb as a pattern recognition receptor with opsonic activity

Highlights

• Zebrafish β-tubulin IVb (TBB4b) gene was inducible by challenge with LPS and LTA.

• Recombinant TBB4b interacted with both LPS and LTA as well as bacteria.

• Recombinant TBB4b stimulated bacterial agglutination in the presence of Ca²⁺.

• Recombinant TBB4b promoted phagocytosis of bacteria by macrophages.

Abstract

Previous studies have shown that tubulins play important role in immune responses of both plants and animals, but no experiments have been performed to study the mode of action of tubulins in immune defense. In addition, there is little convincing experimental evidence of functional commitment for specific tubulin isotypes in animals. In the present, we showed that expression of β-tubulin IVb gene was affected by both LPS and LTA, hinting its involvement in anti-infectious response. We also showed that recombinant zebrafish β-tubulin IVb not only interacted with LPS and LTA as well as Gram-negative and -positive bacteria but also agglutinated both Gram-negative and -positive bacteria in a Ca²⁺-dependent fashion. Interestingly, recombinant β-tubulin IVb could enhance the phagocytosis of bacteria by macrophages. Moreover, we demonstrated that β-tubulin IVb was present extracellularly in the serum of zebrafish and mouse. Collectively, these suggest that β-tubulin IVb may be physiologically involved in the systematic immunity of host via acting as a pattern recognition receptor and an opsonin. This also provides a new angle to understand the roles of β-tubulin IVb.

Introduction

Tubulin, an abundant protein present in all eukaryotic cells, belongs to one of several members of a small family of globular proteins. It was initially identified as the target of colchicine, an alkaloid that is capable of arresting mitosis in mammals (Borisy and Taylor, 1967; Weisenberg et al., 1968). The most common members of the tubulin family are α-tubulin and β-tubulin. Nowadays, in humans 10 α-tubulin and 9 β-tubulin genes have been identified in the tubulin gene family (Findeisen et al., 2014). Both α-tubulin and β-tubulin are the proteins that make up microtubules, major intracellular structures that are involved in the control of fundamental processes such as cell division, polarity of growth, intracellular trafficking and communications (Nogales, 2001; Libusová and Dráber, 2006). It has also become clear that tubulin isotypes are involved in multiple cellular functions without being incorporated into microtubule structures. For example, tubulins have been shown to regulate the permeability of the mitochondrial outer membrane voltage-dependent anion channel (Rostovtseva et al., 2018).

Although regarded as a house-keeping protein, tubulin seems to play some “luxury” roles. In the plant Arabidopsis thaliana, both α-tubulin and β-tubulin have been shown involved in defense responses via acting as lipopolysaccharide (LPS)-interacting proteins (Vilakazi et al., 2017). Similarly, tubulin has also been shown involved in pathogen recognition in the snail Biomphalaria glabrata (Tetreau et al., 2017). In addition, tubulins are apparently affected in both vertebrate (alligator) and invertebrate (shrimp and crab) by challenge with LPS (Merchant et al., 2009) or with bacterium such as Vibrio anguillarum (Zhang et al., 2010) and Spiroplasma eriocheiris (Meng et al., 2014). Recently, we have isolated a protein from zebrafish embryos by lipoteichoic acid (LTA)-conjugated Sepharose CL-4B affinity chromatography, and identified it as β-tubulin IVb (Ni et al., 2019). Overall, these suggest that tubulins play an important role in immune responses. However, detailed study on the mode of action of tubulins in defense remains largely lacking to date. Moreover, there is little convincing experimental evidence of functional commitment for specific tubulin isotypes in animals. The present study is thus undertaken to explore the immunological function of β-tubulin IVb in zebrafish Danio rerio. It demonstrates for the first time that β-tubulin IVb can function as a pattern recognition receptor with opsonic activity.