Innate immune function of serine/threonine-protein kinase from Macrobrachium rosenbergii in response to host-pathogen interactions

Highlights

• Host-pathogen interaction of serine/threonine protein kinase-23 in M. rosenbergii identified.

• At first time the innate immune function of MrSTPK-23 demonstrated through infection.

• Cationic antimicrobial peptide, MrSL-19 derived from MrSTPK-23 and addressed bactericidal activity.

• Membrane interaction of MrSL-19 with the bacterial cell was demonstrated.

• Differential toxicity of the peptide evaluated in kidney cells and in human blood erythrocytes.

Abstract

The occurrences of multiple drug-resistant strains have been relentlessly increasing in recent years. The aquaculture industry has encountered major disease outbreaks and crucially affected by this situation. The usage of non-specific chemicals and antibiotics expedites the stimulation of resistant strains. Triggering the natural defense mechanism would provide an effective and safest way of protecting the host system. Hence, we have investigated the innate immune function of serine/threonine-protein kinase (STPK) in Macrobrachium rosenbergii (Mr). The in-silico protein analysis resulted in the identification of cationic antimicrobial peptide, MrSL-19, with interesting properties from STPK of M. rosenbergii. Antimicrobial assay, FACS and SEM analysis demonstrated that the peptide potentially inhibits Staphylococcus aureus by interacting with its membrane. The toxic study on MrSL-19 demonstrated that the peptide is not toxic against HEK293 cells as well as human erythrocytes. This investigation showed the significant innate immune property of an efficient cationic antimicrobial peptide, MrSL-19 of STPK from M. rosenbergii.

Introduction

In aquaculture farming, the giant freshwater prawn Macrobrachium rosenbergii attained an important place due to its economic value. This prawn species is majorly farmed in many tropical and sub-tropical countries and exceptionally in Asia-Pacific countries. In countries like Malaysia, China, India and Thailand, the production of M. rosenbergii has held a significant part in their economy [1,2]. Due to the high commercial values for freshwater prawn, various countries have initiated different initiatives to increase their production. Conversely, recent reports have shown a significant fall in the production of M. rosenbergii compared to the last decades [[3], [4], [5]]. The bacterial and viral infections in the hatcheries remained a major problem and resulted in a massive loss in the production [6,7]. The usage of antibiotics has been a well-known strategy to fight against bacterial infections in aquaculture. However, excessive usage has resulted in the development of multiple drug-resistant strains [8,9]. Also, the lack of an adaptive immune system in the crustaceans doesn't render the organisms to protect from pathogens with the help of traditional vaccination [10,11]. The usage of organophosphate and other chemicals has caused severe environmental hazardous [12].

The alternative path of identifying the host innate immune response to the pathogen invasion is the only possible mechanism to manage deadly infectious diseases [13]. In general, hemolymph of the crustaceans are rich in various immune molecules including proteases and antimicrobial peptides which are principally associated with pathogen defense mechanisms [[14], [15], [16], [17], [18]]. Previous examinations provided validation for host immune response of different genes from hemolymph and hepatopancreas of prawns. Understanding the regulatory proteins and their involvement in protecting the host from pathogen invasion would provide a new therapeutic strategy [[19], [20], [21], [22]]. The crucial functions of immune genes in shrimp were brought under focus due to their inevitable defense mechanism and resulted in their functional characterization [23,24].

The serine/threonine-protein kinase (STPK) family is one of the most abundant enzyme families with significant function, i.e., phosphorylation of serine/threonine residues of the protein. For the initiation of various cellular signaling transduction pathways, phosphorylation of the residues in the protein is essential [25]. In bacteria, these STPKs play a significant role in host-pathogen interaction, including virulence and stress responses [26,27]. As a significant virulence factor, the STPKs secreted by Yersinia bacterial family play a key role in protecting it from the phagocytic action of macrophages [28]. In other bacterial species like Salmonella, Staphylococcus, Streptococcus, Legionella and Mycobacterium tuberculosis, STPKs play a role in the virulence and hot-pathogen interaction. This ability to interact with host cells has made the STPKs as a typical therapeutic target for anti-infective therapies [60]. Based on their catalytic domain with rich serine/threonine and tyrosine residues, STPK is sub grouped into eight families [29]. One of the STPK family members, protein STPK-23, was recently identified in the genome-wide transcriptomic analysis of crustaceans [[30], [31], [32], [33]]. In M. rosenbergii, biological stress caused a modulation in the expression of STPK-23 [34,35]. Though studies confirm that STPKs perform a crucial role as a virulence factor in bacteria, there are no reports on their functions in crustaceans. The variation in expression of STPK-23 during stress showed that it might have a critical function in the pathogen defense mechanism of M. rosenbergii.

Therefore, in this study, we have investigated the role of STPKs in the host response of M. rosenbergii during pathogenic challenges. As WSSV is one of the significant pathogens which causes severe infection in the M. rosenbergii culture, we have studied the host response during WSSV infection [18,36,37]. Followed by that, another significant bacterial pathogen of aquaculture Vibrio harveyi was also challenged against M. rosenbergii and investigated for the regulation of STPK-23. During the bioinformatics analysis of the STPK-23 protein structure, we predicted that a peptide region, MrSL-19 could have antimicrobial activity. Based upon this prediction, we synthesized and evaluated the in vitro activity, mechanism of action against bacterial pathogens, in addition to cytotoxicity.