Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon

Abstract

Anti-lipopolysaccharide factors (ALFs), originally characterized from horseshoe crabs, have been recently identified from hemocytes of the black tiger shrimp, Penaeus monodon, by a genomic approach. In order to characterize the properties and biological activities of this immune effector in shrimp, ALFPm3, the most abundant isoform found in P. monodon, was expressed in the yeast Pichia pastoris. Large-scale production in fermentor provided 262 mg/l of recombinant ALFPm3 which was purified to homogeneity by single chromatography step on expanded-bed Streamline SP6XL. The rALFPm3 was further characterized in terms of N-terminal sequencing and mass spectrometry. Anti-microbial assays demonstrated that rALFPm3 has a broad spectrum of anti-fungal properties against filamentous fungi, and anti-bacterial activities against both Gram-positive and Gram-negative bacteria, associated with a bactericidal effect. Interestingly, rALFPm3 is highly efficient against various Vibrio species including strains pathogenic for shrimp. Finally, a synthetic peptide corresponding to a part of the putative LPS-binding site of ALFPm3 was shown to display activities mainly directed against Gram-positive bacteria indicating the involvement of the full molecule to the anti-microbial activity for Gram-negative bacteria.

Introduction

Anti-lipopolysaccharide factors (ALFs) have been first isolated from large granule hemocytes of horseshoe crab and they consist of basic proteins of around 100 amino acids [1], [2]. It was shown that exposure of limulus hemocytes to bacterial endotoxins resulted in rapid degranulation and activation of an intracellular coagulation cascade [3]. Limulus ALF is able to inhibit this cascade by binding and neutralizing LPS and was shown to have an anti-bacterial effect on the growth of the Gram-negative bacteria Salmonella minnesota but not on the Gram-positive strain Staphylococcus aureus [1]. Since, the discovery of ALFs, various lipopolysaccharide-binding molecules and derivatives have attracted great interest in mammalian health as candidate therapeutic and/or prophylactic agents for the management of septic shocks [4]. Indeed, in humans, sepsis caused by Gram-positive and Gram-negative bacteria may cause shock that result in inflammatory responses with tissue injury, multi-organ failure and often in death [5], [6]. It is known that lipid A, the conserved hydrophobic region of lipopolysaccharides (LPS) constitutes the bioactive center and toxic component of LPS. Among the molecules evaluated for properties of lipid A neutralization, Limulus anti-lipopolysaccharide factor (LALF) and derived peptides are intensively studied [7], [8], [9]. Comparatively, few studies have been devoted to the anti-microbial properties and the activity spectrum of this effector. Indeed, increased resistance of bacteria towards antibiotic drugs has stimulated intensive effort for discovery and characterization of anti-microbial peptides as sources or templates for the design of new therapeutic antibiotics.

Recently, cDNA clones homologous to the horseshoe crab ALFs were identified from the hemocytes of the black tiger shrimp, Penaeus monodon, and the atlantic white shrimp, Litopenaeus setiferus, by expressed sequence tag (EST) approach [10], [11], [24], [25], [26]. At least five different ALF sequences (ALFPm1–ALFPm5) have been identified in P. monodon hemocytes and ALFPm3 was shown to be the most abundant isoform found in both normal and Vibrio harveyi-infected shrimp hemocyte cDNA libraries, whereas the others were only evidenced in the infected ones [12]. The deduced amino acid sequence of the ALFPm3 (98 amino acids) showed 57–65% homology with those of Tachypleus ALF and Limulus ALF (Fig. 1). All ALF sequences including ALFPm3 have two conserved cysteine residues and the clustering of positive charges mainly within the disulfide loop (30–51 residues) [2], [13]. This positive cluster is defined as the putative LPS-binding site. Until now in shrimp, only penaeidins, a family of anti-microbial peptides, have been intensively studied in terms of biological properties, anti-microbial activities, gene expression and localization in response to infection [14]. The other anti-microbial effectors recently identified such as crustins or ALFs remain to be characterized for their biological properties and immune functions. As done for a lysozyme sequence identified in Marsupenaeus japonicus [15], the production of recombinant molecule is a good alternative to chromatographic purification approaches, particularly when the effector is weakly expressed and produced in shrimp tissues.

In order to further characterize the penaeid shrimp ALF in terms of biological activity, three-dimensional structure and immune functions, a large-scale production system of the molecule was considered. We report the recombinant expression of ALFPm3 in the methylotrophic yeast Pichia pastoris, its purification to homogeneity and characterization. We have studied the anti-microbial activity of rAFLPm3 and compared to that of a synthetic peptide corresponding to the putative LPS-binding domain of the molecule. Finally, the availability of large amount of protein led us to the preparation of a specific antibody for further detection and localization of this immune effector in shrimp hemocytes.