Role of anti-lipopolysaccharide factor from the black tiger shrimp, Penaeus monodon, in protection from white spot syndrome virus infection

The anti-lipopolysaccharide factor (ALF) from the black tiger shrimp, Penaeus monodon, has been shown previously to exhibit a broad spectrum of activity against various strains of bacteria and fungi. Herein, the recombinant ALFPm3 (rALFPm3) protein was examined for its role in the defence against white spot syndrome virus (WSSV) infection in haematopoietic (Hpt) cell cultures of the freshwater crayfish, Pacifastacus leniusculus, as well as in live P. monodon shrimps. Incubation of Hpt cell cultures with a mixture of WSSV and rALFPm3 resulted in a dose-dependent decrease in VP28 gene expression levels, compared with those incubated with WSSV alone, with an rALFPm3 IC(50) value lower than 2.5 mu M. However, pre-treatment of Hpt cells with 5 mu M rALFPm3 showed no induced protection against subsequent WSSV infection, whereas the synthetic crayfish ALF peptide could protect cells at a higher concentration (10 mu M). The in vivo role of ALFPm3 was examined by injection of P. monodon with WSSV pre-treated with rALFPm3 protein. The results clearly showed that rALFPm3 was able to reduce WSSV propagation and prolong the survival of shrimps

Functional Divergence in Shrimp Anti-Lipopolysaccharide Factors (ALFs): From Recognition of Cell Wall Components to Antimicrobial Activity

Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded beta-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a heterologous expression system. Group D ALFs were found to have impaired LPS-binding activities and only limited antimicrobial activity compared to Group B ALFs. Interestingly, all four ALF groups were shown to be simultaneously expressed in an individual shrimp and to follow different patterns of gene expression in response to a microbial infection. Group B was by far the more expressed of the ALF genes. From our results, nucleotide sequence variations in shrimp ALFs result in functional divergence, with significant differences in LPS-binding and antimicrobial activities. To our knowledge, this is the first functional characterization of the sequence diversity found in the ALF family