Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity

Abstract

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 β-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.

Figure 1. Amino acid sequence alignments of mature polypeptides of the four ALF groups (Group A, B, C and D) found in penaeids.

Asterisks (*) indicate residues conserved in each specific group of ALF sequences. Residues conserved in all ALF sequences are highlighted in black. The conserved cysteine bridge is indicated. Residues involved in LPS-binding of Group B ALFs are indicated by arrows. α-helices and β-strands identified in the three-dimensional structure of Penmon ALF-B1 (PDB entry 2JOB) are indicated by blue and red boxes, respectively.

Figure 2. Penaeid shrimp ALFs cluster into four groups.

ALF sequences from penaeid shrimps and horseshoe crabs, and scygonadins from crabs (outgroup) were aligned with MAFFT alignment program prior to phylogenetic analysis. The tree was constructed using the Neighbour-Joining method (Pairwise deletion) in MEGA 4. Bootstrap sampling was reiterated 1,000 times. Sequences included in analyses were the following: (i) Shrimp ALFs: black tiger shrimp Penaeus monodon (Penmon ALF-A1 or ALFPm2: ABP73291; Penmon ALF-A1 or ALFPm1: ABP73290; Penmon ALF-B1 or ALFPm3: ABP73289; Penmon ALF-B2 and -B3 ; Penmon ALF-B4: ADC32520; Penmon ALF-C1 or ALFPm6: ADM21460; Penmon ALF-C2 or ALFPm6: AER45468), kuruma prawn (Marjap ALF-A1 or MjALF2: BAH22585; Marjap ALF-C1 or M-ALF: BAE92940), fleshy prawn Fenneropenaeus chinensis (Fenchi ALF-B1 or ALFFc: AAX63831), Indian prawn F. indicus (Fenind ALF-B1: ADE27980; Fenind ALF-B2: ADK94454), pink shrimp Farfantepenaeus paulensis (Farpau ALF-B1 or ALFFpau: ABQ96193), Atlantic white shrimp Litopenaeus setiferus (Litset ALF-D1: BE846661), Pacific white leg shrimp L. vannamei (Litvan ALF-A1 or LvALF1: EW713395; Litvan ALF-A2: FE087264; Litvan ALF-B1 or ALFLv3: ABB22833; Litvan ALF-B2 or ALFLv2: ABB22832; Litvan ALF-C1: FE153599; Litvan ALF-C2: FE176556; Litvan ALF-C3: FE058235; Litvan ALF-C4: FE079082; Litvan ALF-C5: FE088301; Litvan ALF-C6: FE078559; Litvan ALF-C7: FE079755; Litvan ALF-C8: FE105941; Litvan ALF-C9: FE090668; Litvan ALF-C10: FE052210; Litvan ALF-C11: FE088625; Litvan ALF-C12 or: LvALF2: EW713396; Litvan ALF-D1: FE152534; Litvan ALF-D2: FE151634; Litvan ALF-D3: FE110967; Litvan ALF-D4: FE115964), Southern white shrimp L. schmitti (Litsch ALF-B1 or ALFLsch: ABJ90465) and blue shrimp L. stylirostris (Litsty ALF-B1: AGH32549; Litsty ALF-D1: AAY33769); (ii) Horseshoe crab ALFs: Chinese horseshoe crab Tachypleus tridentatus (TACTR_ALF: P07087; TACTR2_ALF: AAK00651), Atlantic horseshoe crab Limulus polyphemus (LIMPO_ALF: P07086) and Southeast Asian horseshoe crab Carcinoscorpius rotundicauda (CARRO_ALF: CK086627); (iii) Scygonadins (outgroup): giant mud crab Scylla serrata (S serrata scygonadin: AAW57403; S serrata scygonadin-2: ABI96918; S serrata SSAP: ABM05493) and green crab Carcinus maenas (C. maenas scygonadin: DY307310).

Figure 3. Purification of recombinant Litsty ALF-D1.

HPLC profile of recombinant Litsty ALF-D1 on a C18 UP5NEC 25QS column. The acetonitrile percentage corresponding to the biphasic gradient is shown as a grey line. Recombinant Litsty ALF-D1 was observed as an absorbance peak (black line) eluted at 42% acetonitrile. The MALDI-TOF-MS spectrum of the collected peak (inset) showed a single mass at m/z = 10816.9.

Figure 4. Differential LPS-binding properties of shrimp ALFs from Groups B and D.

The ability of Litsty ALF-D1 (Group D, open triangles) to bind LPS was compared to that of Penmon ALF-B1 (Group B, open squares) in the Limulus amoebocyte lysate (LAL) assay. Polymixin B (PmB, black circles) was used as a positive control. Absorbance (405 nm) is indicative of LAL assay activation by LPS. LPS neutralization by LPS-binding peptides prevents LAL assay activation. It takes here 20-fold more Litsty ALF-D1 than Penmon ALF-B1 to neutralize an equivalent amount of LPS.

Figure 5. All four ALF groups are simultaneously transcribed in a single shrimp.

The basal gene expression levels of the four ALF Groups identified in L. vannamei shrimp (Litvan ALF-A to -D) were determined by RT-qPCR on circulating hemocytes of individual shrimps (n = 15) according to the 2^−ΔΔCq method . The ribosomal protein L40 (Litvan-rpl40, GenBank: FE077602) was used as a reference gene. Results are expressed as mean values (central black squares) ±SE (boxes) and ±SD (whiskers) of relative expressions on a logarithmic scale. Significant differences between the basal RNA expression levels of the four ALF Groups (Student’s t-test, p<0.05) are indicated by different lowercase letters (a, b, c and d). The use of a same letter indicates the absence of significant difference, while the use of different letters indicates significant difference.

Figure 6. Differential expression of ALF groups in response to an experimental infection.

The transcript abundance of the four ALF Groups (Litvan ALF-A, -B, -C, and -D) was measured by RT-qPCR on circulating hemocytes of shrimp (three groups of three shrimps per condition) injected with the shrimp pathogen Fusarium solani (black boxes), sterile sea water (SSW, grey boxes) or non-injected (white boxes). Analyses were performed 24 and 48 hours after injection. Relative abundances of ALF transcripts are calculated according the 2^−ΔΔCq method , with the L. vannamei ribosomal protein L40 (Litvan-rpl40, GenBank: FE077602) used as reference gene. Results are expressed as mean values (central squares) ±SE (boxes) and ±SD (whiskers) of relative expressions. Significant differences between conditions for each ALF groups (Student’s t-test, p<0.05) are indicated by different lowercase letters (a, b, and c). The use of a same letter indicates the absence of significant difference, while the use of different letters indicates significant difference.