Anti-lipopolysaccharide factors in the American lobster Homarus americanus: molecular characterization and transcriptional response to Vibrio fluvialis challenge

Abstract

Two partial mRNA sequences predicted to encode anti-lipopolysaccharide factors (ALFs) were identified among expressed sequence tags generated from the American lobster Homarus americanus and complete cDNA sequences were obtained from library clones. Comparison of the translated amino acid sequences to those publicly available confirmed similarity to arthropod anti-lipopolysaccharide factors. Both protein sequences, designated ALFHa-1 and ALFHa-2, contained an N-terminal signal peptide and two half-cysteines participating in a disulfide bridge, features conserved in other ALFs. Predicted secondary structures were similar to that described for the ALF from the horseshoe crab Limulus polyphemus. As part of an exploratory study of immunity in H. americanus, lobsters were injected with the bacterium Vibrio fluvialis and gill, hematopoietic, and hepatopancreas tissues were sampled for analysis of gene expression of ALFHa-1 and ALFHa-2 by quantitative PCR. The relative abundance of ALFHa-2 mRNA was not significantly affected by Vibrio injection in any of the three tissues tested. In contrast, ALFHa-1 mRNA levels in gills were increased by the treatment some 17-fold. Our results support a molecularly specific regulation of antimicrobial proteins in response to bacterial infection in H. americanus.

Keywords: DNA sequencing; antimicrobial peptides; crustacean; gill tissue; hematopoetic tissue; hepatopancreas tissue; real-time quantitative PCR.

Figure 1

Nucleotide and amino acid sequence of anti-lipopolysaccharide factor cDNA ALFHa-1 from the lobster Homarus americanus, derived from the library clone identified by expressed sequence tag CN853488. The predicted amino acid sequence encoded by the open reading frame is indicated by single letter abbreviations, and the 25-amino-acid signal peptide predicted by SignalP 3.0 (Bendtsen et al. 2004) is underlined. Conserved cysteine residues are indicated by arrows, defining a predicted loop region containing the highly cationic KPXXRR motif (˜˜˜˜˜). (Accession No. EU625516.).

Figure 2

Nucleotide and amino acid sequence of anti-lipopolysaccharide factor cDNA ALFHa-2 from the lobster Homarus americanus, derived from the library clone identified by expressed sequence tag FC556430. The predicted amino acid sequence encoded by the open reading frame is indicated by single letter abbreviations, and the 26-amino-acid signal peptide predicted by SignalP 3.0 is underlined. Conserved cysteine residues are indicated by arrows, defining a predicted loop region containing the highly cationic KPXXRR motif (˜˜˜˜˜) (Accession No. EU625517).

Figure 3

Hydrophobicity plots (Kyte et al. 1982) of amino acid sequences of anti-lipopolysaccharide factors ALFHa-1 and ALFHa-2 translated from cDNA sequences from Homarus americanus. The hydrophobicity index was calculated over a window of 9 residues by the ExPaSy ProtScale server (Swiss Institute of Bioinformatics). The signal peptide predicted by SignalP 3.0 (Bendtsen et al. 2004) is indicated by a solid bar.

Figure 4

Secondary structure predictions via PSIPRED (McGuffin et al. 2000) for anti-lipopolysaccharide factors ALFHa-1 and ALFHa-2 from Homarus americanus. α-Helical regions (H) are indicated by cylinders, β-strand regions (E) by block arrows, and random coil (C) by straight lines. Confidence of the prediction is indicated by bars associated with the “Conf” line.

Figure 5

Alignment of representative arthropod anti-lipopolysaccharide protein sequences, selected from chelicerate and decapod crustacean examples. Intensity of color reflects percentage identity calculated via MEGA4 (Tamura et al. 2007) and displayed via GeneDoc (Nicholas et al. 1997). ALFs presented include those from Pacific white shrimp Litopenaeus vannamei (DQ208703), pink shrimp Farfantepenaeus paulensis (EF601051), giant tiger prawn Penaeus monodon (ALFPm2 EF523561, ALFPm3 EF523559), American lobster Homarus americanus (ALFHa-1 EU625516, ALFHa-2 EU625517, this study), freshwater shrimp Macrobrachium olfersii (EU289220), kuruma prawn Marsupenaeus japonicus (AB210110), mud crab Scylla paramamosain (EF207786), and mature ALF peptides in the chelicerates Japanese horseshoe crab Tachypleus tridentatus (AF227150) and Atlantic horseshoe crab Limulus polyphemus (P07086). Arrows indicate conserved cysteine residues participating in a disulfide bridge that forms a cationic loop in the protein.

Figure 6

Neighbor-joining tree of representative arthropod anti-lipopolysaccharide protein sequences based on the alignment in Figure 5, as calculated via MEGA4 (Tamura et al. 2007). Branch lengths were computed using the Poisson correction method and are expressed as number of amino acid substitutions per site. Accession numbers associated with the indicated species of origin are listed in the legend of Figure 5.

Figure 7

Relative expression of two anti-lipopolysaccharide factor mRNAs, ALFHa-1 and ALFHa-2, in hepatopancreas (HEP), hematopoietic tissue (HEM), and gill of Homarus americanus 24 hours following injection with tryptone soya broth medium (TSB) (light bars) or Vibrio fluvialis in TSB (filled bars), determined by quantitative PCR. A statistically significant effect of Vibrio injection was observed only for ALFHa-1 in gill tissue (ANOVA with Bonferroni posttests, N=3).