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Antimicrobial peptides (AMPs), most of which are small proteins, are necessary for innate immunity against pathogens. Anti-lipopolysaccharide factor (ALF) with a conserved lipopolysaccharide binding domain (LBD) can bind to lipopolysaccharide (LPS) and neutralize LPS activity. The antibacterial mechanism of ALF, especially its role in bacteria, needs to be further investigated. In this study, the antibacterial role of an anti-lipopolysaccharide factor (PcALF5) derived from Procambarus clarkii was analyzed. PcALF5 could inhibit the replication of the microbiota in vitro and enhance the bacterial clearance ability in crayfish in vivo. Far-western blot assay results indicated that PcALF5 bound to two proteins of E. coli (approximately 25 kDa and 15 kDa). Mass spectrometry (MS), far-western blot assay, and pull-down results showed that 30S ribosomal protein S4 (RPS4, 25 kD) interacted with PcALF5. Further studies revealed that another E. coli protein binding to PcALF5 could be the large mechanosensitive channel (MscL), which is reported to participate in the transport of peptides and antibiotics. Additional assays showed that PcALF5 inhibited protein synthesis and promoted the transcription of ribosomal component genes in E. coli. Overall, these results indicate that PcALF5 could transfer into E. coli by binding to MscL and inhibit protein synthesis by interacting with RPS4. This study reveals the mechanism underlying ALF involvement in the antibacterial immune response and provides a new reference for the research on antibacterial drugs.

International audience; LBPs (lipopolysaccharide binding proteins) and BPIs (bactericidal permeability increasing proteins) are important proteins involved in defense against bacterial pathogens. We recently discovered a novel biocidal activity of a LBP/BPI from the gastropod Biomphalaria glabrata and demonstrated its role in parental immune protection of eggs, highlighting the importance of LBP/BPIs in invertebrate immunity. Here we characterize four additional LBP/BPI from B. glabrata, presenting conserved sequence architecture and exon-intron structure. Searches of invertebrate genomes revealed that existence of LBP/BPIs is not a conserved feature since they are absent from phyla such as arthropods and platyhelminths. Analyses of LBP/BPI transcripts from selected mollusk species showed recent parallel duplications in some species, including B. glabrata. In this snail species, LBP/BPI members vary in their expression tissue localization as well as their change in expression levels after immune challenges (Gram-negative bacterium; Gram-positive bacterium or yeast). These results, together with the predicted protein features provide evidences of functional specialization of LBP/BPI family members in molluscs.

Xenopus laevis serum lectin XCL1 is a newly identified molecule of the XCGL (or X-lectin) family, a unique group of Ca 2+ -dependent lectins that have a fibrinogen-like domain. The XCL1 protein was purified from lipopolysaccharide (LPS)-stimulated frog sera by sequential affinity chromatography on heparin-acrylic beads and galactose–Sepharose. XCL1 comprises multiple oligomeric proteins consisting of 37-kDa subunit polypeptides, as revealed by sodium dodecyl sulfate–polyacrylamide electrophoresis (SDS–PAGE) and Western blot analyses using the monoclonal antibody (mAb) produced against the recombinant XCL1 polypeptide. In the presence of Ca 2+ , the protein bound to Escherichia coli , Staphylococcus aureus , LPS and galactose and the bound XCL1 was competitively eluted using ribose and xylose, and the elution was as efficient as that using EDTA, whereas elution using hexoses, GalNAc or GlcNAc was less effective. In reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses, XCL1 expression was ubiquitously detected in frog tissues, with relatively high levels in hematopoietic tissues including the spleen, liver and kidney. Intraperitoneal injection of E. coli, S. aureus or 100–300 μg S-type LPS from various bacteria induced several-fold increases in serum XCL1 concentrations on day 3, and the elevated levels retained up to day 12. It also caused a remarkable increase of the splenic XCL1 expression on day 3, followed by a rapid decline to nearly nonstimulated control levels by day 7. The R-type LPS with shortened polysaccharide chains was less effective in inducing the serum XCL1 response, indicating that the sugar chains of LPS were important, if not essential, for the stimulation of XCL1 production. These results suggest that XCL1 is a pathogen recognition molecule involved in antimicrobial innate immunity in Xenopus .

In invertebrates, C-type lectin plays an important role in innate immunity by mediating the recognition of pathogens to host cells and clearing microinvaders. A few C-type lectins have been identified from shrimps, but none of their gene or protein sequences is known to date. In this paper, a C-type lectin (named PmLec) specific for bacterial lipopolysaccharide was purified from the serum of the shrimp Penaeus monodon. The binding of PmLec to lipopolysaccharide was mainly mediated through the O-antigen. PmLec had a strong hemagglutinating and bacterial-agglutinating activity as well as an opsonic effect that enhances hemocyte phagocytosis. The PmLec cDNA sequence was obtained from the cDNA library of P. monodon by polymerase chain reaction with the degenerated primer designed according to the amino-terminal residue sequence of purified PmLec. A 546-bp open reading frame was found to encode a putative protein comprising 182 amino acids and containing a preceding signal peptide of 17 amino acids. A C-type lectin domain existed in PmLec, but no glycosylation site was found. The recombinant PmLec protein expressed in Escherichia coli also showed the same agglutinating activity and opsonic effect as that of the native protein. This is the first report of a lectin cDNA from the shrimp. PmLec functions as a pattern-recognition protein and an opsonin in the shrimp, and it provides a clue to elucidate the role of lectin in the innate immunity of aquatic invertebrates at the molecular level.

A hemagglutinin was isolated from hemocytes of the ascidian, Halocynthia roretzi, by a procedure including extraction and ion-exchange chromatography on CM-cellulose. The molecular weight of the hemagglutinin was estimated to be 120,000 by gel filtration. It was resistant to acid treatment but sensitive to alkali or heat treatment. The hemagglutinating activity was inhibited by heparin, chondroitin sulfate, and lipopolysaccharide (LPS), but not by mono- and disaccharides such as N-acetyl-galactosamine, galactose, and melibiose. The hemagglutinin showed binding ability to heparin and LPS, as demonstrated by heparin-Sepharose chromatography and centrifugation experiments, respectively. It was also found that the hemagglutinin can bind to various bacteria such as Escherichia coli, Bacillus subtilis, Vibrio anguillarum, Pseudomonas perfectomarinus, Achromobacter aquamarinus, and Alteromonas putrefaciens, and can agglutinate all of them.

A lipopolysaccharide (LPS)-binding lectin was recovered from the serum of Limulus polyphemus by ion-exchange chromatography. Electrophoretic analysis of this lectin preparation revealed three poorly migrating bands. When whole serum was incubated with glycolipid obtained from the Rc mutant of Salmonella minnesota prior to electrophoresis, bands corresponding to those seen in the partially purified lectin were missing, suggesting that the recovered material was composed of isolectins. Qualitative precipitin tests revealed no reactivity of this purified lectin with lipid A fractions or with LPS devoid of 2-keto-3-deoxyoctonate (KDO). The agglutination of chicken erythrocytes by this lectin was inhibited by both N-acetyl-neuraminic acid and KDO. Erythrocytes complexed with glycolipid from the Re mutant of S. minnesota were strongly agglutinated by this lectin. We conclude that this LPS-binding lectin is specific for the KDO portion of the molecule and that it is identical to the previously described sialic acid-binding lectin from L. polyphemus. This lectin may play a role in the host defense mechanisms of Limulus.