LPS-binding Domain Research Articles

LPS binding caspase activation and recruitment domains (CARDs) are bipartite lipid binding modules.

Caspase-11 is an innate immune pattern recognition receptor (PRR) that detects cytosolic bacterial lipopolysaccharides (LPS) through its caspase activation and recruitment domain (CARD), triggering inflammatory cell death known as pyroptosis. Caspase-11 also detects eukaryotic ( i.e. self) lipids. This observation raises the question of whether common or distinct mechanisms govern the interactions with self and nonself lipids. In this study, using biochemical, computational, and cell-based assays, we report that the caspase-11 CARD functions as a bipartite lipid-binding module. Distinct regions within the CARD bind to phosphate groups and long acyl chains of self and nonself lipids. Self-lipid binding capability is conserved across numerous caspase-11 homologs and orthologs. The symmetry in self and nonself lipid detection mechanisms enabled us to engineer an LPS-binding domain de novo , using an ancestral CARD-like domain present in the fish Amphilophus citrinellus . These findings offer critical insights into the molecular basis of LPS recognition by caspase-11 and highlight the fundamental and likely inseparable relationship between self and nonself discrimination.

Machine learning and genetic algorithm-guided directed evolution for the development of antimicrobial peptides

IntroductionAntimicrobial peptides (AMPs) are valuable alternatives to traditional antibiotics, possess a variety of potent biological activities and exhibit immunomodulatory effects that alleviate difficult-to-treat infections. Clarifying the structure-activity relationships of AMPs can direct the synthesis of desirable peptide therapeutics. ObjectivesIn this study, the lipopolysaccharide-binding domain (LBD) was identified through machine learning-guided directed evolution, which acts as a functional domain of the anti-lipopolysaccharide factor family of AMPs identified from Marsupenaeus japonicus. MethodsLBDA-D was identified as an output of this algorithm, in which the original LBDMj sequence was the input, and the three-dimensional solution structure of LBDB was determined using nuclear magnetic resonance. Furthermore, our study involved a comprehensive series of experiments, including morphological studies and in vitro and in vivo antibacterial tests. ResultsThe NMR solution structure showed that LBDB possesses a circular extended structure with a disulfide crosslink at the terminus and two 310-helices and exhibits a broad antimicrobial spectrum. In addition, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that LBDB induced the formation of a cluster of bacteria wrapped in a flexible coating that ruptured and consequently killed the bacteria. Finally, coinjection of LBDB, Vibrio alginolyticus and Staphylococcus aureus in vivo improved the survival of M. japonicus, demonstrating the promising therapeutic role of LBDB for treating infectious disease. ConclusionsThe findings of this study pave the way for the rational drug design of activity-enhanced peptide antibiotics.

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression

BackgroundInvertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. ResultsThis study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four β sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. ConclusionAntibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

TurboID biotin-tagging mass spectrometry identifies specific caspase-11-associated proteins regulating non-canonical inflammasome activation

Abstract While it has been demonstrated that cytosolic LPS can directly activate caspase11, the cellular processes regulating the non-canonical inflammasome response remain poorly defined. Caspase11 and caspase1 show substantial structural similarity, however, unlike the activation of caspase1 by NLR inflammasomes, there are no sensor or adaptor proteins known to be involved in transmitting cytosolic LPS signal to caspase11. Also, while caspase11 has been shown to associate with LPS, it lacks a characteristic LPS binding domain as observed in many other LPS binding proteins such as MD2 and LBP. Thus, we hypothesized that other effectors may be required to facilitate cytosolic LPS recognition. Moreover, the pathway is likely to be tightly regulated as caspase11 activation leads to highly inflammatory cell death. To identify novel regulators of caspase11, we generated immortalized macrophages expressing a caspase11-TurboID-DHFR chimeric protein. The destabilizing domain was included to avoid cell death induced by caspase11 over-expression. We used a TurboID biotin-tagging MS assay to detect proteins in close proximity to caspase11 pre and post cytosolic LPS introduction. Importantly, the TurboID assay permits recognition of transient interactions, typically missed by traditional IP. To validate relevance of putative hits, we used siRNA knockdown in BMDM. We’ve identified novel regulators specific for cytosolic LPS triggering. Several proteins interact with caspase11 only in the resting state, suggesting negative regulation to prevent pyroptosis. Among the identified regulators are kinases and proteins with pyrin and LRR domains, both common NLR features. This work was supported by the Intramural Research Program of NIAID, NIH.

A crayfish ALF inhibits the proliferation of microbiota by binding to RPS4 and MscL of E. coli

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.

Molecular and Functional Characterization of an Anti-lipopolysaccharide Factor Mm-ALF from Speckled Shrimp Metapenaeus monoceros.

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides of approximately 100 amino acid residues with a broad spectrum of antimicrobial activity. It is an amphipathic peptide with an N-terminal hydrophobic region and a lipopolysaccharide binding domain (LBD). In the present study, we report an isoform of the anti-lipopolysaccharide factor (Mm-ALF) from the speckled shrimp, Metapenaeus monoceros. A 359bp cDNA encoded 119 amino acids, and the sequence showed 99.16% similarity to ALF from the shrimp Fenneropenaeus indicus. The mature peptide of 94 amino acids has a net charge of +8, molecular weight 10.62kDa, and pI 10.11. The mature peptide Mm-ALF was recombinantly expressed in E. coli Rosetta-gami cells, and the peptide was isolated and purified. The rMm-ALF exhibited notable antibacterial activity against Gram-positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative (Escherichia coli, Edwardsiella tarda, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio proteolyticus, Vibrio cholerae and Vibrio fluvialis) bacteria.

Characterization of a new homologous anti-lipopolysaccharide factor SpALF7 in mud crab Scylla paramamosain

Invertebrates, such as mud crab Scylla paramamosain, rely only on innate immunity. Anti-lipopolysaccharide factors (ALFs), are one of the typical antimicrobial peptides (AMPs) commonly found in crustaceans, playing a vital role in the immune defense against pathogens. In this study, a new homologous ALF, named SpALF7, was identified from S. paramamosain. Its full-length cDNA sequence consists of 640 bp, including 121 deduced amino acid residues. The homology between SpALF7 and the other six reported ALFs in S. paramamosain is about 24.69%–43.59%. Absolute quantitative PCR (qPCR) analysis showed that SpALF7 was widely distributed throughout the embryonic and larval developmental stages and the tissues of adult crabs, with the highest transcription level in the seminal vesicles of adult male crabs. Not surprisingly, SpALF7 gene could be significantly upregulated in hepatopancreas under LPS and Vibrio alginolyticus challenge analyzed by relative qPCR. Through the Pichia pastoris eukaryotic expression system, the SpALF7 recombinant protein (rSpALF7) was successfully obtained and a truncated peptide containing the putative LPS binding domain (sSpALF7) was chemically synthesized. Both of them exhibited potent antibacterial activity against several Gram-positive and Gram-negative bacteria. The bactericidal kinetic curves produced by rSpALF7 showed that it could kill more than 90% Pseudomonas stutzeri and Shigella flexneri within 5 min and 120 min at a concentration of 1 μM and 6 μM, respectively. After rSpALF7 treatment, the disruption of cell membrane integrity in S. flexneri and P. stutzeri was observed by scanning electron microscope, which was further confirmed by the flow cytometry assay with SYTO9 and propidium iodide (PI) stainning. In addition, it was worth noting that although rSpALF7 had no bactericidal effect on the crab pathogen V. alginolyticus, it could significantly reduce the bacterial endotoxin levels. Furthermore, the in vivo studies showed that rSpALF7 could obviously improve the survival of crabs infected by V. alginolyticus. Taken together, these results suggested that SpALF7 might play an important role in the innate immunity of S. paramamosain, and has potential applications in aquaculture.

An anti-lipopolysaccharide factor Md-ALF from the Indian flower tail shrimp, Metapenaeus dobsoni: Molecular and phylogenetic characterization

Antimicrobial peptides play a crucial role in the innate immune defence mechanism of various organisms. Anti-lipopolysaccharide factors (ALFs) and their derivatives are a type of cationic AMPs which exhibit broad spectrum antimicrobial activities. This study identified and characterized an anti-lipopolysaccharide factor (Md-ALF) from the haemocytes of Indian flower tail shrimp, Metapenaeus dobsoni. The cDNA of Md-ALF consisted of 294 bp with 98 deduced amino acids. The deduced protein sequence possessed a LPS binding domain and shared highest sequence identity with shrimp ALFs. It showed an isoelectric point of 9.76 and a net positive charge of +7. The secondary structure analysis revealed the presence of three helices, four β turns and three β hairpin like structures. In silico analysis revealed that Md-ALF has anti-bacterial, anti-viral, anti-fungal and anti-cancer properties. Phylogenetic analysis revealed the ancestral relationship of Md-ALF with other shrimp ALFs. Molecular and physico- chemical characterization of Md-ALF showed similarity with reported ALFs from other crustaceans, suggesting its possible role in the innate defence mechanism of M. dobsoni.

Identification of a group D anti-lipopolysaccharide factor (ALF) from kuruma prawn (Marsupenaeus japonicus) with antibacterial activity against Vibrio parahaemolyticus

Anti-lipopolysaccharide factor (ALF), which belongs to the antimicrobial peptide (AMP) family, has become a relatively new weapon to combat severe infections and has been demonstrated to be active against bacteria, fungi and some viruses. In the present study, a new ALF of group D (MjALF-D; GenBank accession No. MN416688) from Marsupenaeus japonicus was detected. MjALF-D encodes a polypeptide with 124 aa, and the peptide contains a 26-residue signal peptide and a lipopolysaccharide-binding domain (LBD). The structure of MjALF-D was found to consist of three α-helices, four β-sheets and random coils. qRT-PCR analysis revealed that MjALF-D expression was primarily observed in the stomach and was universally upregulated in both the gill and stomach after challenge by lipopolysaccharide (LPS) and Vibrio parahaemolyticus. Moreover, rMjALF-D can inhibit the growth of V. parahaemolyticus. rMjALF-D could destroy the bacterial membrane and lead to cytoplasmic leakage investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which may be the mechanism by which rMjALF-D inhibits V. parahaemolyticus. Additionally, rMjALF-D showed distinct binding or antibacterial ability after direct incubation with V. parahaemolyticus or bacterial genomic DNA and a certain effect on the protein expression of it. Together, these results indicated that rMjALF-D possessed the antibacterial activity against V. parahaemolyticus and the potential involvement in the innate immune response of M. japonicus.

Molecular and antimicrobial characterization of a group G anti-lipopolysaccharide factor (ALF) from Penaeus monodon

Anti-lipopolysaccharide factors (ALFs) are important host-defense molecules of crustaceans. They all contain a lipopolysaccharide-binding domain (LBD) and some ALFs exhibit strong antimicrobial activity. In this research, a Group G ALF from Penaeus monodon (ALFPm11) was studied. It is an anionic peptide specifically having a cationic and highly amphipathic LBD, with five positively charged residues separated by aromatic residues. It was abundantly expressed in the hepatopancreas of P. monodon normally but the expression level in other tissues was relatively low or undetectable. However, in the shrimps challenged by Vibrio, expression of ALFPm11 could be detected in all tissues. Chemically synthesized ALFPm11-LBD displayed high inhibitory activity (minimum inhibition concentration≤ 4 μM) against various bacteria, e.g. Exiguobacterium sp. L33, Bacillus sp. T2, and Acinetobacter sp. L32. It also displayed apparent activity in the agar well diffusion assay. Furthermore, it could efficiently induce agglutination of both Gram-positive and Gram-negative bacteria and cause significant membrane permeabilization of the bacteria. As a comparative study, ALFPm11-LBD showed a better or equal antimicrobial function to ALFPm3-LBD which was reported to possess strong antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. Thus, this research found a new effective ALF in P. monodon and demonstrated its antimicrobial mechanism, suggesting its potential applications in the future.

De novo assembly, characterization of tissue-specific transcriptomes and identification of immune related genes from the scallop Argopecten purpuratus

Antimicrobial peptides (AMPs) are an essential component of innate immunity of invertebrates. Anti-lipopolysaccharide factor (ALF), as a main type of AMPs in crustaceans, attends in the disease prevention in general. In this research, a novel Group D ALF was identified and characterized from Penaeus monodon, named PenmonALF8. It was an anionic peptide, with both the full-length peptide and lipopolysaccharide binding domain (LBD) a low isoelectric point. PenmonALF8, composed of a signal peptide of 26 amino acids and a mature peptide of 98 amino acids, probably contained three alpha helixes and four beta sheets. Moreover, PenmonALF8 was detected in all tested tissues of P. monodon, and the expression level in hemocyte and intestine was relatively high. When challenged by Vibrio parahaemolyticus, PenmonALF8 showed 30–100 times higher expression level in all the tissues except in hemocyte and intestine, indicating that PenmonALF8 played a very important role in the immune response of P. monodon. By fusing to a SUMO protein, PenmonALF8 was successfully over-expressed in E. coli and purified by affinity chromatography. Additionally, the reconstituted PenmonALF8 and its LBD region displayed modest antimicrobial activity. This is the first research about the Group D ALF in P. monodon, which provides more information for humoral immunity study of shrimps.

Evidences of Tenacibaculum maritimum evading strategies against Senegalese sole (Solea senegalensis) primary head-kidney leucocytes

Antimicrobial peptides (AMPs) are an essential component of innate immunity of invertebrates. Anti-lipopolysaccharide factor (ALF), as a main type of AMPs in crustaceans, attends in the disease prevention in general. In this research, a novel Group D ALF was identified and characterized from Penaeus monodon, named PenmonALF8. It was an anionic peptide, with both the full-length peptide and lipopolysaccharide binding domain (LBD) a low isoelectric point. PenmonALF8, composed of a signal peptide of 26 amino acids and a mature peptide of 98 amino acids, probably contained three alpha helixes and four beta sheets. Moreover, PenmonALF8 was detected in all tested tissues of P. monodon, and the expression level in hemocyte and intestine was relatively high. When challenged by Vibrio parahaemolyticus, PenmonALF8 showed 30–100 times higher expression level in all the tissues except in hemocyte and intestine, indicating that PenmonALF8 played a very important role in the immune response of P. monodon. By fusing to a SUMO protein, PenmonALF8 was successfully over-expressed in E. coli and purified by affinity chromatography. Additionally, the reconstituted PenmonALF8 and its LBD region displayed modest antimicrobial activity. This is the first research about the Group D ALF in P. monodon, which provides more information for humoral immunity study of shrimps.

The Immune Activity of PT-Peptide Derived from Anti-Lipopolysaccharide Factor of the Swimming Crab Portunus trituberculatus Is Enhanced when Encapsulated in Milk-Derived Extracellular Vesicles.

PT-peptide is derived from the anti-lipopolysaccharide factor of the swimming crab Portunus trituberculatus. The peptide, consisting of 34 amino acids, contains a lipopolysaccharide binding domain. In this study, we investigated the effect of PT-peptide encapsulated in raw milk-derived extracellular vesicles (EVs), designated as EVs-PT peptide, on immune regulation. The results showed that raw milk-derived EVs efficaciously delivered the PT-peptide into monocytes and elevated immune activity, including reactive oxygen species level, superoxide anion production, and phagocytosis. PT-peptide and EVs-PT peptide also elevated the secretion of cytokines, such as interferon-γ, interleukin-6, and tumor necrosis factor-α in human monocytic THP-1 cells. These results suggest that the PT-peptide could be developed as an immune stimulator.

Characterization of a Lymphoid Organ Specific Anti-lipopolysaccharide Factor From Shrimp Reveals Structure-Activity Relationship of the LPS-Binding Domain.

Anti-lipopolysaccharide factor (ALF) is a kind of important antimicrobial peptides with broad-spectrum antimicrobial activities. The LPS-binding domain (LBD) contributes to the major antimicrobial activity of ALF. However, LBDs from different ALFs share low sequence similarity. The general character of LBDs needs to be elucidated to understand the molecular mechanism of their function and facilitate LBD-original drug design. Here we identified a lymphoid organ specifically expressed ALF, designated as FcALF8, from the Chinese shrimp Fenneropenaeus chinensis. The synthetic LBD peptide of FcALF8 (LBD8) showed strong antibacterial activities to the pathogenic Vibrio, such as Vibrio alginolyticus, Vibrio harveyi, and Photobacterium damselae with a MIC value of 0.5–1, 1–2, and 1–2 μM, respectively. FcALF8 knock-down using dsRNA led to significant increase of the viable bacteria in the lymphoid organ and hepatopancreas of shrimp upon V. harveyi infection. On the contrary, the proliferation of V. harveyi in the shrimp lymphoid organ and hepatopancreas significantly decreased after infected by LBD8 pre-incubated V. harveyi. Sequence alignments showed that the LBDs from 39 ALFs shared only two identical cysteine residues. However, 17 of the total 22 LBD residues showed high similarity when the amino acids were classified into hydrophobic and hydrophilic ones. A further activity analysis on modified LBD8 peptides showed that the antibacterial activity of LBD8 was lost after linearization and apparently weakened after changing the amino acid property at certain positions. The data indicated that the disulfide bond and amino acid property contributed to the conservation of the functional domain. To the best of our knowledge, this is the first identified ALFs specifically expressed in the lymphoid organ of shrimp with strong antibacterial activity. The present data will give creative instructions for the design of LBD-originated antimicrobial agents.

Characterization of a group D anti-lipopolysaccharide factor (ALF) involved in anti-Vibrio response in Penaeus monodon

Antimicrobial peptides (AMPs) are an essential component of innate immunity of invertebrates. Anti-lipopolysaccharide factor (ALF), as a main type of AMPs in crustaceans, attends in the disease prevention in general. In this research, a novel Group D ALF was identified and characterized from Penaeus monodon, named PenmonALF8. It was an anionic peptide, with both the full-length peptide and lipopolysaccharide binding domain (LBD) a low isoelectric point. PenmonALF8, composed of a signal peptide of 26 amino acids and a mature peptide of 98 amino acids, probably contained three alpha helixes and four beta sheets. Moreover, PenmonALF8 was detected in all tested tissues of P. monodon, and the expression level in hemocyte and intestine was relatively high. When challenged by Vibrio parahaemolyticus, PenmonALF8 showed 30–100 times higher expression level in all the tissues except in hemocyte and intestine, indicating that PenmonALF8 played a very important role in the immune response of P. monodon. By fusing to a SUMO protein, PenmonALF8 was successfully over-expressed in E. coli and purified by affinity chromatography. Additionally, the reconstituted PenmonALF8 and its LBD region displayed modest antimicrobial activity. This is the first research about the Group D ALF in P. monodon, which provides more information for humoral immunity study of shrimps.

Identification and molecular characterization of a novel anti-lipopolysaccharide factor (ALF) from red swamp crayfish, Procambarus clarkii.

Anti-lipopolysaccharide factors are a group of small proteins with broad spectrum antiviral property and antibacterial activity. Herein, we obtained the genomic sequence of the Procambarus clarkii anti-lipopolysaccharide factor (PcALF) gene by using polymerase chain reaction to investigate its expression pattern in various tissues and in the immune tissues (Hepatopancreas) following exposure to pathogens. The deduced protein of PcALF was conserved; it displayed the signal peptides and putative lipo-polysaccharide binding domain, particularly the two conserved cysteine amino acid residues at both ends of the domain. The recombinant protein of PcALF was successfully expressed in Escherichia coli and rabbit anti-PcALF polyclonal antibodies were prepared. The qRT-PCR analysis showed unequal distribution of PcALF transcript in the examined tissues, however the transcript level was greatest in hepatopancreas. The challenge with peptidoglycan (PGN), lipo-polysaccharide (LPS) and Poly I:C significantly enhanced expression level of PcALF in hepatopancreas when compared with the PBS control. RNA interference of PcALF affected the mRNA expression levels of immune-related genes. Taken together, our data suggested that PcALF is an inducible protein and could play a key biological role in the innate immune defense of P. clarkii.

Molecular identification and function analysis of bactericidal permeability-increasing protein/LPS-binding protein 1 (BPI/LBP1) from turbot (Scophthalmus maximus)

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) play important roles in host antimicrobial defense. In the present study, we identified one isoform of BPI/LBP gene from turbot (Scophthalmus maximus), designated as SmBPI/LBP1. The full-length cDNA sequence of SmBPI/LBP1 was 1826 bp, which encoding one secreted protein with 480 amino acid residues. Structurally, the SmBPI/LBP1 showed high similarity to its homologs from other vertebrates or invertebrates, which all contained a signal peptide, a BPI/LBP/CETP N-terminal with a LPS-binding domain, and a BPI/LBP/CETP C-terminal domain. The deduced amino acid sequences of SmBPI/LBP1 shared significant similarity to BPI/LBP of Seriola lalandi dorsalis (71%) and Paralichthys olivaceus (69%). Phylogentic analysis further supported that SmBPI/LBP1 act as a new member of vertebrate BPI/LBP family. SmBPI/LBP1 was ubiquitously expressed in all tested tissues, with the highest expression level in spleen tissue. The mRNA expression of SmBPI/LBP1 in spleen and kidney were significantly up-regulated after Vibrio vulnificus challenge. Finally, the recombinant SmBPI/LBP1 showed high affinity to lipopolysaccharide, followed by peptidoglycan and lipoteichoic acid, which is the ubiquitous component of Gram-negative or Gram-positive bacteria. These results indicated that SmBPI/LBP1 probably played important roles in immune response against bacteria infection.

Development of novel antimicrobial peptides derived from anti-lipopolysaccharide factor of the swimming crab, Portunus trituberculatus

Anti-lipopolysaccharide factors (ALFs) are a representative host defense protein in crustaceans. In this study, we successfully developed two novel antimicrobial peptides (AMPs), named crab-ALF2A and crab-ALF6A, which contain changes to the amino acid sequences of the lipopolysaccharide binding domain and signal peptide, respectively, of the ALF of the swimming crab Portunus trituberculatus. The crab-ALF2A peptide showed potent antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentration [MEC] 1.51–1.93 μg/mL) and the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli (MEC 1.87–1.98 μg/mL), with maximal bactericidal activity at a peptide concentration of 5 μg/mL. The crab-ALF6A peptide also showed potent antimicrobial activity against B. cereus, S. aureus, and S. iniae (MEC 1.49–2.3 μg/mL) and P. aeruginosa and E. coli (MEC 1.72–1.19 μg/mL) at a peptide concentration of 5 μg/mL. Notably, the crab-ALF2A and crab-ALF6A peptides exhibited strong activity against Candida albicans (MECs of 2.11 and 1.95 μg/mL, respectively). These activities were stable following heat treatment. Moreover, the effect of crab-ALF2A and crab-ALF6A peptide treatment on microbe cell morphology was confirmed by scanning electron microscopy. Membrane disruption and damage, and the leakage of cytoplasmic content were clearly observed. A downsizing peptide approach illustrated that the hexapeptide ALF6A8 (RVLLRL) was the shortest peptide showing significant antimicrobial activity. Our approach allows for the generation of novel antimicrobial peptides in a cost effective manner as potential next-generation antibiotics.

HMGB1: LPS Delivery Vehicle for Caspase-11-Mediated Pyroptosis

Recognition of cytoplasmic lipopolysaccharide (LPS) by caspase-11 leads to pyroptosis and secretion of inflammatory mediators. In this issue of Immunity, Deng etal. (2018) report that high-mobility group box 1 (HMGB1) secreted by hepatocytes delivers extracellular LPS into the cytoplasm and mediates pyroptosis.

Differentially expressed genes in hemocytes of Litopenaeus vannamei challenged with Vibrio parahaemolyticus AHPND (VPAHPND) and VPAHPND toxin

While toxin-harboring Vibrio parahaemolyticus has been previously established as the causative agent of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, information on the mechanistic processes that happen in the host during infection is still lacking. Here, we examined the expression responses of the shrimp hemocyte transcriptome to V. parahaemolyticus AHPND (VPAHPND) by RNA sequencing (RNA-seq). Using libraries (SRA accession number SRP137285) prepared from shrimp hemocytes under experimental conditions, a reference library was de novo assembled for gene expression analysis of VPAHPND-challenged samples at 0, 3/6, and 48 h post infection (hpi). Using the library from 0-hpi as the control, 359 transcripts were found to be differentially expressed in the 3/6-hpi library, while 429 were differentially expressed in the 48-hpi library. The expression patterns reported in the RNA-seq of 9 representative genes such as anti-lipopolysaccharide factor (LvALF), crustin p (CRU), serpin 3 (SER), C-type lectin 3 (CTL), clottable protein 2 (CLO), mitogen-activated protein kinase kinase 4 (MKK4), P38 mitogen-activated protein kinase (P38), protein kinase A regulatory subunit 1 (PKA) and DNAJ homolog subfamily C member 1-like (DNJ) were validated by qRT-PCR. The expression of these genes was also analyzed in shrimp that were injected with the partially purified VPAHPND toxin. A VPAHPND toxin-responsive gene, LvALF was identified, and its function was characterized by RNA interference. LvALF knockdown resulted in significantly rapid increase of shrimp mortality caused by toxin injection. Protein-protein interaction analysis by molecular docking suggested that LvALF possibly neutralizes VPAHPND toxin through its LPS-binding domain. The data generated in this study provide preliminary insights into the differences in the immune response of shrimp to the bacterial and toxic aspect of VPAHPND as a disease.