Shrimp Anti-lipopolysaccharide Factor Research Articles

First characterization of an anti-lipopolysaccharide factor (ALF) from hydrothermal vent shrimp: Insights into the immune function of deep-sea crustacean ALF

Anti-lipopolysaccharide factor (ALF) is a type of antimicrobial peptides (AMPs) with a vital role in antimicrobial defense. Although a large amount of ALFs have been identified from neritic and fresh water crustacean species, no functional investigation of ALFs from deep-sea animals have been documented. In the present study, we characterized the immune function of an ALF molecule (named RspALF1) from the shrimp Rimicaris sp. residing in the deep-sea hydrothermal vent in Desmos, Manus Basin. RspALF1 shares 51.5%–62.4% overall sequence identities with known shrimp ALFs and contains the conserved LPS binding domain (LBD). Both recombinant RspALF1 (rRspALF1) and the LBD-derived peptide (ALF1P1) bound to the cell wall components of Gram-negative and Gram-positive bacteria and killed a wide range of bacteria, especially those from deep-sea hydrothermal field, by damaging bacterial cellular structures. The bactericidal activities of rRspALF1 and ALF1P1 were optimal and stably maintained from 4 °C to 37 °C, which is comparable to the ambient temperature range of the habitat of Rimicaris sp. In addition to bacteria, rRspALF1 and ALF1P1 also exhibited anti-fungal activity. rRspALF1 and ALF1P1 exhibited high killing efficiencies, which, in terms of MIC values, were ranged between 0.25 μM and 4 μM for bacteria and 4 μM–8 μM for fungi. When introduced in vivo, both rRspALF1 and ALF1P1 effectively inhibited bacterial infection in shrimp and reduced the dissemination of bacterial and viral pathogens in fish. Together, these results provide the first insight into the biological property of deep-sea ALF and indicate that RspALF1 very likely plays a significant role in immune defense by functioning as a highly effective antimicrobial with a broad target range.

Recombinant Expression of a Modified Shrimp Anti-Lipopolysaccharide Factor Gene in Pichia pastoris GS115 and Its Characteristic Analysis.

Anti-lipopolysaccharide factors (ALFs) with a LPS-binding domain (LBD) are considered to have broad spectrum antimicrobial activities and certain antiviral properties in crustaceans. FcALF2 was one isoform of ALFs isolated from the Chinese shrimp Fenneropenaeus chinensis. Our previous study showed that a modified LBD domain (named LBDv) of FcALF2 exhibited a highly enhanced antimicrobial activity. In the present study, a modified FcALF2 gene (mFcALF2), in which the LBD was substituted by LBDv, was designed and synthesized. This gene was successfully expressed in yeast Pichia pastoris GS115 eukaryotic expression system, and the characteristics of the recombinant protein mFcALF2 were analyzed. mFcALF2 exhibited apparent antibacterial activities against Gram-negative bacteria, including Escherichia coli, Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus, and Gram-positive bacteria, including Bacillus licheniformis and Staphylococcus epidermidis. In addition, mFcALF2 could reduce the propagation of white spot syndrome virus (WSSV) in vivo by pre-incubation with virus. The present study paves the way for developing antimicrobial drugs in aquaculture.

A potential application of shrimp antilipopolysaccharide factor in disease control in aquaculture

Antilipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) exhibits broad antimicrobial activities against various microbes, including bacteria, fungi and viruses, suggesting its potential use in disease control in shrimp aquaculture. Herein, the recombinant ALFPm3 protein (rALFPm3) exhibited effective antibacterial action against Vibrio parahaemolyticus, the causative agent of the new severe emerging disease, early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) at the minimum inhibitory concentration value of 1.25–5.00 μM. The morphological changes such as bleb formation and cell distortion of V. parahaemoluticus were introduced after rALFPm3 treatment. Furthermore, to control V. parahaemoluticus infection, shrimp were treated with rALFPm3 by immersion and reduction in the cumulative mortality of V. parahaemoluticus-infected Penaeus vannamei was observed. As a potential immunostimulant, the administration of dietary rALFPm3 enhanced the transcript level of five immune-related genes; ALFPm3, haemocyte homoeostasis-associated protein, phagocytosis-activating protein, prophenoloxidase-activating enzyme 1 and cytosolic manganese superoxide dismutase. In addition, dietary rALFPm3 increased the shrimp survival after white spot syndrome virus (WSSV) injection by >35% compared to the 0% survival for the control group. Taken together, these results indicate the potential use of dietary rALFPm3 as immunostimulant in controlling AHPND and WSSV infections in shrimp aquaculture.

Enhanced Control of Bladder-Associated Tumors Using Shrimp Anti-Lipopolysaccharide Factor (SALF) Antimicrobial Peptide as a Cancer Vaccine Adjuvant in Mice.

Shrimp anti-lipopolysaccharide factor (SALF) is an antimicrobial peptide with reported anticancer activities, such as suppression of tumor progression. In this study, we prepared a potential cancer vaccine comprised of SALF in conjunction with the cell lysate of inactivated murine bladder carcinoma cells (MBT-2), and evaluated its efficacy in a mouse tumor model. Our study shows that SALF added to cell culture media inhibits growth progression of MBT-2, and that SALF together with inactivated MBT-2 lysate elevates the level of inflammasome activity, and modulates the levels of IL-1β, MCP-1, IL-6, IL-12, and TNF-α in mouse macrophages. Immunization of 7, 14, and 21 day-old mice with the vaccine prevented growth of MBT-2 cell-mediated tumors. The vaccine was found to enhance expression of T-cell, cytotoxic T cells, and NK cells in the immunized mice groups. Recruitment of macrophages, T-helper cells, and NK cells was enhanced, but levels of VEGF were decreased in immunized mice. This report provides empirical evidence that our SALF as vaccine adjuvant enhances antitumor immunity in mice.

Salf Regulates Mitochondria-and Caspase-Dependent Apoptotic Pathways in Cervical Cancer Cells

Antimicrobial peptides (AMPs) are a new source of potential anticancer drugs. One such AMP is the shrimp antilipopolysaccharide factor (SALF), an AMP isolated from ＂Penaeus monodon＂; the SALF was previously reported to induce tumor cell death. However, the mechanism by which the SALF regulates these pathways remains unclear. In this study, we identified that the SALF modulates mitochondria- and caspase-dependent apoptotic pathways in human cervical cancer cells. The SALF was found to inhibit the growth of four cervical cancer cell lines: HeLa, HeLa 299, C33A, and Ca ski. In addition, a fluorescence-activated cell sorting (FACS) analysis revealed that the SALF induced death and G2/M phase arrest in HeLa and HeLa 299 cells. The SALF itself was found to be localized to the cytosol and nuclei of HeLa cells by confocal imaging. Staining with acridine orange/ ethidium bromide and analysis of DNA fragmentation confirmed that the SALF induced apoptosis in HeLa cells. Moreover, caspases-3 and -9 were found to be involved in SALF-induced apoptosis, through the use of various caspase inhibitors in vitro and immunohistochemistry in vivo. Finally, the SALF was found to induce depolarization of mitochondrial membranes with associated translocation of the AIF to nuclei, and to affect levels of Bcl-2 family proteins in HeLa cells. Taken together, our findings indicate that the SALF induces mitochondria- and caspase-dependent apoptotic pathways in HeLa cells. We suggest that the SALF may be an effective treatment for cervical cancer, either alone or in combination with traditional therapies.

Shrimp anti-lipopolysaccharide factor (SALF), an antimicrobial peptide, inhibits proinflammatory cytokine expressions through the MAPK and NF-κB pathways in LPS-induced HeLa cells

Recently, an antimicrobial peptide (AMP), the shrimp anti-lipopolysaccharide factor (SALF), was shown to act against vaginal pathogens as demonstrated by a minimum inhibitory concentration (MIC) assay and suggested that the SALF might play a protective role in orchestrating various defensive responses. The demonstration of a protective role of the SALF in cervical cancer epithelial cells (HeLa cells) led us to investigate the anti-inflammatory effects of the SALF by determining its inhibitory effects on proinflammatory markers in LPS-stimulated cervical cancer HeLa cells. The SALF was shown to inhibit the production of inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-1α, and monocyte chemoattractant protein (MCP)-1 according to an ELISA analysis. The SALF also suppressed mRNA levels of il-6, il-8, il-1α, and mcp-1 according to an RT-PCR. We also found that the SALF might regulate vaginal epithelial cell immune responses through the MAPK and NF-κB pathways. These findings suggest that the SALF is a potential drug candidate for treating chronic inflammatory diseases, such as urethritis, vaginitis, cervicitis, and pelvic inflammatory diseases.

The antimicrobial peptide, shrimp anti-lipopolysaccharide factor (SALF), inhibits proinflammatory cytokine expressions through the MAPK and NF-κB pathways in Trichomonas vaginalis adherent to HeLa cells

Trichomonas vaginalis is a parasitic protozoan that causes sexually transmitted infections (STIs) worldwide. The infection is dangerous and easily spreads within a community. Also, some cases of drug resistance were reported. Previously, we reported that the shrimp anti-lipopolysaccharide factor (SALF), an antimicrobial peptide of 24 amino acids, modulates inflammatory responses and inhibits T. vaginalis growth. To date, there is no report on the mechanism of SALF's actions in T. vaginalis’ adherence to HeLa cells. In this research using an ELISA, we found that the SALF downregulated the release of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1) secreted by T. vaginalis which was adhering to HeLa cells. We also performed real-time PCR experiments to determine the roles of the SALF in the expressions of several proinflammatory genes. Through a Western blot analysis, we determined that SALF treatment inhibited T. vaginalis-treated HeLa cells through the p38 and NF-κB pathways. Furthermore, we used different inhibitors to confirm the pathway by ELISA and Western blotting. Taken together, it is apparent that the SALF suppresses T. vaginalis-induced secretion of proinflammatory cytokines by HeLa cells by acting through the p38 and NF-κB pathways.

Shrimp anti-lipopolysaccharide factor peptide enhances the antitumor activity of cisplatin in vitro and inhibits HeLa cells growth in nude mice

The antitumor activity of the shrimp anti-lipopolysaccharide factor (SALF), an antimicrobial peptide, was not previously examined. In this study, a synthetic SALF was tested for antitumor activity using HeLa cells as the study model. We show that the SALF inhibited the proliferation of HeLa cells and reduced colony formation in a soft agar assay. An enhanced effect was observed when the SALF and cisplatin were used in combination, which caused significant inhibition of HeLa cells. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the SALF altered the membrane structure similar to what a lytic peptide does. A flow cytometric analysis, qRT-PCR, and Western blotting showed that the SALF induced apoptosis, activated caspases-6, -7, and -9, and downregulated Bcl-2 and nuclear factor (NF)-κB suggesting that the SALF induces apoptosis through the death receptor/NF-κB signaling pathway. An in vivo analysis revealed that the SALF displayed significant tumor suppressive activity in mice with tumor xenografts. Overall, these results indicated that the SALF possesses the potential to be a novel therapeutic agent for treating cervical cancer.

Antimicrobial peptides (AMP) with antiviral activity against fish nodavirus

Nervous necrosis virus (NNV) is classified as betanodavirus of Nodaviridae, and has caused mass mortality of numerous marine fish species at larval stage. Antimicrobial peptides (AMPs) play an important role of innate immunity either against bacterial pathogens or viruses. Up to date, little is known if any AMP could effectively inhibit fish nodaviruses and its mechanism. In this study, the antiviral activities of three antimicrobial peptides (AMPs) against grouper NNV (GNNV) were screened in the fish cell line. Two of the three AMPs, tilapia hepcidin 1-5 (TH 1-5) and cyclic shrimp anti-lipopolysaccharide factor (cSALF), were able to agglutinate purified NNV particles into clump, and the clumps were further confirmed to be viral proteins by TEM and Western blot. The NNV solution, separately pre-mixed with AMP (TH 1-5 or cSALF) or deionized-distilled water for 1 h, was used to infect GF-1 cells, and the levels of capsid protein in the GNNV-AMP-infected cells at 1 h post infection were much lower than that in the GNNV-H 2O-infected cells, indicating that only a small portion of viral particles in the GNNV-AMP mixture could successfully infected the cells. Treatment of cBB cells with TH 1-5 and cSALF did not induce Mx gene expression; however, grouper epinecidin-1 (CP643-1) could induce the expression of Mx in the pre-treated cBB cells. This study revealed three AMPs with anti-NNV activity through two different mechanisms, and shed light on the future application in aquaculture.

Shrimp ( Penaeus monodon) anti-lipopolysaccharide factor reduces the lethality of Pseudomonas aeruginosa sepsis in mice

We investigated the efficacy of amino acids 55–76 of the synthetic shrimp anti-lipopolysaccharide factor peptide (SALF 55–76 cyclic peptide), the C-terminal part of the shrimp anti-lipopolysaccharide factor. This study was conducted to elucidate the effects of the antiseptic action of this peptide. The SALF 55–76 cyclic peptide was tested against bacterial clinical isolates and showed broad-spectrum antimicrobial activity. Transmission electron microscopic (TEM) examination of SALF 55–76 cyclic peptide-treated Pseudomonas aeruginosa showed that severe swelling preceded cell death and breakage of the outer membrane; the intracellular inclusion was found to have effluxed extracellularly. When mice were treated with the SALF 55–76 cyclic peptide before bacterial challenge with P. aeruginosa, the peptide highly protected mice against death by sepsis. The P. aeruginosa recovered from SALF 55–76 cyclic peptide-treated mice after 4 h exhibited reduced bacterial growth similar to that recovered from vancomycin-treated mice. In addition, the syntheses of inflammatory cytokines, such as interleukin (IL)-2, IL-4, IL-10, IL-12, IL-13, interferon-γ, and tumor necrosis factor [TNF]-α, were significantly upregulated 4 h after SALF 55–76 cyclic peptide treatment except for IL-4 in the liver. The expressions of Toll-like receptor 4 (Tlr4), Irf3, myd88, and Tram, were considerably elevated, but only Tlr4 existed in the spleen 4 h after SALF 55–76 cyclic peptide treatment. The prophylactic administration of SALF 55–76 cyclic peptide was begun the TNF-α response in comparison to untreated mice by an ELISA analysis. Due to its multifunctional properties, the SALF 55–76 cyclic peptide may become an important prophylaxis against and therapy for bacterial infectious diseases, as well as for septic shock.