Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.

Proteomic and Functional Analyses of Outer Membrane Vesicles Secreted by Vibrio splendidus

Vibrio splendidus is an important opportunistic pathogen ubiquitously present in the marine environment,exhibiting virulence to a variety of cultured animals.The extracellular products secreted by V.splendidus are crucial to bacterial survival and virulence.In this study,the secretion of outer membrane vesicles(OMVs)by V.splendidus was determined,purified,and morphologically characterized.The protein composition of OMVs was analyzed by proteomic analysis.The results showed that approximately 120 proteins were contained in these OMVs,including outer membrane proteins,flagellins,ABC transporters,protease,and iron regulation proteins,etc.,which were involved in bacterial motility,formation of biofilms and the cell membrane components,and cellular localization based on their structural molecule activity,passive transmembrane transporter activity,channel activity,neurotransmitter receptor activity,extracellular ligand-gated ion channel activity,glutamate receptor activity,ligand-gated ion channel activity,and transmembrane signaling receptor activity.To explore the biological functions of OMVs in V.splendidus,the effects of OMVs on the bacterial adaption to iron limitation,antibiotic,and the coelomic fluid of the Apostichopus japonicus were confirmed.This study is the first time to show that V.splendidus secretes OMVs,and OMVs carry functional proteins that enhance bacterial survival under various stresses.

Newly Detected Transmission of bla_(KPC-2) by Outer Membrane Vesicles in Klebsiella Pneumoniae

Objective The prevalence of carbapenem-resistant Klebsiella pneumoniae(CR-KP)is a global public health problem.It is mainly caused by the plasmid-carried carbapenemase gene.Outer membrane vesicles(OMVs)contain toxins and other factors involved in various biological processes,includingβ-lactamase and antibiotic-resistance genes.This study aimed to reveal the transmission mechanism of OMV-mediated drug resistance of Klebsiella(K.)pneumoniae.Methods We selected CR-KP producing K.pneumoniae carbapenemase-2(KPC-2)to study whether they can transfer resistance genes through OMVs.The OMVs of CR-KP were obtained by ultracentrifugation,and incubated with carbapenem-sensitive K.pneumoniae for 4 h.Finally,the carbapenem-sensitive K.pneumoniae was tested for the presence of bla_(KPC-2)resistance gene and its sensitivity to carbapenem antibiotics.Results The existence of OMVs was observed by the electron microscopy.The extracted OMVs had bla_(KPC-2)resistance gene.After incubation with OMVs,bla_(KPC-2)resistance gene was detected in sensitive K.pneumoniae,and it became resistant to imipenem and meropenem.Conclusion This study demonstrated that OMVs isolated from KPC-2-producing CR-KP could deliver bla_(KPC-2)to sensitive K.pneumoniae,allowing the bacteria to produce carbapenemase,which may provide a novel target for innovative therapies in combination with conventional antibiotics for treating carbapenem-resistant Enterobacteriaceae.

Bacterial outer membrane vesicle-based cancer nanovaccines

Tumor vaccines,a type of personalized tumor immunotherapy,have developed rapidly in recent decades.These vaccines evoke tumor antigen-specific T cells to achieve immune recognition and killing of tumor cells.Because the immunogenicity of tumor antigens alone is insufficient,immune adjuvants and nanocarriers are often required to enhance anti-tumor immune responses.At present,vaccine carrier development often integrates nanocarriers and immune adjuvants.Among them,outer membrane vesicles(OMVs)are receiving increasing attention as a delivery platform for tumor vaccines.OMVs are natural nanovesicles derived from Gramnegative bacteria,which have adjuvant function because they contain pathogen associated molecular patterns.Importantly,OMVs can be functionally modified by genetic engineering of bacteria,thus laying a foundation for applications as a delivery platform for tumor nanovaccines.This review summarizes 5 aspects of recent progress in,and future development of,OMV-based tumor nanovaccines:strain selection,heterogeneity,tumor antigen loading,immunogenicity and safety,and mass production of OMVs.

Dynamic shielding of bacterial outer membrane vesicles for safe and efficient chemo-immunotherapy against tumors

Bacterial outer membrane vesicles(OMVs)are potent immunostimulants of regulating the tumor microenvironment(TME)for immunotherapy,and can be used to deliver drugs.However,the severe systemic inflammatory response triggered by OMVs upon intravenous(i.v.)injection has limited their application.Here,we developed a safe and effective strategy by conjugating doxorubicin-loaded serum albumin(SA-DOX,AD)onto the surface of OMVs using a matrix metalloproteinase(MMP)-cleavable peptide linker(cL).This approach enabled the dynamic shielding of OMVs to reduce the systemic side effects while simultaneously enhancing the anti-tumor effects through chemo-immunotherapy.Specifically,the resulting OMV-cL-AD formulation exhibited significantly enhanced accumulation at the tumor site after i.v.administration,facilitated by the SA decoration on the OMVs surface.Subsequently,the shield on the OMV-cL-AD was cleaved by the over-expressed MMP in the TME,leading to the release of both OMVs and AD.This process provided OMV-induced immunotherapy and DOX-induced chemotherapy,resulting in synergistic tumor inhibition.In conclusion,our work demonstrated the potential of OMV-cL-AD as an effective immunochemotherapy strategy that can prolong the survival time of mice without inducing side effects.

Bacterial outer membrane vesicles-based therapeutic platform eradicates triple-negative breast tumor by combinational photodynamic/chemo-/immunotherapy

Bacterial outer membrane vesicles(OMVs)are potent immuno-stimulating agents and have the potentials to be bioengineered as platforms for antitumor nanomedicine.In this study,OMVs are demonstrated as promising antitumor therapeutics.OMVs can lead to beneficial M2-to-M1 polarization of macrophages and induce pyroptosis to enhance antitumor immunity,but the therapeutic window of OMVs is narrow for its toxicity.We propose a bioengineering strategy to enhance the tumor-targeting ability of OMVs by macrophage-mediated delivery and improve the antitumor efficacy by co-loading of photosensitizer chlorin e6(Ce6)and chemotherapeutic drug doxorubicin(DOX)into OMVs as a therapeutic platform.We demonstrate that systemic injection of the DOX/Ce6-OMVs@M therapeutic platform,providing combinational photodynamic/chemo-/immunotherapy,eradicates triple-negative breast tumors in mice without side effects.Importantly,this strategy also effectively prevents tumor metastasis to the lung.This OMVs-based strategy with bioengineering may serve as a powerful therapeutic platform for a synergic antitumor therapy.

An AIE-active probe for efficient detection and high-throughput identification of outer membrane vesicles

Rapid detection and quantification of outer membrane vesicle(OMV)are of both scientific value and clinical implications.However,limited tools are available for investigations of OMVs.Herein,we report a novel fluorescent probe with aggregation-induced emission(AIE)characteristics,namely,OEO-TPE-MEM(OTM),for OMV detection.OTM emits faintly in an aqueous medium,but its fluorescence could be effectively turned on upon interacting with bacteria bodies and OMVs produced by Gram-negative bacteria.Notably,OTM could provide quantitative information on bacterial membrane remodeling and OMV secretion and be applied to high-throughput screening of OMV-inducing agents.This study presents a powerful AIE probe for imaging and quantitative analysis of bacteria envelop and derived OMVs,which might be applied for evaluating research and clinical antimicrobial materials in future studies.

Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera

Brush border membrane vesicles （BBMV） isolated from insect midguts have been widely used to study CrylA binding proteins. Sample preparation is important in two- dimensional electrophoresis （2-DE）, so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）. All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2- DE analysis.

Personalized cancer vaccines from bacteria-derived outer membrane vesicles with antibody-mediated persistent uptake by dendritic cells

Nanocarriers with intrinsic immune adjuvant properties can activate the innate immune system while delivering tumor antigen,thus efficiently facilitating antitumor adaptive immunity.Bacteria-derived outer membrane vesicles(OMVs)are an excellent candidate due to their abundance of pathogen associated molecular patterns.However,during the uptake of OMVs by dendritic cells(DCs),the interaction between lipopolysaccharide and toll-like receptor 4 induces rapid DC maturation and uptake blockage,a phenomenon we refer to as“maturation-induced uptake obstruction"(MUO).Herein we decorated OMV with the DC-targeting aDEC205 antibody(OMV-DEC),which endowed the nanovaccine with an uptake mechanism termed as 4<not restricted to maturation via antibody modifying”(Normandy),thereby overcoming the MUO phenomenon.We also proved the applicability of this nanovaccine in identifying the human tumor neoantigens through rapid antigen display.In summary,this engineered OMV represents a powerful nanocarrier for personalized cancer vaccines,and this antibody modification strategy provides a reference to remodel the DC uptake pattern in nanocarrier design.

Role of Outer Membrane Vesicles in Bacterial Physiology and Host Cell Interactions

Outer membrane vesicles(OMVs)are spherical particles shed from the outer membrane of Gram-negative bacteria,which contain the typical components present in the outer membrane,although enrichment of specific molecules may occur,and furthermore a variety of periplasmic components and occasionally some inner membrane or cytoplasmic fractions.Although the detailed mechanisms of OMV biogenesis are not fully illuminated yet,several models have been proposed that demonstrate OMV biogenesis is an orchestrated well-regulated process.OMV secretion offers a way for both intra-and inter-species bacterial communication and for interaction or modulation of the bacterial environment.Therefore,OMVs have proven to be functionally versatile and important for bacterial physiology and survival of the host environment.In the host,OMVs are internalized via host cell endocytosis pathways,allowing them to subsequently trigger a variety of cellular responses.In this review,we discuss the recent advances in establishing the mechanisms involved in OMV biogenesis and the impact of OMVs on bacterial physiology and intracellular modulation of the host.

Transformation of Structure and Properties of Vesicles Induced by Transition Metal Ions

This paper proved that octodecyl propylenediamine could form vesicles in pure water and aqueous solution of CuCl2 or Cu（NO3）2. The structure and morphology of vesicles were different when the copper （Ⅱ） salt was added to the solution. The results showed that both the counterions and the ligands had strong influence on the configuration of coordinated structures and packing model in bilayer membrane of vesicles.

Bacterial extracellular vesicle applications in cancer immunotherapy

Cancer therapy is undergoing a paradigm shift toward immunotherapy focusing on various approaches to activate the host immune system.As research to identify appropriate immune cells and activate anti-tumor immunity continues to expand,scientists are looking at microbial sources given their inherent ability to elicit an immune response.Bacterial extracellular vesicles(BEVs)are actively studied to control systemic humoral and cellular immune responses instead of using whole microorganisms or other types of extracellular vesicles(EVs).BEVs also provide the opportunity as versatile drug delivery carriers.Unlike mammalian EVs,BEVs have already made it to the clinic with the meningococcal vaccine(Bexsero®).However,there are still many unanswered questions in the use of BEVs,especially for chronic systemically administered immunotherapies.In this review,we address the opportunities and challenges in the use of BEVs for cancer immunotherapy and provide an outlook towards development of BEV products that can ultimately translate to the clinic.

Repair of traumatic plasmalemmal damage to neurons and other eukaryotic cells

The repair（sealing） of plasmalemmal damage,consisting of small holes to complete transections,is critical for cell survival,especially for neurons that rarely regenerate cell bodies.We first describe and evaluate different measures of cell sealing.Some measures,including morphological/ultra-structural observations,membrane potential,and input resistance,provide very ambiguous assessments of plasmalemmal sealing.In contrast,measures of ionic current flow and dye barriers can,if appropriately used,provide more accurate assessments.We describe the effects of various substances（calcium,calpains,cytoskeletal proteins,ESCRT proteins,m UNC-13,NSF,PEG） and biochemical pathways（PKA,PKC,PLC,Epac,cytosolic oxidation） on plasmalemmal sealing probability,and suggest that substances,pathways,and cellular events associated with plasmalemmal sealing have undergone a very conservative evolution.During sealing,calcium ion influx mobilizes vesicles and other membranous structures（lysosomes,mitochondria,etc.） in a continuous fashion to form a vesicular plug that gradually restricts diffusion of increasingly smaller molecules and ions over a period of seconds to minutes.Furthermore,we find no direct evidence that sealing occurs through the collapse and fusion of severed plasmalemmal leaflets,or in a single step involving the fusion of one large wound vesicle with the nearby,undamaged plasmalemma.We describe how increases in perikaryal calcium levels following axonal transection account for observations that cell body survival decreases the closer an axon is transected to the perikaryon.Finally,we speculate on relationships between plasmalemmal sealing,Wallerian degeneration,and the ability of polyethylene glycol（PEG） to seal cell membranes and rejoin severed axonal ends – an important consideration for the future treatment of trauma to peripheral nerves.A better knowledge of biochemical pathways and cytoplasmic structures involved in plasmalemmal sealing might provide insights to develop treatments for traumatic nerve injuries,stroke,muscular dystrophy,and other pathologies.

SNARE complex in axonal guidance and neuroregeneration

Through complex mechanisms that guide axons to the appropriate routes towards their targets, axonal growth and guidance lead to neuronal system formation. These mechanisms establish the synaptic circuitry necessary for the optimal performance of the nervous system in all organisms. Damage to these networks can be repaired by neuroregenerative processes which in turn can re-establish synapses between injured axons and postsynaptic terminals. Both axonal growth and guidance and the neuroregenerative response rely on correct axonal growth and growth cone responses to guidance cues as well as correct synapses with appropriate targets. With this in mind, parallels can be drawn between axonal regeneration and processes occurring during embryonic nervous system development. However, when studying parallels between axonal development and regeneration many questions still arise; mainly, how do axons grow and synapse with their targets and how do they repair their membranes, grow and orchestrate regenerative responses after injury. Major players in the cellular and molecular processes that lead to growth cone development and movement during embryonic development are the Soluble N-ethylamaleimide Sensitive Factor （NSF） Attachment Protein Receptor （SNARE） proteins, which have been shown to be involved in axonal growth and guidance. Their involvement in axonal growth, guidance and neuroregeneration is of foremost importance, due to their roles in vesicle and membrane trafficking events. Here, we review the recent literature on the involvement of SNARE proteins in axonal growth and guidance during embryonic development and neuroregeneration.

The emerging tumor microbe microenvironment: From delineation to multidisciplinary approach-based interventions

Intratumoral microbiota has become research hotspots,and emerges as a non-negligent new component of tumor microenvironments(TME),due to its powerful infuence on tumor initiation,metas-tasis,immunosurveillance and prognosis despite in low-biomass.The accumulations of microbes,and their related components and metabolites within tumor tissues,endow TME with additional pluralistic features which are distinct from the conventional one.Therefore,it's definitely necessary to comprehen-sively delineate the sophisticated landscapes of tumor microbe microenvironment,as well as their func-tions and related underlying mechanisms.Herein,in this review,we focused on the fields of tumor microbe microenvironment,including the heterogeneity of intratumor microbiota in different types of tu-mors,the controversial roles of intratumoral microbiota,the basic features of tumor microbe microenvi-ronment(i.e.,pathogen-associated molecular patterns(PAMPs),typical microbial metabolites,autophagy,infammation,multi-faceted immunomodulation and chemoresistance),as well as the multi-disciplinary approach-based intervention of tumor microbiome for cancer therapy by applying wild-type or engineered live microbes,microbiota metabolites,antibiotics,synthetic biology and rationally de-signed biomaterials.We hope our work will provide valuable insight to deeply understand the interplay of cancer-immune-microbial,and facilitate the development of microbes-based tumor-specific treatments.

Extracellular vesicles:Emerging tools as therapeutic agent carriers

Extracellular vesicles(EVs)are secreted by both eukaryotes and prokaryotes,and are present in all biological fluids of vertebrates,where they transfer DNA,RNA,proteins,lipids,and metabolites from donor to recipient cells in cell-to-cell communication.Some EV components can also indicate the type and biological status of their parent cells and serve as diagnostic targets for liquid biopsy.EVs can also natively carry or be modified to contain therapeutic agents(e.g.,nucleic acids,proteins,polysaccharides,and small molecules)by physical,chemical,or bioengineering strategies.Due to their excellent biocompatibility and stability,EVs are ideal nanocarriers for bioactive ingredients to induce signal transduction,immunoregulation,or other therapeutic effects,which can be targeted to specific cell types.Herein,we review EV classification,intercellular communication,isolation,and characterization strategies as they apply to EV therapeutics.This review focuses on recent advances in EV applications as therapeutic carriers from in vitro research towards in vivo animal models and early clinical applications,using representative examples in the fields of cancer chemotherapeutic drug,cancer vaccine,infectious disease vaccines,regenerative medicine and gene therapy.Finally,we discuss current challenges for EV therapeutics and their future development.

Effect of oxide nanoparticles on the morphology and fluidity of phospholipid membranes and the role of hydrogen bonds

Engineered oxide nanoparticles（NPs） are widely applied in insulators,catalyzers,paints,cosmetic products,textiles and semiconductors.Their attachment on cell membrane may lead to cytotoxicity.The effects of Al_2O_3,Fe_2O_3,SiO_2,TiO_2and ZnO NPs on membrane integrity and fluidity were studied using giant or small unilamellar vesicles in this study.Al_2O_3 and SiO_2NPs disrupted the oppositely charged membrane,indicating the important role of electrostatic attraction.However,Fe_2O_3,TiO_2and ZnO NPs did not cause serious membrane disruption as Al_2O_3 and SiO_2 NPs.Membrane fluidity was evaluated by the generalized polarity（GP） values of Laurdan fluorescent emission.SiO_2 NPs induce the membrane gelation of both positively and negatively charged membrane.Al_2O_3 and ZnO NPs induced the gelation of the oppositely charged membrane,but did not cause obvious membrane gelation to the like charged membrane.The phospholipid molecular structural changes after NP exposure were analyzed by Fourier transform infrared（FT-IR） spectroscopy.FT-IR spectra revealed the hydrogen bond formation between NPs and the carbonyl/phosphate groups of phospholipids.Al_2O_3 and SiO_2 NPs showed strongest evidence of hydrogen bonding on their FT-IR spectra.It was consistent with the microscopic observation and fluorescent data that Al_2O_3 and SiO_2 NPs caused more serious membrane disruption and gelation.This study on membrane damage provides further knowledge on the cytotoxicity of nanomaterials and the safety of NP application.

Salmonella-mediated blood-brain barrier penetration,tumor homing and tumor microenvironment regulation for enhanced chemo/bacterial glioma therapy

Chemotherapy is an important adjuvant treatment of glioma,while the efficacy is far from satisfactory,due not only to the biological barriers of blood-brain barrier(BBB)and blood-tumor barrier(BTB)but also to the intrinsic resistance of glioma cells via multiple survival mechanisms such as upregulation of P-glycoprotein(P-gp).To address these limitations,we report a bacteria-based drug delivery strategy for BBB/BTB transportation,glioma targeting,and chemo-sensitization.Bacteria selectively colonized into hypoxic tumor region and modulated tumor microenvironment,including macrophages repolarization and neutrophils infiltration.Specifically,tumor migration of neutrophils was employed as hitchhiking delivery of doxorubicin(DOX)-loaded bacterial outer membrane vesicles(OMVs/DOX).By virtue of the surface pathogen-associated molecular patterns derived from native bacteria,OMVs/DOX could be selectively recognized by neutrophils,thus facilitating glioma targeted delivery of drug with significantly enhanced tumor accumulation by 18-fold as compared to the classical passive targeting effect.Moreover,the P-gp expression on tumor cells was silenced by bacteria typeⅢsecretion effector to sensitize the efficacy of DOX,resulting in complete tumor eradication with 100%survival of all treated mice.In addition,the colonized bacteria were finally cleared by anti-bacterial activity of DOX to minimize the potential infection risk,and cardiotoxicity of DOX was also avoided,achieving excellent compatibility.This work provides an efficient trans-BBB/BTB drug delivery strategy via cell hitchhiking for enhanced glioma therapy.

Functional Interaction of the SNARE Protein NtSyp121 in Ca^2＋ Channel Gating, Ca^2＋ Transientsand ABA Signalling of Stomatal Guard Cells

There is now growing evidence that membrane vesicle trafficking proteins, especially of the superfamily of SNAREs, are critical for cellular signalling in plants. Work from this laboratory first demonstrated that a soluble, inhibitory （dominant-negative） fragment of the SNARE NtSyp121 blocked K^＋ and CI^- channel responses to the stress-related hormone abscisic acid （ABA）, but left open a question about functional impacts on signal intermediates, especially on Ca^2＋-mediated signalling events. Here, we report one mode of action for the SNARE mediated directly through alterations in Ca^2＋ channel gating and its consequent effects on cytosolic-free [Ca^2＋] （[Ca^2＋]i） elevation. We find that expressing the same inhibitory fragment of NtSyp121 blocks ABA-evoked stomatal closure, but only partially suppresses stomatal closure in the presence of the NO donor, SNAP, which promotes [Ca^2＋]i elevation independently of the plasma membrane Ca^2＋ channels. Consistent with these observations, Ca^2＋ channel gating at the plasma membrane is altered by the SNARE fragment in a manner effective in reducing the potential for triggering a rise in [Ca^2＋]i, and we show directly that its expression in vivo leads to a pronounced suppression of evoked [Ca^2＋]i transients. These observations offer primary evidence for the functional coupling of the SNARE with Ca^2＋ channels at the plant cell plasma membrane and, because [Ca^2＋]i plays a key role in the control of K^＋ and CI^- channel currents in guard cells, they underscore an important mechanism for SNARE integration with ion channel regulation during stomatal closure.

Homology Modeling of CrylAc Toxin-binding Alkaline Phosphatase Receptor from Helicoverpa armigera and Its Functional Interpretation

Alkaline phosphatases （ALPs） attached to the midgut membrane with glycosyl phosphotidyl inositol （GPI） have been proposed as the putative CrylAc toxin receptor in Helicoverpa armigera. Activated toxins bind to ALP receptors on the brush border membrane vesicle （BBMV） of the midgut epithelium, which activates intracellular oncotic pathways and leads to cell death. However, with the long-term use of Cry toxin, insects can develop a strong resistance to insecticidal delta-endotoxins. Although the molecular mechanism of insect resistance has not been fully understood, insects develop resistance to biopesticides due to changes of toxins binding to midgut receptors. So, it is a good idea to investigate the molecular mechanism of insect resistance by analyzing ALP receptor from Helicoverpa armigera （Ha-ALP）. Based on crystal structure of shrimp alkaline phosphatase, the three-dimensional structure of the CrylAc toxin-binding Ha-ALP receptor was obtained by homology modeling and the model was further evaluated using PROSA energy and ERRAT. The important role of binding of toxin to GalNAc on Ha-ALP was discussed in the aspect of CrylAc toxicity. Specific recognition sites of the binding of oligosaccharides to Ha-ALP were predicted. Post-translational modification of ALP provides insights into the functional properties of ALP and leads to profound understanding of receptor and toxin interactions.