RESUMO
Agonists of Toll like receptors (TLRs) have attracted interest as adjuvants and immune modulators. A crystal structure of TLR4/MD2 with E. coli LPS indicates that the fatty acid at C-2 of the lipid A component of LPS induces dimerization of two TLR4-MD2 complexes, which in turn initiates cell signaling leading to the production of (pro)inflammatory cytokines. To probe the importance of the (R)-3-hydroxymyristate at C-2 of lipid A, a range of bis- and mono-phosphoryl lipid A derivatives with different modifications at C-2 were prepared by a strategy in which 2-methylnaphthyl ethers were employed as permanent protecting group that could be readily removed by catalytic hydrogenation. The C-2 amine was protected as 9-fluorenylmethyloxycarbamate, which at a later stage could be removed to give a free amine that was modified by different fatty acids. LPS and the synthetic lipid As induced the same cytokines, however, large differences in activity were observed. A compound having a hexanoyl moiety at C-2 still showed agonistic properties, but further shortening to a butanoyl abolished activity. The modifications had a larger influence on monophosphoryl lipid As. The lipid As having a butanoyl moiety at C-2 could selectively antagonize TRIF associated cytokines induced by LPS or lipid A.
Assuntos
Citocinas , Lipídeo A , Lipopolissacarídeos , Lipídeo A/química , Lipídeo A/farmacologia , Lipídeo A/análogos & derivados , Lipídeo A/síntese química , Citocinas/metabolismo , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/química , Humanos , Antígeno 96 de Linfócito/metabolismo , Antígeno 96 de Linfócito/química , Desenho de Fármacos , Relação Estrutura-Atividade , Transdução de Sinais/efeitos dos fármacosRESUMO
Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from Cellulophaga pacifica KMM 3664T isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that C. pacifica KMM 3664T LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial Salmonella typhimurium LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry.
Assuntos
Lipídeo A , Lipídeo A/química , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/química , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química , Animais , Lipopolissacarídeos/química , CamundongosRESUMO
The mortality of ovarian cancer (OC) has long been the highest among gynecological malignancies. Although OC is considered to be an immunogenic tumor, the effect of immunotherapy is not satisfactory. The immunosuppressive microenvironment is one reason for this, and the absence of recognized effective antigens for vaccines is another. Chemotherapy, as one of the most commonly used treatment for OC, can produce chemotherapy-associated antigens (CAAs) during treatment and show the effect of in situ vaccine. Herein, we designed an antigen capture nano-vaccine NP-TP1@M-M with tumor targeting peptide TMTP1 and dendritic cell (DC) receptor mannose assembled on the surface and adjuvant monophosphoryl lipid A (MPLA) encapsulated in the core of poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles. PLGA itself possessed the ability of antigen capture. TMTP1 was a tumor-homing peptide screened by our research team, which held extensive and excellent tumor targeting ability. After these modifications, NP-TP1@M-M could capture and enrich more tumor-specific antigens after chemotherapy, stimulate DC maturation, activate the adaptive immunity and combined with immune checkpoint blockade to maximize the release of the body's immune potential, providing an eutherapeutic strategy for the treatment of OC.
Assuntos
Antígenos de Neoplasias , Antígeno B7-H1 , Vacinas Anticâncer , Nanopartículas , Neoplasias Ovarianas , Feminino , Neoplasias Ovarianas/tratamento farmacológico , Animais , Camundongos , Vacinas Anticâncer/uso terapêutico , Nanopartículas/química , Linhagem Celular Tumoral , Antígenos de Neoplasias/imunologia , Humanos , Células Dendríticas/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Lipídeo A/análogos & derivados , Lipídeo A/química , Lipídeo A/farmacologia , Imunoterapia/métodos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Camundongos Endogâmicos BALB C , Inibidores de Checkpoint Imunológico/farmacologia , NanovacinasRESUMO
The lipopolysaccharide (LPS) that resides on the outermost surface and protects Gram-negative bacteria from host defenses is one of the key components leading to Salmonella infection, particularly the endotoxic lipid A domain of LPS. Lipid A modifications have been associated with several genes such as the arnT that encodes 4-amino-4-deoxy-L-arabinose transferase, which can be critical for bacteria to resist cationic antimicrobial peptides and interfere with host immune recognition. However, the association of arnT with virulence is not completely understood. Thus, this study aimed to elucidate the interrelationship of the major lipid A modification gene arnT with Salmonella Typhimurium virulence. We observed that the arnT-deficient S. Typhimurium (JOL2943), compared to the wild type (JOL401), displayed a significant decrease in several virulence phenotypes such as polymyxin B resistance, intracellular survival, swarming, and biofilm and extracellular polymeric substance (EPS) production. Interestingly, the cell-surface hydrophobicity, adhesion, and invasion characteristics remained unaffected. Additionally, LPS isolated from the mutant induced notably lower levels of endotoxicity-related cytokines in RAW and Hela cells and mice, particularly IL-1ß with a nine-fold decrease, than WT. In terms of in vivo colonization, JOL2943 showed diminished presence in internal organs such as the spleen and liver by more than 60%, while ileal infectivity remained similar to JOL401. Overall, the arnT deletion rendered the strain less virulent, with low endotoxicity, maintained gut infectivity, and reduced colonization in internal organs. With these ideal characteristics, it can be further explored as a potential attenuated Salmonella strain for therapeutics or vaccine delivery systems.
Assuntos
Lipídeo A , Salmonella typhimurium , Humanos , Animais , Camundongos , Salmonella typhimurium/genética , Lipídeo A/química , Lipopolissacarídeos/química , Virulência , Matriz Extracelular de Substâncias Poliméricas , Células HeLa , Proteínas de Bactérias/genéticaRESUMO
Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.
Assuntos
Lipopolissacarídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Camundongos , Macrófagos/metabolismo , Lipídeo A/química , Lipídeo A/isolamento & purificação , Citocinas/metabolismo , Endopeptidase K/metabolismo , Endopeptidase K/química , Eletroforese em Gel de Poliacrilamida/métodosRESUMO
Lipopolysaccharide (LPS), a cell surface component of Gram-negative bacteria, activates innate immunity. Its active principle is the terminal glycolipid lipidâ A. Acetobacter pasteurianus is a Gram-negative bacterium used in the fermentation of traditional Japanese black rice vinegar (kurozu). In this study, we focused on A.â pasteurianus lipidâ A, which is a potential immunostimulatory component of kurozu. The active principle structure of A.â pasteurianus lipidâ A has not yet been identified. Herein, we first systematically synthesized three types of A.â pasteurianus lipidâ As containing a common and unique tetrasaccharide backbone. We developed an efficient method for constructing the 2-trehalosamine skeleton utilizing borinic acid-catalyzed glycosylation to afford 1,1'-α,α-glycoside in high yield and stereoselectivity. A common tetrasaccharide intermediate with an orthogonal protecting group pattern was constructed via [2+2] glycosylation. After introducing various fatty acids, all protecting groups were removed to achieve the first chemical synthesis of three distinct types of A.â pasteurianus lipidâ As. After evaluating their immunological function using both human and murine cell lines, we identified the active principles of A.â pasteurianus LPS. We also found the unique anomeric structure of A.â pasteurianus lipidâ A contributes to its high chemical stability.
Assuntos
Acetobacter , Lipídeo A , Lipídeo A/química , Lipídeo A/imunologia , Lipídeo A/síntese química , Humanos , Camundongos , Acetobacter/química , Animais , Oligossacarídeos/química , Oligossacarídeos/síntese química , GlicosilaçãoRESUMO
The toxic nature of bacterial endotoxins is affected by the structural details of lipid A, including the variety and position of acyl chains and phosphate group(s) on its diglucosamine backbone. Negative-ion mode tandem mass spectrometry is a primary method for the structure elucidation of lipid A, used independently or in combination with separation techniques. However, it is challenging to accurately characterize constitutional isomers of lipid A extracts by direct mass spectrometry, as the elemental composition and molecular mass of these molecules are identical. Thus, their simultaneous fragmentation leads to a composite, so-called chimera mass spectrum. The present study focuses on the phosphopositional isomers of the classical monophosphorylated, hexaacylated Escherichia coli-type lipid A. Collision-induced dissociation (CID) was performed in an HPLC-ESI-QTOF system. Energy-resolved mass spectrometry (ERMS) was applied to uncover the distinct fragmentation profiles of the phosphorylation isomers. A fragmentation strategy applying multi-levels of collision energy has been proposed and applied to reveal sample complexity, whether it contains only a 4'-phosphorylated species or a mixture of 1- and 4'-phosphorylated variants. This comparative fragmentation study of isomeric lipid A species demonstrates the high potential of ERMS-derived information for the successful discrimination of co-ionized phosphorylation isomers of hexaacylated lipid A.
Assuntos
Lipídeo A , Espectrometria de Massas em Tandem , Lipídeo A/química , Isomerismo , Espectrometria de Massas por Ionização por Electrospray , Escherichia coli , Cromatografia Líquida de Alta Pressão , FosforilaçãoRESUMO
The emergence of multidrug resistance has provided a great challenge to treat nosocomial infections, which have become a major health threat around the globe. Lipid A (an active endotoxin component), the final product of the Raetz lipid A metabolism pathway, is a membrane anchor of lipopolysaccharide (LPS) of the gram-negative bacterial outer membrane. It shields bacterial cells and serves as a protective barrier from antibiotics, thereby eliciting host response and making it difficult to destroy. UDP-2,3-diacylglucosamine pyrophosphate hydrolase (LpxH), a crucial peripheral membrane enzyme of the Raetz pathway, turned out to be the potential target to inhibit the production of Lipid A. This review provides a comprehensive compilation of information regarding the structural and functional aspects of LpxH, as well as its analogous LpxI and LpxG. In addition, apart from by providing a broader understanding of the enzyme-inhibitor mechanism, this review facilitates the development of novel drug candidates that can inhibit the pathogenicity of the lethal bacterium.
Assuntos
Bactérias Gram-Negativas , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/efeitos dos fármacos , Pirofosfatases/metabolismo , Pirofosfatases/química , Lipídeo A/química , Lipídeo A/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , HumanosRESUMO
Lipid A plays a crucial role in Vibrio parahaemolyticus. Previously we have reported the diversity of secondary acylation of lipid A in V. parahaemolyticus and four V. parahaemolyticus genes VP_RS08405, VP_RS01045, VP_RS12170, and VP_RS00880 exhibiting homology to the secondary acyltransferases in Escherichia coli. In this study, the gene VP_RS12170 was identified as a specific lipid A secondary hydroxy-acyltransferase responsible for transferring a 3-hydroxymyristate to the 2'-position of lipid A. Four E. coli mutant strains WHL00, WHM00, WH300, and WH001 were constructed, and they would synthesize lipid A with different structures due to the absence of genes encoding lipid A secondary acyltransferases or Kdo transferase. Then V. parahaemolyticus VP_RS12170 was overexpressed in W3110, WHL00, WHM00, WH300, and WH001, and lipid A was isolated from these strains and analyzed by using thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry. The detailed structural changes of lipid A in these mutant strains with and without VP_RS12170 overexpression were compared and conclude that VP_RS12170 can specifically transfer a 3-hydroxymyristate to the 2'-position of lipid A. This study also demonstrated that the function of VP_RS12170 is Kdo-dependent and its favorite substrate is Kdo-lipid IVA. These findings give us better understanding the biosynthetic pathway and the structural diversity of V. parahaemolyticus lipid A.
Assuntos
Lipídeo A , Vibrio parahaemolyticus , Lipídeo A/química , Lipídeo A/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Espectrometria de MassasRESUMO
This study describes the design and synthesis of five TF-based cancer vaccine candidates using a lipid A mimetic as the carrier and a built-in adjuvant. All synthesized conjugates elicited robust and consistent TF-specific immune responses in mice without external adjuvants. Immunological studies subsequently conducted in wild-type and TLR4 knockout C57BL/6 mice demonstrated that the activation of TLR4 was the main reason that the synthesized lipid A mimetics increased the TF-specific immune responses. All antisera induced by these conjugates can specifically recognize, bind to, and induce the lysis of TF-positive cancer cells. Moreover, representative conjugates 2 and 3 could effectively reduce the growth of tumors and prolong the survival time of mice in vivo, and the efficacies were better than glycoprotein TF-CRM197 with alum adjuvant. Lipid A mimetics could therefore be a promising platform for the development of new carbohydrate-based vaccine carriers with self-adjuvanting properties for the treatment of cancer.
Assuntos
Adjuvantes Imunológicos , Vacinas Anticâncer , Desenho de Fármacos , Lipídeo A , Camundongos Endogâmicos C57BL , Animais , Lipídeo A/análogos & derivados , Lipídeo A/química , Lipídeo A/farmacologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Vacinas Anticâncer/síntese química , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/síntese química , Adjuvantes Imunológicos/química , Camundongos , Camundongos Knockout , Humanos , Feminino , Receptor 4 Toll-Like/metabolismo , Linhagem Celular TumoralRESUMO
Lipid vesicles are widely used for drug and gene delivery, but their structural instability reduces in vivo efficacy and requires specialized handling. To address these limitations, strategies like lipid cross-linking and polymer-lipid conjugation are suggested to enhance stability and biological efficacy. However, the in vivo metabolism of these altered lipids remains unclear, necessitating further studies. A new stabilization technique without chemical modification is urgently needed. Here, a bio-mimetic approach for fabricating robust multilamellar lipid vesicles to enhance in vivo delivery and stabilization of protein antigens is presented. This method leverages 1-O-acylceramide, a natural skin lipid, to facilitate the self-assembly of lipid nanovesicles. Incorporating 1-O-acylceramide, anchoring lipid bilayers akin to its role in the stratum corneum, provides excellent stability under environmental stresses, including freeze-thaw cycles. Encapsulating ovalbumin as a model antigen and the adjuvant monophosphoryl lipid A demonstrates the vesicle's potential as a nanovaccine platform. In vitro studies show enhanced immune responses with both unilamellar and multilamellar vesicles, but in vivo analyses highlight the superior efficiency of multilamellar vesicles in inducing higher antibody and cytokine levels. This work suggests ceramide-induced multilamellar lipid vesicles as an effective nanovaccine platform for enhanced antigen delivery and stability.
Assuntos
Ovalbumina , Animais , Camundongos , Ovalbumina/química , Ovalbumina/imunologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Vacinação/métodos , Lipídeo A/química , Lipídeo A/análogos & derivados , Vacinas/química , Vacinas/imunologia , Ceramidas/química , Lipídeos/química , Nanopartículas/química , Feminino , Camundongos Endogâmicos C57BLRESUMO
Polymyxins are gram-negative antibiotics that target lipid A, the conserved membrane anchor of lipopolysaccharide in the outer membrane. Despite their clinical importance, the molecular mechanisms underpinning polymyxin activity remain unresolved. Here, we use surface plasmon resonance to kinetically interrogate interactions between polymyxins and lipid A and derive a phenomenological model. Our analyses suggest a lipid A-catalyzed, three-state mechanism for polymyxins: transient binding, membrane insertion, and super-stoichiometric cluster accumulation with a long residence time. Accumulation also occurs for brevicidine, another lipid A-targeting antibacterial molecule. Lipid A modifications that impart polymyxin resistance and a non-bactericidal polymyxin derivative exhibit binding that does not evolve into long-lived species. We propose that transient binding to lipid A permeabilizes the outer membrane and cluster accumulation enables the bactericidal activity of polymyxins. These findings could establish a blueprint for discovery of lipid A-targeting antibiotics and provide a generalizable approach to study interactions with the gram-negative outer membrane.
Assuntos
Antibacterianos , Lipídeo A , Polimixina B , Ressonância de Plasmônio de Superfície , Polimixina B/farmacologia , Polimixina B/metabolismo , Lipídeo A/metabolismo , Lipídeo A/química , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Testes de Sensibilidade Microbiana , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/efeitos dos fármacos , CinéticaRESUMO
Adjuvant is an integral part of all vaccine formulations but only a few adjuvants with limited efficacies or application scopes are available. Thus, developing more robust and diverse adjuvants is necessary. To this end, a new class of adjuvants having α- and ß-rhamnose (Rha) attached to the 1- and 6'-positions of monophosphoryl lipid A (MPLA) was designed, synthesized, and immunologically evaluated in mice. The results indicated a synergistic effect of MPLA and Rha, two immunostimulators that function via interacting with toll-like receptor 4 and recruiting endogenous anti-Rha antibodies, respectively. All the tested MPLA-Rha conjugates exhibited potent adjuvant activities to promote antibody production against both protein and carbohydrate antigens. Overall, MPLA-α-Rha exhibited better activities than MPLA-ß-Rha, and 6'-linked conjugates were slightly better than 1-linked ones. Particularly, MPLA-1-α-Rha and MPLA-6'-α-Rha were the most effective adjuvants in promoting IgG antibody responses against protein antigen keyhole limpet hemocyanin and carbohydrate antigen sTn, respectively.
Assuntos
Lipídeo A , Ramnose , Lipídeo A/análogos & derivados , Lipídeo A/química , Lipídeo A/farmacologia , Lipídeo A/imunologia , Animais , Ramnose/química , Ramnose/imunologia , Ramnose/farmacologia , Camundongos , Adjuvantes de Vacinas/química , Adjuvantes de Vacinas/farmacologia , Feminino , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/síntese química , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Hemocianinas/química , Hemocianinas/imunologiaRESUMO
The ATP-binding cassette (ABC) transporter, MsbA, plays a pivotal role in lipopolysaccharide (LPS) biogenesis by facilitating the transport of the LPS precursor lipooligosaccharide (LOS) from the cytoplasmic to the periplasmic leaflet of the inner membrane. Despite multiple studies shedding light on MsbA, the role of lipids in modulating MsbA-nucleotide interactions remains poorly understood. Here we use native mass spectrometry (MS) to investigate and resolve nucleotide and lipid binding to MsbA, demonstrating that the transporter has a higher affinity for adenosine 5'-diphosphate (ADP). Moreover, native MS shows the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL) can tune the selectivity of MsbA for adenosine 5'-triphosphate (ATP) over ADP. Guided by these studies, four open, inward-facing structures of MsbA are determined that vary in their openness. We also report a 2.7 Å-resolution structure of MsbA in an open, outward-facing conformation that is not only bound to KDL at the exterior site, but with the nucleotide binding domains (NBDs) adopting a distinct nucleotide-free structure. The results obtained from this study offer valuable insight and snapshots of MsbA during the transport cycle.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Difosfato de Adenosina , Trifosfato de Adenosina , Espectrometria de Massas , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Espectrometria de Massas/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Lipopolissacarídeos/metabolismo , Lipídeo A/metabolismo , Lipídeo A/química , Ligação Proteica , Modelos Moleculares , Cristalografia por Raios X , Lipídeos/química , Escherichia coli/metabolismo , Conformação ProteicaRESUMO
Anticancer chemo-immunotherapy has gained considerable attention across various scientific domains as a prospective approach for the comprehensive eradication of malignant tumors. Recent research has particularly been focused on traditional anthracycline chemo drugs, such as doxorubicin and mitoxantrone. These compounds trigger apoptosis in tumor cells and evoke immunogenic cell death (ICD). ICD is a pivotal initiator of the cancer-immunity cycle by facilitating the release of damage-associated molecular patterns (DAMPs). The resultant DAMPs released from cancer cells effectively activate the immune system, resulting in an increase in tumor-infiltrating T cells. In this study, we have innovated a co-delivery strategy involving folate-modified liposomes to deliver doxorubicin and monophosphoryl lipid A (MPLA) simultaneously to tumor tissue. The engineered liposomes exploit the overexpression of folate receptors within the tumor tissues. Delivered doxorubicin initiates ICD at the tumor cells, further enhancing the immunogenic stimulus. Additionally, MPLA helps T cell priming by activating antigen-presenting cells. This intricate interplay culminates in a synergistic effect, ultimately resulting in an augmented and potentiated anticancer chemo-immunotherapeutic liposomal treatment.