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The detection of pathogenicity and immunogenicity in Vibrio parahaemolyticus poses a significant challenge due to its threat to human health and food safety, which is strongly correlated with lipid A. Lipid A, a critical component found in most Gram-negative bacteria, functions as a hydrophobic anchor for lipopolysaccharide. V. parahaemolyticus synthesizes multiple lipid A species with various secondary acyl chains. In this study, a secondary acyltransferase of lipid A encoded by VP_RS08405 in V. parahaemolyticus was identified. Based on sequence alignment analysis, V. parahaemolyticus VP_RS08405 has high homology to E. coli lpxL, lpxM and lpxP which encode the three secondary acyltransferases of lipid A. Therefore, V. parahaemolyticus VP_RS08405 was cloned into pBAD33, and the resulting pB08405 was introduced in E. coli mutants WHL00 in which lpxL was deleted, WHM00 in which lpxM was deleted, WHP00 in which lpxP was deleted, and WH300 in which lpxL, lpxM and lpxP were deleted. The recombinant strains WHL00/pB08405, WHM00/pB08405, WHP00/pB08405, WH300/pB08405, as well as their vector controls, were grown at normal and low temperatures. Lipid A species were isolated from the above strains and analyzed by using high-performance liquid chromatography-tandem mass spectrometry and thin-layer chromatography. After comparing the secondary acyl alterations of lipid A from different recombinant strains, it is concluded that VP_RS08405 specifically catalyzed the addition of a palmitoleate to the 2'-position of lipid A and its activity is not temperature-sensitive. In addition, to determine the dependence of VP_RS08405 on Kdo, VP_RS08405 was overexpressed in E. coli mutants WH001 in which waaA was deleted, and WH400 in which waaA, lpxL, lpxM and lpxP were deleted. Lipid A species were isolated from WH001/pB08405 and WH400/pB08405, and analyzed. The results show that the function of VP_RS08405 is Kdo-dependent. These findings provide a better understanding of the structural diversity of lipid A in V. parahaemolyticus., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Immunological adjuvants are vaccine components that enhance long-lasting adaptive immune responses to weakly immunogenic antigens. Monophosphoryl lipid A (MPLA) is a potent and safe vaccine adjuvant that initiates an early innate immune response by binding to the Toll-like receptor 4 (TLR4). Importantly, the binding and recognition process is highly dependent on the monomeric state of MPLA. However, current vaccine delivery systems often prioritize improving the loading efficiency of MPLA, while neglecting the need to maintain its monomeric form for optimal immune activation. Here, we introduce a Pickering emulsion-guided MPLA monomeric delivery system (PMMS), which embed MPLA into the oil-water interface to achieve the monomeric loading of MPLA. During interactions with antigen-presenting cells, PMMS functions as a chaperone for MPLA, facilitating efficient recognition by TLR4 regardless of the presence of lipopolysaccharide-binding proteins. At the injection site, PMMS efficiently elicited local immune responses, subsequently promoting the migration of antigen-internalized dendritic cells to the lymph nodes. Within the draining lymph nodes, PMMS enhanced antigen presentation and maturation of dendritic cells. In C57BL/6 mice models, PMMS vaccination provoked potent antigen-specific CD8 + T cell-based immune responses. Additionally, PMMS demonstrated strong anti-tumor effects against E.G7-OVA lymphoma. These data indicate that PMMS provides a straightforward and efficient strategy for delivering monomeric MPLA to achieve robust cellular immune responses and effective cancer immunotherapy., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The cell-envelope of Gram-negative bacteria contains endotoxic lipopolysaccharides (LPS) that are recognized by the innate immune system via Toll-Like Receptors (TLRs). The intestinal mucosal symbiont Akkermansia muciniphila is known to confer beneficial effects on the host and has a Gram-negative architecture. Here we show that A. muciniphila LPS lacks the O-polysaccharide repeating unit, with the resulting lipooligosaccharide (LOS) having unprecedented structural and signaling properties. The LOS consists of a complex glycan chain bearing two distinct undeca- and hexadecasaccharide units each containing three 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residues. The lipid A moiety appears as a mixture of differently phosphorylated and acylated species and carries either linear or branched acyl moieties. Peritoneal injection of the LOS in mice increased higher gene expression of liver TLR2 than TLR4 (100-fold) and induced high IL-10 gene expression. A. muciniphila LOS was found to signal both through TLR4 and TLR2, whereas lipid A only induced TLR2 in a human cell line. We propose that the unique structure of the A. muciniphila LOS allows interaction with TLR2, thus generating an anti-inflammatory response as to compensate for the canonical inflammatory signaling associated with LOS and TLR4, rationalizing its beneficial host interaction., (© 2024. The Author(s).)

The genus Pseudochrobactrum encompasses free-living bacteria phylogenetically close to Ochrobactrum opportunistic pathogens and to Brucella, facultative intracellular parasites causing brucellosis, a worldwide-extended and grave zoonosis. Recently, Pseudochrobactrum strains were isolated from Brucella natural hosts on Brucella selective media, potentially causing diagnostic confusions. Strikingly, P. algeriensis was isolated from cattle lymph nodes, organs that are inimical to bacteria. Here, we analyse P. algeriensis potential virulence factors in comparison with Ochrobactrum and Brucella. Consistent with genomic analyses, Western-Blot analyses confirmed that P. algeriensis lacks the ability to synthesize the N-formylperosamine O-polysaccharide characteristic of the lipopolysaccharide (LPS) of smooth Brucella core species. However, unlike other Pseudochrobactrum but similar to some early diverging brucellae, P. algeriensis carries genes potentially synthetizing a rhamnose-based O-polysaccharide LPS. Lipid A analysis by MALDI-TOF demonstrated that P. algeriensis LPS bears a lipid A with a reduced pathogen-associated molecular pattern, a trait shared with Ochrobactrum and Brucella that is essential to generate a highly stable outer membrane and to delay immune activation. Also, although not able to multiply intracellularly in macrophages, the analysis of P. algeriensis cell lipid envelope revealed the presence of large amounts of cationic aminolipids, which may account for the extremely high resistance of P. algeriensis to bactericidal peptides and could favor colonization of mucosae and transient survival in Brucella hosts. However, two traits critical in Brucella pathogenicity are either significantly different (T4SS [VirB]) or absent (erythritol catabolic pathway) in P. algeriensis. This work shows that, while diverging in other characteristics, lipidic envelope features relevant in Brucella pathogenicity are conserved in Brucellaceae. The constant presence of these features strongly suggests that reinforcement of the envelope integrity as an adaptive advantage in soil was maintained in Brucella because of the similarity of some environmental challenges, such as the action of cationic peptide antibiotics and host defense peptides. This information adds knowledge about the evolution of Brucellaceae, and also underlines the taxonomical differences of the three genera compared., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Antimicrobial resistance (AMR), together with multidrug resistance (MDR), mainly among Gram-negative bacteria, has been on the rise. Colistin (polymyxin E) remains one of the primary available last resorts to treat infections caused by MDR bacteria during the rapid emergence of global resistance. As the exact mechanism of bacterial resistance to colistin remains undetermined, this study warranted elucidation of the underlying mechanisms of colistin resistance and heteroresistance among carbapenem-resistant Klebsiella pneumoniae isolates., Methods: Molecular analysis was carried out on the resistant isolates using a genome-wide characterisation approach, as well as MALDI-TOF mass spectrometry, to identify lipid A., Results: Among the 32 carbapenem-resistant K. pneumoniae isolates, several isolates showed resistance and intermediate resistance to colistin. The seven isolates with intermediate resistance exhibited the "skip-well" phenomenon, attributed to the presence of resistant subpopulations. The three isolates with full resistance to colistin showed ions using MALDI-TOF mass spectrometry at m/z of 1840 and 1824 representing bisphosphorylated and hexa-acylated lipid A, respectively, with or without hydroxylation at position C'-2 of the fatty acyl chain. Studying the genetic environment of mgrB locus revealed the presence of two insertion sequences that disrupted the mgrB locus in the three colistin-resistant isolates: IS1R and IS903B., Conclusions: Our findings show that colistin resistance/heteroresistance was inducible with mutations in chromosomal regulatory networks controlling the lipid A moiety and insertion sequences disrupting the mgrB gene, leading to elevated minimum inhibitory concentration values and treatment failure. Different treatment strategies should be employed to avoid colistin heteroresistance-linked treatment failures, mainly through combination therapy using colistin with carbapenems, aminoglycosides, or tigecycline., Competing Interests: Declaration of competing interests The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

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., (© 2024. The Author(s).)

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.

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., (© 2024. The Author(s).)

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., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

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.

Lipopolysaccharide (LPS), a cell surface component of Gram-negative bacteria, activates innate immunity. Its active principle is the terminal glycolipid lipid 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. pasteurianus lipid A, which is a potential immunostimulatory component of kurozu. The active principle structure of A. pasteurianus lipid A has not yet been identified. Herein, we first systematically synthesized three types of A. pasteurianus lipid 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. pasteurianus lipid As. After evaluating their immunological function using both human and murine cell lines, we identified the active principles of A. pasteurianus LPS. We also found the unique anomeric structure of A. pasteurianus lipid A contributes to its high chemical stability., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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.

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., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

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., (© 2024. The Author(s).)

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 IV A . These findings give us better understanding the biosynthetic pathway and the structural diversity of V. parahaemolyticus lipid A., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

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 β-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-β-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.

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., Competing Interests: Declaration of competing interest No conflict of interest declared., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 3664 T 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 3664 T 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., (© 2024. The Author(s).)

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-1β 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.

Lipopolysaccharides (LPS) are major players in bacterial infection through the recognition by Toll-like receptor 4 (TLR4). The LPS chemical structure, including the oligosaccharide core and the lipid A moiety, can be strongly influenced by adaptation and modulated to assure bacteria protection, evade immune surveillance, or reduce host immune responses. Deep structural understanding of TLRs signaling is essential for the modulation of the innate immune system in sepsis control and inflammation, during bacterial infection. To advance this knowledge, we have employed computational techniques to characterize the TLR4 molecular recognition of atypical LPSs from different opportunistic members of α2-Proteobacteria, including Brucella melitensis, Ochrobactrum anthropi, and Ochrobactrum intermedium, with diverse immunostimulatory activities. We contribute to unraveling the role of uncommon lipid A chemical features such as bearing very long-chain fatty acid chains, whose presence has been rarely reported, on modulating the proper heterodimerization of the TLR4 receptor complex. Moreover, we further evaluated the influence of the different oligosaccharide cores, including sugar composition and net charge, on TLR4 activation. Our studies contribute to elucidating, from the molecular and biological perspectives, the impact of the α2-Proteobacteria LPS cores and the chemical structure of the atypical lipid A for immune system evasion in opportunistic bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Polymyxin antibiotics are the last resort for treating patients in intensive care units infected with multiple-resistant Gram-negative bacteria. Due to their polycationic structure, their mode of action is based on an ionic interaction with the negatively charged lipid A portion of the lipopolysaccharide (LPS). The most prevalent polymyxin resistance mechanisms involve covalent modifications of lipid A: addition of the cationic sugar 4-amino-L-arabinose (L-Ara4N) and/or phosphoethanolamine (pEtN). The modified structure of lipid A has a lower net negative charge, leading to the repulsion of polymyxins and bacterial resistance to membrane disruption. Genes encoding the enzymatic systems involved in these modifications can be transferred either through chromosomes or mobile genetic elements. Therefore, new approaches to resistance diagnostics have been developed. On another note, interfering with these enzymatic systems might offer new therapeutic targets for drug discovery. This literature review focuses on diagnostic approaches based on structural changes in lipid A and on the therapeutic potential of molecules interfering with these changes., (© 2023 Vanessa Rogga et al., published by Sciendo.)

FLAT n is a tandem mass spectrometric technique that can be used to rapidly generate spectral information applicable for structural elucidation of lipids like lipid A from Gram-negative bacterial species from a single bacterial colony. In this study, we extend the scope and capability of FLAT n by tandem MS fragmentation of lithium-adducted molecular lipid A anions and fragments (FLAT n-Li ) that provides additional structural and diagnostic data from FLAT n samples allowing for the discrimination of terminal phosphate modifications in a variety of pathogenic and environmental species. Using FLAT n-Li , we elucidated the lipid A structure from several bacterial species, including novel structures from arctic bacterioplankton of the Duganella and Massilia genera that favor 4-amino-4-deoxy-l-arabinopyranose (Ara4N) modification at the 1-phosphate position and that demonstrate double glycosylation with Ara4N at the 1 and 4' phosphate positions simultaneously. The structures characterized in this work demonstrate that some environmental psychrophilic species make extensive use of this structural lipid A modification previously characterized as a pathogenic adaptation and the structural basis of resistance to cationic antimicrobial peptides. This observation extends the role of phosphate modification(s) in environmental species adaptation and suggests that Ara4N modification can functionally replace the positive charge of the phosphoethanolamine modification that is more typically found attached to the 1-phosphate position of modified lipid A.

To develop a Bordetella bronchiseptica vaccine with reduced endotoxicity, we previously inactivated lpxL1, the gene encoding the enzyme that incorporates a secondary 2-hydroxy-laurate in lipid A. The mutant showed a myriad of phenotypes. Structural analysis showed the expected loss of the acyl chain but also of glucosamine (GlcN) substituents, which decorate the phosphates in lipid A. To determine which structural change causes the various phenotypes, we inactivated here lgmB, which encodes the GlcN transferase, and lpxL1 in an isogenic background and compared the phenotypes. Like the lpxL1 mutation, the lgmB mutation resulted in reduced potency to activate human TLR4 and to infect macrophages and in increased susceptibility to polymyxin B. These phenotypes are therefore related to the loss of GlcN decorations. The lpxL1 mutation had a stronger effect on hTLR4 activation and additionally resulted in reduced murine TLR4 activation, surface hydrophobicity, and biofilm formation, and in a fortified outer membrane as evidenced by increased resistance to several antimicrobials. These phenotypes, therefore, appear to be related to the loss of the acyl chain. Moreover, we determined the virulence of the mutants in the Galleria mellonella infection model and observed reduced virulence of the lpxL1 mutant but not of the lgmB mutant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Gram-negative bacteria have a unique cell surface that can be modified to maintain bacterial fitness in diverse environments. A well-defined example is the modification of the lipid A component of lipopolysaccharide (LPS), which promotes resistance to polymyxin antibiotics and antimicrobial peptides. In many organisms, such modifications include the addition of the amine-containing constituents 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN). Addition of pEtN is catalyzed by EptA, which uses phosphatidylethanolamine (PE) as its substrate donor, resulting in production of diacylglycerol (DAG). DAG is then quickly recycled into glycerophospholipid (GPL) synthesis by the DAG kinase A (DgkA) to produce phosphatidic acid, the major GPL precursor. Previously, we hypothesized that loss of DgkA recycling would be detrimental to the cell when LPS is heavily modified. Instead, we found that DAG accumulation inhibits EptA activity, preventing further degradation of PE, the predominant GPL of the cell. However, DAG inhibition of pEtN addition results in complete loss of polymyxin resistance. Here, we selected for suppressors to find a mechanism of resistance independent of DAG recycling or pEtN modification. Disrupting the gene encoding the adenylate cyclase, cyaA , fully restored antibiotic resistance without restoring DAG recycling or pEtN modification. Supporting this, disruptions of genes that reduce CyaA-derived cAMP formation (e.g., ptsI ) or disruption of the cAMP receptor protein, Crp, also restored resistance. We found that loss of the cAMP-CRP regulatory complex was necessary for suppression and that resistance arises from a substantial increase in l-Ara4N-modified LPS, bypassing the need for pEtN modification. IMPORTANCE Gram-negative bacteria can alter the structure of their LPS to promote resistance to cationic antimicrobial peptides, including polymyxin antibiotics. Polymyxins are considered last-resort antibiotics for treatment against multidrug-resistant Gram-negative organisms. Here, we explore how changes in general metabolism and carbon catabolite repression pathways can alter LPS structure and influence polymyxin resistance., Competing Interests: The authors declare no conflict of interest.

Marine bacteria, which are often described as chemical gold, are considered an exceptional source of new therapeutics. Considerable research interest has been given to lipopolysaccharides (LPSs), the main components of the Gram-negative outer membrane. LPS and its lipid A portion from marine bacteria are known to exhibit a tricky chemistry that has been often associated with intriguing properties such as behaving as immune adjuvants or anti-sepsis molecules. In this scenario, we report the structural determination of the lipid A from three marine bacteria within the Cellulophaga genus, which showed to produce an extremely heterogenous blend of tetra- to hexa-acylated lipid A species, mostly carrying one phosphate and one D-mannose on the glucosamine disaccharide backbone. The ability of the three LPSs in activating TLR4 signaling revealed a weaker immunopotential by C. baltica NNO 15840 T and C. tyrosinoxydans EM41 T , while C. algicola ACAM 630 T behaved as a more potent TLR4 activator., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Colistin (polymyxin E) is a group of cationic antimicrobial cyclic peptides and is recognized as a last-resort defense against lethal infections with carbapenem-resistant pathogens. In addition to the plasmid-borne mobilized phosphoethanolamine (PEA) transferases, the functional expression of lipid A-modifying enzymes encoded on chromosomes has been attributed to intrinsic bacterial colistin resistance. However, the mechanisms of colistin resistance in Riemerella anatipestifer remain unknown. Herein, the GE296_RS09715 gene-encoded Lipid A PEA transferases (RaEptA) was identified in R. anatipestifer. Genetic and structural analyses revealed that the amino acid sequence of RaEptA shared 26.6%-33.1% similarities with the family of Lipid A PEA transferases (EptA) and MCR-like proteins and have defined 12 residues that contribute to the formation of phosphatidylethanolamine (PE)-recognizable cavities. Comparative analyses of colistin resistance in RA-LZ01 and RA-LZ01ΔRaEptA showed the level of colistin has fallen from 96 μg mL -1 down to 24 ~ 32 μg mL -1 . Site-directed mutagenesis assay of the PE-binding cavity and expression of the mutants reveals that K309-rRaEptA can remodel the surface of Escherichia coli and rendering it resistant to colistin, suggesting this point-mutation of P309K is necessary for EptA-mediated lipid A modification. Moreover, the virulence of RA-LZ01ΔRaEptA was attenuated compared with RA-LZ01 both in vivo and vitro. Taken together, the results represent the RaEptA involved in the colistin resistance and pathogenicity, and the P309K mutation might alter bacterial adaptation and increase the spread of colistin resistance from R. anatipestifer to other gram-negative bacteria. The findings of this study suggest another scenario for the spread of colistin resistance genes and should be considered by a wide audience., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Polymyxins are the last-line antibiotics used to treat Gram-negative pathogens. Thus, the discovery and biochemical characterization of the resistance genes against polymyxins are urgently needed for diagnosis, treatment, and novel antibiotic design. Herein, we report novel polymyxin-resistance genes identified from sediment and seawater microbiome. Despite their low sequence identity against the known pmrE and pmrF , they show in vitro activities in UDP-glucose oxidation and l-Ara4N transfer to undecaprenyl phosphate, respectively, which occur as the part of lipid A modification that leads to polymyxin resistance. The expression of pmrE and pmrF also showed substantially high MICs in the presence of vanadate ions, indicating that they constitute polymyxin resistomes. IMPORTANCE Polymyxins are one of the last-resort antibiotics. Polymyxin resistance is a severe threat to combat multidrug-resistant pathogens. Thus, up-to-date identification and understanding of the related genes are crucial. Herein, we performed structure-guided sequence and activity analysis of five putative polymyxin-resistant metagenomes. Despite relatively low sequence identity to the previously reported polymyxin-resistance genes, at least four out of five discovered genes show reactivity essential for lipid A modification and polymyxin resistance, constituting antibiotic resistomes.

Species within the Enterobacter cloacae complex (ECC) include globally important nosocomial pathogens. A three-year study of ECC in Germany identified Enterobacter xiangfangensis as the most common species (65.5%) detected, a result replicated by examining a global pool of 3246 isolates. Antibiotic resistance profiling revealed widespread resistance and heteroresistance to the antibiotic colistin and detected the mobile colistin resistance (mcr)-9 gene in 19.2% of all isolates. We show that resistance and heteroresistance properties depend on the chromosomal arnBCADTEF gene cassette whose products catalyze transfer of L-Ara4N to lipid A. Using comparative genomics, mutational analysis, and quantitative lipid A profiling we demonstrate that intrinsic lipid A modification levels are genospecies-dependent and governed by allelic variations in phoPQ and mgrB, that encode a two-component sensor-activator system and specific inhibitor peptide. By generating phoPQ chimeras and combining them with mgrB alleles, we show that interactions at the pH-sensing interface of the sensory histidine kinase phoQ dictate arnBCADTEF expression levels. To minimize therapeutic failures, we developed an assay that accurately detects colistin resistance levels for any ECC isolate., (© 2023. The Author(s).)

Lipopolysaccharide (LPS), a cell surface component of Gram-negative bacteria, and its active principle, lipid A, have immunostimulatory properties and thus potential to act as adjuvants. However, canonical LPS acts as an endotoxin by hyperstimulating the immune response. Therefore, it is necessary to structurally modify LPS and lipid A to minimize toxicity while maintaining adjuvant effects for use as vaccine adjuvants. Various studies have focused on the chemical synthetic method of lipid As and their structure-activity relationship, which are reviewed in this chapter., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lipid A plays an important role in the pathogenicity and antimicrobial resistance of Vibrio parahaemolyticus, but little is known about the structure and biosynthesis of lipid A in V. parahaemolyticus. In this study, lipid A species were either directly extracted or obtained by the acid hydrolysis of lipopolysaccharide from V. parahaemolyticus ATCC33846 cells and analyzed by thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry. Several lipid A species in V. parahaemolyticus cells were characterized, and two of these species were not connected to polysaccharides. One free lipid A species has the similar structure as the hexa-acylated lipid A in Escherichia coli, and the other is a hepta-acylated lipid A with an additional secondary C16:0 acyl chain. Three lipid A species were isolated by the acid hydrolysis of lipopolysaccharide: the 1 st one has the similar structure as the hexa-acylated lipid A in E. coli, the 2 nd one is a hepta-acylated lipid A with an additional secondary C16:0 acyl chain and a secondary 2-OH C12:0 acyl chain, and the 3 rd one is equal to the 2 nd species with a phosphoethanolamine modification. These results are important for understanding the biosynthesis of lipid A in V. parahaemolyticus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Research in the continuously increasing threat of polymyxin-resistant multidrug-resistant Pseudomonas aeruginosa , which causes severe infection in immunocompromised patients, has resulted in the development of several polymyxin-derived cyclic lipopeptides containing l-α-γ- diamino butyric acid-like FADDI-019 (F19). In this work, F19's insertion into a minimal model of the asymmetric outer membrane of the bacterium, which contained only penta-acylated lipid A (LipA) and lacked keto-d-octulosonic acid and O-antigens, in the top leaflet and phospholipids in the bottom leaflet, was studied. F19 exhibited all of the hallmarks of the self-uptake mechanism into the asymmetric bilayer. While a single monomer of the lipopeptide did not get partitioned into the inside of the bilayer, it competitively displaced Ca 2+ from the membrane surface, observed as a decrease in Ca 2+ coordination number with phosphate groups (1.89 vs 1.718), resulting in membrane destabilization. This resulted in an increment of the average defect size and the probability of interplay between lipid tails and hydrophobic residues of another F19. When more than one monomer was present in the system, the first monomer remained docked on the surface, while other monomers intercalated into the bilayer interior with their hydrophobic moieties "sleeved" by lipid acyl chains. The free energy barrier for partial insertion of the lipopeptide into a bilayer in the presence of surface-docked second F19 was recorded at ∼1.3 kcal/mol using two-dimensional (2D) well-tempered metadynamics, making it a low barrier process at 300 K. This study is an attempt to demonstrate the self-uptake mechanism of F19 during intercalation process into the bilayer interior, which may help in the design of better alternates for polymyxins to work against polymyxin resistance.

The degree of polymyxin B (PMB) resistance was measured in 40 clinical Acinetobacter baumannii isolates obtained from health care facilities. All of the tested isolates possessed a multidrug-resistant (MDR) phenotype against four classes of antibiotics (meropenem, doxycycline, gentamicin, and erythromycin), except for PMB. The bla OXA-23 gene was detected throughout the genetic analysis and experimental assay, indicating that all of the MDR strains were carbapenem-resistant A. baumannii strains. Multilocus sequence typing-based genotyping revealed that nine selected strains belonged to the international clone II lineage. When matrix-assisted laser desorption ionization-time of flight mass spectrometry was performed, intrinsic lipid A modification by phosphoethanolamine (PEtN) incorporation was noticeable only in the PMB-resistant (PMB R ) strains. However, the presence of hexa- and penta-acylated lipid A due to the loss of the laurate (C 12 ) acyl chain was noted in all PMB-susceptible strains but not in the PMB R strains. The reduction of negative surface charges in the PMB R strains was assessed by zeta potential analysis. Fluorescence imaging using dansyl-PMB revealed that, in the PMB R strains, PMB was less likely to bind to the cell surface. IMPORTANCE The widespread presence of MDR pathogens, including A. baumannii, is causing serious hospital-acquired infections worldwide. Extensive surveillance of MDR clinical A. baumannii isolates has been conducted, but the underlying mechanisms for their development of MDR phenotypes are often neglected. Either lipid A modification or loss of lipopolysaccharide in Gram-negative bacteria leads to PMB R phenotypes. The prevalence of intrinsic lipid A modification in PMB R clinical strains was attributed to high levels of basal expression of pmrC and eptA-1 . Our findings suggest that new therapeutic strategies are warranted to combat MDR pathogens due to the emergence of many PMB R clinical strains.

Objectives: Neisseria gonorrhoeae is an exclusively human pathogen that commonly infects the urogenital tract resulting in gonorrhoea. Empirical treatment of gonorrhoea with antibiotics has led to multidrug resistance and the need for new therapeutics. Inactivation of lipooligosaccharide phosphoethanolamine transferase A (EptA), which attaches phosphoethanolamine to lipid A, results in attenuation of the pathogen in infection models. Small molecules that inhibit EptA are predicted to enhance natural clearance of gonococci via the human innate immune response., Methods: A library of small-fragment compounds was tested for the ability to enhance susceptibility of the reference strain N. gonorrhoeae FA1090 to polymyxin B. The effect of these compounds on lipid A synthesis and viability in models of infection were tested., Results: Three compounds, 135, 136 and 137, enhanced susceptibility of strain FA1090 to polymyxin B by 4-fold. Pre-treatment of bacterial cells with all three compounds resulted in enhanced killing by macrophages. Only lipid A from bacterial cells exposed to compound 137 showed a 17% reduction in the level of decoration of lipid A with phosphoethanolamine by MALDI-TOF MS analysis and reduced stimulation of cytokine responses in THP-1 cells. Binding of 137 occurred with higher affinity to purified EptA than the starting material, as determined by 1D saturation transfer difference NMR. Treatment of eight MDR strains with 137 increased susceptibility to polymyxin B in all cases., Conclusions: Small molecules have been designed that bind to EptA, inhibit addition of phosphoethanolamine to lipid A and can sensitize N. gonorrhoeae to killing by macrophages., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Detection of Gram-negative bacterial lipid A by the extracellular sensor, myeloid differentiation 2 (MD2)/Toll-like receptor 4 (TLR4), or the intracellular inflammasome sensors, CASP4 and CASP5, induces robust inflammatory responses. The chemical structure of lipid A, specifically its phosphorylation and acylation state, varies across and within bacterial species, potentially allowing pathogens to evade or suppress host immunity. Currently, it is not clear how distinct alterations in the phosphorylation or acylation state of lipid A affect both human TLR4 and CASP4/5 activation. Using a panel of engineered lipooligosaccharides (LOS) derived from Yersinia pestis with defined lipid A structures that vary in their acylation or phosphorylation state, we identified that differences in phosphorylation state did not affect TLR4 or CASP4/5 activation. However, the acylation state differentially impacted TLR4 and CASP4/5 activation. Specifically, all tetra-, penta-, and hexa-acylated LOS variants examined activated CASP4/5-dependent responses, whereas TLR4 responded to penta- and hexa-acylated LOS but did not respond to tetra-acylated LOS or penta-acylated LOS lacking the secondary acyl chain at the 3' position. As expected, lipid A alone was sufficient for TLR4 activation. In contrast, both core oligosaccharide and lipid A were required for robust CASP4/5 inflammasome activation in human macrophages, whereas core oligosaccharide was not required to activate mouse macrophages expressing CASP4. Our findings show that human TLR4 and CASP4/5 detect both shared and nonoverlapping LOS/lipid A structures, which enables the innate immune system to recognize a wider range of bacterial LOS/lipid A and would thereby be expected to constrain the ability of pathogens to evade innate immune detection.

Immune sensing of the Gram-negative bacterial membrane glycolipid lipopolysaccharide (LPS) is both a critical component of host defense against bacterial infection and a contributor to the hyperinflammatory response, potentially leading to sepsis and death. Innate immune activation by LPS is due to the lipid A moiety, an acylated di-glucosamine molecule that can activate inflammatory responses via the extracellular sensor Toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) or the cytosolic sensor caspase-11 (Casp11). The number and length of acyl chains present on bacterial lipid A structures vary across bacterial species and strains, which affects the magnitude of TLR4 and Casp11 activation. TLR4 and Casp11 are thought to respond similarly to various lipid A structures, as tetra-acylated lipid A structures do not activate either sensor, whereas hexa-acylated structures activate both sensors. However, the precise features of lipid A that determine the differential activation of each receptor remain poorly defined, as direct analysis of extracellular and cytosolic responses to the same sources and preparations of LPS/lipid A structures have been limited. To address this question, we used rationally engineered lipid A isolated from a series of bacterial acyl-transferase mutants that produce novel, structurally defined molecules. Intriguingly, we found that the location of specific secondary acyl chains on lipid A resulted in differential recognition by TLR4 or Casp11, providing new insight into the structural features of lipid A required to activate either TLR4 or Casp11. Our findings indicate that TLR4 and Casp11 sense nonoverlapping areas of lipid A chemical space, thereby constraining the ability of Gram-negative pathogens to evade innate immunity.

TLR4 is a key pattern recognition receptor that can sense pathogen- and danger- associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro-inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species-specific, and barely predictable structure-activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan-based lipid A-mimicking molecules that can activate human and murine TLR4-mediated signaling with picomolar affinity. Exploiting crystal structure - based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible β(1→6)-linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non-reducing βGlcN(1↔1')βGlcN scaffold. Excellent stereoselectivity in a challenging β,β-1,1' glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of β,β-1,1'-linked diglucosamine-derived glycolipids entailing multiple long-chain (R)-3- acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D-molecular shape and conformational rigidity of unnatural β,β-1,1'-linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4-mediated signaling in a species-independent manner., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Sepsis is a serious medical condition characterized by bacterial infection and a subsequent massive systemic inflammatory response. In an effort to identify compounds that block lipopolysaccharide (LPS)-induced inflammation reported herein is the development of simple Lipid-A analogues that lack a disaccharide core yet still possess potent antagonistic activity against LPS. The structure of the new lead compound was developed based on predictive computational experiments. LPS antagonism by the lead compound was not straightforward, and a biphasic effect was observed suggesting a possibility of more than one binding site. An IC 50 value of 13 nM for the new compound was determined for the possible high affinity site. The combination of computational, synthetic, and biological studies revealed new structural determinants of these simplified analogues. It is expected that the acquired information will aid future design of LPS targeting glycopharmaceuticals.

The lipopolysaccharide (LPS) is a characteristic molecule of the outer leaflet of the Gram-negative bacterial outer membrane, which consists of lipid A, core oligosaccharide, and O antigen. The lipid A is embedded in outer membrane and provides an efficient permeability barrier, which is particularly important to reduce the permeability of antibiotics, toxic cationic metals, and antimicrobial peptides. LPS, an important modulator of innate immune responses ranging from localized inflammation to disseminated sepsis, displays a high level of structural and functional heterogeneity, which arise due to regulated differences in the acylation of the lipid A and the incorporation of non-stoichiometric modifications in lipid A and the core oligosaccharide. This review focuses on the current mechanistic understanding of the synthesis and assembly of the lipid A molecule and its most salient non-stoichiometric modifications.

Polymyxin B (PMB) is clinically used as a last-line therapy against life-threatening Gram-negative "superbugs". However, thorough understanding of the membrane actions of PMB at a molecular level is still lacking. In this work, a variety of bacterial membrane mimics with varying lipid compositions were built, and their interactions with PMB were systematically investigated using coarse-grained molecular dynamics simulation. PMB demonstrated characteristic preference to specific lipid species during its interaction with different membrane systems, such as the rough mutant lipipolysacchrides (Re LPS) preference in an outer membrane (OM) or the cardiolipin and POPG affinity in an inner membrane (IM). As a result of the lipid-specific actions, complicated membrane interaction states of PMB were observed, including adsorption on the OM surface. In contrast, for the IM or a mutative OM containing "impurity lipids" like POPE, POPG or lipid A, it could insert into the membrane via its acyl chain. Such actions of PMB influence the structure and lipid mobility of the membrane. In particular, the OM-bound PMB breaks the synchronous movement of Re LPS molecules in the outer leaflet and makes them diffuse more randomly, while its insertion into IM blocks the phospholipid diffusion and makes the membrane more homogeneous in the trajectory space. Our results provide insight into the action mechanism of PMB at a membrane level and a foundation for developing novel and safer polymyxin strategies for better clinical use., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Safe and effective vaccines are the best method to defeat worldwide SARS-CoV-2 and its circulating variants. The SARS-CoV-2 S protein and its subunits are the most attractive targets for the development of protein-based vaccines. In this study, we evaluated three lipophilic adjuvants, monophosphoryl lipid A (MPLA), Toll-like receptor (TLR) 1/2 ligand Pam 3 CSK 4 , and α-galactosylceramide ( α -GalCer), in liposomal and nonliposomal vaccines. The immunological results showed that the MPLA-adjuvanted liposomal vaccine induced the strongest humoral and cellular immunity. Therefore, we further performed a systematic comparison of S-trimer, S-ECD, S1, and RBD as antigens in MPLA-adjuvanted liposomes and found that, although these four vaccines all induced robust specific antibody responses, only S-trimer, S1, and RBD liposomes, but not S-ECD, elicited potent neutralizing antibody responses. Moreover, RBD, S-trimer, and S1 liposomes effectively neutralized variants (B.1.1.7/alpha, B.1.351/beta, P.1/gamma, B.1.617.2/delta, and B.1.1.529/omicron). These results provide important information for the subunit vaccine design against SARS-CoV-2 and its variants.

Enterobacter species are classified as high-priority pathogens due to high prevalence of multidrug resistance from persistent antibiotic use. For Enterobacter infections caused by multidrug-resistant isolates, colistin (polymyxin E), a last-resort antibiotic, is a potential treatment option. Treatment with colistin has been shown to lead to emergence of polymyxin resistance. The primary mechanism for colistin resistance is modification of terminal phosphate moieties of lipid A, leading to decreased membrane electronegativity and reducing colistin binding affinity. Detection of these modifications, including the addition of phosphoethanolamine and 4-amino-4-deoxy-l-arabinose (Ara4N), can be used for prediction of colistin resistance using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The objective of this study was to identify lipid A markers for colistin resistance in Enterobacter species and Klebsiella aerogenes (formerly Enterobacter aerogenes). Using a collection of Enterobacter and Klebsiella aerogenes clinical isolates, broth MICs for colistin were determined initially. Subsequently, killing assays were carried out to determine how the concentration of colistin at which there is approximately 50% survival (kill 50 ) equates to their MICs. Finally, lipid A analysis was conducted via MALDI-TOF MS using the novel rapid extraction method, termed fast lipid analysis technique (FLAT), to correlate MIC and killing efficacy with predictive lipid A modifications. Sensitivity and specificity of the MS assay compared to MIC interpretation were 100% and 53.4%, respectively. A receiver operator characteristic (ROC) demonstrated that MS was highly correlated with killing, with area under the curve of 0.97. This analysis demonstrated the potential utility of MALDI-TOF MS as a rapid diagnostic platform of colistin resistance in Enterobacter species. IMPORTANCE In this study, we develop a novel method for identifying colistin resistance in Enterobacter species and Klebsiella aerogenes without performing antimicrobial susceptibility testing. Typically, susceptibility testing requires an additional 24 to 48 h, while the MS assay described in this study allows for resistant identifications in under 1 h after initial culture. Identification using MALDI-TOF MS would save time and prevent inappropriate use of colistin. MALDI-TOF MS is an easy-to-use, readily available, robust diagnostic tool in clinical laboratories. Furthermore, this study highlights limitations of polymyxin susceptibility testing. Use of a killing assay best captures how colistin treats infection and is shown to be highly correlated with our MS assay; thus, the MS assay in this study effectively predicts how colistin would treat a patient's infection. Use of MALDI-TOF MS for accurate and early identification of antimicrobial resistance can improve antimicrobial stewardship and patient outcomes.

Lipopolysaccharides (LPS) are the main components of the external leaflet of the Gram-negative outer membrane and consist of three different moieties: lipid A, core oligosaccharide, and O-polysaccharide. The lipid A is a glucosamine disaccharide with different levels of acylation and phosphorylation, beside carrying, in certain cases, additional substituents on the sugar backbone. It is also the main immunostimulatory part of the LPS, as its recognition by the host immune system represents a fundamental event for detection of perilous microorganisms. Moreover, an uncontrolled immune response caused by a large amount of circulating LPS can lead to dramatic outcomes for human health, such as septic shock. The immunostimulant properties of an LPS incredibly vary depending on lipid A chemical structure, and for this reason, natural and synthetic variants of the lipid A are under study to develop new drugs that mimic or antagonise its natural effects. Here, we review past and recent findings on the lipid A as an antibiotic target and immune-therapeutic molecule, with a special attention on the crucial role of the chemical structure and its exploitation for conceiving novel strategies for treatment of several immune-related pathologies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aeromonas species are found in the aquatic environment, drinking water, bottled mineral water, and different types of foods, such as meat, fish, seafood, or vegetables. Some of these species are primary or opportunistic pathogens for invertebrates and vertebrates, including humans. Among the pathogenic factors associated with these species, there are the lipopolysaccharides (LPSs). LPSs are the major components of the external leaflet of Gram-negative bacterial outer membrane. LPS is a glycoconjugate, generally composed of three portions: lipid A, core oligosaccharide, and O-specific polysaccharide or O-antigen. The latter, which may be present (smooth LPS) or not (rough LPS), is the most exposed part of the LPS and is involved in the pathogenicity by protecting infecting bacteria from serum complement killing and phagocytosis. The O-antigen is a polymer of repeating oligosaccharide units with high structural variability, particularly the terminal sugar, that confers the immunological specificity to the O-antigen. In this study, we established the structure of the O-chain repeating unit of the LPS from Aeromonas bivalvium strain 868 E T (=CECT 7113 T = LMG 23376 T ), a mesophilic bacterium isolated from cockles ( Cardium sp.) and obtained from a retail market in Barcelona (Spain), whose biosynthesis core LPS cluster does not contain the waaE gene as most of Aeromonas species. After mild acid hydrolysis, the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by chemical analysis and NMR spectroscopy. The polymer consists of a heptasaccharide repeating unit containing D-GalNAc, L-Rha, D-GlcNAc, and D-FucNAc residues.

Whole-cell vaccines against Gram-negative bacteria commonly display high reactogenicity caused by the endotoxic activity of lipopolysaccharide (LPS), one of the major components of the bacterial outer membrane. Underacylation of the lipid A moiety of LPS has been related with reduced endotoxicity in several Gram-negative species. Here, we evaluated whether the inactivation of two genes encoding lipid A acylases of Bordetella bronchiseptica , i.e. pagP and lpxL1 , could be used for the development of less reactogenic vaccines against this pathogen for livestock and companion animals. Inactivation of pagP resulted in the loss of the secondary palmitate chain at position 3' of lipid A, but hardly affected the potency of the LPS to activate the Toll-like receptor 4 (TLR4). Inactivation of lpxL1 resulted in the loss of the secondary 2-hydroxy laurate group present at position 2 of lipid A and, unexpectedly, in the additional loss of the glucosamines that decorate the phosphate groups at positions 1 and 4' and in an increase in LPS molecules carrying O-antigen. The resulting LPS showed greatly reduced potency to activate TLR4 in HEK-Blue reporter cells expressing human or mouse TLR4 as well as in porcine macrophages. Characterization of the lpxL1 mutant revealed many pleiotropic phenotypes, including increased resistance to SDS and rifampicin, increased susceptibility to cationic antimicrobial peptides, decreased auto-aggregation and biofilm formation, and a tendency to decreased infectivity of macrophages, which are all related to the altered LPS structure. We suggest that the lpxL1 mutant will be useful for the generation of safer vaccines.

Opioid use disorders and fatal overdose due to consumption of fentanyl-laced heroin remain a major public health menace in the United States. Vaccination may serve as a promising potential remedy to combat accidental overdose and to mitigate the abuse potential of opioids. We previously reported the heroin and fentanyl monovalent vaccines carrying, respectively, a heroin hapten, 6-AmHap, and a fentanyl hapten, para- AmFenHap, conjugated to tetanus toxoid (TT). Herein, we describe the mixing of these antigens to formulate a bivalent vaccine adjuvanted with liposomes containing monophosphoryl lipid A (MPLA) adsorbed on aluminum hydroxide. Immunization of mice with the bivalent vaccine resulted in IgG titers of >10 5 against both haptens. The polyclonal sera bound heroin, 6-acetylmorphine, morphine, and fentanyl with dissociation constants ( K d ) of 0.25 to 0.50 nM. Mice were protected from the anti-nociceptive effects of heroin, fentanyl, and heroin +9% (w/w) fentanyl. No cross-reactivity to methadone and buprenorphine was observed in vivo . Naloxone remained efficacious in immunized mice. These results highlighted the potential of combining TT-6-AmHap and TT- para- AmFenHap to yield an efficacious bivalent vaccine that could ablate heroin and fentanyl effects. This vaccine warrants further testing to establish its potential translatability to humans.

Gram-negative bacterial cell surface component lipopolysaccharide (LPS) and its active principle, lipid A, exhibit immunostimulatory effects and have the potential to act as adjuvants. However, canonical LPS acts as an endotoxin by hyperstimulating the immune response. Therefore, LPS and lipid A must be structurally modified to minimize their toxic effects while maintaining their adjuvant effect for application as vaccine adjuvants. In the field of chemical ecology research, various biological phenomena occurring among organisms are considered molecular interactions. Recently, the hypothesis has been proposed that LPS and lipid A mediate bacterial-host chemical ecology to regulate various host biological phenomena, mainly immunity. Parasitic and symbiotic bacteria inhabiting the host are predicted to possess low-toxicity immunomodulators due to the chemical structural changes of their LPS caused by co-evolution with the host. Studies on the chemical synthesis and functional evaluation of their lipid As have been developed to test this hypothesis and to apply them to low-toxicity and safe adjuvants.

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium ( Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a β-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

Lipid A, the membrane-bound phosphoglycolipid component of bacteria, is held responsible for the clinical syndrome of gram-negative sepsis. In this study, the fragmentation behavior of a set of synthetic lipid A derivatives was studied by electrospray ionization multistage mass spectrometry (ESI-MS n ), in conjunction with tandem mass spectrometry (MS/MS), using low-energy collision-induced dissociation (CID). Genealogical insight about the fragmentation pathways of the deprotonated 4'-monophosphoryl lipid A structural analogs led to proposals of a number of alternative dissociation routes that have not been reported previously. Each of the fragment ions was interpreted using various possible mechanisms, consistent with the principles of reactions described in organic chemistry. Specifically, the hypothesized mechanisms are: (i) cleavage of the C-3 primary fatty acid leaves behind an epoxide group attached to the reducing sugar; (ii) cleavage of the C-3' primary fatty acid (as an acid) generates a cyclic phosphate connected to the nonreducing sugar; (iii) cleavage of the C-2' secondary fatty acid occurs both in acid and ketene forms; iv) the C-2 and C-2' primary fatty acids are eliminated as an amide and ketene, respectively; (v) the 0,2 A 2 cross-ring fragment contains a four-membered ring (oxetanose); (vi) the 0,4 A 2 ion is consecutively formed from the 0,2 A 2 ion by retro-aldol, retro-cycloaddition, and transesterification; and (vii) formations of H 2 PO 4 - and PO 3 - are associated with the formation of sugar epoxide. An understanding of the relation between 0,2 A 2 and 0,4 A 2 -type sugar fragments and the different cleavage mechanisms of the two ester-linked primary fatty acids is invaluable for distinguishing lipid A isomers with different locations of a single ester-linked fatty acid (i.e., at C-3 or C-3'). Thus, in addition to a better comprehension of lipid A fragmentation processes in mass spectrometers, our observations can be applied for a more precise elucidation of naturally occurring lipid A structures.

Toxoplasma gondii ( T. gondii ) infection causes severe zoonotic toxoplasmosis, which threatens the safety of almost one-third of the human population globally. However, there is no effective protective vaccine against human toxoplasmosis. This necessitates anti- T. gondii vaccine development, which is a main priority of public health. In this study, we optimized the adjuvant system 04 (AS04), a vaccine adjuvant constituted by 3-O-desacyl-4'-monophosphoryl lipid A (a TLR4 agonist) and aluminum salts, by packing it within natural extracts of β-glucan particles (GPs) from Saccharomyces cerevisiae to form a GP-AS04 hybrid adjuvant system. Through a simple mixing procedure, we loaded GP-AS04 particles with the total extract (TE) of T. gondii lysate, forming a novel anti- T. gondii vaccine GP-AS04-TE. Results indicated that the hybrid adjuvant can efficiently and stably load antigens, mediate antigen delivery, facilitate the dendritic uptake of antigens, boost dendritic cell maturation and stimulation, and increase the secretion of pro-inflammatory cytokines. In the mouse inoculation model, GP-AS04-TE significantly stimulated the function of dendritic cells, induced a very strong TE-specific humoral and cellular immune response, and finally showed a strong and effective protection against toxoplasma chronic and acute infections. This work proves the potential of GP-AS04 for exploitation as a vaccine against a range of pathogens.