Lipopolysaccharide Engineering in Neisseria meningitidis

Pupo, Elder; Hamstra, Hendrik-Jan; Meiring, Hugo D.; Ley, Peter van der

doi:10.1074/jbc.m114.554345

Cited by 23 publications

(24 citation statements)

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“…Neisseria meningitidis typically produces hexa-acylated LPS with phosphate and phosphoethanolamine groups appended to the 1 and 4′ position of the lipid A1314. Heterologous expression of LPS modifying enzymes such as PagL or deletion of lipid A biosynthesis enzymes such as LpxL1 and LpxL2 has been used to detoxify the highly active meningococcal LPS1315.…”

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confidence: 99%

“…Heterologous expression of LPS modifying enzymes such as PagL or deletion of lipid A biosynthesis enzymes such as LpxL1 and LpxL2 has been used to detoxify the highly active meningococcal LPS1315. Deletion of LpxL1 was shown to be an advantageous method for detoxifying meningococcal LPS when making meningococcal outer membrane vesicle vaccines, without the need to use a detergent to reduce excess LPS-related reactogenicity16.…”

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confidence: 99%

“…However, the activity of this modified LPS has been reduced to the extent that it barely induces any activation of the TLR4/MD-2 complex on human cells, making it less applicable as a stand-alone vaccine adjuvant17. Heterologous expression of pagL in N. meningitidis results in a different attenuated penta-acylated LPS structure, which is still capable of inducing TLR4 activation and induces a TRIF-biased cytokine production on a human monocytic cell line13. The challenge of LPS-based adjuvants is finding the optimal balance between retaining a sufficient amount of immune activation while limiting toxic side effects.…”

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Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide

Zariri

Pupo

Riet

et al. 2016

Sci Rep

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Neisseria meningitidis contains a very potent hexa-acylated LPS that is too toxic for therapeutic applications. We used systematic molecular bioengineering of meningococcal LPS through deletion of biosynthetic enzymes in combination with induction of LPS modifying enzymes to yield a variety of novel LPS mutants with changes in both lipid A acylation and phosphorylation. Mass spectrometry was used for detailed compositional determination of the LPS molecular species, and stimulation of immune cells was done to correlate this with endotoxic activity. Removal of phosphethanolamine in lipid A by deletion of lptA slightly reduces activity of hexa-acylated LPS, but this reduction is even more evident in penta-acylated LPS. Surprisingly, expression of PagL deacylase in a penta-acylated lpxL1 mutant increased LPS activity, contradicting the general rule that tetra-acylated LPS is less active than penta-acylated LPS. Further modification included expression of lpxP, an enzyme known to add a secondary 9-hexadecenoic acid to the 2’ acyl chain. The LpxP enzyme is temperature-sensitive, enabling control over the ratio of expressed modified hexa- and penta-acylated LPS by simply changing the growth temperature. These LPS derivatives display a broad range of TLR4 activity and differential cytokine induction, which can be exploited for use as vaccine adjuvant or other TLR4-based therapeutics.

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Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide

Zariri

Pupo

Riet

et al. 2016

Sci Rep

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“…MM6 cells do not express DC-SIGN (data not shown), and thus the oligosaccharide would not influence binding, internalization, and activity. The basis for the limited effect of lgtB pagL ϩ nOMVs on the IP-10 response compared to the reduced release of proinflammatory cytokines IL-6 and IL-1␤ is unclear, but this has been previously observed for purified pagL LPS (16). Activation of TLR4 and MD-2 very much depends on how the lipid A structure is situated inside the MD-2 pocket and whether the two dimerization interfaces can be formed (31).…”

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confidence: 99%

“…1). These penta-acylated lipid A forms show reduced endotoxic activity compared to that of hexa-acylated LPS (16). The combination of rmpM deletion and genetic LPS modification thus enables the isolation of high yields of so-called native OMVs (nOMVs) with reduced endotoxicity without the need for deoxycholate treatment.…”

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Meningococcal Outer Membrane Vesicle Composition-Dependent Activation of the Innate Immune Response

et al. 2016

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cMeningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen-associated molecular patterns, including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an extremely potent hexa-acylated LPS, leading to adverse effects when its OMVs are applied as vaccines. To create safe OMV vaccines, detergent treatment is generally used to reduce the LPS content. While effective, this method also leads to loss of protective antigens such as lipoproteins. Alternatively, genetic modification of LPS can reduce its toxicity. In the present study, we have compared the effects of standard OMV isolation methods using detergent or EDTA with those of genetic modifications of LPS to yield a penta-acylated lipid A (lpxL1 and pagL) on the in vitro induction of innate immune responses. The use of detergent decreased both Toll-like receptor 4 (TLR4) and TLR2 activation by OMVs, while the LPS modifications reduced only TLR4 activation. Mutational removal of PorB or lipoprotein factor H binding protein (fHbp), two proteins known to trigger TLR2 signaling, had no effect, indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent-treated OMVs and lpxL1 OMVs showed similar reductions of cytokine profiles in the human monocytic cell line MM6 and human dendritic cells (DCs). OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of proinflammatory cytokines interleukin-6 (IL-6) and IL-1␤ but not of IP-10, a typical TRIF-dependent chemokine. Taken together, these data show that lipid A modification can be used to obtain OMVs with reduced activation of innate immunity, similar to what is found after detergent treatment. G ram-negative bacteria have the ability to naturally shed vesicles from the outer membrane. These spontaneous outer membrane vesicles (sOMVs) are composed of lipids, outer membrane proteins, lipopolysaccharide (LPS), and some periplasmic proteins. Shedding of sOMVs occurs without significant disruption in the integrity of the cell, and release of vesicles is increased by bacterial stress responses (1, 2). Some of the proposed functions of shedding of outer membrane blebs include protection and transport of secreted molecules, interaction with host cells, evasion of immunity by action as a decoy for antibodies and other antibacterial molecules, and action as nucleators in biofilm formation (3).The fact that OMVs are nonreplicating structures that contain many crucial surface components and virulence factors, in combination with pathogen-associated molecular patterns (PAMPs) that trigger innate immune responses, makes them attractive as candidate vaccines. This is evidenced by the successful control of meningococcal serogroup B epidemics using OMV vaccines (4-6) and experimental studies that have explored the vaccine potential of OMVs against other bacterial pathogens (7, 8). The isolation of OMVs from Neiss...

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Nonreducing Sugar Scaffold Enables the Development of Immunomodulatory TLR4‐specific LPS Mimetics with Picomolar Potency

Strobl,

Zucchetta,

Vašíček

et al. 2024

Angewandte Chemie

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Innate immune defense mechanisms against infection and cancer encompass the modulation of pattern recognition receptor (PRR)‐mediated inflammation, including upregulation of various transcription factors and the activation of pro‐inflammatory pathways important for immune surveillance. Dysfunction of PRRs‐mediated signaling has been implicated in cancer and autoimmune diseases, while the overactivation of PRRs‐driven responses during infection can lead to devastating consequences such as acute lung injury or sepsis. We used crystal structure‐based design to develop immunomodulatory lipopolysaccharide (LPS) mimetics targeting one of the ubiquitous PRRs, toll‐like receptor 4 (TLR4). Taking advantage of an exo‐anomeric conformation and specific molecular shape of synthetic nonreducing β,β‐diglucosamine, which was investigated by NMR, we developed two sets of Lipid A mimicking glycolipids capable of either potently activating innate immune responses or inhibiting pro‐inflammatory signaling. Stereoselective 1,1'‐glycosylation towards fully orthogonally protected nonreducing GlcNβ(1↔1')βGlcN followed by stepwise assembly of differently functionalised phosphorylated glycolipids provided biologically active molecules that were evaluated for their ability to trigger or to inhibit cellular innate immune responses. Two LPS mimetics, identified as potent TLR4‐specific inducers of the intracellular signaling pathways, serve as vaccine adjuvant‐ and immunotherapy candidates, while anionic glycolipids with TLR4‐inhibitory potential hold therapeutic promise for the management of acute or chronic inflammation.

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Lipopolysaccharide Engineering in Neisseria meningitidis

Abstract: Background: LPS biosynthesis modification offers the potential to create detoxified variants with improved immunostimulatory properties. Results: Pentaacyl LPS molecules from pagL ϩ and lpxL1 Ϫ meningococcal mutants were structurally characterized and found to

Cited by 23 publications

References 44 publications

Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide

Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide

Meningococcal Outer Membrane Vesicle Composition-Dependent Activation of the Innate Immune Response

Nonreducing Sugar Scaffold Enables the Development of Immunomodulatory TLR4‐specific LPS Mimetics with Picomolar Potency

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