SARS-CoV-2 spike protein as a bacterial lipopolysaccharide delivery system in an overzealous inflammatory cascade

  1. Firdaus Samsudin
  2. Palur Raghuvamsi
  3. Ganna Petruk
  4. Manoj Puthia
  5. Jitka Petrlova
  6. Paul MacAry
  7. Ganesh S Anand
  8. Peter J Bond
  9. Artur Schmidtchen

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Abstract

Accumulating evidence indicates a potential role for bacterial lipopolysaccharide (LPS) in the overactivation of the immune response during SARS-CoV-2 infection. LPS is recognized by Toll-like receptor 4, mediating proinflammatory effects. We previously reported that LPS directly interacts with SARS-CoV-2 spike (S) protein and enhances proinflammatory activities. Using native gel electrophoresis and hydrogen–deuterium exchange mass spectrometry, we showed that LPS binds to multiple hydrophobic pockets spanning both the S1 and S2 subunits of the S protein. Molecular simulations validated by a microscale thermophoresis binding assay revealed that LPS binds to the S2 pocket with a lower affinity compared to S1, suggesting a role as an intermediate in LPS transfer. Congruently, nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells is strongly boosted by S2. Using NF-κB reporter mice followed by bioimaging, a boosting effect was observed for both S1 and S2, with the former potentially facilitated by proteolysis. The Omicron S variant binds to LPS, but with reduced affinity and LPS boosting in vitro and in vivo. Taken together, the data provide a molecular mechanism by which S protein augments LPS-mediated hyperinflammation.

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  1. Version published to 10.1093/jmcb/mjac058
  2. Rapid Reviews Infectious Diseases

    Peter A Ward

    Review 2: "SARS-CoV-2 spike protein as a bacterial lipopolysaccharide delivery system in an overzealous inflammatory cascade"

    This preprint explores binding of SARS-CoV-2 spike protein with lipopolysaccharide (LPS), and its role in increased inflammatory response. Reviewers find presented evidence reliable, while exercising caution with presented generalizations regarding use of drugs blocking TLR4-LPS.

  3. Rapid Reviews Infectious Diseases

    Chiranjib Chakraborty

    Review 1: "SARS-CoV-2 spike protein as a bacterial lipopolysaccharide delivery system in an overzealous inflammatory cascade"

    This preprint explores binding of SARS-CoV-2 spike protein with lipopolysaccharide (LPS), and its role in increased inflammatory response. Reviewers find presented evidence reliable, while exercising caution with presented generalizations regarding use of drugs blocking TLR4-LPS.

  5. ScreenIT

    SciScore for 10.1101/2021.10.29.466401: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies against the His-tag (1:2000, Invitrogen) were followed by secondary HRP conjugated antibodies (1:2000, Dako, Denmark), for detection of both proteins.
    His-tag
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Sf9 cells transfected by following were used to express the S protein by following bac-to-bac baculovirus expression system (Thermo Fisher, SG).
    Sf9
    suggested: CLS Cat# 604328/p700_Sf9, RRID:CVCL_0549)
    SARS-CoV-2 S, SARS-CoV-2 S1 and two variants of SARS-CoV-2 S2 proteins, produced by ACROBiosystems (USA), were used for BN-PAGE, MST and to stimulate THP-1 cells and for in vivo experiments.
    THP-1
    suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse inflammation model and in vivo imaging: BALB/c tg (NF-B-RE-Luc)-Xen reporter mice (Taconic Biosciences, Albany, NY, USA, 10– 12 weeks old) were used to study the immunomodulatory effects of SARS-CoV-2 S protein subunits alone or in combination with LPS.
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Software and Algorithms
    SentencesResources
    The image was obtained using a Gel Doc Imager (Bio-Rad Laboratories,
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    The initial coordinates of E. coli lipid A and LPS were obtained from CHARMM-GUI LPS modeller,62 while the initial coordinates of the S protein were extracted from the cryo-EM structure of S ECD in the closed state (PDB: 6XR8)23 with missing loops constructed using Modeller version 9.21.63 Protein and lipid were parameterized using the CHARMM36 forcefield.
    Modeller
    suggested: (MODELLER, RRID:SCR_008395)
    70 All simulations were performed using GROMACS 201871 and the trajectories visualised in VMD.
    GROMACS
    suggested: (GROMACS, RRID:SCR_014565)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 31. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  6. Version published to 10.1101/2021.10.29.466401v3 on bioRxiv
  7. Version published to 10.1101/2021.10.29.466401v2 on bioRxiv
  8. Version published to 10.1101/2021.10.29.466401v1 on bioRxiv