The consortium of authors from Sweden, Singapore, and Denmark published a series of studies on the specific interaction between the SARS-CoV-2 spike (S) protein and bacterial lipopolysaccharide (LPS). Their findings demonstrated that the S protein binds to LPS through multiple sites on the S1 and S2 subunits. This interaction enhanced the inflammatory response in vitro and in vivo and led to the S protein aggregate formation.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus. Its genome encodes four structural proteins, namely the spike (S), envelope (E), nucleocapsid (N), and membrane (M) protein. The S protein appears to be a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. The S protein is a glycosylated homotrimer with each monomer composed of subunits S1 and S2, separated by host cell proteases. The S1 subunit is composed of the N-terminal domain (NTD), the receptor binding domain (RBD) with a receptor binding motif, and two C-terminal domains (CTD).
The bacterial LPS is the main component of the outer membrane of Gram-negative bacteria. LPS is recognized by the toll-like receptor 4 (TLR4), which leads to activation of the TLR4 pathway and massive release of cytokines. The levels of bacterial LPS are significantly elevated in patients with severe COVID-19 and non-survivors during hospitalization. Patients with metabolic syndrome, associated with a high blood level of bacterial LPS due to gut dysbiosis and translocation of bacterial components into the systemic circulation, are at a higher risk of developing severe COVID-19 with sepsis and acute respiratory distress syndrome. Interestingly, a recent animal study performed in a mouse model that expresses the S1 subunit of the S protein in the olfactory cavity has shown that enhanced systemic inflammation by intraperitoneal injection of LPS resulted in findings specific to GABA-ergic neurons. The results demonstrated markedly reduced expression of calbindin, a marker of γ-aminobutyric acid (GABA)-ergic neurons in mouse brains, and the absence of any changes in other neuronal differentiation markers. https://discovermednews.com/s1-protein-causes-brain-inflammation-and-decreases-the-acetylcholine-levels/
About the studies
In 2020, Petruk G et al. demonstrated a previously unrecognized interaction between the SARS-CoV-2 S protein and LPS. Their results have shown that S protein boosted the LPS response, enhancing inflammation in vitro and in vivo. A combination of the S protein and low concentrations of LPS boosted inflammatory responses in peripheral blood mononuclear cells isolated from human blood and monocytic THP-1 cells, immortalized monocyte-like cell line derived from the peripheral blood of a childhood case of acute monocytic leukemia.
The S protein alone did not increase nuclear factor-kappa B (NF-κB) activation. NF-kB is an ancient protein transcription factor that regulates multiple aspects of innate and adaptive immune functions and serves as a pivotal mediator of inflammatory responses. However, a combination of the S protein and extremely low concentrations of LPS significantly enhanced the activation of NF-κB in monocytic THP-1 cells. In human PBMCs, the combination of the S protein and extremely low concentrations of LPS induced significant cytokine boosting directly dependent on NF-κB activation, such as tumor necrosis factor-alpha and interleukin-6.
To investigate the proinflammatory effects of S1 and S2 subunits of the S protein, researchers incubated monocytic THP-1 cells with increasing concentrations of LPS and a constant amount of S1 or S2 subunits and measured the levels of NF-κB after 20 hours. The S protein preparations were contaminated with LPS, and researchers took this into account. Notably, the intrinsic proinflammatory activity of S2 preparations was correlated with the presence of LPS contaminants, but this effect was not observed for the S1 subunit, alone or in combination with LPS.
To determine whether a stronger proinflammatory effect was found for the S2 subunit because of its contamination with LPS, the experiments in monocytic THP-1 cells were repeated with polymyxin B, a neutralizer of LPS. Polymyxin B suppressed the NF-κB activation by S2 alone or S2 mixed with LPS. However, there was no alteration in the NF-κB activation in THP-1 cells treated with the S1 alone or S1 mixed with LPS and polymyxin B. The authors stated that these findings support the hypothesis that the S protein acts as a mediator rather than a direct cause of hyperinflammation.
Scientists then examined whether an interaction with proteases secreted in the tissue during inflammation, such as neutrophil elastase, could modify the structure of the S1 subunit and alter its affinity to LPS. They incubated THP-1 cells with the digested or undigested S1 subunit in the absence or presence of LPS. Interestingly, the results showed that S1 mixed with LPS and then digested with neutrophil elastase boosted NF-κB activation.
Finally, the researchers investigated the in vivo inflammatory response. Administration of S1 alone did not result in any measurable boosting of NF-κB activation, however, the administration of S1 in combination with subcutaneously administrated LPS resulted in a significant proinflammatory response. According to these results, the S1 subunit displayed a boosting effect on LPS-induced inflammation in vivo but not in vitro, suggesting additional mechanisms in the former.
Notably, the LPS-binding capability and proinflammatory boosting effect of the Omicron S protein were reduced compared to those of the Wuhan strain both in vivo and in vitro.
The molecular mechanism of the S protein aggregation in the presence of LPS
The researchers discovered that LPS binds to multiple hydrophobic pockets in the S1 and S2 subunits. The LPS bound to cryptic pockets in the NTD and RBD in the S1 subunit and to a large groove between the S protein monomers in the S2 subunit. In addition, the LPS binds to the S2 pocket with a lower affinity than to the S1 pockets.
The authors also examined regions with positive aggregation propensity scores to understand the molecular mechanism of the S protein aggregation in the presence of LPS. A positive aggregation propensity score indicates a high propensity for aggregation. Two LPS-binding pockets with positive aggregation propensity scores were found on the S protein, loop 246–250 on the NTD and loop 621–624 near the C-terminal domain 2 (CTD2). Importantly, the loop 621–624 is adjacent to peptide 601–620.
Previous in vitro studies demonstrated that the S protein forms amyloid fibrils. Three peptides 192–211, 601–620, and 1166–1185 meet the criteria for amyloid fibrils. Notably, these findings revealed that loop 621–624 in the CTD2 domain, with a high propensity for aggregation, is adjacent to peptide 601–620.
The authors then used electron and fluorescence microscopy to further investigate the size of S protein aggregates before and after the LPS challenge. In the presence of LPS, the S proteins formed aggregates significantly larger than those formed by the S protein alone.
To investigate whether the aggregation of S protein was dependent on LPS, researchers used the TCP-25 peptide that blocks the LPS-triggering effect. Administration of TCP-25 reversed the S protein aggregation, confirming the role of LPS in this process. According to the authors, these results showed that complexes of the S protein and LPS can form stable aggregates, but further studies are needed to investigate how LPS drives the formation of large S protein aggregates.
Original figure from the article by Samsudin F et al. Journal of Molecular Cell Biology (2022)
Conclusion
These studies have shown that LPS binds to the SARS-CoV-2 S protein at multiple sites, enhancing proinflammatory responses. The molecular mechanism of the interaction between the SARS-CoV-2 S protein and bacterial LPS has been described in detail, suggesting that this interaction leads to enhanced inflammatory responses. Overstimulation of the TLR4 pathway by LPS triggers a hyperinflammatory state, that can lead to sepsis and ARDS.
These findings have established a significant link between excessive inflammation during SARS-CoV-2 infection and comorbidities associated with increased levels of bacterial endotoxins. According to the authors, the S protein acts as an additional transport system for LPS to its receptors. These findings on the synergism between LPS and the S protein are of clinical and therapeutic importance.
Recent computational studies showed that the S protein could bind to several aggregation-prone amyloid proteins such as Aβ, α-synuclein, tau, prions, and TAR DNA binding protein-43. Therefore, it is imperative to investigate whether LPS under certain conditions may similarly trigger the aggregation of S proteins in vivo, and the formation of amyloids via the interaction of S protein/LPS complexes with other amyloidogenic proteins. Understanding the relationship between the S protein aggregation and amyloid formation will have important implications for therapeutic interventions for neuropathologies associated with SARS-CoV-2 infection.
Journal References
Petruk GPM et al. SARS-CoV-2 spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity. J Mol Cell Biol. 2020;12 (12):916–32. (Open Access) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799037/
Petrlova J, et al. SARS-CoV-2 spike protein aggregation is triggered by bacterial lipopolysaccharide. FEBS Letters 596 (2022) 2566–2575. (Open Access) https://febs.onlinelibrary.wiley.com/doi/full/10.1002/1873-3468.14490
Samsudin F et al. SARS-CoV-2 spike protein as a bacterial lipopolysaccharide delivery system in an overzealous inflammatory cascade. Journal of Molecular Cell Biology (2022), 14(9), mjac058 (Open Access) https://academic.oup.com/jmcb/article/14/9/mjac058/6761401
