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 consortium of authors from Sweden, Singapore, and Denmark in several studies investigated the specific interaction between the SARS-CoV-2 spike (S) protein and bacterial lipopolysaccharide (LPS).
The S protein, a major pathogenic factor in the unique pathogenesis of SARS-CoV-2, 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. The toll-like receptor 4 (TLR4) recognizes LPS, activating the TLR4 pathway and massive release of cytokines. Overstimulation of the TLR4 pathway by LPS triggers a hyperinflammatory state, that can lead to sepsis and acute respiratory distress syndrome (ARDS). Previous studies have shown that patients with severe COVID-19 and non-survivors during hospitalization have significantly elevated levels of bacterial LPS. In addition, previous data demonstrated that patients with metabolic syndrome and high blood levels 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 ARDS.
The outer membrane of Gram-negative bacteria
About the Studies and Results
The interaction between the SARS-CoV-2 S protein and lipopolysaccharide
In 2020, Petruk G et al. demonstrated a previously unrecognized interaction between the SARS-CoV-2 S protein and bacterial LPS. They have shown that a combination of the SARS-CoV-2 S protein and low concentrations of LPS boosted inflammatory responses in human peripheral blood mononuclear cells (PBMC) and monocytic THP-1 cells, immortalized monocyte-like cell line from the peripheral blood of a childhood case of acute monocytic leukemia.
The S protein alone did not increase the activation of nuclear factor-kappa B (NF-κB). This ancient protein transcription factor regulates multiple aspects of innate and adaptive immune functions 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 boosting of cytokines directly dependent on NF-κB activation, such as tumor necrosis factor-alpha and interleukin-6.
To investigate the pro-inflammatory effects of S1 and S2 subunits of the S protein, the researchers incubated monocytic THP-1 cells with increasing concentrations of LPS and constant concentrations of S1 or S2 subunits and measured the NF-κB levels after 20 hours. Of note, the S protein preparations were contaminated with LPS, and researchers took this into account.
The results showed that the proinflammatory activity of the S2 subunit correlated with the presence of LPS contaminants, and this effect was not seen for the S1 subunit. To determine whether a stronger proinflammatory effect of the S2 subunit resulted from its contamination with LPS, the experiments in monocytic THP-1 cells were repeated with polymyxin B, a neutralizer of LPS. The administration of polymyxin B suppressed the activation of NF-κB by the S2 subunit alone or the S2 subunit mixed with LPS, but not by the S1 subunit alone or S1 mixed with LPS.
In vivo investigation showed that administration of the S1 subunit alone did not result in any measurable boosting of NF-κB activation, but, the administration of the S1 in combination with subcutaneously administrated LPS resulted in a significant proinflammatory response. The authors then investigated in vitro whether the interaction of the S1 subunit with proteases secreted in the tissue during inflammation, such as neutrophil elastase, affects the affinity of the S1 to LPS. The findings revealed that the S1 subunit mixed with LPS and then digested with neutrophil elastase boosted NF-κB activation. According to these results, the S1 subunit displayed a boosting effect on LPS-induced inflammation in vivo but not in vitro, suggesting some additional mechanisms in the former.
Importantly, 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
One of the studies of this consortium of authors has shown that LPS binds to multiple hydrophobic pockets in the S1 and S2 subunits, with a lower affinity to the S2 pockets than the S1 pockets. They also investigated the molecular mechanism of the S protein aggregation in the presence of LPS.
The researchers explored regions with positive aggregation propensity scores, which indicates a high propensity for aggregation, and found two LPS-binding pockets with positive aggregation propensity scores on the S protein, loop 246–250 on the NTD and loop 621–624 near the C-terminal domain 2 (CTD2). Since the previous in vitro studies demonstrated that the S protein forms amyloid fibrils, it is worth noting that the loop 621–624 with a positive aggregation propensity score is adjacent to peptides 601–620, which is one of three peptides (192–211, 601–620, and 1166–1185) that meet the criteria for amyloid fibrils.
Electron and fluorescence microscopy, used to examine the size of the S protein aggregates before and after the LPS challenge, demonstrated that the S protein aggregates formed in the presence of LPS were significantly larger than those formed by the S protein alone. Administration of TCP-25 peptide that blocks the LPS-triggering effect, reversed the S protein aggregation. This confirmed 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. However, further studies are needed to investigate how LPS drives the formation of large S protein aggregates.
The original figure from the article by Samsudin F et al. Journal of Molecular Cell Biology (2022)
Conclusion
These studies have shown that binding of the bacterial LPS at multiple sites of the SARS-CoV-2 S protein enhanced proinflammatory responses in vitro and in vivo and led to the S protein aggregate formation. According to the authors, the S protein acts as an additional transport system for LPS to its receptors, as a mediator rather than a direct cause of hyperinflammation.
These findings have established a significant link between excessive inflammation during SARS-CoV-2 infection and comorbidities associated with increased levels of bacterial endotoxins. This synergism between LPS and the S protein is of clinical and therapeutic importance.
In addition, as some recent computational studies showed that the S protein could bind to several aggregation-prone amyloid proteins, it is imperative to investigate whether LPS under certain conditions may trigger in vivo aggregation of the S proteins, and the formation of amyloids through the interaction between the S protein/LPS complexes and other amyloidogenic proteins.
These articles were published in the Journal of Molecular and Cell Biology and FEBS Letters
Journal References
Petruk GPM et al. SARS-CoV-2 spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity. Journal of Molecular Cell Biology 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
