Aspergillus Fumigatus Secretes Proteases That Activate SARS-CoV-2 Spike Protein, Assisting COVID-19 Progression!

COVID-19 News: The ongoing battle against SARS-CoV-2 and its associated disease, COVID-19, has taken numerous unexpected turns. Recent studies have unveiled a potential connection between Aspergillus fumigatus, a common fungal pathogen, and the aggravation of COVID-19 progression. This revelation stems from the discovery that A. fumigatus secretes a protease(s) capable of activating the SARS-CoV-2 spike protein. In this COVID-19 News report, we delve into the background, methods, results, and implications of this groundbreaking research conducted by the University of the Free State and Durban University of Technology in South Africa.


The intermolecular interactions of the docked furin protease-SARS-CoV-2 spike protein complex and A. fumigatus alkaline protease 1-SARS-CoV-2 spike protein showing the amino acid residues of each protease that interacted with the spike protein.

Background: COVID-19 and Fungal Co-Infections
As the COVID-19 pandemic unfolded, clinicians observed an alarming trend – the co-manifestation of microbial infections, particularly fungal infections, in patients battling COVID-19. Clinical case-control studies revealed that the intense inflammatory response triggered by COVID-19 facilitates conditions conducive to fungal infections, with a notable association between COVID-19 and aspergillosis caused by A. fumigatus. This fungal infection often emerges within days of intensive care unit admission, and its mortality rate in COVID-19 patients is reported to be a staggering 56%. Recognizing the significance of A. fumigatus as a potential serious complication in COVID-19, the fungus was included in the World Health Organization's fungal priority pathogens list. This study emerged from the need to understand the interaction between A. fumigatus and SARS-CoV-2, particularly the role of fungal proteases in activating the viral spike protein.
 
Methods: Exploring Protease Activity and Molecular Docking
To investigate the hypothesis that A. fumigatus secretes proteases capable of activating the SARS-CoV-2 spike protein, the researchers employed a multi-faceted approach. Initially, the culture media's supernatant was analyzed for protease activity. Subsequently, the researchers utilized a fluorogenic mimetic peptide of the SARS-CoV-2 spike protein, containing the S1/S2 site, to assess proteolytic activity. Complementing these experimental analyses, molecular docking using HADDOCK was employed to predict the binding affinity between A. fumigatus alkaline protease 1 and the SARS-CoV-2 spike protein.
 
Results: A. fumigatus Protease Activation of SARS-CoV-2 Spike Protein
The study's findings provide compelling evidence that A. fumigatus secretes protease(s) capable of activating the SARS-CoV-2 spike protein. The in-silico analysis revealed a strong binding affinity between A. fumigatus alkaline protease 1 and the spike protein, surpassing that of the human furin protease, a reference serine-based protease. Docking scores, electrostatic energy, and Van der Waals energy calculations supported t he notion that A. fumigatus alkaline protease 1 could efficiently bind to the SARS-CoV-2 spike protein.
 
The subsequent enzymatic assays confirmed the proteolytic activity of A. fumigatus supernatant, highlighting its ability to cleave a mimetic peptide resembling the furin cleavage site in the SARS-CoV-2 spike protein. Importantly, the study progressed beyond mimetic peptides, using a full-length spike protein present in pseudovirions to demonstrate A. fumigatus supernatant's capacity to transduce HEK-293T cells more effectively than recombinant furin.
 
In-Depth Analysis: Comparative Insights with Human Furin Protease
To gain a deeper understanding of the potential impact of A. fumigatus alkaline protease 1 on the SARS-CoV-2 spike protein, the researchers conducted comparative analyses with human furin protease. The in-silico data revealed that A. fumigatus alkaline protease 1 exhibited a more negative HADDOCK docking score than furin protease when docked with the SARS-CoV-2 spike protein.

The docking scores are indicative of the orientation and binding affinity of a protein or enzyme at the binding domain of another. In this context, a more negative score suggests a superior binding affinity. Additionally, the buried surface area, reflecting the amount of protein surface not in contact with water upon complexation, was significantly greater for A. fumigatus alkaline protease 1 compared to furin protease. This implies a stronger binding affinity and a larger interaction surface area, further supporting the notion that A. fumigatus alkaline protease 1 could be a potent activator of the SARS-CoV-2 spike protein.
 
Furthermore, amino acid residue analyses revealed distinct interactions between furin protease and A. fumigatus alkaline protease 1 with the spike protein cleavage site. The two proteases exhibited different orientation preferences in the binding site, with furin protease forming more hydrogen bonds in the complex compared to A. fumigatus alkaline protease 1. This suggests that while both proteases interact with the cleavage site, the nature of these interactions differs, potentially influencing their respective abilities to activate the SARS-CoV-2 spike protein.
 
Enzymatic Evidence: Unraveling the Mechanism
The enzymatic evidence presented in the study not only reinforces the computational predictions but also sheds light on the mechanism through which A. fumigatus supernatant activates the SARS-CoV-2 spike protein. The fluorogenic assay demonstrated the biochemical efficiency of A. fumigatus supernatant in cleaving a mimetic peptide that contains a unique amino acid sequence (SPRRAR↓S), positioned at the interface between the S1 and S2 sites of the spike protein. This sequence serves as a cleavage site for human furin protease, indicating the potential of A. fumigatus supernatant to mimic the furin protease activity in activating the spike protein.
 
However, it is essential to note the limitations associated with mimetic peptides, which might not precisely mimic the cleavage patterns of a full-length SARS-CoV-2 spike protein due to conformational differences. To address this concern, the study employed a more physiologically relevant approach by using a full-length spike protein present in pseudovirions for transduction studies. The results demonstrated that A. fumigatus supernatant surpassed recombinant furin in transducing HEK-293T cells with SARS-CoV-2 pseudovirions, further confirming the fungal protease's ability to activate the spike protein and facilitate viral entry into host cells.
 
Discussion: Implications for COVID-19 Management
This research unravels a previously unrecognized aspect of the complex interplay between viral and fungal infections. The potential of A. fumigatus' protease(s) to activate the SARS-CoV-2 spike protein raises critical questions about the impact of fungal co-infections on COVID-19 progression. The in-depth computational and experimental analyses provide a foundation for understanding the molecular mechanisms underlying this interaction.
 
Comparative analyses with human furin protease underscore the potency of A. fumigatus alkaline protease 1 in binding to and potentially activating the SARS-CoV-2 spike protein. The study's findings suggest that, in the presence of A. fumigatus and SARS-CoV-2 co-infection, the fungus could exacerbate COVID-19 development by activating the viral spike protein, thus enhancing viral entry and potentially worsening disease outcomes.
 
Furthermore, the implications of this discovery extend beyond the immediate context of COVID-19. The activation of viral proteins by fungal proteases opens new avenues for understanding viral pathogenesis and host-pathogen interactions. It prompts us to consider the broader spectrum of viral and fungal interactions and their impact on human health. Future research endeavors may explore the potential of other viral proteins, such as the influenza hemagglutinin, to be activated by fungal proteases, leading to enhanced viral infectivity.

In the realm of clinical management, the recognition of fungal co-infections in COVID-19 patients becomes paramount. The identification of A. fumigatus as a potential contributor to COVID-19 progression underscores the importance of vigilant monitoring for fungal infections, especially in critically ill patients. Early detection and treatment of fungal co-infections may mitigate their detrimental effects on COVID-19 outcomes and improve patient survival.
 
Moreover, the study's findings highlight the potential utility of targeted therapeutic interventions to disrupt the interaction between fungal proteases and viral proteins. Strategies aimed at inhibiting fungal protease activity or blocking viral entry pathways mediated by fungal proteases could offer novel approaches to managing COVID-19 and associated complications. Development of protease inhibitor compounds tailored to inhibit fungal proteases involved in viral activation may represent a promising avenue for future therapeutic interventions.
 
However, it is essential to proceed with caution in the development and implementation of such therapies, considering potential side effects and unintended consequences. Comprehensive preclinical studies and clinical trials are warranted to evaluate the safety and efficacy of targeted therapeutic strategies aimed at disrupting fungal-viral interactions in COVID-19 patients.
 
Conclusion: Paving the Way for Future Research and Therapeutic Strategies
In conclusion, this study provides valuable insights into the intricate interplay between fungal infections and COVID-19, revealing the potential of A. fumigatus protease(s) to activate the SARS-CoV-2 spike protein and exacerbate disease progression. The comprehensive approach, integrating computational modeling, enzymatic assays, and transduction studies, offers a robust framework for understanding the molecular mechanisms underlying fungal-viral interactions.
 
As we navigate the complexities of infectious disease dynamics, fueled by the convergence of viral and fungal pathogens, this study serves as a beacon of hope, illuminating new pathways for understanding and combatting COVID-19 and its associated complications. By unraveling the intricate interplay between Aspergillus fumigatus protease(s) and SARS-CoV-2 spike protein, we pave the way for future research endeavors and therapeutic strategies aimed at mitigating the impact of fungal co-infections on COVID-19 progression.
 
The study findings were published in the peer reviewed Virology Journal.
https://virologyj.biomedcentral.com/articles/10.1186/s12985-024-02331-z
 
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