Protein Arginylation Is Regulated during SARS-CoV-2 Infection.

BACKGROUND: In 2019, the world witnessed the onset of an unprecedented pandemic. By February 2022, the infection by SARS-CoV-2 has already been responsible for the death of more than 5 million people worldwide. Recently, we and other groups discovered that SARS-CoV-2 infection induces ER stress and activation of the unfolded protein response (UPR) pathway. Degradation of misfolded/unfolded proteins is an essential element of proteostasis and occurs mainly in lysosomes or proteasomes. The N-terminal arginylation of proteins is characterized as an inducer of ubiquitination and proteasomal degradation by the N-degron pathway. RESULTS: The role of protein arginylation during SARS-CoV-2 infection was elucidated. Protein arginylation was studied in Vero CCL-81, macrophage-like THP1, and Calu-3 cells infected at different times. A reanalysis of in vivo and in vitro public omics data combined with immunoblotting was performed to measure levels of arginyl-tRNA-protein transferase (ATE1) and its substrates. Dysregulation of the N-degron pathway was specifically identified during coronavirus infections compared to other respiratory viruses. We demonstrated that during SARS-CoV-2 infection, there is an increase in ATE1 expression in Calu-3 and Vero CCL-81 cells. On the other hand, infected macrophages showed no enzyme regulation. ATE1 and protein arginylation was variant-dependent, as shown using P1 and P2 viral variants and HEK 293T cells transfection with the spike protein and receptor-binding domains (RBD). In addition, we report that ATE1 inhibitors, tannic acid and merbromine (MER) reduce viral load. This finding was confirmed in ATE1-silenced cells. CONCLUSIONS: We demonstrate that ATE1 is increased during SARS-CoV-2 infection and its inhibition has potential therapeutic value.

Schinus terebinthifolius Leaf Lectin (SteLL) Reduces the Bacterial and Inflammatory Burden of Wounds Infected by Staphylococcus aureus Promoting Skin Repair.

Staphylococcus aureus is commonly found in wound infections where this pathogen impairs skin repair. The lectin isolated from leaves of Schinus terebinthifolius (named SteLL) has antimicrobial and antivirulence action against S. aureus. This study evaluated the effects of topical administration of SteLL on mice wounds infected by S. aureus. Seventy-two C57/BL6 mice (6−8 weeks old) were allocated into four groups: (i) uninfected wounds; (ii) infected wounds, (iii) infected wounds treated with 32 µg/mL SteLL solution; (iv) infected wounds treated with 64 µg/mL SteLL solution. The excisional wounds (64 mm2) were induced on the dorsum and infected by S. aureus 432170 (4.0 × 106 CFU/wound). The daily treatment started 1-day post-infection (dpi). The topical application of both SteLL concentrations significantly accelerated the healing of S. aureus-infected wounds until the 7th dpi, when compared to untreated infected lesions (reductions of 1.95−4.55-fold and 1.79−2.90-fold for SteLL at 32 µg/mL and 64 µg/mL, respectively). The SteLL-based treatment also amended the severity of wound infection and reduced the bacterial load (12-fold to 72-fold for 32 µg/mL, and 14-fold to 282-fold for 64 µg/mL). SteLL-treated wounds show higher collagen deposition and restoration of skin structure than other groups. The bacterial load and the levels of inflammatory markers (IL-6, MCP-1, TNF-α, and VEGF) were also reduced by both SteLL concentrations. These results corroborate the reported anti-infective properties of SteLL, making this lectin a lead candidate for developing alternative agents for the treatment of S. aureus-infected skin lesions.

Schinus terebinthifolia leaf lectin (SteLL) has anti-infective action and modulates the response of Staphylococcus aureus-infected macrophages.

Staphylococcus aureus is recognized as an important pathogen causing a wide spectrum of diseases. Here we examined the antimicrobial effects of the lectin isolated from leaves of Schinus terebinthifolia Raddi (SteLL) against S. aureus using in vitro assays and an infection model based on Galleria mellonella larvae. The actions of SteLL on mice macrophages and S. aureus-infected macrophages were also evaluated. SteLL at 16 µg/mL (8 × MIC) increased cell mass and DNA content of S. aureus in relation to untreated bacteria, suggesting that SteLL impairs cell division. Unlike ciprofloxacin, SteLL did not induce the expression of recA, crucial for DNA repair through SOS response. The antimicrobial action of SteLL was partially inhibited by 50 mM N-acetylglucosamine. SteLL reduced staphyloxathin production and increased ciprofloxacin activity towards S. aureus. This lectin also improved the survival of G. mellonella larvae infected with S. aureus. Furthermore, SteLL induced the release of cytokines (IL-6, IL-10, IL-17A, and TNF-α), nitric oxide and superoxide anion by macrophagens. The lectin improved the bactericidal action of macrophages towards S. aureus; while the expression of IL-17A and IFN-γ was downregulated in infected macrophages. These evidences suggest SteLL as important lead molecule in the development of anti-infective agents against S. aureus.

Betulinic Acid Prevents the Acquisition of Ciprofloxacin-Mediated Mutagenesis in Staphylococcus aureus.

The occurrence of damage on bacterial DNA (mediated by antibiotics, for example) is intimately associated with the activation of the SOS system. This pathway is related to the development of mutations that might result in the acquisition and spread of resistance and virulence factors. The inhibition of the SOS response has been highlighted as an emerging resource, in order to reduce the emergence of drug resistance and tolerance. Herein, we evaluated the ability of betulinic acid (BA), a plant-derived triterpenoid, to reduce the activation of the SOS response and its associated phenotypic alterations, induced by ciprofloxacin in Staphylococcus aureus. BA did not show antimicrobial activity against S. aureus (MIC > 5000 µg/mL), however, it (at 100 and 200 µg/mL) was able to reduce the expression of recA induced by ciprofloxacin. This effect was accompanied by an enhancement of the ciprofloxacin antimicrobial action and reduction of S. aureus cell volume (as seen by flow cytometry and fluorescence microscopy). BA could also increase the hyperpolarization of the S. aureus membrane, related to the ciprofloxacin action. Furthermore, BA inhibited the progress of tolerance and the mutagenesis induced by this drug. Taken together, these findings indicate that the betulinic acid is a promising lead molecule in the development helper drugs. These compounds may be able to reduce the S. aureus mutagenicity associated with antibiotic therapies.

Polysaccharide-Based Formulations for Healing of Skin-Related Wound Infections: Lessons from Animal Models and Clinical Trials.

Skin injuries constitute a gateway for pathogenic bacteria that can be either part of tissue microbiota or acquired from the environmental. These microorganisms (such as Acinetobacter baumannii, Enterococcus faecalis,Pseudomonas aeruginosa, and Staphylococcus aureus) produce virulence factors that impair tissue integrity and sustain the inflammatory phase leading for establishment of chronic wounds. The high levels of antimicrobial resistance have limited the therapeutic arsenal for combatting skin infections. Thus, the treatment of non-healing chronic wounds is a huge challenge for health services worldwide, imposing great socio-economic damage to the affected individuals. This scenario has encouraged the use of natural polymers, such as polysaccharide, in order to develop new formulations (membranes, nanoparticles, hydrogels, scaffolds) to be applied in the treatment of skin infections. In this non-exhaustive review, we discuss the applications of polysaccharide-based formulations in the healing of infected wounds in animal models and clinical trials. The formulations discussed in this review were prepared using alginate, cellulose, chitosan, and hyaluronic acid. In addition to have healing actions per se, these polysaccharide formulations can act as transdermal drug delivery systems, controlling the release of active ingredients (such as antimicrobial and healing agents). The papers show that these polysaccharides-based formulations are efficient in controlling infection and improve the healing, even in chronic infected wounds. These data should positively impact the design of new dressings to treat skin infections.