The toxic substance of Staphylococcus aureus has positive effects: it stimulates tissue regeneration

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Normally they are among the many harmless organisms found in and on the human body: one in four people have millions of Staphylococcus aureus bacteria on their skin and on the mucous membranes of the upper respiratory tract, without being aware of it.

In some cases, however, the harmless bacteria can turn into pathogens, which can lead to skin inflammation and lung infections, or – in the worst cases – sepsis.

“This happens especially when the bacteria multiply too fast, for example when a person’s immune system is weakened by an infection or injury,” says Prof. Oliver Werz of Friedrich Schiller University Jena in Germany.

The Professor for Pharmaceutical Chemistry and his team have studied the molecular defense mechanisms of the human immune system in the fight against such Staphylococcus aureus infections and made a surprising discovery.

As the research team reports in the current issue of the specialist journal Cell Reports, the toxic cocktail with which Staphylococcus aureus damages cells and tissues also has positive effects: specific immune cells are stimulated by the bacterial toxin to produce specialized messenger substances that help to reduce inflammation and to promote tissue healing. Prof. Werz expects this hitherto unknown mechanism to be significant for future treatments of skin inflammation and chronic wounds.

Immune cells produce anti-inflammatory messenger substances

In their latest study, the researchers from the University of Jena, Jena University Hospital and the Leibniz Institute on Aging—Fritz Lipmann Institute (FLI), together with colleagues from Harvard Medical School and the University of Naples, have studied in particular the bacterial toxin “α-Hemolysin” and examined its effect on M2 macrophages.

M2 macrophages are immune cells which, in the later stages of an inflammatory reaction, ensure that bacteria that have been killed, and damaged cell components, are removed, and that the tissue regenerates.

“They are therefore a kind of cellular waste disposal,” says Paul Jordan, doctoral candidate in Werz’s team and lead author of the publication, describing the function of these cells.

The researchers showed that α-hemolysin binds to specific receptor proteins on the surface of M2 macrophages and thus triggers the production of anti-inflammatory messenger substances in the cells, which then cause the inflammation to resolve.

In the study, the scientists were also able to show that these transmitters promote tissue regeneration in an animal model.

The anti-inflammatory messenger substances include resolvins, maresins and protectins that are formed from omega-3 fatty acids.


Staphylococcus aureus (S. aureus) is a critical pathogen that causes a wide spectrum of infections, such as pneumonia, soft tissue infections, wounds, arthroses, and skin infections.

The rapid spread of multidrug-resistant and highly virulent S. aureus strains has resulted in increased morbidity and mortality and great economic loss worldwide. Recurrent infections and the overuse of antibiotics contribute to the development of antibiotic resistance, which in turn promotes the spread of S. aureus (Sampedro et al., 2014).

Meanwhile, this pathogen is capable of forming biofilms in stressful environments and of protecting active cells from the effects of antibiotics and host defense mechanisms. Thus, the frequency of S. aureus infection is increasing, but the available therapeutics are limited.

In addition to evolving various resistance mechanisms, S. aureus also expresses multiple virulence determinants, such as enterotoxins, sortase, hemolysins, and bicomponent leukocidins, for the invasion or modulation of natural host defense mechanisms and the establishment of infection.

These virulence factors have been reported to contribute to the pathogenicity of S. aureus by acting in combination; however, some toxins alone can be sufficient for such contributions.

Among these virulence factors, α-hemolysin (Hla) is a toxin with an indispensable role in various infections, such as pneumonia and skin abscesses (Kennedy et al., 2010). Hla is encoded by a single gene (hla) and is secreted as a water-soluble monomer that is approximately 33 kDa.

When Hla attaches to target cells, such as platelets, epithelial cells, endothelial cells, or leukocytes, the monomers undergo conformation changes and oligomerize, forming β-barrel pores 1–2 nm in diameter through the lipid bilayer, which results in cell death and tissue lesion (Los et al., 2013).

Recently, the metalloproteinase ADAM10 was proposed to be a unique receptor of Hla, and subsequent studies showed that ADAM10 is an indispensable mediator for Hla binding to various types of cell membranes and forming β-pores. Additionally, ADAM10 also contributes to barrier disruption by promoting the cleavage of E-cadherin during S. aureus infection (Inoshima et al., 2011).

Hla is involved in the activation of immune signaling through various means during S. aureus infection, including through Hla-ADAM10-mediated cytotoxicity. Hla coupled with other S. aureus-derived molecules can directly trigger inflammation by activating recognition receptors, such as TLR2.

Indirectly, the ADAM10-mediated pore conformation of Hla causes ion fluxes that are responsible for many intracellular signaling pathways during S. aureus infection. The extracellular Na+ influx and K+ efflux of cells is sufficient to induce the involved immune signaling pathways, including the p38-MAPK, NLRP3-mediated, and c-Fos signaling pathways, stimulating the production of IL-1β, TNF-α, IL-6, and other cytokines (Seilie and Bubeck Wardenburg, 2017).

Additionally, the Ca2+ signaling that precedes cell death is initiated by the disruption of the plasma membrane. However, the inflammation resulting from bacterial infection is a double-edged sword. The contribution of inflammation is dependent on the context and site of infection, which can be protective or detrimental to the host.

Excessive inflammation may lead to tissue lesions and lethality. Previous studies have shown that inhibiting excessive inflammatory signaling is an alternative solution to promote S. aureus clearance (Gonzalez-Juarbe et al., 2015). In contrast, insufficient inflammation may be beneficial for bacterial growth and lead to severe infection. Thus, it is important to balance inflammatory reactions and bacterial infection.

Myricetin is a well-characterized natural flavonoid that widely exists in vegetables, fruits, and some beverages (Hertog et al., 1992; Mu et al., 2016); the major sources of myricetin are vegetables, fruits, and tea (Hertog et al., 1993). Myricetin was previously reported as a promising preventive natural compound with anti-inflammation, antitumor, antiviral, antibacterial, and antivirulence properties (Shih et al., 2009; Phillips et al., 2011; Ding et al., 2012; Tsai et al., 2015; Lopes et al., 2017; Silva et al., 2017).

With the development of nutrition, some dietary bioactive components in food have become increasingly attractive, among which is tea, which naturally triggered our interest in researching the biological activities of myricetin. Here, we illustrated that myricetin is an effective inhibitor of Hla with the potential to protect A549 cells in vitro and alleviate lung injury in vivo during S. aureus infection.

Additionally, studies with immune cells revealed that myricetin influences the Hla-mediated activation of immune signaling and inflammation. Thus, myricetin is proposed to be an effective anti-infection inhibitor against S. aureus by targeting Hla.

Myricetin Simultaneously Inhibits Hla Hemolytic Activity and Production Without Affecting S. aureus Viability

Myricetin (Figure 1A) has been shown to have multiple biological activities, including antioxidant, anti-inflammatory, antimicrobial, and cytoprotective activities (Hu et al., 2018; Kim et al., 2018; Rocha et al., 2018). Here, the addition of myricetin significantly reduced the hemolytic activity of purified Hla.

Statistically, the lysis of rabbit erythrocytes caused by Hla was attenuated by this compound in a dose-dependent manner, and the hemolytic activity of Hla was reduced from 100% to 37.4% ± 1.041 in the presence of 4 μg/ml myricetin. Furthermore, the hemolytic activity of Hla was almost completely abrogated when treated with 32 μg/ml myricetin (Figures 1B,C).

As expected, such inhibition was also observed for the hemolytic activity of culture supernatants of S. aureus co-cultured with various concentrations of myricetin (Figures 1D,E), suggesting that myricetin treatment may directly neutralize Hla activity, inhibit S. aureus growth, or reduce Hla production.

Consistent with our hypothesis, treatment with myricetin at the concentrations required for the inhibition of Hla activity did not visibly inhibit S. aureus viability (Figure 1F). However, myricetin treatment remarkably reduced the production of Hla at the concentrations that didn’t affect S. aureus growth (Figures 1G–I).

Additionally, the MICs of myricetin tested for S. aureus were >256 μg/ml, which greatly exceeded the test concentration in all of the assays. Taken together, these data revealed that myricetin effectively inhibits Hla by simultaneously reducing Hla hemolytic activity and production without affecting S. aureus growth.

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Figure 1
Myricetin inhibits the hemolytic activity of Hla. (A) Chemical structure of myricetin. (B,C) Inhibition of Hla activity by myricetin (Myr). The hemolytic activity of purified Hla (5 μg/ml) pretreated with the indicated concentrations of myricetin was determined using a hemolysis assay. (D,E) Hemolytic activity of the supernatants from S. aureus co-cultured with myricetin. The sample treated with DMSO was regarded as a negative control, and the sample treated with Hla or the supernatants from 8325-4 strain in the presence of DMSO served as a positive control (100% hemolysis). (F) Growth curves of S. aureus in the presence of various concentrations of myricetin. (G) Western blot assay for Hla expression in the supernatants and precipitates of S. aureus cultures treated with or without myricetin. The gray value of Hla expression in the culture supernatants (H) and precipitate (I) of S. aureus cultures treated with or without myricetin using Image-Pro Plus 6.0 software. Each column shows the mean ± SD of three independent experiments and analyzed by the one-way ANOVA. *P < 0.05 and **P < 0.01 compared to the positive group.

Discussion

Antibiotics were the preferred choice for treating S. aureus infection in the early stages of antibiotic development due to their excellent antibacterial activities. However, with the rapid increase in antibiotic-resistant and highly virulent strains, such as methicillin-resistant S. aureus (MRSA), the disease burden is gradually increasing, and the potency of traditional antibiotics is declining.

Thus, novel therapeutic strategies are urgently needed. To end this, alternative strategies have been well-explored, including antivirulence therapy, antibodies, bacteriophages, and vaccines.

Antivirulence therapy is aimed at disarming key virulence factors involved in disease progression rather than killing bacteria, meaning this strategy exerts a milder evolutionary pressure on the development of antibiotic resistance. There are many antivirulence targets for S. aureus due to the high adaptability, versatility, and pathogenicity of this bacteria in hosts, and it is highly attributed to the diversified virulence factors, which are employed to evade the immune system and establish infection (Diep et al., 2016).

Although the combined action of multiple toxins is necessary to enhance virulence, some individual toxins may be sufficient to cause damage and inflammation. Among these toxins, Hla is indispensable for S. aureus infection, rendering Hla an ideal target for the development of antivirulence strategies and agents.

Here, we initially showed that myricetin attenuated the pore formation of Hla by changing the secondary structure of Hla. The hemolytic activity of Hla was reduced from 100 to 37.40% in the sample treated with 4 μg/ml myricetin. Notably, almost no hemolytic activity was observed in the sample treated with 32 μg/ml of myricetin.

Interestingly, myricetin also suppressed the expression of Hla at a relatively lower concentration without affecting S. aureus viability. Although the mechanism for such action has not been completely characterized in our work, 32 μg/ml myricetin treatment led to 46.67% of Hla production in the supernatants and 48.46% in the precipitates of the co-cultures compared to the positive control without myricetin (Figures 1, ​,2).2).

Additionally, we confirmed that myricetin inhibited the biofilm formation in a Hla-dependent way, as evident by the fact that no inhibition was observed by myricetin for the DU1090 strain (Figure 3). To evaluate whether myricetin is capable of protecting cells, we applied A549 cells to perform an LDH assay; the results demonstrated that myricetin prevented S. aureus-mediated cell injury by targeting Hla at a low concentration of 4 μg/ml (Figure 4).

Host innate immune responses to S. aureus and bacteria-associated virulence determinants are aimed at clearing bacteria and minimizing tissue damage during bacterial infection. However, excessive inflammatory responses could aggravate bacterial infection by facilitating bacterial escape from the immune system or contributing to tissue damage.

For example, autophagy is regarded as a conserved pathway that confers resistance to bacteria or other pathogens (Maurer et al., 2015a,b). However, recent studies have demonstrated that autophagy is exploited by S. aureus to promote replication, escape, and host cell killing (Schnaith et al., 2007; Zhu et al., 2018).

Hla- and IL-1β-mediated pathological injury is thought to be responsible for the high morbidity and mortality of S. aureus infection, especially pneumonia (Goodman et al., 2003). Here, we showed that myricetin, as an agent that targets Hla, could downregulate the cascade of host responses triggered by S. aureus in a Hla-dependent way and reduce the secretion of IL-1β (Figure 5), which is associated with the recruitment of immune cells, the predisposition of acute lung injury, and systemic inflammation (Goodman et al., 2003).

Here, we propose that myricetin not only protects epithelial cells by inhibiting Hla activity but also reduces the enhanced secretion of IL-1β from immune cells by modulating MAPK and NF-κB pathways, and thereby alleviates lung injury in vivo during S. aureus infection (Figures 6, ​,77).

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Figure 7
Scheme summarizing the protective effects of Myricetin on Staphylococcus aureus infection. Myricetin is an effective inhibitor of Hla as it simultaneously targets Hla production and Hla activity, and thereby downregulates the inflammatory response in S. aureus infected cells by suppressing NF-κB and MAPK signaling pathways in vitro, which helps alleviate lung injury in vivo during S. aureus infection.

As a natural dietary flavonoid, myricetin has many sources, including tea, vegetables, fruits, and berries, thus, the cost of myricetin is much lower than the cost of developing new antibiotics or vaccines; myricetin has also been reported to have multiple biological activities, such as anti-inflammatory, antioxidant, ion homeostasis regulation, anti-tumor, and obesity prevention activities (Mu et al., 2016).

Here, we found that myricetin protected A549 cells against S. aureus by targeting Hla and excessive inflammation caused by S. aureus, which is beneficial to relieving lung injury, and myricetin treatment did not exhibit anti-S. aureus activity, which will slow the development of antibiotic resistance to some extent.

Although pharmacokinetic-pharmacodynamic studies were not conducted herein, our results indicate that myricetin is an attractive candidate for further testing as an adjunctive therapy for S. aureus infections.

With the development of antibiotics, many resistance determinants and mechanisms have gradually emerged, such as β-lactamases, enzymatic modification and inactivation, and efflux pump systems (Allen, 2008; Fast and Sutton, 2013; Vestergaard et al., 2019).

Unfortunately, antibiotic resistance spreads faster than the discovery of new compounds, leading to a public health concern. The combination of some inhibitors that target β-lactamases and antibiotics is an optional first-line method for the treatment of bacterial infections (Drawz and Bonomo, 2010; Ejim et al., 2011; Wang et al., 2018).

Here, we proposed that the combination of myricetin with antibiotics may improve the treatment of S. aureus infection and slow the development of antibiotic resistance by targeting bacterial virulence and the bacterium itself. In addition, this combination would decrease the usage of antibiotics and expand the life of antibiotics. In conclusion, myricetin is a promising candidate in the pharmaceutical industries for treating S. aureus infection by targeting Hla.

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390952/


More information: Paul M. Jordan et al, Staphylococcus aureus-Derived α-Hemolysin Evokes Generation of Specialized Pro-resolving Mediators Promoting Inflammation Resolution, Cell Reports (2020). DOI: 10.1016/j.celrep.2020.108247

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