Dosage‐Dependent Antimicrobial Activity of DNA‐Histone Microwebs Against Staphylococcus Aureus

Abstract

Biofilms are surface-bound microbe assemblies encased in an extracellular matrix including extracellular DNA (eDNA), proteins, and polysaccharides, which allow bacteria to resist host immune clearing mechanisms. [1] One critical biofilm component is eDNA, which helps maintain biofilm structural integrity, [2] as well as counter host antimicrobial peptides [3] and antibiotic treatments. [4] Although eDNA can engage positively with pathogens, it is known to have an important role in the innate immune response, where neutrophils may be triggered to release neutrophil extracellular traps (NETs), [5,6] cobweb-structured DNA-histone complexes that capture and disarm microorganisms. [7,8] The observed antimicrobial activity of NET-based eDNA is due, at least in part, to their chelation with bacterial membrane proteins and subsequent lysis. [9] Histones, key NET components that modulate the self-assembly of DNA, also contribute to overall antimicrobial activity by destabilizing bacterial cell walls and chromosome organization. [10,11] It is also reported that histone inhibits biofilm formation in a dosage-dependent manner. [12] Previous studies have indicated that the antimicrobial activity of DNA or histone largely depends on their surface charges, and blockage of the surface charge results in declined antimicrobial activity. [9,11] Staphylococcus aureus is an opportunistic pathogen that can cause persistent biofilm infections in soft tissues and bones. [13] Planktonic S. aureus induces neutrophils to produce NETs that can effectively trap and suppress the bacteria. [7] On the other hand, S. aureus biofilms can thwart the antimicrobial functions of NETs by secreting nucleases to degrade the eDNA in NETs; [5,14,15] they also utilize fibronectin-binding protein B (FnBPB) and proteases to neutralize histone-mediated killing. [10,16] Producing DNA-degrading nucleases that protect them from NETs within hours of surface attachment, [5] S. aureus biofilms actively induce neutrophil NETosis to protect themselves from other granulocytic killing mechanisms. [17] In contrast to the many studies that study how biofilms affect neutrophils, NETosis, and NETs, there is no systematic analysis of how different types of NETs may impact planktonic S. aureus response in forming biofilms. This may be important because NET composition including protein content and DNA sources Neutrophil extracellular traps (NETs) are antimicrobial cobweb-structured materials produced by immune cells for clearance of pathogens in the body, but are paradoxically associated with biofilm formation and exacerbated lung infections. To provide a better materials perspective on the pleiotropic roles played by NETs at diverse compositions/concentrations, a NETs-like material (called "microwebs", abbreviated as μwebs) is synthesized for decoding the antimicrobial activity of NETs against Staphylococcus aureus in infection-relevant conditions. It is shown that μwebs composed of low-to-intermediate concentrations of DNA-histone complexes successfully trap and inhibit S...

Synthetic neutrophil extracellular traps dissect bactericidal contribution of NETs under regulation of α-1-antitrypsin

“…In summary, a material synthesis approach is introduced to dissect the antimicrobial activity of NETs. While we have previously reported on the synthesis of minimal NET-mimetic mesh-like materials composed of DNA and histones, this report further incorporates other crucial NET proteins such as NE and MPO as well as tissue environment factors that affect NET function, such as AAT, into the functional biomaterial modeling of NETs ( 9 , 12 , 53 , 54 ). Placing our focus on antibacterial activity against PAO1, we identified histone, a critical protein in epigenetics often overlooked in antibacterial research, as the most potent antimicrobial among the major NET proteins.…”

“…Many of these NET proteins are bactericidal at minimum inhibitory concentrations (MICs) for specific bacteria but are present at sub-MIC levels in NETs ( 8 ). In addition, NETs are continuously degraded by enzymes and phagocytes in the physiological environment, making the quantification of NET proteins and elucidation of their contributions a challenging task ( 9 ). Although specific NET proteins have been isolated for study, their antimicrobial activity may deviate from that when incorporated in NETs.…”

Visualization of Nuclease- and Serum-Mediated Chromatin Degradation with DNA–Histone Mesostructures

“…Our group previously described a minimal NET-mimetic microweb, comprised of DNA and histones, for their ability to mimic aspects of the bacteria-suppressive features of NETs [ 10 , 11 ]. We have also described a surface-attached NET-mimetic biomaterial we call DHMs, which have a defined mesoscale structure also comprised of DNA and histones, for their NET-mimetic, immune-cell-activating features [ 9 ].…”

“…DNA–histone mesostructures (DHMs) [ 9 ] and microwebs [ 10 , 11 ] resemble aspects of the chromatin backbone structure of NETs. For example, we previously reported that deoxyribonuclease I (DNase I) degrades both DHMs [ 9 ] and microwebs [ 10 ] in a manner that resembles the nuclease-mediated degradation that NETs undergo.…”

Engineering in vitro models of cystic fibrosis lung disease using neutrophil extracellular trap inspired biomaterials

“…To do this, we used a synthetic NET (sNET) biomaterial based on previous work to mimic the chromatin scaffold of endogenous NETs. 26,30,31 This cell-free protocol can be used to generate NET-like biomaterials without the difficulties associated with in vitro generation and isolation of endogenous NETs or co-culture of neutrophils within the HAE model. To understand the impact of NETs on the viscoelastic properties of mucus, we incorporated the sNETs into a synthetic mucus hydrogel as described in our prior work [32][33][34] and mucus produced by HAE cells.…”