Sepsis remains a major clinical challenge due to the limited efficacy of existing therapies in controlling excessive inflammation. The engineered stapled peptide sHVF18, derived from an evolutionarily conserved thrombin innate fold, binds both lipopolysaccharide (LPS) and the LPS-binding groove of CD14, enabling dual targeting of bacterial components and host immune signaling. To define structural prerequisites for this dual action, we combined evolutionary analysis, in silico modeling, and experimental methods. Substituting the N-terminal histidine with lysine (K) or arginine (R) improved solubility, reduced aggregation, and enhanced interactions with LPS. However, unexpectedly, K-substitutions impaired CD14 binding, whereas R variants retained weaker affinity, possibly through cation-π interactions. The essential role of the evolutionarily conserved N-terminal histidine for CD14 interactions and therapeutic efficacy was demonstrated using LPS-induced shock and polymicrobial sepsis models. While the K variant exhibited superior efficacy in LPS-induced shock, its disrupted CD14 interactions rendered it ineffective in polymicrobial sepsis. In contrast, sHVF18, by engaging both LPS and CD14, effectively reduced inflammation and improved survival in polymicrobial sepsis. These findings highlight that targeting of both LPS and CD14 is essential for therapeutic efficacy, underscoring multivalency as a key principle for future sHVF18-based sepsis therapeutics.

Apolipoprotein E (APOE) is distributed across various human tissues and plays a crucial role in lipid metabolism. Recent investigations have uncovered an additional facet of APOE's functionality, revealing its role in host defense against bacterial infections. To assess the antibacterial attributes of APOE3 and APOE4, we conducted antibacterial assays using Pseudomonas aeruginosa and Escherichia coli. Exploring the interaction between APOE isoforms and lipopolysaccharides (LPSs) from E. coli, we conducted several experiments, including gel shift assays, CD, and fluorescence spectroscopy. Furthermore, the interaction between APOE isoforms and LPS was further substantiated through atomic resolution molecular dynamics simulations. The presence of LPS induced the aggregation of APOE isoforms, a phenomenon confirmed through specific amyloid staining, as well as fluorescence and electron microscopy. The scavenging effects of APOE3/4 isoforms were studied through both in vitro and in vivo experiments. In summary, our study established that APOE isoforms exhibit binding to LPS, with a more pronounced affinity and complex formation observed for APOE4 compared with APOE3. Furthermore, our data suggest that APOE isoforms neutralize LPS through aggregation, leading to a reduction of local inflammation in experimental animal models. In addition, both isoforms demonstrated inhibitory effects on the growth of P. aeruginosa and E. coli. These findings provide new insights into the multifunctionality of APOE in the human body, particularly its role in innate immunity during bacterial infections.

Recently, mass spectrometry based peptidomics studies have proven useful in the identification of biomarkers and bioactive peptide-based therapeutics. Here, we present a dataset comprised of temporal wound fluid peptidomics data from highly defined porcine models. Wound fluids from porcine wounds infected with Staphylococcus aureus and Pseudomonas aeruginosa, and uninfected controls, were sampled at different timepoints of the infection. Peptides were extracted from the samples, followed by liquid chromatography tandem mass spectrometry analysis in data dependent acquisition mode. The resulting spectra and searched files have been deposited to online repositories and made easily accessible to enable further investigations of the infected and uninfected wound fluid peptidome.

The emergence of antibiotic resistance necessitates effective strategies for evaluating antimicrobial agents. Bioluminescent bacteria, either naturally occurring or engineered with modified reporter genes like bacterial luciferase, provide real-time assessment of bacterial viability through light emission. We investigated the antibacterial effects of cefotaxime and doxycycline using bioluminescent strains of S. aureus and P. aeruginosa, combining optical density measurements with bioluminescence monitoring. Treatment with cefotaxime resulted in a significant reduction of the bioluminescent signal in P. aeruginosa compared to untreated controls, while doxycycline induced a delayed growth curve. Both antimicrobials demonstrated strong efficacy against S. aureus, as evidenced by decreased bioluminescence signals. Results from bioluminescence assays and classical minimum inhibitory concentration and minimum bactericidal concentration methods showed consistent alignment, validating the bioluminescence approach. This study demonstrates that bioluminescence-based methods offer a reliable, real-time alternative to traditional bacterial viability assays for evaluating antimicrobial efficacy.

The concept of intratumoral microbiota is gaining attention in current research. Tumor-associated microbiota can activate oncogenic signaling pathways such as NF-kappa B, thereby promoting tumor development and progression. Numerous studies have demonstrated that curcumin and its analogs possess strong antitumor effects by targeting the NF-kappa B signaling pathway, along with potent antibacterial properties. In this study, we tested the antibacterial activity of two curcuminoids, Py-cPen and V-cPen, against the Gram-negative bacterial strains Pseudomonas aeruginosa and Escherichia coli and the Gram-positive bacterial strain Streptococcus aureus using in vitro assays and fluorescent microscopy. We observed that both Py-cPen and V-cPen reduced NF-kappa B activation upon lipopolysacharide (LPS) challenge in cell assays. In addition, our findings indicate that Py-cPen and V-cPen interact with LPS, as demonstrated by transmission electron microscopy and confirmed using in silico analyses, thereby modulating LPS activity. Overall, our data indicate that Py-cPen and V-cPen exhibit strong antibacterial and antiinflammatory properties, suggesting their potential as candidates for new multitarget therapeutic strategies.

Glycosaminoglycans (GAGs) such as heparin and heparan sulfate (HS) play crucial roles in inflammation and wound healing, serving as regulators of growth factors and pro-inflammatory mediators. In this study, we investigated the influence of heparin/HS on thrombin proteolysis and its interaction with the generated 11 kDa thrombin-derived C-terminal peptides (TCPs). Employing various biochemical and biophysical methods, we demonstrated that 11 kDa TCPs aggregate in the presence of GAGs, including heparin, heparan sulfate, and chondroitin sulfate-B. Circular dichroism analysis demonstrated that 11 kDa TCPs, in the presence of GAGs, adopt a beta-sheet structure, a finding supported by thioflavin T1 (ThT) fluorescence measurements and visualization of 11 kDa TCP-heparin complexes using transmission electron microscopy (TEM). Furthermore, our investigations revealed a stronger binding affinity between 11 kDa TCPs and GAGs with higher sulfate group contents. Congruently, in silico simulations showed that interactions between 11 kDa TCPs and heparin/HS are predominantly electrostatic in nature. Collectively, our study suggests that 11 kDa TCPs have the capacity to aggregate in the presence of GAGs, shedding light on their potential roles in inflammation and wound healing.

Surgical site infections (SSI) are a clinical and economic burden. Suture-associated SSI may develop when bacteria colonize the suture surface and form biofilms that are resistant to antibiotics. Thrombin-derived C-terminal peptide (TCP)-25 is a host defense peptide with a unique dual mode of action that can target both bacteria and the excessive inflammation induced by bacterial products. The peptide demonstrates therapeutic potential in preclinical in vivo wound infection models. In this study, the authors set out to explore whether TCP-25 can provide a new bioactive innate immune feature to hydrophilic polyglactin sutures (Vicryl). Using a combination of biochemical, biophysical, antibacterial, biofilm, and anti-inflammatory assays in vitro, in silico molecular modeling studies, along with experimental infection and inflammation models in mice, a proof-of-concept that TCP-25 can provide Vicryl sutures with a previously undisclosed host defense capacity, that enables targeting of bacteria, biofilms, and the accompanying inflammatory response, is shown.

Bacterial lipopolysaccharide (LPS) induces rapid protein aggregation in human wound fluid. We aimed to characterize these LPS-induced aggregates and their functional implications using a combination of mass spectrometry analyses, biochemical assays, biological imaging, cell experiments, and animal models. The wound-fluid aggregates encompass diverse protein classes, including sequences from coagulation factors, annexins, histones, antimicrobial proteins/peptides, and apolipoproteins. We identified proteins and peptides with a high aggregation propensity and verified selected components through Western blot analysis. Thioflavin T and Amytracker staining revealed amyloid-like aggregates formed after exposure to LPS in vitro in human wound fluid and in vivo in porcine wound models. Using NF-κB-reporter mice and IVIS bioimaging, we demonstrate that such wound-fluid LPS aggregates induce a significant reduction in local inflammation compared with LPS in plasma. The results show that protein/peptide aggregation is a mechanism for confining LPS and reducing inflammation, further emphasizing the connection between host defense and amyloidogenesis.

There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.