Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus.

Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities.

Variation of Proteolytic Cleavage Sites towards the N-Terminal End of the S2 Subunit of the Novel SARS-CoV-2 Omicron Sublineage BA.2.12.1.

The prevalence of novel SARS-CoV-2 variants is also accompanied by an increased turnover rate and additional cleavage sites at the positions necessary for priming the Spike (S) protein. Of these priming sites, the proteolytically sensitive polybasic sequence of the activation loop at the S1/S2 interface and the S2' location within the S2 subunit of the S protein are cleaved by furin and TMPRSS2, which are important for the infection of the target cell. Neutrophils, migrating to the site of infection, secrete serine proteases to fight against pathogens. The serine proteases encompass neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG), which can hydrolyze the peptide bond adjacent to the S1/S2 interface. SARS-CoV-2 might take the opportunity to hijack proteases from an immune response to support viral entry to the cell. The region near S704L within the S2 subunit, a novel amino acid substitution of SARS-CoV-2 Omicron sublineage BA.2.12.1, is located close to the S1/S2 interface. We found that NE, PR3, and CatG digested the peptide within this region; however, the S704L amino acid substitution altered cleavage sites for PR3. In conclusion, such an amino acid substitution modifies S2 antigen processing and might further impact the major histocompatibility complex (MHC) binding and T cell activation.

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity.

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton's tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1ß release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.

Distinct Lugdunins from a New Efficient Synthesis and Broad Exploitation of Its MRSA-Antimicrobial Structure.

A new solid-phase peptide synthesis and bioprofiling of the antimicrobial activity of lugdunin, a fibupeptide, enable a comprehensive structure-activity relationship (SAR) study (MRSA Staphylococcus aureus). Distinct lugdunin analogues with variation of the three important amino acids Val2, Trp3, and Leu4 are readily available based on the established high-output synthesis. This efficient synthesis concept takes advantage of the presynthesized thiazolidine building block. To gain further knowledge of SAR, d-Val2, and d-Leu4 were replaced with aliphatic amino acids. For l-Trp3 derivatization, a set of non-natural aromatic amino acids with manifold substitution and annulation patterns precisely shows structural imperatives, starting from the exchange of d-Val6 → d-Trp6 with a 2-fold improved biological activity. d-Trp6-lugdunin analogues with additional variation of d-Val2 and d-Leu4 residues were designed and synthesized followed by antimicrobial profiling. For the first time, these SAR studies deliver valuable information on the tolerance of other amino acids to d-Val2, l-Trp3, and d-Leu4 in the sequence of lugdunin.

An ECF-type transporter scavenges heme to overcome iron-limitation in Staphylococcus lugdunensis.

Energy-coupling factor type transporters (ECF) represent trace nutrient acquisition systems. Substrate binding components of ECF-transporters are membrane proteins with extraordinary affinity, allowing them to scavenge trace amounts of ligand. A number of molecules have been described as substrates of ECF-transporters, but an involvement in iron-acquisition is unknown. Host-induced iron limitation during infection represents an effective mechanism to limit bacterial proliferation. We identified the iron-regulated ECF-transporter Lha in the opportunistic bacterial pathogen Staphylococcus lugdunensis and show that the transporter is specific for heme. The recombinant substrate-specific subunit LhaS accepted heme from diverse host-derived hemoproteins. Using isogenic mutants and recombinant expression of Lha, we demonstrate that its function is independent of the canonical heme acquisition system Isd and allows proliferation on human cells as sources of nutrient iron. Our findings reveal a unique strategy of nutritional heme acquisition and provide the first example of an ECF-transporter involved in overcoming host-induced nutritional limitation.

Synthesis of a Novel Curcumin Derivative as a Potential Imaging Probe in Alzheimer's Disease Imaging.

BACKGROUND: Curcumin has been of interest in the field of Alzheimer's disease. Early studies on transgenic mice showed promising results in the reduction of amyloid plaques.However, curcumin is very poorly soluble in aqueous solutions and not easily accessible to coupling as it contains only phenolic groups as potential coupling sites. For these reasons only few imaging studies using curcumin bound as an ester were performed and curcumin is mainly used as nutritional supplement. METHODS: In the present study we produced an aminoethyl ether derivative of curcumin using a nucleophilic substitution reaction. This is a small modification and should not impact the properties of curcumin while introducing an easily accessible reactive amino group. This novel compound could be used to couple curcumin to other molecules using the standard methods of peptide synthesis. We studied the aminoethyl-curcumin compound and a tripeptide carrying this aminoethyl-curcumin and the fluorescent dye fluorescein (FITC-curcumin) in vitro on cell culture using confocal laser scanning microscopy and flow cytometry. Then these two substances were tested ex vivo on brain sections prepared from transgenic mice depicting Alzheimer-like ß-amyloid plaques. RESULTS: In the in vitro CLSM microscopy and flow cytometry experiments we found dot-like unspecific uptake and only slight cytotoxicity correlating with this uptake. As these measurements were optimized for the use of fluorescein as dye we found that the curcumin at 488nm fluorescence excitation was not strong enough to use it as a fluorescence marker in these applications. In the ex vivo sections CLSM experiments both the aminoethyl-curcumin and the FITC-curcumin peptide bound specifically to ß- amyloid plaques. CONCLUSION: In conclusion we successfully produced a novel curcumin derivative which could easily be coupled to other imaging or therapeutic molecules as a sensor for amyloid plaques.

Lugdunin amplifies innate immune responses in the skin in synergy with host- and microbiota-derived factors.

Recently our groups discovered lugdunin, a new cyclic peptide antibiotic that inhibits Staphylococcus aureus epithelial colonization in humans and rodents. In this work, we analyzed its immuno-modulatory and antimicrobial potential as a single agent or in combination with other microbiota- or host-derived factors. We show that pretreatment of primary human keratinocytes or mouse skin with lugdunin in combination with microbiota-derived factors results in a significant reduction of S. aureus colonization. Moreover, lugdunin increases expression and release of LL-37 and CXCL8/MIP-2 in human keratinocytes and mouse skin, and results in the recruitment of monocytes and neutrophils in vivo, both by a TLR/MyD88-dependent mechanism. Interestingly, S. aureus elimination by lugdunin is additionally achieved by synergistic antimicrobial activity with LL-37 and dermcidin-derived peptides. In summary, our results indicate that lugdunin provides multi-level protection against S. aureus and may thus become a promising treatment option for S. aureus skin infections in the future.

Synthetic Lugdunin Analogues Reveal Essential Structural Motifs for Antimicrobial Action and Proton Translocation Capability.

Lugdunin, a novel thiazolidine cyclopeptide, exhibits micromolar activity against methicillin-resistant Staphylococcus aureus (MRSA). For structure-activity relationship (SAR) studies, synthetic analogues obtained from alanine and stereo scanning as well as peptides with modified thiazolidine rings were tested for antimicrobial activity. The thiazolidine ring and the alternating d- and l-amino acid backbone are essential. Notably, the non-natural enantiomer displays equal activity, thus indicating the absence of a chiral target. The antibacterial activity strongly correlates with dissipation of the membrane potential in S. aureus. Lugdunin equalizes pH gradients in artificial membrane vesicles, thereby maintaining membrane integrity, which demonstrates that proton translocation is the mode of action (MoA). The incorporation of extra tryptophan or propargyl moieties further expands the diversity of this class of thiazolidine cyclopeptides.

The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size.

Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N-acetyl-glucosamine) oligomers, we here identify six-subunit-long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size-dependent system of immuno-modulation that appears conserved in plants and humans. Since blocking of the chitin-TLR2 interaction effectively prevents chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin-TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease.

Human commensals producing a novel antibiotic impair pathogen colonization.

The vast majority of systemic bacterial infections are caused by facultative, often antibiotic-resistant, pathogens colonizing human body surfaces. Nasal carriage of Staphylococcus aureus predisposes to invasive infection, but the mechanisms that permit or interfere with pathogen colonization are largely unknown. Whereas soil microbes are known to compete by production of antibiotics, such processes have rarely been reported for human microbiota. We show that nasal Staphylococcus lugdunensis strains produce lugdunin, a novel thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus, and a rare example of a non-ribosomally synthesized bioactive compound from human-associated bacteria. Lugdunin is bactericidal against major pathogens, effective in animal models, and not prone to causing development of resistance in S. aureus. Notably, human nasal colonization by S. lugdunensis was associated with a significantly reduced S. aureus carriage rate, suggesting that lugdunin or lugdunin-producing commensal bacteria could be valuable for preventing staphylococcal infections. Moreover, human microbiota should be considered as a source for new antibiotics.