Development of novel antimicrobial agents is a top priority in the fight against multidrug-resistant (MDR) and persistent bacteria. We developed a panel of synthetic antimicrobial and antibiofilm peptides (SAAPs) with enhanced antimicrobial activities compared to the parent peptide, human antimicrobial peptide LL-37. Our lead peptide SAAP-148 was more efficient in killing bacteria under physiological conditions in vitro than many known preclinical- and clinical-phase antimicrobial peptides. SAAP-148 killed MDR pathogens without inducing resistance, prevented biofilm formation, and eliminated established biofilms and persister cells. A single 4-hour treatment with hypromellose ointment containing SAAP-148 completely eradicated acute and established, biofilm-associated infections with methicillin-resistant and MDR from wounded ex vivo human skin and murine skin in vivo. Together, these data demonstrate that SAAP-148 is a promising drug candidate in the battle against antibiotic-resistant bacteria that pose a great threat to human health.
Posttranslational modification (PTM) of islet autoantigens can cause lack of central tolerance in type 1 diabetes (T1D). Tissue transglutaminase (tTG), involved in PTM of gluten antigens in celiac disease, creates negatively charged peptides favored by T1D-predisposing HLA-DQ molecules, offering an attractive candidate modifying islet autoantigens in T1D. The highly predisposing HLA-DQ8cis/trans molecules share preferences for negatively charged peptides, as well as distinct peptide-binding characteristics that distinguish their peptide-binding repertoire. We screened islet autoantigens with the tTG substrate motif for candidate-modified epitopes binding to HLA-DQ8cis/trans and identified 31 candidate islet epitopes. Deamidation was confirmed for 28 peptides (90%). Two of these epitopes preferentially bound to HLA-DQ8cis and six to HLA-DQ8trans upon deamidation, whereas all other peptides bound equally to HLA-DQ8cis/trans. HLA-DQ8cis-restricted T cells from a new-onset T1D patient could only be generated against a deamidated proinsulin peptide, but cross-reacted with native proinsulin peptide upon restimulation. The rate of T-cell autoreactivity in recent-onset T1D patients extended from 42% to native insulin to 68% adding responses to modified proinsulin, versus 20% and 37% respectively, in healthy donors. Most patients responded by interferon-g, whereas most healthy donors produced interleukin-10 only. Thus, T-cell autoreactivity exists to modified islet epitopes that differs in quality and quantity between patients and healthy donors.
Targeting CD8+ T cells to recurrent tumor-specific mutations can profoundly contribute to cancer treatment. Some of these mutations are potential tumor antigens although they can be displayed by non-spliced epitopes only in a few patients, because of the low affinity of the mutated non-spliced peptides for the predominant HLA class I alleles. Here, we describe a pipeline that uses the large sequence variety of proteasome-generated spliced peptides and identifies spliced epitope candidates, which carry the mutations and bind the predominant HLA-I alleles with high affinity. They could be used in adoptive T cell therapy and other anti-cancer immunotherapies for large cohorts of cancer patients. As a proof of principle, the application of this pipeline led to the identification of a KRAS G12V mutation-carrying spliced epitope candidate, which is produced by proteasomes, transported by TAPs and efficiently presented by the most prevalent HLA class I molecules, HLA-A*02:01 complexes.
Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence.Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease. Following the identification of a CD2830 cleavage site in human HSP90, a series of synthetic peptide substrates was used to identify the favorable CD2830 cleavage motif. This motif was characterized by a high prevalence of proline residues. Intriguingly, CD2830 has a preference for cleaving Pro-Pro bonds, unique among all hitherto described proteases. Strikingly, within the C. difficile proteome two putative adhesion molecules, CD2831 and CD3246, were identified that contain multiple CD2830 cleavage sites (13 in total). We subsequently found that CD2830 efficiently cleaves CD2831 between two prolines at all predicted cleavage sites. Moreover, native CD2830, secreted by live cells, cleaves endogenous CD2831 and CD3246.These findings highlight CD2830 as a highly specific endoproteinase with a preference for proline residues Clostridium difficile is an anaerobic, Gram-positive, sporeforming bacterium. Human intake of spores occurs through the fecal-oral route. Upon leaving the stomach and becoming exposed to bile acids in the small intestine, the C. difficile spores germinate. Once germinated, the vegetative cells in the colon encounter an unreceptive environment (1). Competition with the normal flora, immune responses, gastric fluids (2), and specialized antimicrobial peptides all act against a developing infection. Moreover, the physical barrier formed by a layer of glycoproteins (mucins) covering the underlying epithelial cells forms a major hurdle for firm adhesion.Many enteric pathogens express factors that reduce competition, allow evasion of host immune responses, and promote adhesion and/or invasion of tissues. These virulence factors are located in the cell membrane or wall (controlling adhesion and protection) or are secreted (modifying the surrounding environment). The best studied virulence factors in C. difficile are the toxins TcdA and TcdB (3, 4), which cause destruction of the intestinal barrier by disrupting the epithelial actin cytoskeleton. It is speculated that the subsequent increased permeability of the intestinal epithelium leads to increased exudation of fluids, including nutritional substances. Damage to the intestinal mucosa causes the main symptoms of C. difficile infection, including pseudomembranous colitis (1).Data illustrating how C. difficile circumvents host defense mechanisms are limited. As an example, in response to attack by antimicrobial peptides, C. difficile expresses a set of genes that change the surface charge, thereby diminishing the interaction of cationic antimicrobial peptides on th...
OP-145, a synthetic antimicrobial peptide developed from a screen of the human cathelicidin LL-37, displays strong antibacterial activities and is--at considerably higher concentrations--lytic to human cells. To obtain more insight into its actions, we investigated the interactions between OP-145 and liposomes composed of phosphatidylglycerol (PG) and phosphatidylcholine (PC), resembling bacterial and mammalian membranes, respectively. Circular dichroism analyses of OP-145 demonstrated a predominant α-helical conformation in the presence of both membrane mimics, indicating that the different membrane-perturbation mechanisms are not due to different secondary structures. Membrane thinning and formation of quasi-interdigitated lipid-peptide structures was observed in PG bilayers, while OP-145 led to disintegration of PC liposomes into disk-like micelles and bilayer sheets. Although OP-145 was capable of binding lipoteichoic acid and peptidoglycan, the presence of these bacterial cell wall components did not retain OP-145 and hence did not interfere with the activity of the peptide toward PG membranes. Furthermore, physiological Ca++ concentrations did neither influence the membrane activity of OP-145 in model systems nor the killing of Staphylococcus aureus. However, addition of OP-145 at physiological Ca++-concentrations to PG membranes, but not PC membranes, resulted in the formation of elongated enrolled structures similar to cochleate-like structures. In summary, phospholipid-driven differences in incorporation of OP-145 into the lipid bilayers govern the membrane activity of the peptide on bacterial and mammalian membrane mimics.
The present study aims to develop an implant coating releasing novel antimicrobial agents to prevent biomaterial-associated infections. The LL-37-derived synthetic antimicrobial and anti-biofilm peptides (SAAP)-145 and SAAP-276 exhibit potent bactericidal and anti-biofilm activities against clinical and multidrug-resistant Staphylococcus aureus strains by rapid membrane permeabilization, without inducing resistance. Injection of SAAP-145, but not SAAP-276, along subcutaneous implants in mice reduces S. aureus implant colonization by approximately 2 log, but does not reduce bacterial numbers in surrounding tissue. To improve their efficacy, SAAP-145 and SAAP-276 are incorporated in a polymer-lipid encapsulation matrix (PLEX) coating, providing a constant release of 0.6% daily up to 30 d after an initial burst release of >50%. In a murine model for biomaterial-associated infection, SAAP-145-PLEX and SAAP-276-PLEX coatings significantly reduce the number of culture positive implants and show ≥3.5 and ≥1.5 log lower S. aureus implant and tissue colonization, respectively. Interestingly, these peptide coatings are also highly effective against multidrug-resistant S. aureus, both reducing implant colonization by ≥2 log. SAAP-276-PLEX additionally reduces tissue colonization by 1 log. Together, the peptide-releasing PLEX coatings hold promise for further development as an alternative to coatings releasing conventional antibiotics to prevent biomaterial-associated infections.
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