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Cyclic peptides are receiving significant attention thanks to their antimicrobial activity and high serum stability, which is useful to develop and design novel antimicrobial agents. Antimicrobial peptides appear to be key components of innate defences against bacteria, viruses, and fungi. Among the others, defensins possess a strong microbicidial activity. Defensins are cationic and amphipathic peptides with six cysteine residues connected by three disulfide bonds found in plants, insects, and mammals; they are divided in three families: α-, β-, and θ-defensins. α-Defensins are contained in the primary granules of human neutrophils; β-defensins are expressed in human epithelia; and θ-defensins are pseudo-cyclic defensins not found in humans, but in rhesus macaques. The structural diversities among the three families are reflected in a different antimicrobial action as well as in serum stability. The engineering of these peptides is an exciting opportunity to obtain more functional antimicrobial molecules highlighting their potential as therapeutic agents. The present review reports the most recent advances in the field of cyclic peptides with a specific regard to defensin analogs.

This research was funded internally by the Servicio para el Control de la Esterilización (SCE)/Laboratorio de Microbiología Oral (LMO) of the University of Oviedo (FUO-19-258, FUO-21-304) and the Fundación Sira Carrasco para la Ayuda a la Fibrosis Quística (SV-03-FSCARRASCO). M.T.A. was granted funding by SCE/LMO. P.F. was a short-term visiting researcher during her residency training (MIR).

Human α and β-defensins are cationic antimicrobial peptides characterized by three disulfide bonds with a triple stranded β-sheet motif. It is presumed that interaction with the bacterial cell surface and membrane permeabilization by defensins is an important step in the killing process. In this study, we have compared interactions of three human α-defensins HNP3, HNP4, HD5 and human β-defensins HBD1-4 that are active against Escherichia coli, with its cell surface and inner membrane as well as negatively charged model membranes. We have also included the inactive α-defensin HD6 in the study. Among the α-defensins, HNP4, HD5 and HD6 were more effective in increasing the zeta potential as compared to HNP3. Among the β-defensins, HBD1 was the least effective in increasing the zeta potential. The zeta potential modulation data indicate variations in the surface charge neutralizing ability of α- and β-defensins. Comparison of E. coli inner membrane and model membrane permeabilizing abilities indicated that HD5, HD6 and HBD1 do not permeabilize membranes. Although HBD4 does not permeabilize model membranes, considerable damage to the inner membrane of E. coli is observed. Our data indicate that mammalian defensins do not kill E. coli by a simple mechanism involving membrane permeabilization though their antibacterial potencies are very similar.

Today, Candida albicans is still the most common cause of both local and life-threatening systemic candidiasis. The spread of resistant fungal strains has resulted in an urgent need to search for new promising antimycotics. Here, we investigated the antifungal action of the tobacco defensin NaD1 against susceptible and resistant to azoles and echinocandins strains of C. albicans. We demonstrated that NaD1 was equally effective and fungicidal against all tested strains. The MIC and MFC values were 6.25 and 12.5 µM, respectively. We showed for the first time that NaD1 could act synergistically not only with caspofungin but also with human host defense antimicrobial peptides cathelicidin LL-37 and β-defensin-2 (HBD2) against susceptible and resistant fungal strains. Using flow cytometry, we demonstrated that NaD1 in combinations with LL-37 or HBD2 can reinforce each other by enhancing membrane disruption. Using the Caco-2 cell monolayer model, we demonstrated that NaD1 impaired the adhesion of C. albicans cells to the human epithelium. Moreover, NaD1 inhibited the formation of fungal biofilms in Sabouraud broth and less markedly in nutrient-rich RPMI-1640 medium, and enhanced the antibiofilm activity of caspofungin. Thus, we hypothesized that NaD1 might affect the development of candidiasis in vivo, including that caused by resistant fungal strains.

Staphylococcus aureus causes large numbers of both relatively benign and serious human infections, which are mediated in large part by the organisms’ secreted exotoxins. Since 1921, it has been known that lysozyme and, as shown later in the 1900s, other innate immune peptides, including human neutrophil α-defensin-1 (HNP-1) and human β-defensin 1 (HBD-1), are either not antistaphylococcal or are only weakly inhibitory to growth. Innate immune molecules, including antimicrobial peptides (for example, defensins) and lysozyme, function to delay or prevent bacterial infections. These molecules are commonly found on mucosal and skin surfaces. Staphylococcus aureus is a common pathogen and causes millions of infections annually. It is well known that innate immune molecules, such as defensins and lysozyme, either poorly inhibit or do not inhibit the growth of S. aureus . Our current studies show that the α-defensin human neutrophil α-defensin-1 (HNP-1) and lysozyme inhibit exotoxin production, both hemolysins and superantigens, which are required for S. aureus infection. HNP-1 inhibited exotoxin production at concentrations as low as 0.001 μg/mL. Lysozyme inhibited exotoxin production at 0.05 to 0.5 μg/mL. Both HNP-1 and lysozyme functioned through at least one two-component system (SrrA/B). The β-defensin human β-defensin 1 (HBD-1) inhibited hemolysin but not superantigen production. The cation chelator S100A8/A9 (calprotectin), compared to EDTA, was tested for the ability to inhibit exotoxin production. EDTA at high concentrations inhibited exotoxin production; these were the same concentrations that interfered with staphylococcal growth. S100A8/A9 at the highest concentration tested (10 μg/mL) had no effect on S. aureus growth but enhanced exotoxin production. Lower concentrations had no effect on growth or exotoxin production. Lysostaphin is regularly used to lyse S. aureus . The lytic concentrations of lysostaphin were the only concentrations that also inhibited growth and exotoxin production. Our studies demonstrate that a major activity of innate defensin peptides and lysozyme is inhibition of staphylococcal exotoxin production but not inhibition of growth. IMPORTANCE Staphylococcus aureus causes large numbers of both relatively benign and serious human infections, which are mediated in large part by the organisms’ secreted exotoxins. Since 1921, it has been known that lysozyme and, as shown later in the 1900s, other innate immune peptides, including human neutrophil α-defensin-1 (HNP-1) and human β-defensin 1 (HBD-1), are either not antistaphylococcal or are only weakly inhibitory to growth. Our study confirms those findings but, importantly, shows that at subgrowth inhibitory concentrations, these positively charged innate immune peptides inhibit exotoxin production, including both hemolysins and the superantigen toxic shock syndrome toxin-1. The data show that the principal activity of innate immune peptides in the host is likely to be inhibition of exotoxin production required for staphylococcal mucosal or skin colonization rather than growth inhibition.

ObjectivesAntimicrobial peptides (AMPs) represent important facets of the immune system controlling infectious diseases. However, pathogens show varying susceptibilities to AMPs. This study investigates the susceptibilities of strains of Streptococcus mutans (SM), Actinomyces naeslundii (AN), and Lactobacillus spp. (LB) towards AMPs and if there are correlations between the appearance of such high-risk strains and clinical caries status.Material and methodsPlaque samples were collected from patients along with clinical examinations. Bacterial strains were identified via selective media, matrix-assisted laser desorption/ionization analysis-time of flight (MALDI-TOF), and arbitrary-primed-PCR (AP-PCR). Each strain was tested for susceptibility to LL-37, HBD-2, HNP-1, and HNP-3 or phosphate-buffered saline as negative control in a biofilm model on hydroxylapatite discs. Survival rates and resulting risk classification for each strain were determined. Correlations were calculated between the number of high-risk strains (all/S. mutans) appearing in patients and their clinical caries status.ResultsForty-seven patients were included with mean DMFT values of 11.4 ± 8.7. A total of 8 different SM, 30 LB, and 47 AN strains were detected. One-way ANOVA indicated that type/concentration of AMPs had major influence on reductions of Lactobacilli and Actinomyces. Seventeen strains of AN, 2 of SM, and 6 of LB had low susceptibilities to AMPs. The number of such strains in patients showed significant positive correlations to the DMFT values (all p = 0.001; r = 0.452; S. mutans p < 0.0001, r = 0.558).ConclusionThe occurrence of low susceptible strains to AMPs seems to correlate with the individual caries status.Clinical relevanceThe results may lead to new ways to identify individuals with increased caries risk.

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.

Defensins are small, cysteine-rich peptides involved in plant defense, though their insecticidal properties remain largely unexplored. Previously, based on transcriptome we identified a defensin gene in black gram in response to bruchid ( Callosobruchus maculatus ) infestation. In the present study, we cloned and sequenced full-length cDNAs of defensin genes from multiple legumes and conducted phylogenetic analyses. Two sequence variants were identified, exhibiting 95–98% homology with a previously reported insecticidal defensin gene (Accession no. AF326687). Variant 1 (DefV1) was present in black gram, pea, cowpea, and common bean, whereas variant 2 (DefV2) was identified in mung bean, chickpea, and pigeon pea. Computational analysis, including molecular docking, visualization, and molecular dynamics (MD) simulations, demonstrated enhanced interactions between DefV1 and bruchid α-amylase, suggesting a “Cork in the Bottle” inhibitory mechanism. Additionally, insect bioassays using artificial seeds supplemented with DefV1 showed no adult emergence. These findings highlight black gram defensin as a promising insecticidal agent and a potential candidate for genetic improvement of bruchid resistance in legumes.

The last decade led to the discovery and characterization of several human beta-defensins. Analysis of genomic information indicates that the number of beta-defensin-like molecules encoded by the human genome may number in the tens. Growing interest in beta-defensins steadily enhances our knowledge about various aspects of their gene location, expression patterns and the transcription factors involved in their regulation in vivo. The hallmark property of beta-defensins, their antimicrobial activity, is clearly only the tip of the iceberg in the extensive network of inter-relations within the immune system in which these peptides function. Structural studies of beta-defensins provide the molecular basis for a better understanding of their properties, functions and their potential for practical applications. In this review, we present some recent advances in the studies of human beta-defensins, with an emphasis on possible correlations between their structural and functional properties.

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli . The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea , the cysteine-stabilized αβ-defensins (Csαβ-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.