Antifungal activity of human antimicrobial peptides targeting apoptosis in Candida auris.

Introduction. Innovative antifungal therapies are of crucial importance to combat the potentially life-threatening infections linked to the multidrug-resistant fungal pathogen Candida auris. Induction of regulated cell death, apoptosis, could provide an outline for future therapeutics. Human antimicrobial peptides (AMPs), well-known antifungal compounds, have shown the ability to induce apoptosis in pathogenic fungi.Hypothesis/Gap Statement . Although it is known that AMPs possess antifungal activity against C. auris, their ability to induce apoptosis requires further investigations.Aim. This study evaluated the effects of AMPs on the induction of apoptosis in C. auris.Methods. Human neutrophil peptide-1 (HNP-1), human ß-Defensins-3 (hBD-3) and human salivary histatin 5 (His 5) were assessed against two clinical C. auris isolates. Apoptosis hallmarks were examined using FITC-Annexin V/PI double labelling assay and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick-end labelling (TUNEL) to detect phosphatidylserine externalization and DNA fragmentation, respectively. Then, several intracellular triggers were studied using JC-10 staining, spectrophotometric assay and 2',7'-dichlorofluorescin diacetate staining to measure the mitochondrial membrane potential, cytochrome-c release and reactive oxygen species (ROS) production, respectively.Results and conclusion. FITC-Annexin V/PI staining and TUNEL analysis revealed that exposure of C. auris cells to HNP-1 and hBD-3 triggered both early and late apoptosis, while His 5 caused significant necrosis. Furthermore, HNP-1 and hBD-3 induced significant mitochondrial membrane depolarization, which resulted in substantial cytochrome c release. In contrast to His 5, which showed minimal mitochondrial depolarization and no cytochrome c release. At last, all peptides significantly increased ROS production, which is related to both types of cell death. Therefore, these peptides represent promising and effective antifungal agents for treating invasive infections caused by multidrug-resistant C. auris.

Impact of metal coordination and pH on the antimicrobial activity of histatin 5 and the products of its hydrolysis.

This work focuses on the relationship between the coordination chemistry and antimicrobial activity of Zn(II) and Cu(II) complexes of histatin 5 and the products of its hydrolysis: its N-terminal fragment (histatin 5-8) and C-terminal fragment (histatin 8). Cu(II) coordinates in an albumin-like binding mode and Zn(II) binds to up to 3 His imidazoles. The antimicrobial activity of histatins and their metal complexes (i) strongly depends on pH - they are more active at pH 5.4 than at 7.4; (ii) the complexes and ligands alone are more effective in eradicating Gram-positive bacteria than the Gram-negative ones, and (iii) Zn(II) coordination is able to change the structure of the N-terminal region of histatin 5 (histatin 5-8) and moderately increase all of the studied histatins' antimicrobial potency.

Histatin 5-Inspired Short-Chain Peptides Selectively Combating Pathogenic Fungi with Multifaceted Mechanisms.

Short-chain antifungal peptides (AFPs) inspired by histatin 5 have been designed to address the problem of antifungal drug resistance. These AFPs demonstrate remarkable antifungal activity, with a minimal inhibitory concentration as low as 2 µg mL-1. Notably, these AFPs display a strong preference for targeting fungi rather than bacteria and mammalian cells. This is achieved by binding the histidine-rich domains of the AFPs to the Ssa1/2 proteins in the fungal cell wall, as well as the reduced membrane-disrupting activity due to their low amphiphilicity. These peptides disrupt the nucleus and mitochondria once inside the cells, leading to reactive oxygen species production and cell damage. In a mouse model of vulvovaginal candidiasis, the AFPs demonstrate not only antifungal activity, but also promote the growth of beneficial Lactobacillus spp. This research provides valuable insights for the development of fungus-specific AFPs and offers a promising strategy for the treatment of fungal infectious diseases.

Improving Photocleavage Efficiency of Photocleavable Protein for Antimicrobial Peptide Histatin 1 Expression.

BACKGROUND: Antimicrobial peptides (AMPs) are promising alternative agents for antibiotics to overcome antibiotic resistance problems. But, it is difficult to produce large-scale antimicrobial research due to the toxicity towards expression hosts or degradation by peptidases in the host. Therefore, heterologous recombinant expression of antimicrobial peptides has always been a challenging issue. OBJECTIVES: To overcome toxicity to the expression host and low expression level, a new photocleavable protein fusion expression method for antimicrobial peptides is provided.3 Methods: Through directed evolution and high throughput screening, a photocleavable protein mutant R6-2-6-4 with a higher photocleavage efficiency was obtained. The DNA coding sequence of antimicrobial peptide Histatin 1 was fused within the sequence of R6-2-6-4 gene. The fusion gene was successfully expressed in Escherichia coli expression system. RESULTS: Antimicrobial peptide Histatin 1 could be successfully expressed and purified by fusing within PhoCl mutant R6-2-6-4. The antimicrobial activity was rarely affected, and the MIC value was 33 ug/mL, which was basically equivalent to 32 ug/mL of the chemically synthesized Histatin 1. After amplification in a 5 L fermenter, the expression of PhoCl mutant (R6-2-6-4)-Histatin1 improved up to 87.6 mg/L in fermenter, and Histatin1 obtained by photocleavage also could up to 11 mg/L. The prepared Histatin1 powder remained stable when stored at 4oC for up to 4 months without any degradation. In addition, the expression and photocleavage of ß -Defensin105 and Lysostaphin verified the certain universality of the PhoCl mutant fusion expression system. CONCLUSION: Antimicrobial peptides Histatin 1, ß -Defensin 105 and Lysostaphin were successfully expressed and purified by photocleavable protein mutant. This may provide a novel strategy to express and purify antimicrobial peptides in the Escherichia coli expression system.

Histatin-5 interacts with cellular copper to promote antifungal activity against Candida albicans.

Histatin-5 (Hist-5) is an antimicrobial peptide found in human saliva that functions to defend the oral cavity from microbial infections, such as those caused by the fungal pathogen Candida albicans (C. albicans). Hist-5 can bind Cu in multiple oxidation states, Cu2+ and Cu+in vitro, and supplemental Cu2+ has been shown to improve the fungicidal activity of the peptide against C. albicans in culture. However, the exact role of Cu on the antifungal activity of Hist-5 and whether direct peptide-Cu interactions occur intracellularly has yet to be fully determined. Here, we used a combination of fluorescence spectroscopy and confocal microscopy experiments to show reversible Cu-dependent quenching of a fluorescent Hist-5 analogue, Hist-5*, indicating a direct interaction between Hist-5 and intracellular Cu. X-ray fluorescence microscopy images revealed peptide-induced changes to cellular Cu distribution and cell-associated Cu content. These data support a model in which Hist-5 can facilitate the hyperaccumulation of Cu in C. albicans and directly interact with Cu intracellularly to increase the fungicidal activity of Hist-5.

The salivary peptide histatin-1 enhances bone repair in vivo.

The salivary peptide histatin-1 was recently described as a novel osteogenic factor that stimulates cell adhesion, migration, and differentiation in bone-lineage cells. Since these cell responses collectively contribute to bone regeneration, we hypothesized that histatin-1 harbors the capacity to enhance bone tissue repair at the preclinical level. By using a model of monocortical bone defect, we explored the effects of histatin-1 in tibial mineralization and organic matrix formation in vivo. To this end, different amounts of histatin-1 were embedded in one-mm3 collagen sponges and then applied to tibial monocortical defects in C57bl/6 mice. After seven days, mice were euthanized, and samples were processed for subsequent analysis. Micro-computed tomography screening showed that histatin-1 increased intraosseous mineralization, and this phenomenon was accompanied by augmented collagen matrix deposition and closure of cortical defect edges, as determined by Hematoxylin-Eosin and Masson's Trichrome staining. Moreover, immunohistochemical analyses showed that histatin-1 increased the expression of the osteogenic marker alkaline phosphatase, which was accompanied by augmented blood vessel formation. Collectively, our findings show that histatin-1 itself promotes bone regeneration in an orthotopic model, proposing this molecule as a therapeutic candidate for use in bone regenerative medicine.

Anti-biofilm effect of salivary histatin 5 on Porphyromonas gingivalis.

This study aimed to investigate the effects of salivary histatin 5 (Hst5) on Porphyromonas gingivalis (P. gingivalis) biofilms in vitro and in vivo and the possible mechanisms. In in vitro experiments, P. gingivalis biomass was determined by crystal violet staining. Polymerase chain reaction, scanning electron microscopy, and confocal laser scanning microscopy were used to determine the Hst5 concentration. A search for potential targets was performed using transcriptomic and proteomic analyses. In vivo experimental periodontitis was established in rats to evaluate the effects of Hst5 on periodontal tissues. Experimental results showed that 25 µg/mL Hst5 effectively inhibited biofilm formation, and increased concentrations of Hst5 increased the inhibitive effect. Hst5 might bind to the outer membrane protein RagAB. A combination of transcriptomic and proteomic analyses revealed that Hst5 could regulate membrane function and metabolic processes in P. gingivalis, in which RpoD and FeoB proteins were involved. In the rat periodontitis model, alveolar bone resorption and inflammation levels in periodontal tissues were reduced by 100 µg/mL Hst5. This study showed that 25 µg/mL Hst5 inhibited P. gingivalis biofilm formation in vitro by changing membrane function and metabolic process, and RpoD and FeoB proteins might play important roles in this process. Moreover, 100 µg/mL Hst5 inhibited periodontal inflammation and alveolar bone loss in rat periodontitis via its antibacterial and anti-inflammatory effects. KEY POINTS: • Anti-biofilm activity of histatin 5 on Porphyromonas gingivalis was investigated. • Histatin 5 inhibited Porphyromonas gingivalis biofilm formation. • Histatin 5 showed inhibitory effects on the occurrence of rat periodontitis.

Combating fungal phytopathogens with human salivary antimicrobial peptide histatin 5 through a multi-target mechanism.

Blast disease caused by Magnaporthe oryzae is a major contributor to decreased crop yield and rice production globally. The use of chemical fungicides to combat crop pathogens is not only unsafe but also promotes the emergence of pathogenic variants, leading to recurrent host infections. To address plant diseases, antimicrobial peptides have emerged as a promising alternative as they are effective, safe, and biodegradable antifungal agents. This study examines the antifungal activity and mechanism of action of the human salivary peptide histatin 5 (Hst5) on M. oryzae. Hst5 causes morphogenetic defects in the fungus, including non-uniform chitin distribution on the fungal cell wall and septa, deformed hyphal branching, and cell lysis. Importantly, a pore-forming mechanism of Hst5 in M. oryzae was ruled out. Furthermore, the interaction of Hst5 with the M. oryzae genomic DNA suggests that the peptide may also influence gene expression in the blast fungus. In addition to its effects on morphogenetic defects and cell lysis, Hst5 also inhibits conidial germination, appressorium formation, and the appearance of blast lesions on rice leaves. The elucidated multi-target antifungal mechanism of Hst5 in M. oryzae provides an environmentally friendly alternative to combating blast infections in rice by preventing fungal pathogenicity. The promising antifungal characteristics of the AMP peptide may also be explored for other crop pathogens, making it a potential biofungicide for the future.

Effect of Non-Surgical Periodontal Therapy on Clinical Parameters of Periodontitis, Oral Candida spp. Count and Lactoferrin and Histatin Expression in Saliva and Gingival Crevicular Fluid of HIV-Infected Patients.

BACKGROUND: Periodontitis (PDT) has gained attention in the literature with the increase in life expectancy of people living with HIV on combined antiretroviral therapy (cART). Thus, the search for inflammatory biomarkers could be useful to understand the pathophysiology of chronic oral diseases in the cART era. OBJECTIVE: The aim of this study was to evaluate the impact of non-surgical periodontal therapy (NSPT) on clinical parameters of PDT, Candida spp. count and expression of lactoferrin (LF) and histatin (HST) in saliva and gingival crevicular fluid (GCF) of HIV-infected patients. METHODS: Bleeding index (BI), probing depth (PD), clinical attachment level (CAL), colonyforming units (CFUs) of Candida spp, and LF and HST levels were measured in saliva and GCF of both groups at three different times: baseline (before treatment), and 30 and 90 days after the NSPT. Clinical, mycological and immunoenzymatic analyses were also performed. RESULTS: Twenty-two HIV-infected patients and 25 non-HIV-infected patients with PDT participated in the study. NSPT was effective in improving periodontal clinical parameters, including ≤ 4 sites with PD ≤ 5mm and BI ≤ 10%. Significant change in oral Candida spp. count occurred neither between the two groups nor after NSPT. And the salivary and GCF levels of LF and HST were not influenced by the NSPT; by contrast, except for salivary LF, HST and LF were shown to exhibit significantly higher levels in HIV-infected than in non-HIV-infected patients. CONCLUSION: NSPT was effective in improving periodontal disease parameters in HIV-infected patients, but did not affect LF and HST expression in saliva and GCF of HIV-infected patients.

GPCR/endocytosis/ERK signaling/S2R is involved in the regulation of the internalization, mitochondria-targeting and -activating properties of human salivary histatin 1.

Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)-a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.

Zinc Binding Inhibits Cellular Uptake and Antifungal Activity of Histatin-5 in Candida albicans.

Histatin-5 (Hist-5) is a polycationic, histidine-rich antimicrobial peptide with potent antifungal activity against the opportunistic fungal pathogen Candida albicans. Hist-5 can bind metals in vitro, and metals have been shown to alter the fungicidal activity of the peptide. Previous reports on the effect of Zn2+ on Hist-5 activity have been varied and seemingly contradictory. Here, we present data elucidating the dynamic role Zn2+ plays as an inhibitory switch to regulate Hist-5 fungicidal activity. A novel fluorescently labeled Hist-5 peptide (Hist-5*) was developed to visualize changes in internalization and localization of the peptide as a function of metal availability in the growth medium. Hist-5* was verified for use as a model peptide and retained antifungal activity and mode of action similar to native Hist-5. Cellular growth assays showed that Zn2+ had a concentration-dependent inhibitory effect on Hist-5 antifungal activity. Imaging by confocal microscopy revealed that equimolar concentrations of Zn2+ kept the peptide localized along the cell periphery rather than internalizing, thus preventing cytotoxicity and membrane disruption. However, the Zn-induced decrease in Hist-5 activity and uptake was rescued by decreasing the Zn2+ availability upon addition of a metal chelator EDTA or S100A12, a Zn-binding protein involved in the innate immune response. These results lead us to suggest a model wherein commensal C. albicans may exist in harmony with Hist-5 at concentrations of Zn2+ that inhibit peptide internalization and antifungal activity. Activation of host immune processes that initiate Zn-sequestering mechanisms of nutritional immunity could trigger Hist-5 internalization and cell killing.

Histatin-1 alleviates high-glucose injury to skin keratinocytes through MAPK signaling pathway.

BACKGROUND: Damage to keratinocytes and other skin cells in a high-glucose environment has been proven to be an important reason for the poor wound healing ability of chronic diabetes mellitus. Histatin-1 has been preliminarily proven to stimulate the wound healing process of the oral and non-oral mucosa and has been found to be related to the activation of extracellular signal-regulated kinase (ERK). AIM OF THE STUDY: The purpose of this study was to investigate the effect of histatin-1 on high-glucose-injured keratinocytes and the role of the Ras-Raf-MEK-ERK signaling pathway on the effect of histatin-1 to improve diabetic wound healing. METHODS: A human keratinocyte model damaged by high glucose was constructed, cell proliferation was detected by the Cell Counting Kit-8 assay, and cell apoptosis was detected by flow cytometry. The expression level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was detected by ELISA, and the mitogen-activated protein kinase (MAPK) signaling pathway protein expression level was detected by Western blot. C-fos mRNA expression was detected by real-time PCR. RESULTS: The results indicated that histatin-1 promoted proliferation and reduced the rate of apoptosis and 8-OHdG content in keratinocytes with high-glucose injury. In addition, histatin-1 down-regulated MEK phosphorylation in keratinocytes with high-glucose injury. However, with the extension of the intervention, the effect of histatin-1 on c-fos mRNA expression was different. At the early stage of high-glucose injury (12 h), the expression of c-fos mRNA was not increased in high-glucose-injured keratinocytes treated with histatin-1 but then c-fos mRNA expression was gradually upregulated. CONCLUSION: Histatin-1 could alleviate keratinocyte injury caused by high glucose levels and promoted wound healing in vitro. In addition, histatin-1 could exert anti-apoptotic and antioxidant damage effects under high-glucose injury states. These effects of histatin-1 may be related to its regulation of the MAPK signaling pathway. Therefore, these findings provide an essential theoretical basis for histatin-1 to become a safe and effective new peptide biological agent to promote wound healing in patients with diabetes.

Acellular dermal matrix decorated with collagen-affinity peptide accelerate diabetic wound healing through sustained releasing Histatin-1 mediated promotion of angiogenesis.

Treating diabetic ulcers is a major challenge in clinical practice, persecuting millions of patients with diabetes and increasing the medical burden. Recombinant growth factor application can accelerate diabetic wound healing via angiogenesis. The local administration of recombinant growth factors has no robust clinical efficiency because of the degradation of append short duration of the molecules in the hostile inflammatoryenvironment.The present study focused on the pathophysiology of impaired neovascularization and growth factor short duration in the diabetic wound. We prepared a collagen-binding domain (CBD)-fused recombinant peptide (C-Histatin-1) that had both pro-angiogenesis capacity and collagen-affinity properties. Next, we created a biocompatible acellular dermal matrix (ADM) as a drug delivery carrier that featured collagen-richness, high porosity, and non-cytotoxicity. C-Histatin-1 was then tethered on ADM to obtain a sustained-release effect. Finally, a functional scaffold (C-Hst1/ADM) was developed. C-Hst1/ADM can sustain-release Histatin-1 to promote the adhesion, migration, and angiogenesisof vascular endothelial cells in vitro. Using a diabetic wound model, we showed that C-Hst1/ADM could significantly promote angiogenesis, reduce scar widths, and improve extracellular collagen accumulation. Therefore, the results of this study provide a foundation for the clinical application of C-Hst1/ADM covering scaffold in the treatment of diabetic wounds.

Identification of VEGFR2 as the Histatin-1 receptor in endothelial cells.

Histatin-1 is a salivary peptide with antimicrobial and wound healing promoting activities, which was previously shown to stimulate angiogenesis in vitro and in vivo via inducing endothelial cell migration. The mechanisms underlying the proangiogenic effects of Histatin-1 remain poorly understood and specifically, the endothelial receptor for this peptide, is unknown. Based on the similarities between Histatin-1-dependent responses and those induced by the prototypical angiogenic receptor, vascular endothelial growth factor receptor 2 (VEGFR2), we hypothesized that VEGFR2 is the Histatin-1 receptor in endothelial cells. First, we observed that VEGFR2 is necessary for Histatin-1-induced endothelial cell migration, as shown by both pharmacological inhibition studies and siRNA-mediated ablation of VEGFR2. Moreover, Histatin-1 co-immunoprecipitated and co-localized with VEGFR2, associating spatial proximity between these proteins with receptor activation. Indeed, pulldown assays with pure, tagged and non-tagged proteins showed that Histatin-1 and VEGFR2 directly interact in vitro. Optical tweezers experiments permitted estimating kinetic parameters and rupture forces, indicating that the Histatin-1-VEGFR2 interaction is transient, but specific and direct. Sequence alignment and molecular modeling identified residues Phe26, Tyr30 and Tyr34 within the C-terminal domain of Histatin-1 as relevant for VEGFR2 binding and activation. This was corroborated by mutation and molecular dynamics analyses, as well as in direct binding assays. Importantly, these residues were required for Histatin-1 to induce endothelial cell migration and angiogenesis in vitro. Taken together, our findings reveal that VEGFR2 is the endothelial cell receptor of Histatin-1 and provide insights to the mechanism by which this peptide promotes endothelial cell migration and angiogenesis.

Histatin-1 Attenuates LPS-Induced Inflammatory Signaling in RAW264.7 Macrophages.

Macrophages play a critical role in the inflammatory response to environmental triggers, such as lipopolysaccharide (LPS). Inflammatory signaling through macrophages and the innate immune system are increasingly recognized as important contributors to multiple acute and chronic disease processes. Nitric oxide (NO) is a free radical that plays an important role in immune and inflammatory responses as an important intercellular messenger. In addition, NO has an important role in inflammatory responses in mucosal environments such as the ocular surface. Histatin peptides are well-established antimicrobial and wound healing agents. These peptides are important in multiple biological systems, playing roles in responses to the environment and immunomodulation. Given the importance of macrophages in responses to environmental triggers and pathogens, we investigated the effect of histatin-1 (Hst1) on LPS-induced inflammatory responses and the underlying molecular mechanisms in RAW264.7 (RAW) macrophages. LPS-induced inflammatory signaling, NO production and cytokine production in macrophages were tested in response to treatment with Hst1. Hst1 application significantly reduced LPS-induced NO production, inflammatory cytokine production, and inflammatory signaling through the JNK and NF-kB pathways in RAW cells. These results demonstrate that Hst1 can inhibit LPS-induced inflammatory mediator production and MAPK signaling pathways in macrophages.

Histatin 1 enhanced the speed and quality of wound healing through regulating the behaviour of fibroblast.

OBJECTIVES: Histatin 1(Hst 1) has been proved to promote wound healing. However, there was no specific study on the regulation made by Hst 1 of fibroblasts in the process of wound healing. This research comprehensively studied the regulation of Hst 1 on the function of fibroblasts in the process of wound healing and preliminary mechanism about it. MATERIALS AND METHODS: The full-thickness skin wound model was made on the back of C57/BL6 mice. The wound healing, collagen deposition and fibroblast distribution were detected on days 3, 5 and 7 after injury. Fibroblast was cultured in vitro and stimulated with Hst 1, and then, their biological characteristics and functions were detected. RESULTS: Histatin 1 can effectively promote wound healing, improve collagen deposition during and after healing and increase the number and function of fibroblasts. After healing, the mechanical properties of the skin also improved. In vitro, the migration ability of fibroblasts stimulated by Hst 1 was significantly improved, and the fibroblasts transformed more into myofibroblasts, which improved the function of contraction and collagen secretion. In fibroblasts, mTOR signalling pathway can be activated by Hst 1. CONCLUSIONS: Histatin 1 can accelerate wound healing and improve the mechanical properties of healed skin by promoting the function of fibroblasts. The intermolecular mechanisms need to be further studied, and this study provides a direction about mTOR signalling pathway.

A Synergic Potential of Antimicrobial Peptides against Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) is the pathogenic agent responsible for the bacterial canker of kiwifruit (BCK) leading to major losses in kiwifruit productions. No effective treatments and measures have yet been found to control this disease. Despite antimicrobial peptides (AMPs) having been successfully used for the control of several pathogenic bacteria, few studies have focused on the use of AMPs against Psa. In this study, the potential of six AMPs (BP100, RW-BP100, CA-M, 3.1, D4E1, and Dhvar-5) to control Psa was investigated. The minimal inhibitory and bactericidal concentrations (MIC and MBC) were determined and membrane damaging capacity was evaluated by flow cytometry analysis. Among the tested AMPs, the higher inhibitory and bactericidal capacity was observed for BP100 and CA-M with MIC of 3.4 and 3.4-6.2 µM, respectively and MBC 3.4-10 µM for both. Flow cytometry assays suggested a faster membrane permeation for peptide 3.1, in comparison with the other AMPs studied. Peptide mixtures were also tested, disclosing the high efficiency of BP100:3.1 at low concentration to reduce Psa viability. These results highlight the potential interest of AMP mixtures against Psa, and 3.1 as an antimicrobial molecule that can improve other treatments in synergic action.

Effects and mechanisms of histatins as novel skin wound-healing agents.

Wound healing is a complex and important physiological process that maintains the integrity of skin after various injuries. Abnormal wound healing, especially of chronic wounds, impairs normal physical function. Therefore, the search for effective and safe healing agents is one of the main concerns. Histatins are histidine-rich low molecular weight peptides that are expressed in the saliva of both humans and higher primates. Histatins have two main biological effects, cell stimulation and bacteria killing, with the former playing an important role in wound healing by promoting epithelial cell and fibroblast migration and angiogenesis and enhancing the re-epithelialization of the wounded area. Because of these biological effects, histatins have been shown to be promising agents of improved wound healing. Histatins are categorized into many subtypes, of which histatin 1 and its hydrolysates are the most effective in promoting wound healing. This review addresses the bioactivity of histatins in wound healing, such as their stimulatory effects on epithelial cells and fibroblasts, and elucidates the possible mechanisms by which histatin subtypes induce their biological effects.

Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in Candida glabrata.

Candida glabrata is an emerging fungal pathogen whose success depends on its ability to resist antifungal drugs but also to thrive against host defenses. In this study, the predicted multidrug transporter CgTpo4 (encoded by ORF CAGL0L10912g) is described as a new determinant of virulence in C. glabrata, using the infection model Galleria mellonella. The CgTPO4 gene was found to be required for the C. glabrata ability to kill G. mellonella. The transporter encoded by this gene is also necessary for antimicrobial peptide (AMP) resistance, specifically against histatin-5. Interestingly, G. mellonella's AMP expression was found to be strongly activated in response to C. glabrata infection, suggesting AMPs are a key antifungal defense. CgTpo4 was also found to be a plasma membrane exporter of polyamines, especially spermidine, suggesting that CgTpo4 is able to export polyamines and AMPs, thus conferring resistance to both stress agents. Altogether, this study presents the polyamine exporter CgTpo4 as a determinant of C. glabrata virulence, which acts by protecting the yeast cells from the overexpression of AMPs, deployed as a host defense mechanism.

Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation.

Histatin-1 is a salivary antimicrobial peptide involved in the maintenance of enamel and oral mucosal homeostasis. Moreover, Histatin-1 has been shown to promote re-epithelialization in soft tissues, by stimulating cell adhesion and migration in oral and dermal keratinocytes, gingival and skin fibroblasts, endothelial cells and corneal epithelial cells. The broad-spectrum activity of Histatin-1 suggests that it behaves as a universal wound healing promoter, although this is far from being clear yet. Here, we report that Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation. Specifically, Histatin-1 promoted cell adhesion, spreading, and migration of SAOS-2 cells and MC3T3-E1 preosteoblasts in vitro, when placed on a fibronectin matrix. Besides, Histatin-1 induced the expression of osteogenic genes, including osteocalcin, osteopontin, and Runx2, and increased both activity and protein levels of alkaline phosphatase. Furthermore, Histatin-1 promoted mineralization in vitro, as it augmented the formation of calcium deposits in both SAOS-2 and MC3T3-E1 cells. Mechanistically, although Histatin-1 failed to activate ERK1/2, FAK, and Akt, which are signaling proteins associated with osteogenic differentiation or cell migration, it triggered nuclear relocalization of ß-catenin. Strikingly, the effects of Histatin-1 were recapitulated in cells that are nonosteogenically committed, since it promoted surface adhesion, migration, and the acquisition of osteogenic markers in primary mesenchymal cells derived from the apical papilla and dental pulp. Collectively, these observations indicate that Histatin-1 is a novel osteogenic factor that promotes bone cell differentiation, surface adhesion and migration, as crucial events required for bone tissue regeneration.