Systematic Screening of Penetratin’s Protein Targets by Yeast Proteome Microarrays

Cell-penetrating peptides (CPPs) have distinct properties to translocate across cell envelope. The key property of CPPs to translocation with attached molecules has been utilized as vehicles for the delivery of several potential drug candidates that illustrate the significant effect in in-vitro experiment but fail in in-vivo experiment due to selectively permeable nature of cell envelop. Penetratin, a well-known CPP identified from the third α-helix of Antennapedia homeodomain of Drosophila, has been widely used and studied for the delivery of bioactive molecules to treat cancers, stroke, and infections caused by pathogenic organisms. Few studies have demonstrated that penetratin directly possesses antimicrobial activities against bacterial and fungal pathogens; however, the mechanism is unknown. In this study, we have utilized the power of high-throughput Saccharomyces cerevisiae proteome microarrays to screen all the potential protein targets of penetratin. Saccharomyces cerevisiae proteome microarrays assays of penetratin followed by statistical analysis depicted 123 Saccharomyces cerevisiae proteins as the protein targets of penetratin out of ~5800 Saccharomyces cerevisiae proteins. To understand the target patterns of penetratin, enrichment analyses were conducted using 123 protein targets. In biological process: ribonucleoprotein complex biogenesis, nucleic acid metabolic process, actin filament-based process, transcription, DNA-templated, and negative regulation of gene expression are a few significantly enriched terms. Cytoplasm, nucleus, and cell-organelles are enriched terms for cellular component. Protein-protein interactions network depicted ribonucleoprotein complex biogenesis, cortical cytoskeleton, and histone binding, which represent the major enriched terms for the 123 protein targets of penetratin. We also compared the protein targets of penetratin and intracellular protein targets of antifungal AMPs (Lfcin B, Histatin-5, and Sub-5). The comparison results showed few unique proteins between penetratin and AMPs. Nucleic acid metabolic process and cellular component disassembly were the common enrichment terms for penetratin and three AMPs. Penetratin shows unique enrichment items that are related to DNA biological process. Moreover, motif enrichment analysis depicted different enriched motifs in the protein targets of penetratin, LfcinB, Histatin-5, and Sub-5.

The salivary, metal-binding peptide histatin-5 buffers extracellular copper availability

The family of human salivary histidine-rich peptides known as histatins bind zinc (Zn) and copper (Cu), but whether they contribute to nutritional immunity by influencing Zn and/or Cu availability has not been examined. We hypothesised that histatin-5 (Hst5) limits Zn availability (and promotes bacterial Zn starvation) and/or raises Cu availability (and promotes bacterial Cu poisoning). To test this hypothesis, Group A Streptococcus (GAS), which colonises the human oropharynx, was used as a model bacterium. Contrary to our hypothesis, Hst5 did not strongly influence Zn availability. This peptide did not induce expression of Zn uptake genes in GAS, nor did it suppress growth of an ΔadcAI mutant strain that is impaired in Zn uptake. Equilibrium competition measurements confirmed that Hst5 binds Zn weakly and does not compete with the high-affinity Zn uptake protein AdcAI for binding Zn. By contrast, Hst5 bound Cu with a high affinity and strongly influenced Cu availability. However, contrary to our hypothesis, Hst5 did not promote Cu toxicity. Instead, this peptide suppressed expression of Cu-inducible genes in GAS, stopped intracellular accumulation of Cu, and rescued growth of a ΔcopA mutant strain that is impaired in Cu efflux in the presence of added Cu. These findings led us to propose a new role for Hst5 and salivary histatins as major Cu buffers in saliva that reduce the potential negative effects of Cu exposure to microbes. We speculate that histatins promote oral and oropharyngeal health by contributing to microbial homeostasis in these host niches.

Liposomes functionalized with fungi-targeting peptide demonstrate increased interaction with Candida albicans

Candida albicans infections can be challenging to treat, as current antifungal drugs exhibit poor water solubility and host toxicity. To overcome these issues, new methods of drug delivery are needed. Liposomes have been shown to be an effective method for administrating antifungals and can increase bioavailability and solubility while decreasing toxicity. However, existing antifungal liposomal formulations lack infection specificity. For example, AmBisome, a liposomal formulation of amphotericin B, relies on passive accumulation to infection sites. We have developed antifungal liposomes that display fungi-targeting moieties to promote interaction with Candida;we predict that these formulations can increase fungal eradication and decrease off-site toxicity. Here, the C. albicans-targeting peptide P-113Q2.10 (AQRHHGYKRQFH), a derivative of the antifungal peptide histatin 5, was incorporated into liposomes via conjugation to palmitic acid (PA). PA-P-113Q2.10 conjugates were synthesized using solid phase peptide synthesis, confirmed by liquid-chromatography-mass spectrometry. Liposomes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and cholesterol with 1% w/w PA-P-113Q2.10 were formed via thin film-hydration and extrusion, yielding ∼100 nm liposomes with a polydispersity index of ∼0.1. Flow cytometry demonstrated that interaction with C. albicans SC5314 was enhanced for P-113Q2.10 liposomes, increasing from ∼60% in cells incubated with liposomes lacking peptide to ∼79%. These liposomes preferentially interact with C. albicans compared to NIH 3T3 murine fibroblasts; on average, only ∼15% of fibroblasts incubated with liposomes (with and without peptide) showed positive liposome interaction. This liposome formulation has the potential to serve as an antifungal delivery platform that selectively targets C. albicans cells for increased efficacy in treatment of fungal infections.

Envisaging Antifungal Potential of Histatin 5: A Physiological Salivary Peptide

Fungi are reported to cause a range of superficial to invasive human infections. These often result in high morbidity and at times mortality. Conventional antifungal agents though effective invariably exhibit drug interactions, treatment-related toxicity, and fail to elicit significant effect, thus indicating a need to look for suitable alternatives. Fungi thrive in humid, nutrient-enriched areas. Such an environment is well-supported by the oral cavity. Despite this, there is a relatively low incidence of severe oral and periodontal fungal infections, attributed to the presence of antimicrobial peptides hosted by saliva, viz. histatin 5 (Hstn 5). It displays fungicidal activity against a variety of fungi including Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, and unicellular yeast-like Saccharomyces cerevisiae. Candida albicans alone accounts for about 70% of all global fungal infections including periodontal disease. This review intends to discuss the scope of Hstn 5 as a novel recourse for the control of fungal infections.

A Novel Role for Histatin 5 in Combination with Zinc to Promote Commensalism in C. albicans Survivor Cells

Candida albicans is maintained as a commensal by immune mechanisms at the oral epithelia. Oral antifungal peptide Histatin 5 (Hst 5) may function in innate immunity, but the specific role Hst 5 plays in C. albicans commensalism is unclear. Since Zn-binding potentiates the candidacidal activity of Hst 5, we hypothesized that Hst 5+Zn would elicit a unique fungal stress response to shape interactions between C. albicans and oral epithelial cells (OECs). We found that Hst 5+Zn but not Hst 5 alone resulted in the activation of cell wall integrity (CWI) signaling, and deletion mutants were then used to determine that CWI-mediated chitin synthesis was protective against killing. Using flow cytometry, we confirmed that Hst 5+Zn-treated cells had significantly elevated levels of cell-wall chitin, mannan and β-1,3 glucan compared to Hst 5-treated cells. We then tested the activation of host signaling components involved in C. albicans cell-wall recognition. The immunoblot assay of C. albicans-exposed oral epithelial cells showed increased activation of EphA2 and NF-κB but not EGFR. Interestingly, C. albicans treated with Hst 5+Zn induced the global suppression of pro-inflammatory cytokine release from OECs, but an increase in negative regulator IL-10. Hst 5+Zn-treated cells were more adherent but ultimately less invasive to OECs than control cells, thus indicating lowered virulence. Therefore, Hst 5+Zn-treated C. albicans cells are discerned by epithelial monolayers, but are less virulent and promote anti-inflammatory signaling, suggesting that Hst 5+Zn in combination could play a role in regulating commensalism of oral C. albicans through cell wall reorganization.

Histatin 5 Metallopeptides and Their Potential against Candida albicans Pathogenicity and Drug Resistance

Usually caused by Candida albicans, buccal candidiasis begins with the morphological transition between yeast and hyphal cells. Over time and without the correct treatment, it can be disseminated through the bloodstream becoming a systemic infection with high mortality rates. C. albicans already shows resistance against antifungals commonly used in treatments. Therefore, the search for new drugs capable of overcoming antifungal resistance is essential. Histatin 5 (Hst5) is an antimicrobial peptide of the Histatin family, that can be found naturally in human saliva. This peptide presents high antifungal activity against C. albicans. However, Hst5 action can be decreased for interaction with enzymes and metal ions present in the oral cavity. The current work aims to bring a brief review of relevant aspects of the pathogenesis and resistance mechanisms already reported for C. albicans. In addition, are also reported here the main immune responses of the human body and the most common antifungal drugs. Finally, the most important aspects regarding Histatin 5 and the benefits of its interaction with metals are highlighted. The intention of this review is to show the promising use of Hst5 metallopeptides in the development of effective drugs.

Association between Antimicrobial Peptide Histatin 5 Levels and Prevalence of Candida in Saliva of Patients with Down Syndrome

There are no studies on Candida colonization and micropeptides of saliva in any patient. Therefore, we studied the effects of the salivary antimicrobial peptide histatin 5 on oral fungal colonization; subjects were subdivided into Down syndrome (D) and normal (N) groups by age: N-1 and D-1, age <20 years; N-2 and D-2, age >40 years. Histatin 5 concentration in saliva was measured by enzyme-linked immunosorbent assay. Oral Candida species were identified using CHROMagar Candida. Candida colonization was significantly enhanced in the D-1 and D-2 groups compared to the N-1 and N-2 groups. There was no predominant difference in salivary histatin 5 concentration between the D-1 and N-1 groups, but it was significantly lower in the D-2 group than in the N-2 group. Only in the N-2 group was there a correlation between the concentration of histatin 5 and total protein, while no correlation was found in the other groups. In elderly patients with Down syndrome, the decrease in histatin 5 shown in this study may lead to oral Candida colony formation. Therefore, the results of this study suggest that a deficiency of the antimicrobial peptide histatin 5 could possibly induce oral Candida infection in DS.

Inhibition of Candida albicans and Mixed Salivary Bacterial Biofilms on Antimicrobial Loaded Phosphated Poly(methyl methacrylate)

Biofilms play a crucial role in the development of Candida-associated denture stomatitis. Inhibition of microbial adhesion to poly(methyl methacrylate) (PMMA) and phosphate containing PMMA has been examined in this work. C. albicans and mixed salivary microbial biofilms were compared on naked and salivary pre-conditioned PMMA surfaces in the presence or absence of antimicrobials (Cetylpyridinium chloride [CPC], KSL-W, Histatin 5 [His 5]). Polymers with varying amounts of phosphate (0–25%) were tested using four C. albicans oral isolates as well as mixed salivary bacteria and 24 h biofilms were assessed for metabolic activity and confirmed using Live/Dead staining and confocal microscopy. Biofilm metabolism was reduced as phosphate density increased (15%: p = 0.004; 25%: p = 0.001). Loading of CPC on 15% phosphated disks showed a substantial decrease (p = 0.001) in biofilm metabolism in the presence or absence of a salivary pellicle. Salivary pellicle on uncharged PMMA enhanced the antimicrobial activity of CPC only. CPC also demonstrated remarkable antimicrobial activity on mixed salivary bacterial biofilms under different conditions displaying the potent efficacy of CPC (350 µg/mL) when combined with an artificial protein pellicle (Biotene half strength).