Metabolic Plasticity of Candida albicans in Response to Different Environmental Conditions.

The ubiquitous commensal Candida albicans, part of the human microbiota, is an opportunistic pathogen able to cause a wide range of diseases, from cutaneous mycoses to life-threatening infections in immunocompromised patients. Candida albicans adapts to different environments and survives long-time starvation. The ability to switch from yeast to hyphal morphology under specific environmental conditions is associated with its virulence. Using hydrogen nuclear magnetic resonance spectroscopy, we profiled the intracellular and extracellular metabolome of C. albicans kept in water, yeast extract-peptone-dextrose (YPD), and M199 media, at selected temperatures. Experiments were carried out in hypoxia to mimic a condition present in most colonized niches and fungal infection sites. Comparison of the intracellular metabolites measured in YPD and M199 at 37 °C highlighted differences in specific metabolic pathways: (i) alanine, aspartate, glutamate metabolism, (ii) arginine and proline metabolism, (iii) glycerolipid metabolism, attributable to the diverse composition of the media. Moreover, we hypothesized that the subtle differences in the M199 metabolome, observed at 30 °C and 37 °C, are suggestive of modifications propaedeutic to a subsequent transition from yeast to hyphal form. The analysis of the metabolites' profiles of C. albicans allows envisaging a molecular model to better describe its ability to sense and adapt to environmental conditions.

Therapeutic Effect of an Antibody-Derived Peptide in a Galleria mellonella Model of Systemic Candidiasis.

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans. A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

Wickerhamomyces Yeast Killer Toxins' Medical Applications.

Possible implications and applications of the yeast killer phenomenon in the fight against infectious diseases are reviewed, with particular reference to some wide-spectrum killer toxins (KTs) produced by Wickerhamomyces anomalus and other related species. A perspective on the applications of these KTs in the medical field is provided considering (1) a direct use of killer strains, in particular in the symbiotic control of arthropod-borne diseases; (2) a direct use of KTs as experimental therapeutic agents; (3) the production, through the idiotypic network, of immunological derivatives of KTs and their use as potential anti-infective therapeutics. Studies on immunological derivatives of KTs in the context of vaccine development are also described.

In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity.

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

In Vitro and In Vivo Anti-Candida Activity and Structural Analysis of Killer Peptide (KP)-Derivatives.

The previously described decapeptide AKVTMTCSAS (killer peptide, KP), derived from the variable region of a recombinant yeast killer toxin-like anti-idiotypic antibody, proved to exert a variety of antimicrobial, antiviral, and immunomodulatory activities. It also showed a peculiar self-assembly ability, likely responsible for the therapeutic effect in animal models of systemic and mucosal candidiasis. The present study analyzed the biological and structural properties of peptides derived from KP by substitution or deletion of the first residue, leaving unchanged the remaining amino acids. The investigated peptides proved to exert differential in vitro and/or in vivo anti-Candida activity without showing toxic effects on mammalian cells. The change of the first residue in KP amino acidic sequence affected the conformation of the resulting peptides in solution, as assessed by circular dichroism spectroscopy. KP-derivatives, except one, were able to induce apoptosis in yeast cells, like KP itself. ROS production and changes in mitochondrial transmembrane potential were also observed. Confocal and transmission electron microscopy studies allowed to establish that selected peptides could penetrate within C. albicans cells and cause gross morphological alterations. Overall, the physical and chemical properties of the first residue were found to be important for peptide conformation, candidacidal activity and possible mechanism of action. Small antimicrobial peptides could be exploited for the development of a new generation of antifungal drugs, given their relative low cost and ease of production as well as the possibility of devising novel delivery systems.

Anti-Infective Antibody-Derived Peptides Active against Endogenous and Exogenous Fungi.

Mycoses still represent relevant opportunistic infections worldwide, although overshadowed in recent years by other severe and more widespread infections. Moreover, deep-seated mycoses are often accompanied by unacceptably high mortality rates. Etiologic agents include endogenous components of the mycobiota, Candida and Malassezia species above all, and exogenous species, both yeasts and filamentous fungi. Old and new fungal pathogens are increasingly characterized by resistance to the existing antifungal agents, making imperative the search for effective and safe new therapeutics. Among the candidate molecules proposed in recent decades, synthetic peptides derived from the complementarity determining and constant regions of diverse antibodies (Abs), as well as the translated products of Ab-encoding genes, have proved of considerable interest. Their anti-infective activities, regardless of the specificity and isotype of the originating Ab, will be briefly presented and discussed in the light of their different mechanisms of action. Intriguing suggestions on the possible function of Abs after their half-life will be presented, following the recent detection, in human serum, of an antimicrobial Ab-derived peptide. Overall, Abs could represent a source of biologically active, highly flexible peptides, devoid of detectable toxicity, which can be easily synthesized and manipulated to be used, alone or in association with already available drugs, for new anti-infective strategies.

A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo.

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597-609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

An engineered anti-idiotypic antibody-derived killer peptide (KP) early activates swine inflammatory monocytes, CD3+CD16+ natural killer T cells and CD4+CD8α+ double positive CD8ß+ cytotoxic T lymphocytes associated with TNF-α and IFN-γ secretion.

This study evaluated the early modulation of the phenotype and cytokine secretion in swine immune cells treated with an engineered killer peptide (KP) based on an anti-idiotypic antibody functionally mimicking a yeast killer toxin. The influence of KP on specific immunity was investigated using porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) as ex vivo antigens. Peripheral blood mononuclear cells (PBMC) from healthy pigs were stimulated with KP and with a scramble peptide for 20 min, 1, 4 and 20 h or kept unstimulated. The cells were analyzed using flow cytometry and ELISA. The same time-periods were used for KP pre-incubation/co-incubation to determine the effect on virus-recalled interferon-gamma (IFN-γ) secreting cell (SC) frequencies and single cell IFN-γ productivity using ELISPOT. KP induced an early dose-dependent shift to pro-inflammatory CD172α+CD14+high monocytes and an increase of CD3+CD16+ natural killer (NK) T cells. KP triggered CD8α and CD8ß expression on classical CD4-CD8αß+ cytotoxic T lymphocytes (CTL) and double positive (DP) CD4+CD8α+ Th memory cells (CD4+CD8α+low CD8ß+low). A fraction of DP cells also expressed high levels of CD8α. The two identified DP CD4+CD8α+high CD8ß+low/+high CTL subsets were associated with tumor necrosis factor alpha (TNF-α) and IFN-γ secretion. KP markedly boosted the reactivity and cross-reactivity of PRRSV type-1- and PCV2b-specific IFN-γ SC. The results indicate the efficacy of KP in stimulating Th1-biased immunomodulation and support studies of KP as an immunomodulator or vaccine adjuvant.

Dissection of the Structural Features of a Fungicidal Antibody-Derived Peptide.

The synthetic peptide T11F (TCRVDHRGLTF), derived from the constant region of human IgM antibodies, proved to exert a significant activity in vitro against yeast strains, including multidrug resistant isolates. Alanine substitution of positively charged residues led to a decrease in candidacidal activity. A more dramatic reduction in activity resulted from cysteine replacement. Here, we investigated the conformational properties of T11F and its alanine-substituted derivatives by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Peptide interaction with Candida albicans cells was studied by confocal and scanning electron microscopy. T11F and most of its derivatives exhibited CD spectra with a negative band around 200 nm and a weaker positive band around 218 nm suggesting, together with NMR coupling constants, the presence of a polyproline II (PPII) helix, a conformational motif involved in a number of biological functions. Analysis of CD spectra revealed a critical role for phenylalanine in preserving the PPII helix. In fact, only the F11A derivative presented a random coil conformation. Interestingly, the loss of secondary structure influenced the rate of killing, which turned out to be significantly reduced. Overall, the obtained results suggest that the PPII conformation contributes in characterising the cell penetrating and fungicidal properties of the investigated peptides.

Discovering a new class of antifungal agents that selectively inhibits microbial carbonic anhydrases.

Infections caused by pathogens resistant to the available antimicrobial treatments represent nowadays a threat to global public health. Recently, it has been demonstrated that carbonic anhydrases (CAs) are essential for the growth of many pathogens and their inhibition leads to growth defects. Principal drawbacks in using CA inhibitors (CAIs) as antimicrobial agents are the side effects due to the lack of selectivity toward human CA isoforms. Herein we report a new class of CAIs, which preferentially interacts with microbial CA active sites over the human ones. The mechanism of action of these inhibitors was investigated against an important fungal pathogen, Cryptococcus neoformans, revealing that they are also able to inhibit CA in microbial cells growing in vitro. At our best knowledge, this is the first report on newly designed synthetic compounds selectively targeting ß-CAs and provides a proof of concept of microbial CAs suitability as an antimicrobial drug target.

Novel Activity of a Synthetic Decapeptide Against Toxoplasma gondii Tachyzoites.

The killer peptide KP is a synthetic decapeptide derived from the sequence of the variable region of a recombinant yeast killer toxin-like microbicidal single-chain antibody. KP proved to exert significant activities against diverse microbial and viral pathogens through different mechanisms of action, but little is known of its effect on apicomplexan protozoa. The aim of the present study was to evaluate the in vitro activity of KP against Toxoplasma gondii, a globally widespread protozoan parasite of great medical interest. The effect of KP treatment and its potential mechanism of action on T. gondii were evaluated by various methods, including light microscopy, quantitative PCR, flow cytometry, confocal microscopy, and transmission electron microscopy. In the presence of KP, the number of T. gondii tachyzoites able to invade Vero cells and the parasite intracellular proliferation were significantly reduced. Morphological observation and analysis of apoptotic markers suggested that KP is able to trigger an apoptosis-like cell death in T. gondii. Overall, our results indicate that KP could be a promising candidate for the development of new anti-Toxoplasma drugs with a novel mechanism of action.

Antiviral Activity of Synthetic Peptides Derived from Physiological Proteins.

BACKGROUND/AIMS: New antivirals are needed to supplement or replace currently used drugs. The aim of this study was to evaluate the antiviral activity of synthetic peptides derived from physiological proteins. METHODS: Vero cell monolayers were infected with herpes simplex virus 1, vesicular stomatitis virus, adenovirus, and coxsackievirus B5 strains in the presence of different concentrations of the selected peptides and viral yield was determined by plaque reduction assays to evaluate the antiviral activity of the peptides. Virucidal activity was evaluated by determining the residual infectivity of viral suspensions treated for 1 h with the peptides at the same concentrations as in the viral yield assays. RESULTS: Among the investigated peptides, the killer peptide proved to exert a considerable antiviral activity against herpes simplex virus, attributable to a direct effect on virus particles, while its derivative K10S showed to be effective against the four investigated virus strains only at the highest concentration tested, yet, the inhibitory effects were only partial. CONCLUSION: Overall, initial evidence is provided on the antiviral activity of several peptides, as well as of their derivatives. Further investigation is warranted to ascertain the mechanism of action in order to develop new potential antiviral drugs.

Fungicidal activity of peptides encoded by immunoglobulin genes.

Evidence from previous works disclosed the antimicrobial, antiviral, anti-tumour and/or immunomodulatory activity exerted, through different mechanisms of action, by peptides expressed in the complementarity-determining regions or even in the constant region of antibodies, independently from their specificity and isotype. Presently, we report the selection, from available databases, of peptide sequences encoded by immunoglobulin genes for the evaluation of their potential biological activities. Synthetic peptides representing the translated products of J lambda and J heavy genes proved to act in vitro against pathogenic fungi, entering yeast cells and causing their death, and exerted a therapeutic effect in a Galleria mellonella model of infection by Candida albicans. No haemolytic, cytotoxic and genotoxic effects were observed on mammalian cells. These findings raise the hypothesis that antibodies could be the evolutionary result of the adaptive combination of gene products ancestrally devoted to innate antimicrobial immunity.

The synthetic killer peptide KP impairs Candida albicans biofilm in vitro.

Candida albicans is a commensal organism, commonly inhabiting mucosal surfaces of healthy individuals, as a part of the resident microbiota. However, in susceptible hosts, especially hospitalized and/or immunocompromised patients, it may cause a wide range of infections. The presence of abiotic substrates, such as central venous or urinary catheters, provides an additional niche for Candida attachment and persistence, particularly via biofilm development. Furthermore, Candida biofilm is poorly susceptible to most antifungals, including azoles. Here we investigated the effects of a synthetic killer peptide (KP), known to be active in vitro, ex vivo and/or in vivo against different pathogens, on C. albicans biofilm. Together with a scrambled peptide used as a negative control, KP was tested against Candida biofilm at different stages of development. A reference strain, two fluconazole-resistant and two fluconazole-susceptible C. albicans clinical isolates were used. KP-induced C. albicans oxidative stress response and membrane permeability were also analysed. Moreover, the effect of KP on transcriptional profiles of C. albicans genes involved in different stages of biofilm development, such as cell adhesion, hyphal development and extracellular matrix production, was evaluated. Our results clearly show that the treatment with KP strongly affected the capacity of C. albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in C. albicans oxidative stress response and membrane permeability; also, biofilm-related genes expression was significantly reduced. Comparable inhibitory effects were observed in all the strains employed, irrespective of their resistance or susceptibility to fluconazole. Finally, KP-mediated inhibitory effects were observed also against a catheter-associated C. albicans biofilm. This study provides the first evidence on the KP effectiveness against C. albicans biofilm, suggesting that KP may be considered as a potential novel tool for treatment and prevention of biofilm-related C. albicans infections.

Idiotypic Antifungal Vaccination: Immunoprotection by Antiidiotypic Antibiotic Antibodies.

As implied by the idiotypic network theory, the interaction between the functional epitope of a microbicidal molecule (X) and its specific cell-wall receptor (RX) on sensitive microorganisms may be imaged by the bond between the idiotype (Id) of a neutralizing monoclonal antibody (anti-X Ab) and its anti-idiotype (anti-Id) X-like Ab (anti-anti-X Ab). Consequently, anti-X Ab Id may mimic RX acting as a vaccine (idiotypic vaccination) for the elicitation of protective anti-Id Abs with antibiotic activity (antibiobodies).

Phenotypic modulation of porcine CD14+ monocytes, natural killer/natural killer T cells and CD8αß+ T cell subsets by an antibody-derived killer peptide (KP).

An engineered killer peptide (KP) based on a recombinant anti-idiotypic antibody representing the functional image of a yeast killer toxin (KT) was demonstrated to mediate antimicrobial effects against fungi and viruses. KP binds to murine dendritic cells and macrophages and up-regulate co-receptor expression, thus sustaining CD4+ lymphocyte activation. No immunological data are available in domestic animals thus KP-induced immunomodulation was evaluated in porcine monocyte and lymphocyte subsets. PBMC from healthy adult pigs were stimulated with KP or a scramble peptide (SP), or kept unstimulated for 24, 48 and 72h, and subsequently analyzed by flow cytometry. In monocytes, KP induced a strong dose-dependent shift from a major fraction of CD172α+CD14+low cells to a predominant fraction of CD172α+CD14+high cells, known to sustain leukocyte activation/differentiation and inflammatory responses. The CD16+ cell percentages, specifically the CD3+CD16+ natural killer T (NKT) cell fraction and CD16 expression showed an intense and stable dose-dependent increase while the CD3-CD16+ NK cell fraction decreased. CD4+ and CD8+ T cells increased and CD8α and CD8ß expression were up-regulated. CD8ß+ cytotoxic T cells and CD16+ cells comparably increased. A marked stimulation of activated CD16+CD25+ and CD8ß+CD25+ cells was observed at 24h. The increase of CD8α+ cells and CD8α expression were due to increased CD4+CD8α+ (memory T helper) cells, also showing a CD8α+high phenotype. Concomitantly, the CD4+CD8α- T helper lymphocyte fraction significantly decreased. Overall, KP induced a wide modulation of innate immune and T cells that can exert regulatory and cytotoxic functions, which are fundamental for an efficient Th1 response.

A Naturally Occurring Antibody Fragment Neutralizes Infectivity of Diverse Infectious Agents.

A phosphorylated peptide, named K40H, derived from the constant region of IgMs was detected in human serum by liquid chromatography coupled to high-resolution mass spectrometry. Synthetic K40H proved to exert a potent in vitro activity against fungal pathogens, and to inhibit HIV-1 replication in vitro and ex vivo. It also showed a therapeutic effect against an experimental infection by Candida albicans in the invertebrate model Galleria mellonella. K40H represents the proof of concept of the innate role that naturally occurring antibody fragments may exert against infectious agents, shedding a new light upon the posthumous role of antibodies and opening a new scenario on the multifaceted functionality of humoral immunity.

Natural and synthetic peptides with antifungal activity.

In recent years, the increase of invasive fungal infections and the emergence of antifungal resistance stressed the need for new antifungal drugs. Peptides have shown to be good candidates for the development of alternative antimicrobial agents through high-throughput screening, and subsequent optimization according to a rational approach. This review presents a brief overview on antifungal natural peptides of different sources (animals, plants, micro-organisms), peptide fragments derived by proteolytic cleavage of precursor physiological proteins (cryptides), synthetic unnatural peptides and peptide derivatives. Antifungal peptides are schematically reported based on their structure, antifungal spectrum and reported effects. Natural or synthetic peptides and their modified derivatives may represent the basis for new compounds active against fungal infections.

Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins.

Synthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.