Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats.

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

A review on lactoferrin as a proton pump inhibitor.

Lactoferrin (Lf) is a versatile natural milk-derived protein that exhibits multiple interesting biological activities. Since it is safe for human administration and currently manufactured using low cost and well-established large-scale processes, the Lf scientific community has been devoted at dissecting its mechanisms of action towards its more rational and efficient use for various applications. Emerging literature has identified proton pumping ATPases as molecular targets of Lf in different cellular models linked to distinct activities of this natural protein. Information on this subject has not been systematically analysed before, hence herein we review the current state of art on the effect of Lf on proton pumping ATPases. Though structurally different, we propose that Lf holds a proton pump inhibitor (PPI)-like activity based on the functional resemblance with the classical inhibitors of the stomach H+/K+-ATPase. The downstream events and outcomes of the PPI-like activity of Lf, as well as its impact for the development of improved Lf applications are also discussed.

Lactoferrin perturbs lipid rafts and requires integrity of Pma1p-lipid rafts association to exert its antifungal activity against Saccharomyces cerevisiae.

Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs ergosterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma1-10 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi.

Cytosolic Acidification Is the First Transduction Signal of Lactoferrin-induced Regulated Cell Death Pathway.

In yeast, we reported the critical role of K+-efflux for the progress of the regulated cell death (RCD) induced by human lactoferrin (hLf), an antimicrobial protein of the innate immune system that blocks Pma1p H+-ATPase. In the present study, the K+ channel Tok1p was identified as the K+ channel-mediating K+-efflux, as indicated by the protective effect of extracellular K+ (30 mM), K+-channel blockers, and the greater hLf-resistance of TOK1-disrupted strains. K+-depletion was necessary but not sufficient to induce RCD as inferred from the effects of valinomycin, NH4Cl or nigericin which released a percentage of K+ similar to that released by lactoferrin without affecting cell viability. Cytosolic pH of hLf-treated cells decreased transiently (0.3 pH units) and its inhibition prevented the RCD process, indicating that cytosolic acidification was a necessary and sufficient triggering signal. The blocking effect of lactoferrin on Pma1p H+-ATPase caused a transitory decrease of cytosolic pH, and the subsequent membrane depolarization activated the voltage-gated K+ channel, Tok1p, allowing an electrogenic K+-efflux. These ionic events, cytosolic accumulation of H+ followed by K+-efflux, constituted the initiating signals of this mitochondria-mediated cell death. These findings suggest, for the first time, the existence of an ionic signaling pathway in RCD.

Antifungal Mechanism of Action of Lactoferrin: Identification of H+-ATPase (P3A-Type) as a New Apoptotic-Cell Membrane Receptor.

Human lactoferrin (hLf) is a protein of the innate immune system which induces an apoptotic-like process in yeast. Determination of the susceptibility to lactoferrin of several yeast species under different metabolic conditions, respiratory activity, cytoplasmic ATP levels, and external medium acidification mediated by glucose assays suggested plasma membrane Pma1p (P3A-type ATPase) as the hLf molecular target. The inhibition of plasma membrane ATPase activity by hLf and the identification of Pma1p as the hLf-binding membrane protein confirmed the previous physiological evidence. Consistent with this, cytoplasmic ATP levels progressively increased in hLf-treated Candida albicans cells. However, oligomycin, a specific inhibitor of the mitochondrial F-type ATPase proton pump (mtATPase), abrogated the antifungal activity of hLf, indicating a crucial role for mtATPase in the apoptotic process. We suggest that lactoferrin targeted plasma membrane Pma1p H(+)-ATPase, perturbing the cytoplasmic ion homeostasis (i.e., cytoplasmic H(+) accumulation and subsequent K(+) efflux) and inducing a lethal mitochondrial dysfunction. This initial event involved a normal mitochondrial ATP synthase activity responsible for both the ATP increment and subsequent hypothetical mitochondrial proton flooding process. We conclude that human lactoferrin inhibited Pma1p H(+)-ATPase, inducing an apoptotic-like process in metabolically active yeast. Involvement of mitochondrial H(+)-ATPase (nonreverted) was essential for the progress of this programmed cell death in which the ionic homeostasis perturbation seems to precede classical nonionic apoptotic events.

Antimicrobial mechanism of action of transferrins: selective inhibition of H+-ATPase.

Two bacterial species with different metabolic features, namely, Pseudomonas aeruginosa and Lactococcus lactis, were used as a comparative experimental model to investigate the antimicrobial target and mechanism of transferrins. In anaerobiosis, P. aeruginosa cells were not susceptible to lactoferrin (hLf) or transferrin (hTf). In aerobiosis, the cells were susceptible but O(2) consumption was not modified, indicating that components of the electron transport chain (ETC) were not targeted. However, the respiratory chain inhibitor piericidin A significantly reduced the killing activity of both proteins. Moreover, 2,6-dichlorophenolindophenol (DCIP), a reducing agent that accepts electrons from the ETC coupled to H(+) extrusion, made P. aeruginosa susceptible to hLf and hTf in anaerobiosis. These results indicated that active cooperation of the cell was indispensable for the antimicrobial effect. For L. lactis cells lacking an ETC, the absence of a detectable transmembrane electrical potential in hLf-treated cells suggested a loss of H(+)-ATPase activity. Furthermore, the inhibition of ATPase activity and H(+) translocation (inverted membrane vesicles) provided direct evidence of the ability of hLf to inhibit H(+)-ATPase in L. lactis. Based on these data, we propose that hLf and hTf also inhibit the H(+)-ATPase of respiring P. aeruginosa cells. Such inhibition thereby interferes with reentry of H(+) from the periplasmic space to the cytoplasm, resulting in perturbation of intracellular pH and the transmembrane proton gradient. Consistent with this hypothesis, periplasmic H(+) accumulation was prevented by anaerobiosis or by piericidin A or was induced by DCIP in anaerobiosis. Collectively, these results indicate that transferrins target H(+)-ATPase and interfere with H(+) translocation, yielding a lethal effect in vitro.

Human lactoferrin induces apoptosis-like cell death in Candida albicans: critical role of K+-channel-mediated K+ efflux.

Human lactoferrin (hLf) induced an apoptosis-like phenotype in Candida albicans cells, which includes phosphatidylserine externalization, nuclear chromatin condensation, DNA degradation, and increased reactive oxygen species (ROS) production. Intracellular ROS accumulation was seen to correlate with candidacidal activity in hLf-treated cells. Mitochondrial activity was involved as indicated by mitochondrial depolarization and increased hLf resistance of cells preincubated with sordarin or erythromycin, the latter of which inhibits protein synthesis in mitoribosomes. Interestingly, Cl(-)- and K(+)-channel blockers prevented the hLf antimicrobial activity, but only when cells were pretreated with the blocking agent (tetraethylammonium) prior to the hLf-induced K(+)-release period. These results indicate for the first time that K(+)-channel-mediated K(+) efflux is required for the progression of apoptosis-like process in yeast, suggesting that this essential apoptotic event of higher eukaryotes has been evolutionary conserved among species ranging from yeasts to humans.

The gamma-core motif correlates with antimicrobial activity in cysteine-containing kaliocin-1 originating from transferrins.

Kaliocin-1 is a 31-residue peptide derived from human lactoferrin, and with antimicrobial properties that recapitulate those of its 611 amino acid parent holoprotein. As kaliocin-1 is a cysteine-stabilized peptide, it was of interest to determine whether it contained a multidimensional gamma-core signature recently identified as common to virtually all classes of disulfide-stabilized antimicrobial peptides. Importantly, sequence and structural analyses identified an iteration of this multidimensional antimicrobial signature in kaliocin-1. Further, the gamma-core motif was found to be highly conserved in the transferrin family of proteins across the phylogenetic spectrum. Previous studies suggested that the mechanism by which kaliocin-1 exerts anti-candidal efficacy depends on mitochondrial perturbation without cell membrane permeabilization. Interestingly, results of a yeast two-hybrid screening analysis identified an interaction between kaliocin-1 and mitochondrial initiation factor 2 in a Saccharomyces cerevisiae model system. Taken together, these data extend the repertoire of antimicrobial peptides that contain gamma-core motifs, and suggest that the motif is conserved within large native as well as antimicrobial peptide subcomponents of transferrin family proteins. Finally, these results substantiate the hypothesis that antimicrobial activity associated with host defense effector proteins containing a gamma-core motif may correspond to targets common to fungal mitochondria or their bacterial ancestors.

Molecular genetics of berry colour variation in table grape.

The genetics and biochemistry of anthocyanins and flavonol biosynthesis and their role in plant organ pigmentation is well established in model species. However, the genetic basis of colour variation is species specific and understanding this variation is very relevant in many fruit and flower crop species. Among grape cultivars, there is a wide genetic variation for berry colour ranging from yellow-green ("white" cultivars) to dark blue berries. Berry colour results from the synthesis and accumulation of anthocyanins in the berry skin, which in plants is commonly regulated by transcription factors belonging to the MYB and bHLH families. In this work, we aimed to identify the major genetic determinants of berry colour variation in a large collection of table grape cultivars and somatic variants. The genetic analyses of berry colour in a few grape segregating progenies had previously identified a single locus on linkage group 2 responsible for colour variation. Furthermore, somatic variation for berry skin colour in cultivar Italia had been associated with the presence of a Gret1 retrotransposon in the promoter region of VvmybA1, a Myb gene whose expression is associated to skin colouration. The results show that VvmybA1 is the gene underlying the mapped locus controlling berry colour in grape. Additionally, the molecular analyses indicate that genetic and somatic berry colour variation can be associated to molecular variation at VvmybA1 in more than 95% of the analyzed cultivars. Thus, VvmybA1 is a major determinant of berry colour variation in table grape and its instability is the major cause of somatic variation for this trait.

Different anti-Candida activities of two human lactoferrin-derived peptides, Lfpep and kaliocin-1.

The synthetic peptides Lfpep and kaliocin-1 include the sequences from positions 18 to 40 and 153 to 183 of human lactoferrin, respectively. Lfpep is a cationic peptide with bactericidal and giardicidal effects, whereas kaliocin-1 is a novel bactericidal peptide that corresponds to a highly homologous sequence present in the transferrin family of proteins. Both peptides presented fungicidal activity against Candida spp., including fluconazole- and amphotericin B-resistant clinical isolates. Lfpep exhibited higher antifungal activity (8- to 30-fold) and salt resistance than kaliocin-1. The killing activity of Lfpep was mediated by its permeabilizing activity on Candida albicans cells, whereas kaliocin-1 was unable to disrupt the cytoplasmic membrane, as indicated by its inability to allow permeation of propidium iodide and the small amount of K+ released. The amino acid sequence of kaliocin-1 includes the "multidimensional antimicrobial signature" conserved in disulfide-containing antimicrobial peptides and a striking similarity to brevinin-1Sa, an antimicrobial peptide from frog skin secretions, exhibiting a "Rana box"-like sequence. These features may be of interest in the design of new antifungals.

Antibiotic tolerance induced by lactoferrin in clinical Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Lactoferrin-induced cell depolarization and a delayed tobramycin-killing effect on Pseudomonas aeruginosa cells were correlated. This antibiotic tolerance effect (ATE) reflects the ability of a defense protein to modify the activity of an antibiotic as a result of its modulatory effect on bacterial physiology. P. aeruginosa isolates from cystic fibrosis patients showed higher ATE values (< or = 6-fold) than other clinical strains.

Effects of human lactoferrin on the cytoplasmic membrane of Candida albicans cells related with its candidacidal activity.

Human lactoferrin is an innate host defence protein with antimicrobial activity that exerts a candidacidal effect in a cation concentration-dependent manner. We investigated the ability of this cationic protein (with an isoelectric point of 8.7) to permeabilize the cytoplasmic membrane of Candida albicans cells. Despite minor K(+)-release in lactoferrin-treated C. albicans cells, the killing effect was not related to an extensive membrane permeabilization, as indicated by: (a) the non-release of macromolecular cytosolic constituents; (b) the non-permeabilization for extracellular propidium iodide nor for intracellular accumulated calcein; and (c) the inability to disrupt the phospholipid bilayer of 8-aminonaphthalene-1,3,6, trisulfonic acid/p-xylene-bis-pyridiniumbromide-loaded liposomes. These results suggest that lactoferrin exerts its candidacidal effect through a mechanism different from membrane permeabilization described for other cationic peptides.

Modulation of in vitro fungicidal activity of human lactoferrin against Candida albicans by extracellular cation concentration and target cell metabolic activity.

The anti-Candida activity of the innate defense protein human lactoferrin was investigated. Lactoferrin displayed a clear fungicidal effect against Candida albicans only under low-strength conditions. This candidacidal activity was inversely correlated with the extracellular concentration of the monovalent cations and was prevented by Na(+) and K(+) (> or 30 mM) and by divalent cations (Ca(2+) and Mg(2+) at > or 4 mM). A slight cellular release of K(+), cytosolic acidification, and a change in the membrane potential were observed in C. albicans cells treated with lactoferrin, suggesting that this protein directly or indirectly interacts with the cytoplasmic membrane. Mitochondrial inhibitors (carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, azide, and antimycin) as well as anaerobic conditions significantly reduced the killing effect of lactoferrin. These results suggest that low-strength conditions and the cellular metabolic state may modulate the candidacidal activity of human lactoferrin.