Fighting pathogenic yeasts with plant defensins and anti-fungal proteins from fungi.

Fungal infections represent a significant health risk worldwide. Opportunistic infections caused by yeasts, particularly by Candida spp. and their virulent emerging isolates, have become a major threat to humans, with an increase in fatal cases of infections attributed to the lack of effective anti-yeast therapies and the emergence of fungal resistance to the currently applied drugs. In this regard, the need for novel anti-fungal agents with modes of action different from those currently available is undeniable. Anti-microbial peptides (AMPs) are promising candidates for the development of novel anti-fungal biomolecules to be applied in clinic. A class of AMPs that is of particular interest is the small cysteine-rich proteins (CRPs). Among CRPs, plant defensins and anti-fungal proteins (AFPs) of fungal origin constitute two of the largest and most promising groups of CRPs showing anti-fungal properties, including activity against multi-resistant pathogenic yeasts. In this review, we update and compare the sequence, structure, and properties of plant defensins and AFPs with anti-yeast activity, along with their in vitro and in vivo potency. We focus on the current knowledge about their mechanism of action that may lead the way to new anti-fungals, as well as on the developments for their effective biotechnological production. KEY POINTS: • Plant defensins and fungal AFPs are alternative anti-yeast agents • Their multi-faceted mode of action makes occurrence of resistance rather improbable • Safe and cost-effective biofactories remain crucial for clinical application.

Application of recyclable CRISPR/Cas9 tools for targeted genome editing in the postharvest pathogenic fungi Penicillium digitatum and Penicillium expansum.

Penicillium digitatum and Penicillium expansum are plant pathogenic fungi that cause the green and blue mold diseases, respectively, leading to serious postharvest economic losses worldwide. Moreover, P. expansum can produce mycotoxins, which are hazardous compounds to human and animal health. The development of tools that allow multiple and precise genetic manipulation of these species is crucial for the functional characterization of their genes. In this sense, CRISPR/Cas9 represents an excellent opportunity for genome editing due to its efficiency, accuracy and versatility. In this study, we developed protoplast generation and transformation protocols and applied them to implement the CRISPR/Cas9 technology in both species for the first time. For this, we used a self-replicative, recyclable AMA1-based plasmid which allows unlimited number of genomic modifications without the limitation of integrative selection markers. As test case, we successfully targeted the wetA gene, which encodes a regulator of conidiophore development. Finally, CRISPR/Cas9-derived ΔwetA strains were analyzed. Mutants showed reduced axenic growth, differential pathogenicity and altered conidiogenesis and germination. Additionally, P. digitatum and P. expansum ΔwetA mutants showed distinct sensitivity to fungal antifungal proteins (AFPs), which are small, cationic, cysteine-rich proteins that have become interesting antifungals to be applied in agriculture, medicine and in the food industry. With this work, we demonstrate the feasibility of the CRISPR/Cas9 system, expanding the repertoire of genetic engineering tools available for these two important postharvest pathogens and open up the possibility to adapt them to other economically relevant phytopathogenic fungi, for which toolkits for genetic modifications are often limited.

A Novel Secreted Cysteine-Rich Anionic (Sca) Protein from the Citrus Postharvest Pathogen Penicillium digitatum Enhances Virulence and Modulates the Activity of the Antifungal Protein B (AfpB).

Antifungal proteins (AFPs) from ascomycete fungi could help the development of antimycotics. However, little is known about their biological role or functional interactions with other fungal biomolecules. We previously reported that AfpB from the postharvest pathogen Penicillium digitatum cannot be detected in the parental fungus yet is abundantly produced biotechnologically. While aiming to detect AfpB, we identified a conserved and novel small Secreted Cysteine-rich Anionic (Sca) protein, encoded by the gene PDIG_23520 from P. digitatum CECT 20796. The sca gene is expressed during culture and early during citrus fruit infection. Both null mutant (Δsca) and Sca overproducer (Scaop) strains show no phenotypic differences from the wild type. Sca is not antimicrobial but potentiates P. digitatum growth when added in high amounts and enhances the in vitro antifungal activity of AfpB. The Scaop strain shows increased incidence of infection in citrus fruit, similar to the addition of purified Sca to the wild-type inoculum. Sca compensates and overcomes the protective effect of AfpB and the antifungal protein PeAfpA from the apple pathogen Penicillium expansum in fruit inoculations. Our study shows that Sca is a novel protein that enhances the growth and virulence of its parental fungus and modulates the activity of AFPs.

The Antifungal Protein AfpB Induces Regulated Cell Death in Its Parental Fungus Penicillium digitatum.

Filamentous fungi produce small cysteine-rich proteins with potent, specific antifungal activity, offering the potential to fight fungal infections that severely threaten human health and food safety and security. The genome of the citrus postharvest fungal pathogen Penicillium digitatum encodes one of these antifungal proteins, namely AfpB. Biotechnologically produced AfpB inhibited the growth of major pathogenic fungi at minimal concentrations, surprisingly including its parental fungus, and conferred protection to crop plants against fungal infections. This study reports an in-depth characterization of the AfpB mechanism of action, showing that it is a cell-penetrating protein that triggers a regulated cell death program in the target fungus. We prove the importance of AfpB interaction with the fungal cell wall to exert its killing activity, for which protein mannosylation is required. We also show that the potent activity of AfpB correlates with its rapid and efficient uptake by fungal cells through an energy-dependent process. Once internalized, AfpB induces a transcriptional reprogramming signaled by reactive oxygen species that ends in cell death. Our data show that AfpB activates a self-injury program, suggesting that this protein has a biological function in the parental fungus beyond defense against competitors, presumably more related to regulation of the fungal population. Our results demonstrate that this protein is a potent antifungal that acts through various targets to kill fungal cells through a regulated process, making AfpB a promising compound for the development of novel biofungicides with multiple fields of application in crop and postharvest protection, food preservation, and medical therapies.IMPORTANCE Disease-causing fungi pose a serious threat to human health and food safety and security. The limited number of licensed antifungals, together with the emergence of pathogenic fungi with multiple resistance to available antifungals, represents a serious challenge for medicine and agriculture. Therefore, there is an urgent need for new compounds with high fungal specificity and novel antifungal mechanisms. Antifungal proteins in general, and AfpB from Penicillium digitatum in particular, are promising molecules for the development of novel antifungals. This study on AfpB's mode of action demonstrates its potent, specific fungicidal activity through the interaction with multiple targets, presumably reducing the risk of evolving fungal resistance, and through a regulated cell death process, uncovering this protein as an excellent candidate for a novel biofungicide. The in-depth knowledge on AfpB mechanistic function presented in this work is important to guide its possible future clinical and agricultural applications.

The Penicillium chrysogenum Q176 Antimicrobial Protein PAFC Effectively Inhibits the Growth of the Opportunistic Human Pathogen Candida albicans.

Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes promise treatment alternatives to licensed antifungal drugs. In this study, we characterized the Penicillium chrysogenum Q176 antifungal protein C (PAFC), which is phylogenetically distinct to the other two Penicillium antifungal proteins, PAF and PAFB, that are expressed by this biotechnologically important ascomycete. PAFC is secreted into the culture broth and is co-expressed with PAF and PAFB in the exudates of surface cultures. This observation is in line with the suggested role of AMPs in the adaptive response of the host to endogenous and/or environmental stimuli. The in silico structural model predicted five ß-strands stabilized by four intramolecular disulfide bonds in PAFC. The functional characterization of recombinant PAFC provided evidence for a promising new molecule in anti-Candida therapy. The thermotolerant PAFC killed planktonic cells and reduced the metabolic activity of sessile cells in pre-established biofilms of two Candidaalbicans strains, one of which was a fluconazole-resistant clinical isolate showing higher PAFC sensitivity than the fluconazole-sensitive strain. Candidacidal activity was linked to severe cell morphology changes, PAFC internalization, induction of intracellular reactive oxygen species and plasma membrane disintegration. The lack of hemolytic activity further corroborates the potential applicability of PAFC in clinical therapy.

Improving Health-Promoting Effects of Food-Derived Bioactive Peptides through Rational Design and Oral Delivery Strategies.

Over the last few decades, scientific interest in food-derived bioactive peptides has grown as an alternative to pharmacological treatments in the control of lifestyle-associated diseases, which represent a serious health problem worldwide. Interest has been directed towards the control of hypertension, the management of type 2 diabetes and oxidative stress. Many food-derived antihypertensive peptides act primarily by inhibiting angiotensin I-converting enzyme (ACE), and to a lesser extent, renin enzyme activities. Antidiabetic peptides mainly inhibit dipeptidyl peptidase-IV (DPP-IV) activity, whereas antioxidant peptides act through inactivation of reactive oxygen species, free radicals scavenging, chelation of pro-oxidative transition metals and promoting the activities of intracellular antioxidant enzymes. However, food-derived bioactive peptides have intrinsic weaknesses, including poor chemical and physical stability and a short circulating plasma half-life that must be addressed for their application as nutraceuticals or in functional foods. This review summarizes the application of common pharmaceutical approaches such as rational design and oral delivery strategies to improve the health-promoting effects of food-derived bioactive peptides. We review the structural requirements of antihypertensive, antidiabetic and antioxidant peptides established by integrated computational methods and provide relevant examples of effective oral delivery systems to enhance solubility, stability and permeability of bioactive peptides.

Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus.

Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy-to-use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.

Three Antifungal Proteins From Penicillium expansum: Different Patterns of Production and Antifungal Activity.

Antifungal proteins of fungal origin (AFPs) are small, secreted, cationic, and cysteine-rich proteins. Filamentous fungi encode a wide repertoire of AFPs belonging to different phylogenetic classes, which offer a great potential to develop new antifungals for the control of pathogenic fungi. The fungus Penicillium expansum is one of the few reported to encode three AFPs each belonging to a different phylogenetic class (A, B, and C). In this work, the production of the putative AFPs from P. expansum was evaluated, but only the representative of class A, PeAfpA, was identified in culture supernatants of the native fungus. The biotechnological production of PeAfpB and PeAfpC was achieved in Penicillium chrysogenum with the P. chrysogenum-based expression cassette, which had been proved to work efficiently for the production of other related AFPs in filamentous fungi. Western blot analyses confirmed that P. expansum only produces PeAfpA naturally, whereas PeAfpB and PeAfpC could not be detected. From the three AFPs from P. expansum, PeAfpA showed the highest antifungal activity against all fungi tested, including plant and human pathogens. P. expansum was also sensitive to its self-AFPs PeAfpA and PeAfpB. PeAfpB showed moderate antifungal activity against filamentous fungi, whereas no activity could be attributed to PeAfpC at the conditions tested. Importantly, none of the PeAFPs showed hemolytic activity. Finally, PeAfpA was demonstrated to efficiently protect against fungal infections caused by Botrytis cinerea in tomato leaves and Penicillium digitatum in oranges. The strong antifungal potency of PeAfpA, together with the lack of cytotoxicity, and significant in vivo protection against phytopathogenic fungi that cause postharvest decay and plant diseases, make PeAfpA a promising alternative compound for application in agriculture, but also in medicine or food preservation.

Rational Design and Biotechnological Production of Novel AfpB-PAF26 Chimeric Antifungal Proteins.

Antimicrobial peptides (AMPs) have been proposed as candidates to develop new antimicrobial compounds for medicine, agriculture, and food preservation. PAF26 is a synthetic antifungal hexapeptide obtained from combinatorial approaches with potent fungicidal activity against filamentous fungi. Other interesting AMPs are the antifungal proteins (AFPs) of fungal origin, which are basic cysteine-rich and small proteins that can be biotechnologically produced in high amounts. A promising AFP is the AfpB identified in the phytopathogen Penicillium digitatum. In this work, we aimed to rationally design, biotechnologically produce and test AfpB::PAF26 chimeric proteins to obtain designed AFPs (dAfpBs) with improved properties. The dAfpB6 and dAfpB9 chimeras could be produced using P. digitatum as biofactory and a previously described Penicillium chrysogenum-based expression cassette, but only dAfpB9 could be purified and characterized. Protein dAfpB9 showed subtle and fungus-dependent differences of fungistatic activity against filamentous fungi compared to native AfpB. Significantly, dAfpB9 lost the fungicidal activity of PAF26 and AfpB, thus disconnecting this activity from the fungistatic activity and mapping fungicidal determinants to the exposed loop L3 of AfpB, wherein modifications are located. This study provides information on the design and development of novel chimeric AFPs.

Mapping and Identification of Antifungal Peptides in the Putative Antifungal Protein AfpB from the Filamentous Fungus Penicillium digitatum.

Antifungal proteins (AFPs) from Ascomycetes are small cysteine-rich proteins that are abundantly secreted and show antifungal activity against non-producer fungi. A gene coding for a class B AFP (AfpB) was previously identified in the genome of the plant pathogen Penicillium digitatum. However, previous attempts to detect the AfpB protein were not successful despite the high expression of the corresponding afpB gene. In this work, the structure of the putative AfpB was modeled. Based on this model, four synthetic cysteine-containing peptides, PAF109, PAF112, PAF118, and PAF119, were designed and their antimicrobial activity was tested and characterized. PAF109 that corresponds to the γ-core motif present in defensin-like antimicrobial proteins did not show antimicrobial activity. On the contrary, PAF112 and PAF118, which are cationic peptides derived from two surface-exposed loops in AfpB, showed moderate antifungal activity against P. digitatum and other filamentous fungi. It was also confirmed that cyclization through a disulfide bridge prevented peptide degradation. PAF116, which is a peptide analogous to PAF112 but derived from the Penicillium chrysogenum antifungal protein PAF, showed activity against P. digitatum similar to PAF112, but was less active than the native PAF protein. The two AfpB-derived antifungal peptides PAF112 and PAF118 showed positive synergistic interaction when combined against P. digitatum. Furthermore, the synthetic hexapeptide PAF26 previously described in our laboratory also exhibited synergistic interaction with the peptides PAF112, PAF118, and PAF116, as well as with the PAF protein. This study is an important contribution to the mapping of antifungal motifs within the AfpB and other AFPs, and opens up new strategies for the rational design and application of antifungal peptides and proteins.

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses.

BACKGROUND: Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure-function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. RESULTS: The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI-MS, ECD and NMR spectroscopy and growth inhibition assays. CONCLUSION: This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure-function analyses.

Occurrence and function of fungal antifungal proteins: a case study of the citrus postharvest pathogen Penicillium digitatum.

Antifungal proteins (AFPs) of fungal origin have been described in filamentous fungi. AFPs are small, highly stable, cationic cysteine-rich proteins (CRPs) that are usually secreted in high amounts and show potent antifungal activity against non-self fungi. The role of AFPs in the biology of the producer fungus remains unclear. AFPs have been proposed as promising lead compounds for the development of new antifungals. The analyses of available antifungal CRP sequences from fungal origin and their phylogenetic reconstruction led us to propose a new classification of AFPs in three distinct classes: A, B and C. We initiate for the first time the characterization of an AFP in a fungal pathogen, by analysing the functional role of the unique afpB gene in the citrus fruit pathogen Penicillium digitatum. Null ΔafpB mutants revealed that this gene is dispensable for vegetative growth and fruit infection. However, strains that artificially express afpB in a constitutive way (afpB (C)) showed a phenotype of restricted growth, distortion of hyphal morphology and strong reduction in virulence to citrus fruits. These characteristics support an antifungal role for AfpB. Surprisingly, we did not detect the AfpB protein in any of the P. digitatum strains and growth conditions that were analysed in this study, regardless of high gene expression. The afpB (C) phenotype is not stable and occasionally reverts to a wild type-like phenotype but molecular changes were not detected with this reversion. The reduced virulence of afpB (C) strains correlated with localized fruit necrosis and altered timing of expression of fruit defence genes.

Central cell-derived peptides regulate early embryo patterning in flowering plants.

Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning in Arabidopsis. EMBRYO SURROUNDING FACTOR 1 (ESF1) peptides accumulate before fertilization in central cell gametes and thereafter in embryo-surrounding endosperm cells. Biochemical and structural analyses revealed cleavage of ESF1 propeptides to form biologically active mature peptides. Further, these peptides act in a non-cell-autonomous manner and synergistically with the receptor-like kinase SHORT SUSPENSOR to promote suspensor elongation through the YODA mitogen-activated protein kinase pathway. Our findings demonstrate that the second female gamete and its sexually derived endosperm regulate early embryonic patterning in flowering plants.

The myosin motor domain-containing chitin synthase PdChsVII is required for development, cell wall integrity and virulence in the citrus postharvest pathogen Penicillium digitatum.

Chitin is an essential component of the fungal cell wall and a potential target in the development of new antifungal compounds, due to its presence in fungi and not in plants or vertebrates. Chitin synthase genes (chs) constitute a complex family in filamentous fungi and are involved in fungal development, morphogenesis, pathogenesis and virulence. In this study, additional chs genes in the citrus postharvest pathogen Penicillium digitatum have been identified. Comparative analyses included each PdChs in each one of the classes I to VII previously established, and support the grouping of these into three divisions. Disruption of the gene coding PdChsVII, which contains a short version of a myosin motor domain, has been achieved by using Agrobacterium tumefaciens-mediated transformation and revealed its role in the life cycle of the fungus. Disruption strains were viable but showed reduced growth and conidia production. Moreover, Pdchs mutants developed morphological defects as balloon-like enlarged cells and increased chitin content, indicative of an altered cell wall structure. Gene disruption also increased susceptibility to antifungal compounds such as calcofluor white (CFW), sodium dodecyl sulfate (SDS), hydroxide peroxide (H2O2) and commercial fungicides, but significantly no change was observed in the sensitivity to antifungal peptides. The PdchsVII mutants were able to infect citrus fruit and produced tissue maceration, although had reduced virulence and most importantly were greatly impaired in the production of visible mycelium and conidia on the fruit.

Spatio-temporal expression patterns of Arabidopsis thaliana and Medicago truncatula defensin-like genes.

Plant genomes contain several hundred defensin-like (DEFL) genes that encode short cysteine-rich proteins resembling defensins, which are well known antimicrobial polypeptides. Little is known about the expression patterns or functions of many DEFLs because most were discovered recently and hence are not well represented on standard microarrays. We designed a custom Affymetrix chip consisting of probe sets for 317 and 684 DEFLs from Arabidopsis thaliana and Medicago truncatula, respectively for cataloging DEFL expression in a variety of plant organs at different developmental stages and during symbiotic and pathogenic associations. The microarray analysis provided evidence for the transcription of 71% and 90% of the DEFLs identified in Arabidopsis and Medicago, respectively, including many of the recently annotated DEFL genes that previously lacked expression information. Both model plants contain a subset of DEFLs specifically expressed in seeds or fruits. A few DEFLs, including some plant defensins, were significantly up-regulated in Arabidopsis leaves inoculated with Alternaria brassicicola or Pseudomonas syringae pathogens. Among these, some were dependent on jasmonic acid signaling or were associated with specific types of immune responses. There were notable differences in DEFL gene expression patterns between Arabidopsis and Medicago, as the majority of Arabidopsis DEFLs were expressed in inflorescences, while only a few exhibited root-enhanced expression. By contrast, Medicago DEFLs were most prominently expressed in nitrogen-fixing root nodules. Thus, our data document salient differences in DEFL temporal and spatial expression between Arabidopsis and Medicago, suggesting distinct signaling routes and distinct roles for these proteins in the two plant species.

Novel antihypertensive hexa- and heptapeptides with ACE-inhibiting properties: from the in vitro ACE assay to the spontaneously hypertensive rat.

Bioactive ACE inhibiting peptides are gaining interest in hypertension treatment. We have designed and screened six synthetic heptapeptides (PACEI48 to PACEI53) based on two hexapeptide leads (PACEI32 and PACEI34) to improve ACE inhibitory properties and assess their antihypertensive effects. ACE activity was assayed in vitro and ex vivo. Selected peptides were administered to spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. In vitro cytotoxicity was assessed with the MTT reduction test. The six heptapeptides at low micromolar concentration produced different degrees of in vitro inhibition of ACE activity using the synthetic substrate HHL or the natural substrate angiotensin I; and ex vivo inhibition of ACE-dependent, angiotensin I-induced vasoconstriction, but not angiotensin II-induced vasoconstriction. Oral administration of the hexapeptide PACEI32L, and the heptapeptides PACEI50L and PACEI52L, induced reductions in systolic blood pressure lasting up to 3h in SHRs but not in WKY rats. Intravenous injection of PACEI32L and PACEI50L, but not PACEI52L, induced acute transient reductions in mean blood pressure of SHRs. d-Amino acid peptides showed five-fold less ACE inhibitory potency, no inhibitory effect on angiotensin I-induced vasoconstriction, and antihypertensive effect in SHRs after i.v. injection, but not after oral administration. The toxicity of peptides to reduce the viability of cultured cells was in the millimolar range. In conclusion, we have obtained novel rationally designed heptapeptides with improved ACE inhibitory properties when compared to lead hexapeptides. One selected hexapeptide and two heptapeptides show oral antihypertensive effects in SHRs and appear safe in cytotoxicity assays.

Antihypertensive properties of lactoferricin B-derived peptides.

A set of eight lactoferricin B (LfcinB)-derived peptides was examined for inhibitory effects on angiotensin I-converting enzyme (ACE) activity and ACE-dependent vasoconstriction, and their hypotensive effect in spontaneously hypertensive rats (SHR). Peptides were derived from different elongations both at the C-terminal and N-terminal ends of the representative peptide LfcinB(20-25), which is known as the LfcinB antimicrobial core. All of the eight LfcinB-derived peptides showed in vitro inhibitory effects on ACE activity with different IC(50) values. Moreover, seven of them showed ex vivo inhibitory effects on ACE-dependent vasoconstriction. No clear correlation between in vitro and ex vivo inhibitory effects was found. Only LfcinB(20-25) and one of its fragments, F1, generated after a simulated gastrointestinal digestion, showed significant antihypertensive effects in SHR after oral administration. Remarkably, F1 did not show any effect on ACE-dependent vasoconstriction in contrast to the inhibitory effect showed by LfcinB(20-25). In conclusion, two LfcinB-derived peptides lower blood pressure and exhibit potential as orally effective antihypertensive compounds, yet a complete elucidation of the mechanism(s) involved deserves further ongoing research.

Comparative study of antimicrobial peptides to control citrus postharvest decay caused by Penicillium digitatum.

The objective of this study was to investigate and compare the in vitro efficacy and in vivo potential of eight distinct short antimicrobial peptides to control the postharvest green mold disease of oranges caused by the fungus Penicillium digitatum. The L-amino acid versions of the four peptides PAF26, PAF38, PAF40, and BM0, previously obtained by combinatorial approaches, were examined. The study included two antibacterial peptides formerly identified by rational design, BP15 and BP76, and it is demonstrated that they also have in vitro antifungal properties. The natural antimicrobial peptides melittin and indolicidin were also selected for comparison, due to their well-known properties and modes of action. In vitro and in vivo results indicated differential behaviors among peptides, regarding the inhibitory potency in growth media, selectivity against distinct microorganisms, fungicidal activity towards nongerminated conidia of P. digitatum, and efficacy in fruit inoculation assays. Interestingly, a high in vitro inhibitory activity did not necessarily associate with an effective control of fruit infection by P. digitatum. The short tryptophan-rich cationic peptides PAF26, PAF38, PAF40, and BM0 were lethal to conidia of P. digitatum, and this property is correlated with better protection in the decay control test.

Lactoferricin-related peptides with inhibitory effects on ACE-dependent vasoconstriction.

A selection of lactoferricin B (LfcinB)-related peptides with an angiotensin I-converting enzyme (ACE) inhibitory effect have been examined using in vitro and ex vivo functional assays. Peptides that were analyzed included a set of sequence-related antimicrobial hexapeptides previously reported and two representative LfcinB-derived peptides. In vitro assays using hippuryl-L-histidyl-L-leucine (HHL) and angiotensin I as substrates allowed us to select two hexapeptides, PACEI32 (Ac-RKWHFW-NH2) and PACEI34 (Ac-RKWLFW-NH2), and also a LfcinB-derived peptide, LfcinB17-31 (Ac-FKCRRWQWRMKKLGA-NH2). Ex vivo functional assays using rabbit carotid arterial segments showed PACEI32 (both D- and L-enantiomers) and LfcinB17-31 have inhibitory effects on ACE-dependent angiotensin I-induced contraction. None of the peptides exhibited in vitro ACE inhibitory activity using bradykinin as the substrate. In conclusion, three bioactive lactoferricin-related peptides exhibit inhibitory effects on both ACE activity and ACE-dependent vasoconstriction with potential to modulate hypertension that deserves further investigation.