Same same, but different: exploring the enigmatic role of the pituitary adenylate cyclase-activating polypeptide (PACAP) in invertebrate physiology.

Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.

Rational Design of Antifungal Peptides Based on the γ-Core Motif of a Neosartorya (Aspergillus) fischeri Antifungal Protein to Improve Structural Integrity, Efficacy, and Spectrum.

Antifungal peptides offer promising alternative compounds for the treatment of fungal infections, for which new antifungal compounds are urgently needed. Constant and broad antifungal spectra of these peptides play essential roles in their reliable therapeutic application. It has been observed that rationally designed peptides using the evolutionarily conserved γ-core region (GXC-X3-9-C) of an antifungal protein from Neosartorya (Aspergillus) fischeri highly inhibit the growth of fungi. The cysteines in these peptides have free sulfhydryl groups, which allow cyclization and dimerization under oxidative conditions, thereby impairing antifungal efficacy. To overcome this problem, one or two cysteine residues were substituted by serines or S-tert-butyl was applied as a cysteine-protecting group. Furthermore, structural integrity and antifungal efficacy investigations before and after oxidative exposure revealed that substituting both cysteines with serines and S-tert-butylation helped maintain the structural integrity. However, it slightly decreased the antifungal efficacy against a yeast, Candida albicans. Interestingly, S-tert-butylation maintained the efficacy and could extend the antifungal activity to a mold, Aspergillus fumigatus. Usually, cyclization and dimerization did not influence the antifungal efficacy of most peptides. Additionally, hemolysis tests and Galleria mellonella toxicity model experiments indicated that none of the applied modifications made the peptides harmful to animals.

Proline cis/trans Isomerization in Intrinsically Disordered Proteins and Peptides.

BACKGROUND: Intrinsically disordered proteins and protein regions (IDPs/IDRs) are important in diverse biological processes. Lacking a stable secondary structure, they display an ensemble of conformations. One factor contributing to this conformational heterogeneity is the proline cis/trans isomerization. The knowledge and value of a given cis/trans proline ratio are paramount, as the different conformational states can be responsible for different biological functions. Nuclear Magnetic Resonance (NMR) spectroscopy is the only method to characterize the two co-existing isomers on an atomic level, and only a few works report on these data. METHODS: After collecting the available experimental literature findings, we conducted a statistical analysis regarding the influence of the neighboring amino acid types (i ± 4 regions) on forming a cis-Pro isomer. Based on this, several regularities were formulated. NMR spectroscopy was then used to define the cis-Pro content on model peptides and desired point mutations. RESULTS: Analysis of NMR spectra prove the dependence of the cis-Pro content on the type of the neighboring amino acid-with special attention on aromatic and positively charged sidechains. CONCLUSIONS: Our results may benefit the design of protein regions with a given cis-Pro content, and contribute to a better understanding of the roles and functions of IDPs.

Confirmation of the Disulfide Connectivity and Strategies for Chemical Synthesis of the Four-Disulfide-Bond-Stabilized Aspergillus giganteus Antifungal Protein, AFP.

Emerging fungal infections require new, more efficient antifungal agents and therapies. AFP, a protein from Aspergillus giganteus with four disulfide bonds, is a promising candidate because it selectively inhibits the growth of filamentous fungi. In this work, the reduced form of AFP was prepared using native chemical ligation. The native protein was synthesized via oxidative folding with uniform protection for cysteine thiols. AFP's biological activity depends heavily on the pattern of natural disulfide bonds. Enzymatic digestion and MS analysis provide proof for interlocking disulfide topology (abcdabcd) that was previously assumed. With this knowledge, a semi-orthogonal thiol protection method was designed. By following this strategy, out of a possible 105, only 6 disulfide isomers formed and 1 of them proved to be identical with the native protein. This approach allows the synthesis of analogs for examining structure-activity relationships and, thus, preparing AFP variants with higher antifungal activity.

Enhanced Antibacterial Activity of Substituted Derivatives of NCR169C Peptide.

Medicago truncatula in symbiosis with its rhizobial bacterium partner produces more than 700 nodule-specific cysteine-rich (NCR) peptides with diverse physicochemical properties. Most of the cationic NCR peptides have antimicrobial activity and the potential to tackle antimicrobial resistance with their novel modes of action. This work focuses on the antibacterial activity of the NCR169 peptide derivatives as we previously demonstrated that the C-terminal sequence of NCR169 (NCR169C17-38) has antifungal activity, affecting the viability, morphology, and biofilm formation of various Candida species. Here, we show that NCR169C17-38 and its various substituted derivatives are also able to kill ESKAPE pathogens such as Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. The replacement of the two cysteines with serines enhanced the antimicrobial activity against most of the tested bacteria, indicating that the formation of a disulfide bridge is not required. As tryptophan can play role in the interaction with bacterial membranes and thus in antibacterial activity, we replaced the tryptophans in the NCR169C17-38C12,17/S sequence with various modified tryptophans, namely 5-methyl tryptophan, 5-fluoro tryptophan, 6-fluoro tryptophan, 7-aza tryptophan, and 5-methoxy tryptophan, in the synthesis of NCR169C17-38C12,17/S analogs. The results demonstrate that the presence of modified fluorotryptophans can significantly enhance the antimicrobial activity without notable hemolytic effect, and this finding could be beneficial for the further development of new AMPs from the members of the NCR peptide family.

Synthesis of the extracellular domain of GLP-1R by chemical and biotechnological approaches.

The extracellular domain of the glucagon-like peptide-1 receptor, GLP-1R, is responsible for the binding of GLP-1, and a handful of additional agonists (such as exenatide, lixisenatide, and liraglutide) used daily for treating type II diabetes mellitus. Lead discovery and optimization, however, require binding studies, which, in turn, necessitate the total synthesis of GLP-1R, comprising 108 residues. A protein domain of 10-15 kDa size could be obtained either by expression in E. coli or by ligating solid-phase peptide synthesis (SPPS)-made fragments. However, direct overexpression fails to give a properly folded protein, as GLP-1R forms an inclusion body, which fails to refold due to improper disulfide pairing. Several bacterial strains, constructs, and fusion partners were probed and it was found that only co-expression with MBP gave a 3D-fold allowing the native disulfide bond pattern formation. Some fusion partners can act as covalently linked or in situ chaperones for guiding the refolding of GLP-1R toward success. Therefore, the bottleneck to preparing GPCR extracellular domains is the correct pairing of the Cys residues. As a proof-of-concept model, nGLP1-R was made by SPPS to form the purified full-length polypeptide chain, subjected to self-guided or spontaneous Cys pairing. However, the formation of correct SS-pairs was lagging behind any protocol in use support, and the bottleneck of large-scale protein production relies on the risky step of proper refolding, which is sometimes possible only if a suitable fusion partner effectively helps and catalysis of the correct disulfide formation.

Legume Plant Peptides as Sources of Novel Antimicrobial Molecules Against Human Pathogens.

Antimicrobial peptides are prominent components of the plant immune system acting against a wide variety of pathogens. Legume plants from the inverted repeat lacking clade (IRLC) have evolved a unique gene family encoding nodule-specific cysteine-rich NCR peptides acting in the symbiotic cells of root nodules, where they convert their bacterial endosymbionts into non-cultivable, polyploid nitrogen-fixing cells. NCRs are usually 30-50 amino acids long peptides having a characteristic pattern of 4 or 6 cysteines and highly divergent amino acid composition. While the function of NCRs is largely unknown, antimicrobial activity has been demonstrated for a few cationic Medicago truncatula NCR peptides against bacterial and fungal pathogens. The advantages of these plant peptides are their broad antimicrobial spectrum, fast killing modes of actions, multiple bacterial targets, and low propensity to develop resistance to them and no or low cytotoxicity to human cells. In the IRLC legumes, the number of NCR genes varies from a few to several hundred and it is possible that altogether hundreds of thousands of different NCR peptides exist. Due to the need for new antimicrobial agents, we investigated the antimicrobial potential of 104 synthetic NCR peptides from M. truncatula, M. sativa, Pisum sativum, Galega orientalis and Cicer arietinum against eight human pathogens, including ESKAPE bacteria. 50 NCRs showed antimicrobial activity with differences in the antimicrobial spectrum and effectivity. The most active peptides eliminated bacteria at concentrations from 0.8 to 3.1 µM. High isoelectric point and positive net charge were important but not the only determinants of their antimicrobial activity. Testing the activity of shorter peptide derivatives against Acinetobacter baumannii and Candida albicans led to identification of regions responsible for the antimicrobial activity and provided insight into their potential modes of action. This work provides highly potent lead molecules without hemolytic activity on human blood cells for novel antimicrobial drugs to fight against pathogens.

sVmKTx, a transcriptome analysis-based synthetic peptide analogue of Vm24, inhibits Kv1.3 channels of human T cells with improved selectivity.

Kv1.3 K+ channels play a central role in the regulation of T cell activation and Ca2+ signaling under physiological and pathophysiological conditions. Peptide toxins targeting Kv1.3 have a significant therapeutic potential in the treatment of autoimmune diseases; thus, the discovery of new toxins is highly motivated. Based on the transcriptome analysis of the venom gland of V. mexicanus smithi a novel synthetic peptide, sVmKTx was generated, containing 36 amino acid residues. sVmKTx shows high sequence similarity to Vm24, a previously characterized peptide from the same species, but contains a Glu at position 32 as opposed to Lys32 in Vm24. Vm24 inhibits Kv1.3 with high affinity (Kd = 2.9 pM). However, it has limited selectivity (~1,500-fold) for Kv1.3 over hKv1.2, hKCa3.1, and mKv1.1. sVmKTx displays reduced Kv1.3 affinity (Kd = 770 pM) but increased selectivity for Kv1.3 over hKv1.2 (~9,000-fold) as compared to Vm24, other channels tested in the panel (hKCa3.1, hKv1.1, hKv1.4, hKv1.5, rKv2.1, hKv11.1, hKCa1.1, hNav1.5) were practically insensitive to the toxin at 2.5 µM. Molecular dynamics simulations showed that introduction of a Glu instead of Lys at position 32 led to a decreased structural fluctuation of the N-terminal segment of sVmKTx, which may explain its increased selectivity for Kv1.3. sVmKTx at 100 nM concentration decreased the expression level of the Ca2+ -dependent T cell activation marker, CD40 ligand. The high affinity block of Kv1.3 and increased selectivity over the natural peptide makes sVmKTx a potential candidate for Kv1.3 blockade-mediated treatment of autoimmune diseases.

Functional characterization and related evolutionary implications of invertebrate gonadotropin-releasing hormone/corazonin in a well-established model species.

In vertebrates, gonadotropin-releasing hormone (GnRH) peptide is the central mediator of reproduction. Homologous peptides have previously also been identified in molluscan species. However, emerging evidence suggests that these molecules might serve diverse regulatory functions and proposes to consider them as corazonin (CRZ). We previously isolated the full-length cDNA of the invGnRH/CRZ peptide (termed ly-GnRH/CRZ) in the well-established invertebrate model species, the great pond snail Lymnaea stagnalis; however, its predicted functions remain to be verified. In this study, we first confirmed the presence of the deduced active peptide from the central nervous system of L. stagnalis. Further, we performed in vivo and in vitro studies to explore the functions of ly-GnRH/CRZ. Injection of sexually mature specimens with synthetic active peptide had an inhibitory effect on locomotion and an acceleratory effect on egg-laying, but had no effect on feeding. The previously predicted modulatory effect of ly-GnRH/CRZ was supported by its identified co-localization with serotonin on the surface of the heart atria. Lastly, we demonstrated not only the presence of ly-GnRH/CRZ in the penial complex but also that ly-GnRH/CRZ-containing neurons project to the efferent penis nerve, suggesting ly-GnRH/CRZ may directly modulate the motor output of this peripheral tissue. Overall, our findings strongly support that ly-GnRH/CRZ is a multifunctional neuropeptide. These results contribute to the understanding of the GnRH superfamily and, more broadly, disciplines such as comparative endocrinology and neurobiology.

Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species.

The increasing rate of fungal infections causes global problems not only in human healthcare but agriculture as well. To combat fungal pathogens limited numbers of antifungal agents are available therefore alternative drugs are needed. Antimicrobial peptides are potent candidates because of their broad activity spectrum and their diverse mode of actions. The model legume Medicago truncatula produces >700 nodule specific cysteine-rich (NCR) peptides in symbiosis and many of them have in vitro antimicrobial activities without considerable toxicity on human cells. In this work we demonstrate the anticandidal activity of the NCR335 and NCR169 peptide derivatives against five Candida species by using the micro-dilution method, measuring inhibition of biofilm formation with the XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, and assessing the morphological change of dimorphic Candida species by microscopy. We show that both the N- and C-terminal regions of NCR335 possess anticandidal activity as well as the C-terminal sequence of NCR169. The active peptides inhibit biofilm formation and the yeast-hypha transformation. Combined treatment of C. auris with peptides and fluconazole revealed synergistic interactions and reduced 2-8-fold the minimal inhibitory concentrations. Our results demonstrate that shortening NCR peptides can even enhance and broaden their anticandidal activity and therapeutic potential.

The Neosartorya fischeri Antifungal Protein 2 (NFAP2): A New Potential Weapon against Multidrug-Resistant Candida auris Biofilms.

Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312-0.5, 0.155-0.5, 0.037-0.375, 0.064-0.375, and 0.064-0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 µM2%, 2.16 µM2%, 33.31 µM2%, 10.72 µM2%, and 111.19 µM2% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms.

Solution Structure, Dynamics, and New Antifungal Aspects of the Cysteine-Rich Miniprotein PAFC.

The genome of Penicillium chrysogenum Q176 contains a gene coding for the 88-amino-acid (aa)-long glycine- and cysteine-rich P. chrysogenum antifungal protein C (PAFC). After maturation, the secreted antifungal miniprotein (MP) comprises 64 aa and shares 80% aa identity with the bubble protein (BP) from Penicillium brevicompactum, which has a published X-ray structure. Our team expressed isotope (15N, 13C)-labeled, recombinant PAFC in high yields, which allowed us to determine the solution structure and molecular dynamics by nuclear magnetic resonance (NMR) experiments. The primary structure of PAFC is dominated by 14 glycines, and therefore, whether the four disulfide bonds can stabilize the fold is challenging. Indeed, unlike the few published solution structures of other antifungal MPs from filamentous ascomycetes, the NMR data indicate that PAFC has shorter secondary structure elements and lacks the typical ß-barrel structure, though it has a positively charged cavity and a hydrophobic core around the disulfide bonds. Some parts within the two putative γ-core motifs exhibited enhanced dynamics according to a new disorder index presentation of 15N-NMR relaxation data. Furthermore, we also provided a more detailed insight into the antifungal spectrum of PAFC, with specific emphasis on fungal plant pathogens. Our results suggest that PAFC could be an effective candidate for the development of new antifungal strategies in agriculture.

Stability Test of PACAP in Eye Drops.

PACAP is a neuropeptide with widespread distribution and diverse biological functions. It has strong cytoprotective effects mediated mainly through specific PAC1 receptors. Experimental data show protective effects of PACAP in the retina and cornea in several pathological conditions. Although intravitreal injections are a common practice in some ocular diseases, delivery of therapeutic agents in the form of eye drops would be more convenient and would lead to fewer side effects. We have previously shown that PACAP, in the form of eye drops, is able to pass through the ocular barriers and can exert retinoprotective effects. As eye drops represent a promising form of administration of PACAP in ocular diseases, it is important to investigate the stability of PACAP in solutions used in eye drops. In this study, the stability of PACAP1-27 and PACAP1-38 in eye drops was measured in four common media and a commercially available artificial tear solution at both room temperature and +4 °C. Mass spectrometry results show that the highest stability was gained with PACAP1-38 in water and 0.9% saline solution at +4 °C, representing 80-90% drug persistence after 2 weeks. PACAP1-38 in the artificial tear showed very fast degradation at room temperature, but was stable at +4 °C. In summary, PACAP1-38 has higher stability than PACAP1-27, with highest stability at +4 °C in water solution, but both peptides in each medium can be stored for relatively longer periods without significant degradation. These data can provide reference for future therapeutic use of PACAP in eye drops.

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.

The potential use of the Penicillium chrysogenum antifungal protein PAF, the designed variant PAFopt and its γ-core peptide Pγopt in plant protection.

The prevention of enormous crop losses caused by pesticide-resistant fungi is a serious challenge in agriculture. Application of alternative fungicides, such as antifungal proteins and peptides, provides a promising basis to overcome this problem; however, their direct use in fields suffers limitations, such as high cost of production, low stability, narrow antifungal spectrum and toxicity on plant or mammalian cells. Recently, we demonstrated that a Penicillium chrysogenum-based expression system provides a feasible tool for economic production of P. chrysogenum antifungal protein (PAF) and a rational designed variant (PAFopt ), in which the evolutionary conserved γ-core motif was modified to increase antifungal activity. In the present study, we report for the first time that γ-core modulation influences the antifungal spectrum and efficacy of PAF against important plant pathogenic ascomycetes, and the synthetic γ-core peptide Pγopt , a derivative of PAFopt , is antifungal active against these pathogens in vitro. Finally, we proved the protective potential of PAF against Botrytis cinerea infection in tomato plant leaves. The lack of any toxic effects on mammalian cells and plant seedlings, as well as the high tolerance to harsh environmental conditions and proteolytic degradation further strengthen our concept for applicability of these proteins and peptide in agriculture.

Potent Chimeric Antimicrobial Derivatives of the Medicago truncatula NCR247 Symbiotic Peptide.

In Rhizobium-legume symbiosis, the bacteria are converted into nitrogen-fixing bacteroids. In many legume species, differentiation of the endosymbiotic bacteria is irreversible, culminating in definitive loss of their cell division ability. This terminal differentiation is mediated by plant peptides produced in the symbiotic cells. In Medicago truncatula more than ∼700 nodule-specific cysteine-rich (NCR) peptides are involved in this process. We have shown previously that NCR247 and NCR335 have strong antimicrobial activity on various pathogenic bacteria and identified interaction of NCR247 with many bacterial proteins, including FtsZ and several ribosomal proteins, which prevent bacterial cell division and protein synthesis. In this study we designed and synthetized various derivatives of NCR247, including shorter fragments and various chimeric derivatives. The antimicrobial activity of these peptides was tested on the ESKAPE bacteria; Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli as a member of Enterobacteriaceae and in addition Listeria monocytogenes and Salmonella enterica. The 12 amino acid long C-terminal half of NCR247, NCR247C partially retained the antimicrobial activity and preserved the multitarget interactions with partners of NCR247. Nevertheless NCR247C became ineffective on S. aureus, P. aeruginosa, and L. monocytogenes. The chimeric derivatives obtained by fusion of NCR247C with other peptide fragments and particularly with a truncated mastoparan sequence significantly increased bactericidal activity and altered the antimicrobial spectrum. The minimal bactericidal concentration of the most potent derivatives was 1.6 µM, which is remarkably lower than that of most classical antibiotics. The killing activity of the NCR247-based chimeric peptides was practically instant. Importantly, these peptides had no hemolytic activity or cytotoxicity on human cells. The properties of these NCR derivatives make them promising antimicrobials for clinical use.

Immunomodulatory Effects of the Neuropeptide Pituitary Adenylate Cyclase-Activating Polypeptide in Acute Toxoplasmosis.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is an endogenous neuropeptide with distinct functions including the regulation of inflammatory processes. PACAP is able to modify the immune response by directly regulating macrophages and monocytes inhibiting the production of inflammatory cytokines, chemokines and free radicals. Here, we analyzed the effect of exogenous PACAP on peripheral immune cell subsets upon acute infection with the parasite Toxoplasma gondii (T. gondii). PACAP administration was followed by diminished innate immune cell recruitment to the peritoneal cavity of T. gondii-infected mice. PACAP did not directly interfere with parasite replication, instead, indirectly reduced parasite burden in mononuclear cell populations by enhancing their phagocytic capacity. Although proinflammatory cytokine levels were attenuated in the periphery upon PACAP treatment, interleukin (IL)-10 and Transforming growth factor beta (TGF-ß) remained stable. While PACAP modulated VPAC1 and VPAC2 receptors in immune cells upon binding, it also increased their expression of brain-derived neurotrophic factor (BDNF). In addition, the expression of p75 neurotrophin receptor (p75NTR) on Ly6Chi inflammatory monocytes was diminished upon PACAP administration. Our findings highlight the immunomodulatory effect of PACAP on peripheral immune cell subsets during acute Toxoplasmosis, providing new insights about host-pathogen interaction and the effects of neuropeptides during inflammation.

In Vivo Applicability of Neosartorya fischeri Antifungal Protein 2 (NFAP2) in Treatment of Vulvovaginal Candidiasis.

As a consequence of emerging numbers of vulvovaginitis cases caused by azole-resistant and biofilm-forming Candida species, fast and efficient treatment of this infection has become challenging. The problem is further exacerbated by the severe side effects of azoles as long-term-use medications in the recurrent form. There is therefore an increasing demand for novel and safely applicable effective antifungal therapeutic strategies. The small, cysteine-rich, and cationic antifungal proteins from filamentous ascomycetes are potential candidates, as they inhibit the growth of several Candida spp. in vitro; however, no information is available about their in vivo antifungal potency against yeasts. In the present study, we investigated the possible therapeutic application of one of their representatives in the treatment of vulvovaginal candidiasis, Neosartorya fischeri antifungal protein 2 (NFAP2). NFAP2 inhibited the growth of a fluconazole (FLC)-resistant Candida albicans strain isolated from a vulvovaginal infection, and it was effective against both planktonic cells and biofilm in vitro We observed that the fungal cell-killing activity of NFAP2 is connected to its pore-forming ability in the cell membrane. NFAP2 did not exert cytotoxic effects on primary human keratinocytes and dermal fibroblasts at the MIC in vitro. In vivo murine vulvovaginitis model experiments showed that NFAP2 significantly decreases the number of FLC-resistant C. albicans cells, and combined application with FLC enhances the efficacy. These results suggest that NFAP2 provides a feasible base for the development of a fundamental new, safely applicable mono- or polytherapeutic topical agent for the treatment of superficial candidiasis.

Structure and Synthesis of Antifungal Disulfide ß-Strand Proteins from Filamentous Fungi.

The discovery and understanding of the mode of action of new antimicrobial agents is extremely urgent, since fungal infections cause 1.5 million deaths annually. Antifungal peptides and proteins represent a significant group of compounds that are able to kill pathogenic fungi. Based on phylogenetic analyses the ascomycetous, cysteine-rich antifungal proteins can be divided into three different groups: Penicillium chrysogenum antifungal protein (PAF), Neosartorya fischeri antifungal protein 2 (NFAP2) and "bubble-proteins" (BP) produced, for example, by P. brevicompactum. They all dominantly have ß-strand secondary structures that are stabilized by several disulfide bonds. The PAF group (AFP antifungal protein from Aspergillus giganteus, PAF and PAFB from P. chrysogenum, Neosartorya fischeri antifungal protein (NFAP)) is the best characterized with their common ß-barrel tertiary structure. These proteins and variants can efficiently be obtained either from fungi production or by recombinant expression. However, chemical synthesis may be a complementary aid for preparing unusual modifications, e.g., the incorporation of non-coded amino acids, fluorophores, or even unnatural disulfide bonds. Synthetic variants up to ca. 6⁻7 kDa can also be put to good use for corroborating structure determination. A short overview of the structural peculiarities of antifungal ß-strand disulfide bridged proteins will be given. Here, we describe the structural propensities of some known antifungal proteins from filamentous fungi which can also be prepared with modern synthetic chemistry methods.

The Evolutionary Conserved γ-Core Motif Influences the Anti-Candida Activity of the Penicillium chrysogenum Antifungal Protein PAF.

Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes represent ideal bio-molecules for the development of next-generation antifungal therapeutics. They are promising candidates to counteract resistance development and may complement or even replace current small molecule-based antibiotics in the future. In this study, we show that a 14 amino acid (aa) long peptide (Pγ) spanning the highly conserved γ-core motif of the Penicillium chrysogenum antifungal protein (PAF) has antifungal activity against the opportunistic human pathogenic yeast Candida albicans. By substituting specific aa we elevated the positive net charge and the hydrophilicity of Pγ and created the peptide variants Pγvar and Pγopt with 10-fold higher antifungal activity than Pγ. Similarly, the antifungal efficacy of the PAF protein could be significantly improved by exchanging the respective aa in the γ-core of the protein by creating the protein variants PAFγvar and PAFγopt. The designed peptides and proteins were investigated in detail for their physicochemical features and mode of action, and were tested for cytotoxicity on mammalian cells. This study proves for the first time the important role of the γ-core motif in the biological function of an AMP from ascomycetes. Furthermore, we provide a detailed phylogenetic analysis that proves the presence and conservation of the γ-core motif in all AMP classes from Eurotiomycetes. We emphasize the potential of this common protein motif for the design of short antifungal peptides and as a protein motif in which targeted aa substitutions enhance antimicrobial activity.