Production of an engineered killer peptide in Nicotiana benthamiana by using a potato virus X expression system

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.     

From <Emphasis Type="Italic">Pichia anomala</Emphasis> killer toxin through killer antibodies to killer peptides for a comprehensive anti-infective strategy

“Antibiobodies”, antibodies (Abs) with antibiotic activity, internal image of a Pichia anomala killer toxin (PaKT) characterized by microbicidal activity against microorganisms expressing β-glucans cell-wall receptors (PaKTRs), were produced by idiotypic vaccination with a PaKT-neutralizing monoclonal Ab (PaKT-like Abs) or induced by a protein-conjugated β-glucan. Human natural PaKT-like Abs (PaKTAbs) were found in the vaginal fluid of women infected with KT-sensitive microorganisms. Monoclonal and recombinant PaKT-like Abs, and PaKTAbs proved to be protective against experimental candidiasis, cryptococcosis and aspergillosis. A killer decapeptide (KP), synthesized from the sequence of a recombinant PaKT-like Ab or produced in transgenic plants, showed a microbicidal activity in vitro, neutralized by β-glucans, a therapeutic effect in vivo, against experimental mucosal and systemic mycoses, and a prophylactic role in planta, against phytopathogenic microorganisms, respectively. KP showed fungicidal properties against all the defective mutants of a Saccharomyces cerevisiae library, inclusive of strains recognized to be resistant to conventional antifungal drugs. KP inhibited in vitro, ex vivo and/or in vivo HIV-1 and Influenza A virus replication, owing to down-regulation of CCR5 co-receptors, physical block of the gp120-receptor interaction and reduction in the synthesis of glycoproteins, HA and M1 in particular. KP modulated the expression of costimulatory and MHC molecules on murine dendritic cells, improving their capacity to induce lymphocyte proliferation. KP, proven to be devoid of cytotoxicity on human cells, showed self-assembly-releasing hydrogel-like properties, catalyzed by β 1,3 glucan. PaKT’s biotechnological derivatives may represent the prototypes of novel antifungal vaccines and anti-infective drugs characterized by different mechanisms of action.     

Immunoadjuvant effect of inactivated Cryptococcus neoformans

Cryptococcus neoformans disseminated into the central nervous system (CNS) of intraperitoneally inoculated adult ICR mice, but did not potentiate penetration of concurrently given Bhanja virus (Bunyaviridae) into the CNS. Likewise, Bhanja virus infection did not affect significantly the course of murine cryptococcosis. However, formalin-killed C. neoformans cells non-specifically increased production of antibodies against the virus infection in the animals. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var.<Emphasis Type="Italic">neoformans</Emphasis> serotype A and A/D in samples from Egypt

The cryptococcal polysaccharide antigen was detected in 10 cerebrospinal fluid (CSF) and 23 serum samples from cryptococcal meningitis and intestinal cryptococcosis by the cryptococcal antigen latex agglutination system (CALAS). CALAS titers in CSF and serum samples of cryptococcal meningitis ranged over 8-2048 and 32-2048, respectively, while in cases of intestinal cryptococcosis, serum titers ranged over 8-2048. The isolates of yeast Cryptococcus neoformans were determined to be of serotype A or of the A/D pair. The total leukocyte count and biochemical parameters in CSF were significantly increased as indicators of microbial infection. Furthermore, the in vitro change of the teleomorph (sexual state) to the anamorph (asexual state) was also detected and the teleomorph state changed in vivo to the encapsulated anamoph state which is more virulent during infection in vivo than the yeast-like noncapsulated form. Two primers for internal transcribed spacer (ITS) regions of ribosomal DNA were used for molecular detection of C. neoformans. After PCR amplification, a DNA band of 415 bp, visualized on agarose gel, indicated the presence of C. neoformans cells in the tested CSF and serum samples. The primer sensitivity was also characterized using purified yeast chromosomal DNA as template; it was about 20 pg or more chromosomal DNA which represents about 10 cells of C. neoformans. The primers were also specific for ITS regions of C. neoformans and gave negative results with Candida albicans and E. coli chromosomal DNA templates.     

Production of an Engineered Killer Peptide in Nicotiana benthamiana by Using a Potato virus X Expression System

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.     

Effects of microplusin, a copper-chelating antimicrobial peptide, against Cryptococcus neoformans

Microplusin is an antimicrobial peptide isolated from the cattle tick Rhipicephalus (Boophilus) microplus. Its copper-chelating ability is putatively responsible for its bacteriostatic activity against Micrococcus luteus as microplusin inhibits respiration in this species, which is a copper-dependent process. Microplusin is also active against Cryptococcus neoformans (MIC(50) = 0.09 μM), the etiologic agent of cryptococcosis. Here, we show that microplusin is fungistatic to C. neoformans and this inhibitory effect is abrogated by copper supplementation. Notably, microplusin drastically altered the respiratory profile of C. neoformans. In addition, microplusin affects important virulence factors of this fungus. We observed that microplusin completely inhibited fungal melanization, and this effect correlates with the inhibition of the related enzyme laccase. Also, microplusin significantly inhibited the capsule size of C. neoformans. Our studies reveal, for the first time, a copper-chelating antimicrobial peptide that inhibits respiration and growth of C. neoformans and modifies two major virulence factors: melanization and formation of a polysaccharide capsule. These features suggest that microplusin, or other copper-chelation approaches, may be a promising therapeutic for cryptococcosis.     

Activity of an engineered synthetic killer peptide on Leishmania major and Leishmania infantum promastigotes

This study was undertaken to analyze the effect of an engineered, killer decapeptide (KP) on Leishmania major and Leishmania infantum promastigotes. The KP was synthesized on the basis of the sequence of a recombinant, single-chain anti-idiotypic antibody acting as a functional internal image of a yeast killer toxin. The evaluation of in vitro inhibitory activity of KP on L. major and L. infantum, release of intracellular green fluorescent protein (GFP) molecules by L. major, DNA fragmentation, and ultrastructural analysis (TEM) of L. infantum upon KP treatment were performed. KP presented antiproliferative and leishmanicidal activity with LC(50)/1 day of 58 and 72 microM for L. major and L. infantum, respectively. A dose-dependent decrease in proliferation and increase of killing of promastigotes was seen after KP treatment. No DNA fragmentation in L. infantum promastigotes or release of intracellular GFP molecules on peptide treatment of a GFP expressing L. major clone was demonstrated. Moreover the plasma-membrane was not disrupted, but, by TEM analysis, intracellular damage was observed.     

Role for Golgi reassembly and stacking protein (GRASP) in polysaccharide secretion and fungal virulence

Secretion of virulence factors is a critical mechanism for the establishment of cryptococcosis, a disease caused by the yeast pathogen Cryptococcus neoformans. One key virulence strategy of C. neoformans is the release of glucuronoxylomannan (GXM), a capsule-associated immune-modulatory polysaccharide that reaches the extracellular space through secretory vesicles. Golgi reassembly and stacking protein (GRASP) is required for unconventional protein secretion mechanisms in different eukaryotic cells, but its role in polysaccharide secretion is unknown. This study demonstrates that a C. neoformans functional mutant of a GRASP orthologue had attenuated virulence in an animal model of cryptococcosis, in comparison with wild-type (WT) and reconstituted cells. Mutant cells manifested altered Golgi morphology, failed to produce typical polysaccharide capsules and showed a reduced ability to secrete GXM both in vitro and during animal infection. Isolation of GXM from cultures of WT, reconstituted or mutant strains revealed that the GRASP orthologue mutant produced polysaccharides with reduced dimensions. The mutant was also more efficiently associated to and killed by macrophages than WT and reconstituted cells. These results demonstrate that GRASP, a protein involved in unconventional protein secretion, is also required for polysaccharide secretion and virulence in C. neoformans.     

The effects of Cryptococcus neoformans-secreted antigens on tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression on human lung epithelial cells

Since primary infection with Cryptococcus neoformans usually occurs in the lungs, and since pulmonary cryptococcosis involves interactions between yeasts and alveolar epithelial cells, we have begun to study the effects of C. neoformans and its secreted antigens (SA) on epithelial reactions potentially associated with localized inflammation. We report here that SAs from encapsulated and acapsular strains of C. neoformans caused significant reductions in tumor necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression on A549 lung epithelial cells in culture. We also present evidence that the reduction in ICAM-1 expression was not associated with SA-induced shedding of this adhesion molecule.     

A dual role for TGF-beta1 in the control and persistence of fungal pneumonia

TGF-beta1 (TGF) has been implicated in the pathogenesis of several chronic infections and is thought to promote microbial persistence by interfering with macrophage function. In rats with experimental pulmonary cryptococcosis, increased lung levels of TGF were present at 12 mo of infection. Within the lung, expression of TGF localized to epithelioid cells and foamy macrophages in areas of inflammation. Increased TGF expression was also observed in the lungs of experimentally infected mice and a patient with pulmonary cryptococcosis. TGF reduced Ab and serum-mediated phagocytosis of Cryptococcus neoformans by rat alveolar macrophages (AM) and peripheral blood monocytes, and this was associated with decreased chemokine production and oxidative burst. Interestingly, TGF-treated rat AM limited both intracellular and extracellular growth of C. neoformans. Control of C. neoformans growth by TGF-treated rat AM was due to increased secretion of lysozyme, a protein with potent antifungal activity. The effects of TGF on the course of infection were dependent on the timing of TGF administration relative to the time of infection. TGF treatment of chronically infected rats resulted in reduced lung fungal burden, while treatment early in the course of infection resulted in increased fungal burden. In summary, our studies suggest a dual role for TGF in persistent fungal pneumonia whereby it contributes to the local control of infection by enhancing macrophage antifungal efficacy through increased lysozyme secretion, while limiting inflammation by inhibiting macrophage/monocyte phagocytosis and reducing associated chemokine production and oxidative burst.     

Immune stimulating and therapeutic potential of tuftsin-incorporated nystatin liposomes against Cryptococcus neoformans in leukopenic BALB/C mice

Cryptococcus neoformans infection is a common fungal infection in persons infected with human immune deficiency virus (HIV) or those with defective cell-mediated immunity. Since treatment of cryptococcal meningitis poses a big challenge, the present study aimed to develop a novel liposomal therapeutic formulation against cryptococcosis. Treatment with tuftsin-incorporated liposomes increased the anti-cryptococcal activity of murine peritoneal macrophages. Prophylactic treatment of mice with tuftsin-incorporated liposomes reduced the dissemination of C.?neoformans to brain tissues. Moreover, the co-administration of tuftsin with nystatin liposomes augmented the anti-cryptococcal activity of nystatin, as mice treated with tuftsin-incorporated nystatin liposomes showed the highest survival and least fungal burden in their brain tissues. The results of the present study favour the use of immune-stimulating molecules along with antifungal agents in the treatment of opportunistic fungal infections.     

Cryptococcus neoformans activates bone marrow-derived conventional dendritic cells rather than plasmacytoid dendritic cells and down-regulates macrophages

Induction of IL-12 and IL-23 is essential for protective immunity against Cryptococcusneoformans. The contribution of dendritic cells vs. macrophages to IL-12/23 production in response to C. neoformans infection is unclear. Activation of conventional bone marrow-derived dendritic cells (BMDC), plasmacytoid BMDC, and bone marrow-derived macrophages (BMMPhi) was assessed by analyzing cytokine responses and the expression of MHC-II, CD86, and CD80 in each cell type. Cryptococcus neoformans induced the release of IL-12/23p40 by BMDC, but not by BMMPhi, in a TLR2- and TLR4-independent but MyD88-dependent manner. Conventional BMDC rather than plasmacytoid BMDC up-regulated MHC-II and CD86, while BMMPhi down-regulated MHC-II and CD86 in response to C. neoformans. The up-regulation of MHC-II and CD86 on BMDC required MyD88. Our data point to conventional DC as critical IL-12/23-producing antigen-presenting cells during cryptococcosis.     

Experimental cryptococcosis in guinea pigs

A guinea pig model was used to evaluate immune response to Cryptococcus neoformans. This model shared characteristics with cryptococcosis in humans. Twenty five guinea pigs injected intraperitoneally with 10(7) viable C. neoformans cells developed disseminated disease. Forty days after infection all guinea pigs were killed and autopsy performed. C. neoformans growth in the lungs, brains, livers and spleen of the infected animals were determined. Furthermore, the immune response was characterized by moderate degree of delayed-type hypersensitivity and humoral response. In some organs was observed neutrophil and lymphocyte infiltration with presence of cryptococci cells. The infiltration observed in the organs was probably a consequence of an immune reaction.     

An innate immune system cell is a major determinant of species-related susceptibility differences to fungal pneumonia

Rats and mice are considered resistant and susceptible hosts, respectively, for experimental cryptococcosis. For both species, alveolar macrophages (AM) are central components of the host response to pulmonary Cryptococcus neoformans infection. We explored the role of AM in three strains of mice and three strains of rats during cryptococcal infection by comparing the outcome of infection after macrophage depletion using liposomal clodronate. AM depletion was associated with enhancement and amelioration of disease in rats and mice, respectively, as measured by lung fungal burden. The apparent protective role for AM in rats correlated with enhanced anti-cryptococcal activity as measured by phagocytic activity, oxidative burst, lysozyme secretion, and ability to limit intracellular growth of C. neoformans. Furthermore, rat AM were more resistant to lysis in association with intracellular infection. In summary, differences in AM function in rats and mice suggest an explanation for the species differences in susceptibility to C. neoformans based on the inherent efficacy of a central effector cell of the innate immune system.     

Fungicidal activity of IFN-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast-form fungus which causes pulmonary and meningeal infections preferentially in the immunocompromised host. It is thought that cell-mediated immunity is important for acquired resistance against cryptococcosis with activated macrophages as the final effector cells. However, specific polysaccharides in the capsule of C. neoformans protect the fungus from adherence to phagocytes and from subsequent phagocytosis. We have studied extracellular killing of C. neoformans by IFN-gamma-activated macrophages and their products. Murine bone marrow-derived macrophages stimulated with rIFN-gamma for 24 h were able to effectively suppress the growth of C. neoformans and the effect of IFN-gamma was augmented by LPS. Killing of C. neoformans was also achieved by cell-free supernatants from bone marrow-derived macrophages stimulated with IFN-gamma plus LPS. Our results indicate that killing of C. neoformans by activated macrophages is independent from toxic oxygen radicals and mediated by secreted protein(s) of apparent molecular mass of 15 and 30 kDa. These findings indicate that activated macrophages play a major role in host defense, although the fungus resists phagocytosis and remains in the extracellular milieu.     

Laccase activity in Cryptococcus gattii strains isolated from goats

Cryptococcosis is a life-threatening infection in humans and animals caused by encapsulated yeasts of the genus Cryptococcus. Cryptococcus neoformans and Cryptococcus gattii are the main agents of this mycosis. Until 2002 C. gattii was classified as a variety of C. neoformans but now is accepted as an independent species. The laccase (phenoloxydase) enzyme produced by these yeasts is considered one of the main pathogenic factors for its ability to induce melanin from dihydroxyphenolic compounds. The vast majority of the studies in laccase and melanin synthesis have been developed using isolates of C. neoformans. The main objective of this study was to evaluate laccase activity in strains of C. gattii, serotype B isolated from immunocompetent goats that died of lung and disseminated cryptococcosis, in several outbreaks occurring in Spain. The laccase activities of these isolates were compared with those of other strains of C. gattii and C. neoformans. After fungal cell rupture, the supernatant of each isolate was analyzed for its laccase activity using as substrate an L-dopa 20 mM solution. The degree of enzymatic activity was assessed according to its absorbance at 450 nm and scored using Enzymatic Units (EU). The maximum values were observed in three strains of C. gattii from goats (EU > 12). The smallest values were observed in one environmental isolate of C. gattii serotype C (EU = 0.7). The highest recorded value for C. neoformans was 6.3 EU in a serotype A isolate from one human case of meningitis. C. gattii serotype B obtained from goats showed different degrees of laccase activity, being the highest in those isolated from severe outbreaks of cryptococcosis. This enzyme appears to represent a major, though nonexclusive, pathogenic factor for Cryptococcus gattii.     

Antifungal activity from Ocimum gratissimum L. towards Cryptococcus neoformans

Cryptococcal infection had an increased incidence in last years due to the explosion of acquired immune deficiency syndrome epidemic and by using new and effective immunosuppressive agents. The currently antifungal therapies used such as amphotericin B, fluconazole, and itraconazole have certain limitations due to side effects and emergence of resistant strains. So, a permanent search to find new drugs for cryptococcosis treatment is essential. Ocimum gratissimum, plant known as alfavaca (Labiatae family), has been reported earlier with in vitro activity against some bacteria and dermatophytes. In our work, we study the in vitro activity of the ethanolic crude extract, ethyl acetate, hexane, and chloroformic fractions, essential oil, and eugenol of O. gratissimum using an agar dilution susceptibility method towards 25 isolates of Cryptococcus neoformans. All the extracts of O. gratissimum studied showed activity in vitro towards C. neoformans. Based on the minimal inhibitory concentration values the most significant results were obtained with chloroformic fraction and eugenol. It was observed that chloroformic fraction inhibited 23 isolates (92%) of C. neoformans at a concentration of 62.5 microg/ml and eugenol inhibited 4 isolates (16%) at a concentration of 0.9 microg/ml. This screening may be the basis for the study of O. gratissimum as a possible antifungal agent.     

Respiratory cryptococcosis in HIV positive patients.

Although the lungs are the portal of entry of the infection, respiratory manifestations of AIDS related cryptococcosis have not been very well studied. The lack of typical findings in clinical and roentgenographic studies and the difficulties in the interpretation of the isolation of Cryptococcus neoformans from bronchial secretions, is probably the explanation for the lack of interest on this subject. The clinical and microbiological findings of 22 HIV positive patients, who presented C. neoformans in their respiratory tract clinical samples, are presented. Seventeen were males and 5 females, their age average was 30.8 years (21-50 years) and the following risk factors for HIV infection were detected: intravenous drug abuse 18, heterosexuals with several sexual partners two, one female prostitute and 1 homosexual man. All patients, except three, showed less than 100 CD4+ cells per microl. The following symptoms were observed: fever, cough, mucoid expectoration and chest ache. Roengenographic studies presented diffuse infiltrative patches in eleven cases, pulmonary cavities in three, pseudotumoral nodules in two, pneumonic infiltration in two and pleural effusion in four patients. C. neoformans was observed and/or isolated from sputum in nine patients, from bronchoalveolar lavage in seven, from lung biopsy in one and from pleural effusion in four cases. Blood cultures for C. neoformans were positive in 13 cases, urine cultures in 10 and in 11 patients C. neoformans was isolated from C.S.F. The latex agglutination tests for C. neoformans capsular polysaccharide rendered positive results in serum samples from 19 patients and from C.S.F. in 14 cases. Seven cases also presented active tuberculosis. According to these findings, it seems that the isolation of C. neoformans from bronchial secretion of HIV positive patients is a signal of disseminated cryptococcosis. It is important to isolate C. neoformans or detect its capsular antigen from other clinical samples in order to confirm the diagnosis of disseminated cryptococcosis. As observed in other studies, pleuropulmonary cryptococcosis does not present a typical clinical pattern.     

An ectophosphatase activity in Cryptococcus neoformans

There is increasing evidence in the literature showing that fungal pathogens express biologically active ectoenzymes. The expression of surface phosphatases at the cell surface of Cryptococcus neoformans, the etiologic agent of cryptococcosis, was evaluated in the present study. Different isolates of C. neoformans express ectophosphatase activity, which is not influenced by capsule size or serotype. The cryptococcal enzyme is an acid phosphatase, inhibited by classic inhibitors of ectophosphatases, including ammonium molybdate and sodium salts of fluoride and orthovanadate. Only the inhibition of enzyme activity caused by sodium orthovanadate has been shown to be irreversible. The cryptococcal ectoenzyme is also inhibited by Zn2+ and inorganic phosphate, the final product of reactions catalyzed by phosphatases. The ectophosphatase from C. neoformans efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate removal when phosphothreonine is used as a substrate. Yeast cells with irreversibly inhibited ectophosphatases are less capable of adhering to animal epithelial cells than fungi fully expressing enzyme activity, suggesting that ectoenzyme expression can contribute to the pathogenesis of C. neoformans.     

The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C. neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C. neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C. neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.