1 Universidade Federal de São Paulo, Unidade de Oncologia Experimental, Departamento de Microbiologia, Imunologia e Parasitologia, 04023062 São Paulo, Brazil; 2 Dipartimento di Patologia e Medicina di Laboratorio, Sezione di Microbiologia, Università degli Studi di Parma, Viale Gramsci 14, 43100 Parma, Italy
Received 4 June 2004; returned 22 July 2004; revised 12 August 2004; accepted 16 August 2004
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Abstract |
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Methods: Fungicidal activity of KP was assessed in vitro and in vivo by inhibition of colony forming units and by histological examination, 8 days after infection, of organs from mice intravenously injected with a virulent strain of P. brasiliensis (3 x 106 yeast cells) and intraperitoneally treated with KP (3.3 µg/g body weight, three doses), in comparison with control animals equally administered with a scrambled decapeptide (SP).
Results: KP but not SP was fungicidal in vitro at 39 ng/multiply-budding yeast cell and less efficiently in its D-isomeric form (0.31 µg/multiply-budding yeast cell). It was also able to markedly reduce the fungal load in organs (liver, lung, spleen) of infected animals.
Conclusions: The therapeutic effect observed opens the way for using the antifungal peptide as an alternative control of PCM in association with conventional antifungal drugs.
Keywords: anti-idiotypic fragments , killer mimotopes , paracoccidioidomycosis
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Introduction |
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Glucans, chitin and mannoproteins in addition to the plasma membrane are natural targets for antifungal drugs. Additional targets are, however, being recognized that are accessible to different ligands which affect cell growth and differentiation. Recently, ceramide monohexosides present at the cell wall of Cryptococcus neoformans, Candida albicans and Pseudallescheria boydii were found to be targets of human antibodies that inhibited bud and germ tube formation and ultimately fungal growth.3 Other targets are melanin, adhesion factors, and cell wall enzymes.
Vaccination against PCM is now a prospective goal after P10 and four other peptides derived from the major diagnostic antigen gp43 were found to be promiscuously presented by several HLA-DR antigens.4 Such a vaccine could function as an adjuvant to chemotherapy significantly reducing the time of treatment.
Another creative approach in this field is the use of monoclonal (MAb) and recombinant single-chain (scFv) anti-idiotypic antibodies produced to represent the internal image of a yeast killer toxin (KT) characterized by a wide spectrum of antimicrobial activity (KTMAb and KTscFv). Pathogenic eukaryotic and prokaryotic microorganisms, such as C. albicans,5 Aspergillus fumigatus,6 multidrug-resistant Mycobacterium tuberculosis,7 antibiotic-resistant Gram-positive cocci,8 and Leishmania major,9 presenting specific KT-cell wall binding sites, were killed in vitro by KTMAb and KTscFv. KTMAb and KTscFv exerted a therapeutic effect in vivo in experimental models of candidiasis, aspergillosis, and pneumocystosis by mimicking the functional activity of protective antibodies naturally produced in humans against surface components of infecting microorganisms. A killer decapeptide (KP) has been recently synthesized and engineered on the basis of the sequenced KTscFv gene. KP demonstrated a strong candidacidal activity in vitro and was able to cure rat vaginal infections caused by fluconazole-susceptible and -resistant C. albicans strains, and to protect immunocompetent as well as SCID mice against systemic candidiasis, thus acting as a functional mimotope of KTscFv.10
In the present work, we report on the fungicidal activity of KP in vitro against P. brasiliensis and its therapeutic activity in an animal model of systemic infection.
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Materials and methods |
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P. brasiliensis Pb18, isolated from a human case of PCM, was used throughout the experiments. Fungal cells were grown at 35°C in a modified PYG medium (mPYG; yeast extract 5 g/L, Bacto peptone 5 g/L, dextrose 15 g/L, agar 15 g/L). Isolated colonies were subcultured in mPYG and exponentially growing cells were used in both in vitro and in vivo experiments.
Peptides
An engineered synthetic killer decapeptide (KP) was used in this study. The synthesis of KP based on the sequence of a single-chain recombinant anti-idiotypic antibody acting as a functional mimotope of a microbicidal yeast killer toxin, and its optimization through alanine scanning were described in detail elsewhere.10 The D-isomeric form of KP (D-KP) was also tested. A scrambled decapeptide (SP), containing the same amino acids as KP but in a different sequence,10 was included as a negative control.
In vitro evaluation of KP activity on P. brasiliensis
The in vitro antifungal activity of KP against P. brasiliensis was manifested by inhibition of cfu after treatment and plating on enriched BHI-agar medium, containing per litre, 37 g of Bacto-Brain Heart Infusion (Difco), 20 g Bacto-Agar (Difco), 40 mL of fetal calf serum, 50 mL of spent culture medium of P. brasiliensis Pb339, and ampicillin/streptomycin at 100 IU/mL and 100 µg/mL, respectively. Briefly, logarithmically growing multiply-budding yeast cells were incubated for 12 h with the peptide in PBS, final volume of 200 µL in an Eppendorf tube, at 37°C with shaking. Peptides (KP, D-KP and SP) were added at 125, 62.5, 31.25, 15.6 and 7.8 µg/mL. After incubation, the entire suspension volumes were plated for cfu determination. Plates were incubated at 35°C and read from day 8 to 20. The results are expressed as the lowest peptide concentration per multiply-budding yeast cell, able to completely inhibit growth.
Fungal challenge and peptide treatment
For evaluation of therapeutic activity, KP and SP (100 µg/200 µL/dose) were administered intraperitoneally to experimentally infected mice 1, 24 and 48 h after the fungal challenge. Yeast cells (3 x 106) were injected intravenously (iv) in the tails of 30 g, male B10A mice from UNIFESP animal facility, in 100 µL suspensions in PBS/animal. Control animals were injected with PBS. After 8 days of infection, organs (lung, spleen, liver) were removed, macerated, homogenized in PBS and plated for cfu determination per gram of tissue. Procedures involving animals and their care were conducted in conformity with national and international laws and policies.
Histopathology
Organs from mice challenged iv with P. brasiliensis (3 x 106) cells were removed after 8 days of infection and routinely treated for histopathology. Lung, spleen and liver tissues from mice treated with KP, SP or PBS were stained by haematoxylineosin (HE) and Gomori for visualizing fungal cells.
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Results and discussion |
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To assess the therapeutic activity of KP, B10A mice were infected iv with 3 x 106 P. brasiliensis Pb18 cells and subsequently treated intraperitoneally with KP or SP, at 3.3 µg/g, 1 h after infection, and 1 and 2 days later. The course of infection was evaluated in terms of cfu recovered from target organs and histology. The results in Table 1 represent cfu from the lung, spleen and liver after 8 days of fungal challenge. The absence of cfu counts in the KP-treated mice, compared with the controls, suggested a direct inhibitory effect of KP on P. brasiliensis yeast cells.
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Acknowledgements |
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Footnotes |
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References |
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