JAC
Journal of Antimicrobial Chemotherapy (2004) 54, 956–958 DOI: 10.1093/jac/dkh430 Advance Access publication 24 September 2004
Therapeutic activity of a killer peptide against experimental paracoccidioidomycosis Luiz R. Travassos1, Luis S. Silva1, Elaine G. Rodrigues1, Stefania Conti2, Antonella Salati2, Walter Magliani2 and Luciano Polonelli2* 1
Universidade Federal de Sa˜o Paulo, Unidade de Oncologia Experimental, Departamento de Microbiologia, Imunologia e Parasitologia, 04023– 062 Sa˜o Paulo, Brazil; 2Dipartimento di Patologia e Medicina di Laboratorio, Sezione di Microbiologia, Universita` degli Studi di Parma, Viale Gramsci 14, 43100 Parma, Italy
Objectives: To evaluate whether an engineered synthetic decapeptide (KP) derived from the sequence of a recombinant anti-idiotypic antibody, that represents the internal image of a Pichia anomala killer toxin, could be fungicidal in vitro and therapeutic in vivo against Paracoccidioides brasiliensis and paracoccidioidomycosis (PCM). 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 3 106 yeast cells) and intraperitoneally treated with KP (3.3 mg/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 mg/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
Introduction Paracoccidioidomycosis (PCM), the prevalent systemic mycosis in South America, is caused by the dimorphic fungus Paracoccidioides brasiliensis and is endemic among individuals living and working in rural areas. Environmental conidia probably act as infectious propagules upon inhalation into the lungs, where they transform into the pathogenic yeast forms.1 The alveolar lesions usually involve a granulomatous inflammatory response with formation of epithelioid tubercles, which is the most effective defence mechanism against the invading fungus. The infection can evolve into different clinical forms from fatal acute to chronic infection, which are associated with various degrees of suppressed cell-mediated immunity. In the chronic progressive forms, dissemination of the fungus to mucocutaneous sites and other organs is accompanied by a vigorous cellular immune
response. The most severe cases of PCM may lead to depression of cellular immunity and to anergy. The current chemotherapy of PCM is based on sulphonamides, amphotericin B, and azole derivatives, mainly itraconazole. Because of the toxicity of antifungal drugs, and the recent identification in P. brasiliensis of genes involved in transport-mediated azole resistance,2 new treatment approaches are needed. 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.
..........................................................................................................................................................................................................................................................................................................................................................................................................................
*Corresponding author. Tel: +39-521-988885; Fax: +39-521-993620; E-mail:
[email protected] ..........................................................................................................................................................................................................................................................................................................................................................................................................................
956 JAC vol.54 no.5 q The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.
Downloaded from http://jac.oxfordjournals.org/ by guest on February 5, 2015
Received 4 June 2004; returned 22 July 2004; revised 12 August 2004; accepted 16 August 2004
Killer peptide against paracoccidioidomycosis Table 1. Colonization of organs from mice challenged intravenously with virulent P. brasiliensis (3 106 yeast cells) and treated with KP and SPa Organ Treatment KP SP PBS a b
lungb
spleenb
liverb
0 2005 ± 790 2533 ± 435
0 1304 ± 649 1187 ± 335
0 229 ± 165 330 ± 178
Groups of three B10A mice; time of infection: 8 days. Colonization is expressed in terms of cfu/g of tissue ± standard deviation.
Materials and methods Fungal strain P. brasiliensis Pb18, isolated from a human case of PCM, was used throughout the experiments. Fungal cells were grown at 358C 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),
Figure 1. Histopathology of liver (a) and lung (b) tissues of mice experimentally infected with Paracoccidioides brasiliensis (yeast form) and treated with killer (right-hand panels) and scrambled (left-hand panels) peptides (Gomori, 400).
957
Downloaded from http://jac.oxfordjournals.org/ by guest on February 5, 2015
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.
L. R. Travassos et al. 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 mg/mL, respectively. Briefly, logarithmically growing multiply-budding yeast cells were incubated for 12 h with the peptide in PBS, final volume of 200 mL in an Eppendorf tube, at 378C with shaking. Peptides (KP, D -KP and SP) were added at 125, 62.5, 31.25, 15.6 and 7.8 mg/mL. After incubation, the entire suspension volumes were plated for cfu determination. Plates were incubated at 358C 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.
For evaluation of therapeutic activity, KP and SP (100 mg/200 mL/dose) were administered intraperitoneally to experimentally infected mice 1, 24 and 48 h after the fungal challenge. Yeast cells (3 106) were injected intravenously (iv) in the tails of 30 g, male B10A mice from UNIFESP animal facility, in 100 mL 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 (3 106) 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 haematoxylin – eosin (HE) and Gomori for visualizing fungal cells.
Results and discussion After 12 h of incubation with KP, multiply-budding yeast cells were completely inhibited at 39 ng of peptide/yeast. The D -isomeric form of KP showed a similar effect at a minimal inhibitory concentration of 0.31 mg/yeast. No inhibition at any concentration used was observed with the scrambled peptide (SP). To assess the therapeutic activity of KP, B10A mice were infected iv with 3 106 P. brasiliensis Pb18 cells and subsequently treated intraperitoneally with KP or SP, at 3.3 mg/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. The SP-treated and infected animals showed several granulomas in the liver, numerous fungal cells at portal and centrolobular
Acknowledgements Financial support: grant from the National AIDS Project, contract number 50D.26 (L.P., S.C., A.S. and W.M.); PronexCNPq/COLSAN grant no. 536 (L.R.T., L.S.S. and E.G.R.).
References 1. Borges-Walmsley, M. I., Chen, D., Shu, X. et al. (2002). The pathobiology of Paracoccidioides brasiliensis. Trends in Microbiology 10, 80–7. 2. Gray, C. H., Borges-Walmsley, M. I., Evans, G. J. et al. (2003). The pfr1 gene from the human pathogenic fungus Paracoccidioides brasiliensis encodes a half-ABC transporter that is transcribed in response to treatment with fluconazole. Yeast 20, 865– 80. 3. Rodrigues, M. L., Travassos, L. R., Miranda, K. R. et al. (2000). Human antibodies against a purified glucosylceramide from Cryptococcus neoformans inhibit cell budding and fungal growth. Infection and Immunity 68, 7049–60. 4. Iwai, L. K., Yoshida, M., Sidney, J. et al. (2003). In silico prediction of peptides binding to multiple HLA-DR molecules accurately identifies immunodominant epitopes from gp43 of Paracoccidioides brasiliensis frequently recognized in primary peripheral blood mononuclear cell responses from sensitized individuals. Molecular Medicine 9, 209–19. 5. Magliani, W., Conti, S., De Bernardis, F. et al. (1997). Therapeutic potential of antiidiotypic single chain antibodies with yeast killer toxin activity. Nature Biotechnology 15, 155–8. 6. Cenci, E., Mencacci, A., Spreca, A. et al. (2002). Killer antiidiotypes protect from early invasive aspergillosis in a murine model of allogeneic T-cell-depleted bone marrow transplantation. Infection and Immunity 70, 2375– 82. 7. Conti, S., Fanti, F., Magliani, W. et al. (1998). Mycobactericidal activity of human natural, monoclonal, and recombinant yeast killer toxin-like antibodies. Journal of Infectious Diseases 177, 807– 11. 8. Conti, S., Magliani, W., Arseni, S. et al. (2000). In vitro activity of monoclonal and recombinant yeast killer toxin-like antibodies against antibiotic-resistant Gram-positive cocci. Molecular Medicine 6, 613–9. 9. Savoia, D., Avanzini, C., Conti, S. et al. (2002). In vitro leishmanicidal activity of a monoclonal antibody mimicking a yeast killer toxin. Journal of Eukaryotic Microbiology 49, 319–23. 10. Polonelli, L., Magliani, W., Conti, S. et al. (2003). Therapeutic activity of an engineered synthetic killer antiidiotypic antibody fragment against experimental mucosal and systemic candidiasis. Infection and Immunity 71, 6205–12.
958
Downloaded from http://jac.oxfordjournals.org/ by guest on February 5, 2015
Fungal challenge and peptide treatment
regions, and at the organ periphery. Great cellular infiltration was observed in the lungs with numerous fungi, and few areas of normal lung architecture. Fungi were also detected in limited foci in the spleen. Every aspect was less pronounced in the KP-treated mice. The liver granulomas were smaller and fewer with no visible fungi. The lungs had little infiltration with extensive areas of normal alveoli and lymphoid cells. No visible fungi were seen. The spleens were little affected with no fungi (Figure 1). We demonstrate, therefore, that KP is strongly effective in inhibiting growth of P. brasiliensis yeast forms in vitro and in vivo. The biotechnological method used in the production of KP10 provides an approach for engineering new antibiotics. Wide-spectrum antimicrobial peptides, such as KP, might be used as an adjuvant to chemotherapy and the projected peptide vaccine,4 to shorten the time of treatment and as an alternative in cases of anergy and drug resistance.
