XVIII. E ect of a killer toxin of Pichia anomala to Pneumocystis. Perspectives in the control of pneumocystosis

Transcription

1 FEMS Immunology and Medical Microbiology 22 (1998) 145^149 XVIII. E ect of a killer toxin of Pichia anomala to Pneumocystis. Perspectives in the control of pneumocystosis Nathalie Seèguy a; *, Luciano Polonelli b, Eduardo Dei-Cas a;c, Jean-Charles Cailliez a;d a Deèpt. Microbiol. Ecosysteémes, Institut Pasteur de Lille, 1 rue Prof. Calmette, BP. 245, Lille, France b Istituto di Microbiologia, Universitaé degli Studi di Parma, Parma, Italy c Faculteè de Meèdecine, Centre Hospitalier Reègional Universitaire de Lille, Lille, France d Faculteè Libre des Sciences (FLS), Universiteè Catholique de Lille, Lille, France Abstract Despite the development of drugs in the prophylaxis of pneumocystosis, Pneumocystis carinii remains a major opportunistic microorganism in immunosuppressed individuals, especially in human immunodeficiency virus-infected patients. Since side effects were frequently observed after administration of trimethoprim-sulfamethoxazole or pentamidine, the drugs which are mainly used in treating human P. carinii pneumonia (PCP), new therapeutic strategies should be developed. Over the last years, the inhibitory effect of a Pichia anomala killer toxin (PaKT), a molecule with a wide spectrum of antimicrobial activity, was characterized on P. carinii. The susceptibility of mouse and rat-derived Pneumocystis to PaKT has been demonstrated by in vitro attachment tests and in vivo infectivity assays. Nevertheless, PaKT is strongly antigenic, toxic and could not be used directly as a therapeutic agent. Then, a new strategy using killer toxin-like anti-idiotypic antibodies (KT-antiIds) mimicking the fungal toxin activity has been developed. Different KT-antiIds were obtained by idiotypic immunization with a monoclonal antibody (mabkt4). This mabkt4 neutralized the killer properties of the PaKT. KT-antiIds were produced by immunization against the variable domain (idiotype) of mabkt4 (internal image of the killer toxin receptor), or they were obtained directly from vaginal fluid of patients affected by recurrent vaginal candidiosis. In this last case, such natural KT-antiIds were immunopurified by affinity-chromatography with mabkt4 and their anti-p. carinii activity was then evaluated. Our results showed that both the in vitro attachment of rat-derived parasites and their infectivity to nude rats were inhibited by the KTantiIds. With regard to KT-antiIds obtained by immunization, the antimicrobial activity of a monoclonal KT-antiIds (mabk10) has been evaluated by using a PCP experimental nude rat model treated by mabk10 administered by aerosol. The pneumocystosis extension was significantly reduced in this model. The monoclonal KT-antiIds were effective against P. carinii in reducing parasite proliferation in lungs of nude rats. Further experiments are in progress to study the in vivo anti-p. carinii activity of KT-antiIds by using recombinant single-chain of the variable fragment of KT-antiIds. Yeast killer toxin-like recombinant molecules could provide the basis for a new therapeutic strategy towards the control of pneumocystosis. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Antiidiotypic antibody; Killer toxin; Nude rat; Pichia anomala; Pneumocystis carinii; Pneumocystosis * Corresponding author. Tel.: +33 (3) ; Fax: +33 (3) ; nathalie.seguy@pasteur-lille.fr / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (98)

2 146 N. Seèguy et al. / FEMS Immunology and Medical Microbiology 22 (1998) 145^ Introduction The development of new therapeutic strategies against Pneumocystis carinii pneumonia (PCP) remains a priority in the eld of opportunistic diseases [1]. Indeed, P. carinii is known to be susceptible to a small number of antimicrobial agents which frequently induce unwanted e ects. Over the last years, a new strategy in the control of P. carinii infections is being explored in our laboratory based on a yeast killer toxin secreted by a Pichia anomala strain. Yeast toxins are proteins [2] or glycoproteins [3] that have a lethal [4] or inhibitory e ect [5] against sensitive strains of yeasts. The yeast killer phenomenon is described in many yeast genera such as Saccharomyces, Ustilago, Kluyveromyces, Candida or Pichia. Unrelated groups of eukaryotic (yeast, hyphomycetes) and prokaryotic (bacteria, algae) microorganisms are sensitive to the antimicrobial e ect of yeast killer toxins. The biological interest of the yeast killer phenomenon has been shown by several studies [6^8], mainly regarding the modalities of the killer toxin xation on speci c cell wall receptors of sensitive target cells. A killer toxin secreted by a strain of P. anomala (PaKT) was selected for its antimicrobial properties against a large panel of pathogenic microorganisms [9]. Its killer activity is depending on ph (4.6) and temperature (25^28³C) conditions. This killer toxin was also selected because its killer activity can be neutralized by a monoclonal antibody (mabkt4) [10]. 2. Interaction between Pneumocystis and the killer toxin of Pichia anomala (PaKT) With regard to large PaKT spectrum activity on yeast [11] and considering that Pneumocystis is a fungus, the Pneumocystis susceptibility to the antifungal properties of PaKT was investigated. Attachment of P. carinii on pulmonary cells is an important step for the development of PCP. This is why a co-culture system was selected to study PaKT interaction. Our data showed that PaKT induced a marked and signi cant inhibitory e ect on the P. carinii in vitro attachment (40%) [12,13]. The inhibitory e ect of PaKT was blocked by a preincubation of this killer molecule with mabkt4. These data showed that the decrease in P. carinii attachment to lung epithelial cells was due to a speci c action of PaKT [13]. In these experiments it was unclear if the inhibition of the attachment was due to the death of the parasite or to a speci c inhibitory e ect of PaKT on the adhesion mechanisms of P. carinii. For these reasons a direct e ect of PaKT on Pneumocystis viability has been explored by examining the ability of the toxin to inhibit in vitro the uptake of a folate precursor ([ 3 H]-pABA). The authors demonstrated the real killer e ect of PaKT on the viability of P. carinii [14]. Ex vivo experiments were developed to establish if PaKT was able to inhibit the parasite infectivity in animals. These manipulations consisted in the instillation of animals with killer toxin treated parasites. Two animals models were used: SCID mice and nude rats, non-latently a ected by Pneumocystis. SCID mice were nasally instillated with mice-derived Pneumocystis, and nude rats were inoculated by the intratracheal route with rat-derived Pneumocystis. Before inoculation, the parasites were incubated in media di erent from the killer toxin. The results indicated that PaKT had a strong inhibitory e ect on the infectivity of Pneumocystis. Indeed, the percentage of inhibition was 75^99% in SCID mice [15], and about 87% in nude rats [16]. The inhibition of Pneumocystis organism infectivity was not due to the acidity of the killer toxin medium because parasites preincubated in complete DMEM, ph 4.6, induced a pneumocystosis as severe as that induced by parasites in DMEM, ph 7.4 [13]. In summary, our rst assays have demonstrated the inhibitory e ect of PaKT on the in vitro attachment of P. carinii organisms derived from mice or rats. This inhibitory e ect could be speci cally blocked by neutralizing the toxin activity with mabkt4. Moreover, our experiments in animal models have shown that PaKT was able to inhibit mouse-derived P. carinii infectivity in SCID mice or rat-derived P. carinii infectivity in nude rats, con- rming the antimicrobial activity of the toxin against the parasite.

3 N. Seèguy et al. / FEMS Immunology and Medical Microbiology 22 (1998) 145^ Interactions between Pneumocystis and yeast killer toxin-like anti-idiotypic antibodies (KT-antiIds) 3.1. Killer toxin-like polyclonal anti-idiotypic antibodies (polyclonal KT-antiIds) The PaKT proved to be very labile at physiological temperature and ph [11]. Moreover, it was not used as a systemic antibody because, as might be expected of a large, foreign protein, it is both toxic and antigenic. Then a new therapeutic approach was considered. Indeed, it was possible to obtain antiidiotypic antibodies (antiids), produced against a monoclonal anti-killer toxin antibody (mabkt4), and mimicking biologic properties of the killer toxin. Thus, these antiids should exercise identical antimicrobial e ects to that of fungal toxin of P. anomala. MabKT4, which neutralized killer properties of PaKT, was used as immunogen to produce rabbit polyclonal antiids [17]. The experiments show a killer e ect of polyclonal antiids (KT-antiIds) on the in vitro growth of Candida albicans cells [17]. Apparently, these KT-antiIds mimicked the killer e ect of PaKT. Moreover, polyclonal KT-antiIds have been raised in sera and vaginal secretions of mice and rats developing C. albicans mycosis, through parenteral and intravaginal immunization with mabkt4. They conferred immunoprotection against systemic or mucosal experimental candidiasis [18,19]. These were the rst reports of antimicrobial protection (idiotypic vaccination) using the properties of killer toxin-like antiids which presumably acted in vivo as antibiotics [20] The natural human killer toxin-like anti-idiotypic antibodies (human KT-antiIds) In the same way, Polonelli et al. [21] puri ed natural human PaKT-like antibodies from the vaginal secretions of patients infected with Candida, by using a nity chromatography with mabkt4. These PaKT-like antibodies appeared to be active in vitro against C. albicans. The anti-candida activity of human PaKT-like antibodies was neutralized by previous reaction with mabkt4, con rming the high speci city of their killer e ect. The human PaKTlike antibodies were de ned then as a mirror of mabkt4, and had similar activity as PaKT; then we could consider them as human anti-idiotypic antibodies (KT-antiIds). Human natural puri ed KTantiIds were also able to transfer passive immunity to naive animals experimentally infected with C. albicans cells [21]. As Pneumocystis was sensitive to PaKT like C. albicans, then the activity of human natural candidacidal KT-antiIds was evaluated against P. carinii. They were tested for their ability to inhibit the in vitro attachment of P. carinii organisms to cultured cells [22] as well as the infectivity of P. carinii to nude rats [16]. In these experiments, the PaKT was used to control the sensitivity of P. carinii cells to the killer e ect of human KT-antiIds. In summary, human natural KT-antiIds induced a signi cant marked inhibition (55% against 40% when we used PaKT) of P. carinii attachment to human epithelial lung cells [22]. The speci city of the human KT-anti- Ids e ect on P. carinii was controlled ex vivo, by preincubating parasites in several inocula. Our data showed that rat-derived parasites preincubated in KT-antiIds-depleted vaginal secretions from patients induced severe pneumocystosis in nude rats. On the contrary, when P. carinii organisms were preincubated with puri ed human KT-antiIds from the same patients, their infectivity was strongly inhibited (74.2%) [16] The monoclonal anti-idiotypic antibody (mabk10) Previous observations con rmed that the activity of a microbiocid toxin can be mirrored through the idiotypic network [18,19]. Thus, a monoclonal killer toxin-like anti-idiotypic antibody (mabk10) has been produced. It was obtained from idiotypic immunization with mabkt4 in the rat model. So, the internal image of mabk10 was able to bind mabkt4 by the idiotype region. First, the evaluation of the antifungal e ect of mabk10 was carried out in vitro against a C. albicans sensitive strain. MabK10 inhibited its growth very signi cantly (90%) [23]. In order to evaluate the therapeutic e ect of mabk10 against extensive PCP, a nebulization protocol using mabk10 was developed in nude rats developing PCP. The last few years e cient nebulizers have been developed [24]. They are able to deliver

4 148 N. Seèguy et al. / FEMS Immunology and Medical Microbiology 22 (1998) 145^149 aerosols in the size range predicted to deposit e ciently in the alveoli. After aerosol administration of mabk10, the extension of rat-p. carinii infection in nude rats was reduced (45.3%) in comparison with that recorded in animals treated with saline solution. No obvious ill e ects were observed in rats receiving aerosolized mabk10 [25]. These data con rmed the real interest of the anti-idiotype approach in developing therapies against PCP when conventional idiotype vaccine strategies are di cult. Moreover, they suggested a feasible experimental approach for producing molecules with anti-pneumocystis activity. 4. The potential anti-pneumocystis activity of recombinant KT-antiIds Recently, a new approach in molecular biology (recombinant DNA methodology) has been initiated [26]. Nucleotide primers have been used to amplify DNA sequences corresponding to the variable domains of mouse immunoglobulin light and heavy chains, thus allowing the DNA encoding the antiids variable domains to be cloned and sequenced. Single chain fragment antiids (ScFv-antiIds) were produced and selected for their antifungal properties against C. albicans strains sensitive to the P. anomala killer toxin or KT-antiIds. These ScFv-antiIds displayed both an in vitro antibiotic e ect against killer toxin-sensitive yeast strain and an e ective therapeutic activity in an experimental model of rat candidal vaginitis. The production of such small synthetic peptides corresponding to arti cial anti-idiotype and avoiding undesired side e ects to the host might constitute a new way for the treatment and prophylaxis of P. carinii infections. 5. Perspectives of the killer phenomenon in the control of pneumocystosis Among new antimicrobial molecules which are tested for the control of PCP, yeast killer toxin-like anti-idiotype antibodies are inhibitors of Pneumocystis attachment and infectivity. Moreover, the same KT-antiIds mimic the properties of the PaKT against sensitive eukaryotic or prokaryotic microorganisms. Since the severity of PCP at the time of treatment may have a signi cant e ect on the outcome of aerosol therapy, pharmacokinetic studies of aerosolized monoclonal or recombinant anti-idiotypic antibodies in patients appear warranted prior to human clinical trials [27]. Furthermore, the monoclonality of KTantiIds should allow the carefully evaluation of their therapeutic potential against a large panel of PaKTsensitive pathogen microorganisms. Isolation and puri cation of parasite killer toxin receptors (KTRs) would allow to give a new insight into the production of natural KT-antiIds (anti-ktrs antibodies) in humans during infection with KTRs-bearing microorganisms. Acknowledgments Our investigations were developed in the framework of an European BIOMED-1 Concerted Action (ref. no. PL941118). N.S. was supported by a SI- DACTION grant (ref. no. ID ). References [1] Orenstein, J.M., Fox, C. and Wahl, S.M. (1997) Macrophages as a source of HIV during opportunistic infections. Science 276, 1857^1861. [2] Palfree, R.G.E. and Bussey, H. (1979) Yeast killer toxin: puri cation and characterization of the protein toxin from Saccharomyces cerevisiae. Eur. J. Biochem. 93, 487^493. [3] Sugisaki, Y., Gunge, N., Sakaguchi, K., Yamasaki, M. and Tamura, G. (1993) Kluyveromyces lactis killer toxin inhibits adenylate cyclase of sensitive yeast cells. Nature 304, 464^ 466. [4] Middelbeek, E.J., Stumm, C. and Vogels, G.D. (1980) E ects of Pichia kluyveri killer toxin on sensitive cells. Antonie van Leeuwenhoek 46, 205^220. [5] Sugisaki, Y., Gunge, N., Sakaguchi, K., Yamasaki, M. and Tamura, G. (1984) Characterization of a novel killer toxin encoded by a double-stranded linear DNA plasmid of Kluyveromyces lactis. Eur. J. Biochem. 141, 241^245. [6] Makower, M. and Bevan, E.A. (1963) The inheritance of a killer character in yeast (Saccharomyces cerevisiae). In: Genetics Today, vol. 1, Proceedings of the 11th International Congress of Genetics (Geert, Ed.), pp. 202^203, MacMillan, The Hague. [7] De La Pena, P., Barros, F., Gascon, S., Lazo, P.S. and Ramos, S. (1980) Primary e ects of yeast killer toxin. Biochem. Biophys. Res. Commun. 96, 544^550. [8] Polonelli, L., Lorenzini, R., De Bernardis, F. and Morace, G.

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