Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice

In the present work we analyze the antigenicity of Leishmania major ribosomal proteins (LRP) in infected BALB/c mice. We show that BALB/c mice vaccinated with LRP in the presence of CpG oligodeoxynucleotides (CpG-ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load after challenge with L. major. This protection was associated with the induction of an IL-12 dependent specific-IFN-gamma response mediated mainly by CD4(+) T cell, albeit a minor contribution of CD8(+) T cells cannot be ruled out. Induction of Th1 responses against LRP also resulted in a reversion of the Th2 responses associated with susceptibility. A marked reduction of IgG1 antibody titer against parasite antigens besides an impaired IL-4 and IL-10 cytokine production by parasite specific T cells was observed. In addition, we show that the administration of the LRP plus CpG-ODN preparation also conferred protection in the naturally resistant C57BL/6 mice. In this strain protection was associated with a LRP specific IFN-gamma production in lymph nodes draining the challenge site. We believe that these evolutionary conserved proteins, combined with adjuvants that favor Th1 responses, may be relevant components of a pan-Leishmania vaccine.     

Ricin and alpha-sarcin alter the conformation of 60S ribosomal subunits at neighboring but different sites

The effects of ricin and alpha-sarcin separately or in combination on the conformation of rat liver ribosomes were investigated by measuring the relative accessibility of individual ribosomal proteins to N-ethylmaleimide after 80S ribosomes were treated with these toxins. By using a double-labelling technique in which ribosomes were incubated with the toxins and then treated with 3H-labelled or 14C-labelled N-ethylmaleimide, it was found that labelling of protein L14 was specifically reduced by treatment with ricin, and that of proteins L3 and L4 by treatment with alpha-sarcin, suggesting that the toxins alter the conformation of ribosomes in the vicinity of these proteins. When ribosomes were treated with both ricin and alpha-sarcin, the extent of labelling of protein L3 was reduced compared to that observed after treatment with alpha-sarcin alone. These results are discussed in relation to previous observations showing that these three proteins are neighbours in the 60S ribosomal subunit and probably play important roles in protein biosynthesis, and in the actions of ricin and alpha-sarcin on 28S rRNA.     

Involvement of the amino-terminal beta-hairpin of the Aspergillus ribotoxins on the interaction with membranes and nonspecific ribonuclease activity

Ribotoxins are a family of potent cytotoxic proteins from Aspergillus whose members display a high sequence identity (85% for about 150 amino acid residues). The three-dimensional structures of two of these proteins, alpha-sarcin and restrictocin, are known. They interact with phospholipid bilayers, according to their ability to enter cells, and cleave a specific phosphodiester bond in the large subunit of ribosome thus inhibiting protein biosynthesis. Two nonconservative sequence changes between these proteins are located at the amino-terminal beta-hairpin of alpha-sarcin, a characteristic structure that is absent in other nontoxic structurally related microbial RNases. These two residues of alpha-sarcin, Lys 11 and Thr 20, have been substituted with the equivalent amino acids in restrictocin. The single mutants (K11L and T20D) and the corresponding K11L/T20D double mutant have been produced in Escherichia coli and purified to homogeneity. The spectroscopic characterization of the purified proteins reveals that the overall native structure is preserved. The ribonuclease and lipid-perturbing activities of the three mutants and restrictocin have been evaluated and compared with those of alpha-sarcin. These proteins exhibit the same ability to specifically inactivate ribosomes, although they show different activity against nonspecific substrate analogs such as poly(A). The mutant variant K11L and restrictocin display a lower phospholipid-interacting ability correlated with a decreased cytotoxicity. The results obtained are interpreted in terms of the involvement of the amino-terminal beta-hairpin in the interaction with both membranes and polyadenylic acid.     

Analysis of the Dynamics of Infiltrating CD4+ T Cell Subsets in the Heart during Experimental Trypanosoma cruzi Infection

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects several million people in Latin America. Myocarditis, observed during both the acute and chronic phases of the disease, is characterized by an inflammatory mononuclear cell infiltrate that includes CD4⁺ T cells. It is known that Th1 cytokines help to control infection. The role that T_reg and Th17 cells may play in disease outcome, however, has not been completely elucidated. We performed a comparative study of the dynamics of CD4⁺ T cell subsets after infection with the T. cruzi Y strain during both the acute and chronic phases of the disease using susceptible BALB/c and non-susceptible C57BL/6 mice infected with high or low parasite inocula. During the acute phase, infected C57BL/6 mice showed high levels of CD4⁺ T cell infiltration and expression of Th1 cytokines in the heart associated with the presence of T_reg cells. In contrast, infected BALB/c mice had a high heart parasite burden, low heart CD4⁺ T cell infiltration and low levels of Th1 and inflammatory cytokines, but with an increased presence of Th17 cells. Moreover, an increase in the expression of IL-6 in susceptible mice was associated with lethality upon infection with a high parasite load. Chronically infected BALB/c mice continued to present higher parasite burdens than C57BL/6 mice and also higher levels of IFN-γ, TNF, IL-10 and TGF-β. Thus, the regulation of the Th1 response by T_reg cells in the acute phase may play a protective role in non-susceptible mice irrespective of parasite numbers. On the other hand, Th17 cells may protect susceptible mice at low levels of infection, but could, in association with IL-6, be pathogenic at high parasite loads.     

Lactococcus lactis as a vehicle for the heterologous expression of fungal ribotoxin variants with reduced IgE-binding affinity

Fungal ribotoxins are a family of extracellular ribonucleases which inhibit protein biosynthesis by inactivating the ribosomes. This inactivation results in the induction of cell death by apoptosis. Ribotoxins show antitumoral properties based on their ability to cross the membrane of some transformed cells. Unfortunately, they also show an unspecific cytotoxicity which has greatly impaired their potential clinical uses. alpha-Sarcin, produced by Aspergillus giganteus, is the best-characterized ribotoxin. Asp f 1, another ribotoxin produced by A. fumigatus, is indeed one of its major allergens. In this work, the Lactococcus lactis MG1363 strain has been engineered to produce and secrete not only wild-type Asp f 1 and alpha-sarcin but also three different mutants with reduced cytotoxicity and/or IgE-binding affinity. The proteins were secreted in native and active form when the extracellular medium employed was buffered at pH values around 8.0. Strains producing the wild-type natural alpha-sarcin were proved to be innocuous when administered intragastrically to mice for a period of 14 days. Overall, the results presented are discussed in terms of its potential application as a vehicle of oral delivery of hypoallergenic variants as well as a starting point to approach the design of strategies to accomplish the safe delivery of these proteins as antitumoral agents.     

Leucine 145 of the ribotoxin alpha-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of alpha-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type alpha-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.     

The action mode of the ribosome-inactivating protein α-sarcin

Based on the tertiary structure of the ribosome-inactivating protein alpha-sarcin, domains that are responsible for hydrolyzing ribosomes and naked RNA have been dissected. In this study, we found that the head-to-tail interaction between the first amino beta-strand and the last carboxyl beta-strand is not involved in catalyzing the hydrolysis of ribosomes or ribonucleic acids. Instead, a four-strand pleated beta-sheet is indispensable for catalyzing both substrates, suggesting that alpha-sarcin and ribonuclease T1 (RNase T1) share a similar catalytic center. The integrity of an amino beta-hairpin and that of the loop L3 in alpha-sarcin are crucial for recognizing and hydrolyzing ribosomes in vitro and in vivo. However, a mutant protein without the beta-hairpin structure, or with a disrupted loop L3, is still capable of digesting ribonucleic acids. The functional involvement of the beta-hairpin and the loop L3 in the sarcin stem/loop RNA of ribosomes is demonstrated by a docking model, suggesting that the two structures are in essence naturally designed to distinguish ribosome-inactivating proteins from RNase T1 to inactivate ribosomes.     

Alpha-sarcin causes a specific cut in 28 S rRNA when microinjected into Xenopus oocytes

The toxin alpha-sarcin specifically cuts 28 S rRNA at a single position 393 nucleotides from its 3' end in isolated rat liver polysomes, provided the ribosomes are pretreated with EDTA or puromycin (Endo, Y. & Wool, I. G. (1982) J. Biol. Chem. 257, 9054-9060). In addition, alpha-sarcin behaves as a purine-specific RNase on deproteinized RNA, cleaving on the 3' side of purines in both single- and double-stranded RNA (Endo, Y., Huber, P. W., and Wool, I. G. (1983) J. Biol. Chem. 258, 2662-2667). Since alpha-sarcin does not readily enter tissue culture cells, we have injected it into Xenopus oocytes in order to determine whether the toxin cleaves after all purines or if it specifically makes a single cut in 28 S rRNA in intact cells. We report here that in oocytes alpha-sarcin specifically cuts 28 S rRNA 377 nucleotides from its 3' end, even when used at concentrations that would degrade deproteinized RNA. alpha-Sarcin does not behave as a general nuclease when injected into Xenopus oocytes nor does it operate by another means such as initiating proteolytic digestion of endogenous oocyte proteins. We demonstrate that injected alpha-sarcin causes a rapid decline in oocyte protein synthesis for soluble cytoplasmic proteins, similar in effect to injection of cycloheximide or puromycin.     

Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system.     

Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin alpha-sarcin.

Alpha-sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three-dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of alpha-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome-inactivating activity of alpha-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of alpha-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of alpha-sarcin and presumably of the other fungal ribotoxins.     

Alpha-sarcin cleaves ribosomal RNA at the alpha-sarcin site in the absence of ribosomal proteins

alpha-Sarcin is capable of specifically cleaving the single phosphodiester bond in the "alpha-sarcin site" of both Escherichia coli and Saccharomyces cerevisiae large rRNAs in the absence of ribosomal proteins. With both sources of rRNA, the rate of cleavage was comparable with and without ribosomal proteins but more complete cleavage was observed in the absence of ribosomal proteins. These observations contrast with earlier findings and indicate that ribosomal proteins are not essential to the unique specificity of the cleavage of rRNA by alpha-sarcin.     

Cytotoxic mechanism of the ribotoxin alpha-sarcin. Induction of cell death via apoptosis.

Alpha-sarcin is a ribosome-inactivating protein that has been well characterized in vitro, but little is known about its toxicity in living cells. We have analyzed the mechanism of internalization of alpha-sarcin into human rhabdomyosarcoma cells and the cellular events that result in the induction of cell death. No specific cell surface receptor for alpha-sarcin has been found. The toxin is internalized via endocytosis involving acidic endosomes and the Golgi, as deduced from the ATP requirement and the effects of NH4Cl, monensin and nigericin on its cytotoxicity. Specific cleavage of 28S rRNA in cultured rhabdomyosarcoma cells, associated with protein biosynthesis inhibition, has been detected. alpha-Sarcin kills rhabdomyosarcoma cells via apoptosis: incubation of cells with alpha-sarcin at a concentration below its IC50 induces internucleosomal genomic DNA fragmentation, reversion of membrane asymmetry, activation of caspase-3-like activity and cleavage of poly(ADP-ribose)polymerase. Apoptosis is not a general direct consequence of protein biosynthesis inhibition, as deduced from the comparative analysis of the effects of alpha-sarcin and cycloheximide; the latter does not induce apoptosis even at concentrations far beyond its IC50, where protein biosynthesis is null. Experiments with a catalytically inactive alpha-sarcin mutant, neither toxic nor apoptotic, reveal that induced apoptosis is directly related to the effects of catalytic activity of the toxin on the ribosomes. The caspase inhibitor z-VAD-fmk does not suppress protein synthesis inhibition by alpha-sarcin. Together, these data suggest that alpha-sarcin-induced caspase activation is a pathway downstream of the 28S rRNA catalytic cleavage and consequent protein biosynthesis inhibition.     

Leishmania donovani Infection of a Susceptible Host Results in Apoptosis of Th1-Like Cells: Rescue of Anti-Leishmanial CMI by Providing Th1-Specific Bystander Costimulation

A protective immune response against Leishmania donovani infection is mediated by T-helper type 1 (Th1) cells. Th1 induced cell-mediated immunity (CMI), as assessed by anti-leishmanial DTH response, is lost in a susceptible host such as BALB/c mice. Although the impaired Th1 function eventuates in unhindered parasite growth and in manifestation of the susceptible phenotype, the mechanism of down-regulation of the Th1 function is yet to be elucidated. Here, we provide evidence that the parasite downregulates the expression of a Th1-specific costimulatory molecule, M150, on the surface of infected BALB/c mice-derived macrophages. Th cells are rendered unresponsive to anti-CD3 Ab-mediated stimulation after interaction with infected macrophages. The anergized T cells produce much less IL-2, IL-4 and IFN-γ compared to those T cells which were costimulated using normal macrophages. The defect in proliferation, anti-CD3 Ab induced unresponsiveness and IFN-γ but not IL-4 production can be restored by providing bystander costimulation through M150. These results not only unfold a novel immune evasion strategy used by the parasite but also clarify the mechanism of Th1 cell debilitation during the disease. Recovery of Th1 cytokine production by bystander costimulation through M150 may help in formulating a new strategy for the elimination of intracellular parasites.     

External ATP permeabilizes transformed cells to macromolecules

External ATP under certain ionic conditions render transformed cells permeable to the translation inhibitor hygromycin B. With this method the protein toxin alpha-sarcin selectively penetrates into 3T6 cells, as compared to 3T3 cells. This entry is enhanced by ATP synthesis blockers such as CCCP. Other proteins, such as horseradish peroxidase and luciferase, also pass selectively in 3T6 cells under permeabilization conditions.     

Parasite-induced Th2 polarization is associated with down-regulated dendritic cell responsiveness to Th1 stimuli and a transient delay in T lymphocyte cycling

The nature of the signals that bias Th effector choice is still not completely understood. Using parasite extracts from pathogens known to induce polarized Th1 or Th2 responses and an in vitro experimental model for priming murine CD4(+) cells, we demonstrated that splenic dendritic cells (DC), but not B cells, promote Th1/Th2 differentiation of naive CD4(+) lymphocytes. Th polarization in this system was found not to depend on DC secretion of the polarizing cytokines IL-12/IL-4, but instead correlated with distinct states of DC activation induced by the different parasite preparations. As expected, conditioning of DC for Th1 development was associated with up-regulation of costimulatory molecules and enhanced chemokine production and required intact MyD88 signaling. In contrast, conditioning of DC for Th2 differentiation correlated with down-regulation of many of the same functions and was MyD88 independent. This dampened DC activation was accompanied in the cocultures by a reduction in the frequency of CD4(+) lymphocytes exiting the first division of the cell cycle. When the latter was mimicked by drug-induced arrest of peptide-primed CD4(+) cells after the S phase of the first cycle, a marked Th2 polarization was also observed. Together, these findings suggest that the emergence of IL-4-producing CD4(+) lymphocytes results from a suppression in DC function leading to a temporary delay in initial T cell cycling.     

TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii

TLRs expressed by a variety of cells, including epithelial cells, B cells, and dendritic cells, are important initiators of the immune response following stimulation with various microbial products. Several of the TLRs require the adaptor protein, MyD88, which is an important mediator for the immune response following Toxoplasma gondii infection. Previously, TLR9-mediated innate immune responses were predominantly associated with ligation of unmethylated bacterial CpG DNA. In this study, we show that TLR9 is required for the Th1-type inflammatory response that ensues following oral infection with T. gondii. After oral infection with T. gondii, susceptible wild-type (WT; C57BL/6) but not TLR9(-/-) (B6 background) mice develop a Th1-dependent acute lethal ileitis; TLR9(-/-) mice have higher parasite burdens than control WT mice, consistent with depressed IFN-gamma-dependent parasite killing. A reduction in the total T cell and IFN-gamma-producing T cell frequencies was observed in the lamina propria of the TLR9(-/-) parasite-infected mice. TLR9 and type I IFN production was observed by cells from infected intestines in WT mice. TLR9 expression by dendritic cell populations is essential for their expansion in the mesenteric lymph nodes of infected mice. Infection of chimeric mice deleted of TLR9 in either the hemopoietic or nonhemopoietic compartments demonstrated that TLR9 expression by cells from both compartments is important for efficient T cell responses to oral infection. These observations demonstrate that TLR9 mediates the innate response to oral parasite infection and is involved in the development of an effective Th1-type immune response.     

Murine immune responses to a novel schistosome egg antigen, SmEP25

Pathology in schistosomiasis consists of granuloma formation around parasite eggs. There is considerable variation in the severity of disease in individuals with schistosomiasis, which may result from differential responses to egg antigens. The egg-induced immunopathology is mediated by CD4+ T helper cells sensitised to egg antigens. In this study, cellular responses to a 25-kDa fraction of egg proteins identified a novel T-cell antigen, SmEP25. The native SmEP25 elicited significant proliferative responses as well as gamma interferon (IFN-gamma), IL-2, IL-4, and IL-5 secretion in CD4+ cells from 8.5-week infected CBA and C57BL/6 mice. In C57BL/6 mice, proliferative responses to SmEP25 were relatively stronger than those directed against the major egg antigen Sm-p40, whereas in CBA mice the reverse was found. SmEP25 elicited stronger Th2 type response than Sm-p40 in both mouse strains. By comparison, recombinant SmEP25 elicited a smaller, Th1-polarised response, with significant IFN-gamma, low levels of IL-5 and essentially no IL-4. B-cell responses to SmEP25 coincided with the start of parasite egg production and SmEP25 protein was restricted to parasite eggs. The systematic identification of T-cell-sensitising egg components will lead to a better understanding of the processes involved in granuloma formation.     

IL-2 and autocrine IL-4 drive the in vivo development of antigen-specific Th2 T cells elicited by nematode parasites

The intestinal nematode parasite, Nippostrongylus brasiliensis, triggers potent type 2 immunity. Using OVA peptide as a model Ag, we have examined the adjuvant effects of this parasite on the in vivo development of Ag-specific Th2 cells from naive DO11.10 T cells. Our findings show that Th2 cells can develop from transferred naive OVA-specific DO11.10 T cells in recipient IL-4-/- mice inoculated with N. brasiliensis plus OVA. However, autocrine IL-4 is required for in situ Th2 cell differentiation since transferred IL-4Ralpha-deficient DO11.10 T cells showed greatly reduced Th2 cell development in inoculated IL-4-/- recipient mice. Surprisingly, we also found that IL-2 blockade promoted B7-dependent T cell cycling, but inhibited the development of OVA-specific Th2 cells. Furthermore, the effects of IL-2 occurred independently of CD25+ T regulatory cells. These studies establish a previously unrecognized requirement for autocrine IL-4 and IL-2 in Th2 responses elicited by nematode parasites.     

EmTIP,a T-Cell Immunomodulatory Protein Secreted by the Tapeworm Echinococcus multilocularis Is Important for Early Metacestode Development

Background

Alveolar echinococcosis (AE), caused by the metacestode of the tapeworm Echinococcus multilocularis, is a lethal zoonosis associated with host immunomodulation. T helper cells are instrumental to control the disease in the host. Whereas Th1 cells can restrict parasite proliferation, Th2 immune responses are associated with parasite proliferation. Although the early phase of host colonization by E. multilocularis is dominated by a potentially parasitocidal Th1 immune response, the molecular basis of this response is unknown.

Principal Findings

We describe EmTIP, an E. multilocularis homologue of the human T-cell immunomodulatory protein, TIP. By immunohistochemistry we show EmTIP localization to the intercellular space within parasite larvae. Immunoprecipitation and Western blot experiments revealed the presence of EmTIP in the excretory/secretory (E/S) products of parasite primary cell cultures, representing the early developing metacestode, but not in those of mature metacestode vesicles. Using an in vitro T-cell stimulation assay, we found that primary cell E/S products promoted interferon (IFN)-γ release by murine CD4+ T-cells, whereas metacestode E/S products did not. IFN-γ release by T-cells exposed to parasite products was abrogated by an anti-EmTIP antibody. When recombinantly expressed, EmTIP promoted IFN-γ release by CD4+ T-cells in vitro. After incubation with anti-EmTIP antibody, primary cells showed an impaired ability to proliferate and to form metacestode vesicles in vitro.

Conclusions

We provide for the first time a possible explanation for the early Th1 response observed during E. multilocularis infections. Our data indicate that parasite primary cells release a T-cell immunomodulatory protein, EmTIP, capable of promoting IFN-γ release by CD4+ T-cells, which is probably driving or supporting the onset of the early Th1 response during AE. The impairment of primary cell proliferation and the inhibition of metacestode vesicle formation by anti-EmTIP antibodies suggest that this factor fulfills an important role in early E. multilocularis development within the intermediate host.