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Leishmania donovanicauses anthroponotic visceral leishmaniasis, responsible for about 50,000 annual deaths worldwide. Current therapies have considerable side effects. Drug resistance has been reported and no vaccine is available nowadays. The development of undifferentiated promastigotes in the sand fly vector’s gut leads to the promastigote form that is highly infective to the mammalian host. Fully differentiated promastigotes play a crucial role in the initial stages of mammalian host infection before internalization in the host phagocytic cell. Therefore, the study of protein levels in the promastigote stage is relevant for disease control, and proteomics analysis is an ideal source of vaccine candidate discovery. This study aims to get insight into the protein levels during the differentiation process of promastigotes by 2DE-MALDI-TOF/TOF. This partial proteome analysis has led to the identification of 75 proteins increased in at least one of the L. donovanipromastigote differentiation and growth phases. This study has revealed the differential abundance of said proteins during growth and differentiation. According to previous studies, some are directly involved in parasite survival or are immunostimulatory. The parasite survival–related proteins are ascorbate peroxidase; cystathionine β synthase; an elongation factor 1β paralog; elongation factor 2; endoribonuclease L-PSP; an iron superoxide dismutase paralog; GDP-mannose pyrophosphorylase; several heat shock proteins—HSP70, HSP83-17, mHSP70-rel, HSP110; methylthioadenosine phosphorylase; two thiol-dependent reductase 1 paralogs; transitional endoplasmic reticulum ATPase; and the AhpC thioredoxin paralog. The confirmed immunostimulatory proteins are the heat shock proteins, enolase, and protein kinase C receptor analog. The potential immunostimulatory molecules according to findings in patogenic bacteria are fructose-1,6-diphophate aldolase, dihydrolipoamide acetyltransferase, isocitrate dehydrogenase, pyruvate dehydrogenase E1α and E1β subunits, and triosephosphate isomerase. These proteins may become disease control candidates through future intra-vector control methods or vaccines.

ABSTRACTWe have previously demonstrated that oral delivery of a disease-promoting particulated antigen of Leishmania amazonensis(LaAg) partially protects mice against cutaneous leishmaniasis. In the present work, we sought to optimize a mucosal vaccine by using the intranasal route for delivery of different antigen preparations, including (i) LaAg, (ii) soluble recombinant p36/LACK leishmanial antigen (LACK), and (iii) plasmid DNA encoding LACK (LACK DNA). BALB/c mice that received two intranasal doses of 10 μg of LaAg and were challenged 1 week postvaccination with L. amazonensisdeveloped delayed but effective control of lesion growth. A diminished parasite burden was accompanied by enhancement of both gamma interferon (IFN-γ) and interleukin-10 levels in the lesion-draining lymph nodes. The vaccine efficacy improved with time. At 4 months postvaccination, when a strong parasite-specific TH1-type response was present in vivo, the infection was controlled for at least 5 months after challenge. In contrast to the particulated LaAg, soluble LACK (10 μg/dose) had no effect. Interestingly, LACK DNA (30 μg/dose), but not empty DNA, promoted rapid and durable protective immunity. Parasite growth was effectively controlled, and at 5 months after challenge LACK-reactive cells in both the mucosal and lesion-draining lymph nodes produced high levels of IFN-γ. These results demonstrate for the first time the feasibility of using the intranasal route for long-lived memory vaccination against cutaneous leishmaniasis with adjuvant-free crude antigens or DNA.

Abstract: Leishmania infantum, the protozoan parasite responsible for leishmaniasis in Europe, is capable of undergoing developmental changes in vitro and provides an excellent model for the study of cell differentiation processes. We have cloned the gene encoding the L17 ribosomal protein. The LiL17 protein family belongs to the macrolide binding site, related to the peptidyl transferase center of the ribosome. Its comparison with other members of the protein family shows several structural differences that may reflect functional variations. The protein kinase C phosphorylation sites display an intermediate pattern involving differences in location and type of residue with respect to all the species considered. Gene-structural analysis suggests the existence of two different encoding genes. The expression of the genes seem to be different with the distinct growth phases of the parasite.

The family of the RACK molecules (receptors for activated C kinases) are present in all the species studied so far. In the genus Leishmania, these molecules also induce a strong immune reaction against the infection. We have cloned and characterised the gene that encodes the RACK analogue from the parasite trypanosomatid Crithidia fasciculata(CACK). The molecule seems to be encoded by two genes. The sequence analysis of the cloned open reading frame indicates the existence of a high degree of conservation not only with other members of the Trypanosomatidae but also with mammalians. The study of the protein kinase C phosphorylation sites shows the presence of three of them, shared with the mammalian species, additional to those present in the other protozoa suggesting a certain phylogenetic distance between the protozoon Crithidia fasciculataand the rest of the Trypanosomatidae. The CACK‐encoded polypeptide shows an additional sequence of four amino acids at the carboxy‐terminal end, which produces a different folding of the fragment with the presence of an α‐helix instead of the β‐sheet usual in all the other species studied. A similar result is elicited at the amino‐terminal end by the change of three amino acid residues. The immunolocalisation experiments show that the CACK displays a pattern with a distribution mainly at the plasma membrane, different from that of the related Leishmaniaspecies used as control, that displays a distribution close to the nucleus. Altogether, the data suggest that the existence of the structural differences found may have functional consequences.

We describe here the major protein components of a highly purified human neutrophil plasma membrane fraction analyzed by uni- and two-dimensional gel electrophoresis, as well as their glycoprotein nature as determined by PAS staining, [125I]-Con A binding and [3H]-sodium borohydride labelling. A polypeptide of about 150kDa appeared as the main Con A binding protein. The topology of the polypeptides has also been determined by protein labelling from the outside of the cell surface by lactoperoxidase catalyzed iodination and from within the bilayer by using the hydrophobic reagent [1251]-iodonaphtylazide. The antigenic features of some cell surface polypeptides have also been determined by the use of monoclonal antibodies. In this context we have detected by immunoprecipitation in human neutrophils the antigen MAC 120, previously found in monocytes and putatively associated with antigen presenting function.

Abstract: Antisera were raised in rabbits to two alloforms of ATPase isolated from two substrains ofMicrococcus lysodeikticus. These alloforms show a similar amino acid composition but differ in the associated carbohydrate components. Similarities and differences in the immunological behavior of the two forms have been assessed by immunodiffusion, immunoelectrophoresis, and crossed immunoelectrophoresis. The ATPase forms show a great extent of homology as might be expected from their relatedness in amino acid composition. Differences in immunological properties are also evident. They do not seem primarily to reflect the differences in the glycan constituents, but do result from the polymorphism of the purified ATPase molecule of each form. This heterogeneity (microheterogeneity) is a consequence of the lability of the ATPase molecule and influences its behavior as antigen and immunogen.

The combined use of proteolytic digestion and lactoperoxidase catalyzed labelling with [125I] applied to membrane-bound or soluble pure F1-ATPase from Micrococcus lysodeikticus has allowed us to establish the topography of its a, ß, ? and d subunits within the protein molecule and with respect to the plane of the membrane.

Antigen presentation by synoviocytes to arthritis-related T-cell clones was studied in Lewis rats. Freshly isolated synoviocytes may be able to take up and process mycobacterial antigens but they seem inefficient as antigen presenting cells. Furthermore, synovial cells inhibited the specific proliferative responses of T lymphocytes by a mechanism which apparently was not antigen-specific or mediated by secreted cytokines. Synovial cells isolated from rats at the period of developing adjuvant arthritis showed a lower inhibitory capacity associated with a lower degree of antigen clearance. These results suggest that synoviocytes might inhibit T-cell responses in normal joints and that this negative control diminishes following arthritis induction.

We have analyzed the cellular and humoral immunity to the mycobacterial 65 KDa heat shock protein (hsp65) in a group of Freund's Adjuvant-immunized rats with a limited susceptibility to Adjuvant arthritis. According to the arthritis indices during the period of study (35 days). two different groups of rats could be distinguished: a) autoimmune Adjuvant arthritic rats (AA), and b) Non-arthritic animals (NA), including both rats which did not display any disease symptoms and rats suffering mild transient inflammation. The cellular response to the immunizing agent (Mycobacterium tuberculosis) or the mitogen Concanavalin A was comparable between both groups of rats. However, we detected an impaired cellular response to the individual hsp65 antigen in the animals that did not develop the disease. On the contrary, the level of hsp65-specific antibodies was much higher in NA animals than in AA rats suggesting a protective role for the hsp65 specific antibodies.

We have cloned the S6 ribosomal protein encoding gene from aLeishmania infantumcDNA library. This parasite protozoon, responsible for leishmaniasis in Europe, is able to undergo developmental changesin vitroand results a good model to study cell differentiation processes. The LiS6 protein sequence indicates its pertinence to the S6 protein family, related to the early mechanisms of cell division, differentiation and activation, and shows an intermediate position between the yeasts and higher eukaryotes. Thus, LiS6 protein has the same amino acid length as that of the higher eukaryotes and certain common features such nucleus entrance sequences and several kinase phosphorylation sites. However, the key functional protein kinase C phosphorylation sites are at different locations and present several threonine instead of the usual serine residues. The gene structural analysis suggest the presence of three different encoding genes that do not present remarkable changes along the different phases of the parasite.

The membrane-bound ATPase activity from two substrains ofMicrococcus lysodeikticus, designated as A and B, was inhibited by antibodies raised against the two forms of purified F1-ATPase. Form B of the enzyme, which behaved as a poorer immunogen than form A, also showed less reactivity as an antigen, independent of the physical state of the F1-ATPase form. Antibodies were raised against the two major subunits (a and ß) isolated fromM. lysodeikticus F1-ATPase form A, which was the most stable form of the enzyme. Anti-(a-subunit) serum strongly inhibited the ATPase activity of membrane-bound ATPase but showed little inhibition of the purified, soluble F1-ATPase. The anti-(ß-subunit) serum inhibited the soluble F1-ATPase, but to a lesser extent than the membrane-bound enzyme. In any event, the effect of anti-ß antibodies on the membrane-bound ATPase was smaller than that of anti-a antibodies. It was postulated that the a subunit ofM. lysodeikticus F1-ATPase plays an essential and regulatory role in the expression of the immunochemical properties of the protein.

1.Treatment of Streptomyces albus G with lysozyme (mg dry weight mycelium/mg lysozyme = 2–3/1) leads to the isolation of membrane fractions whose yields amount to 12–20% of the total celullar protein depending upon the age and state of the bacterial culture.2.The isolated fractions are composed of 52.7 per cent protein and 41 per cent lipid with minor amounts of hexose (2.3–2.4%), hexosamines (1.6–2.1%), RNA (2%) and DNA (0.45%). They possess a chemical composition similar to that of other membrane systems.3.NADH oxidase activities are associated with the membrane fractions from cells of 18–20 h of age. These activities are not detected in the membrane fractions from older cells.4.All membrane fractions shape small vesicles. Differences in size and shape are, however, found between membrane preparations from cells of distinct ages.5.dd-carboxypeptidase is not selectively localized in a membrane fraction of a certain age. The subcellular distribution of this activity is similar to that of other lytic endopeptidases of S. albus G.6.The amounts of dd-carboxypeptidase associated with the various membrane fractions are variable and never very high. The specificity of the dd-carboxypeptidase activity associated with membrane fractions differs from that of the soluble enzyme.7.These results are discussed in relationship with the carboxypeptidase-transpeptidase hypothesis (Leyh-Bouille et al., 1970).