Cloning and Characterization of Trypanosoma congolense and T. vivax Nucleoside Transporters Reveal the Potential of P1-Type Carriers for the Discovery of Broad-Spectrum Nucleoside-Based Therapeutics against Animal African Trypanosomiasis.

African Animal Trypanosomiasis (AAT), caused predominantly by Trypanosoma brucei brucei, T. vivax and T. congolense, is a fatal livestock disease throughout Sub-Saharan Africa. Treatment options are very limited and threatened by resistance. Tubercidin (7-deazaadenosine) analogs have shown activity against individual parasites but viable chemotherapy must be active against all three species. Divergence in sensitivity to nucleoside antimetabolites could be caused by differences in nucleoside transporters. Having previously characterized the T. brucei nucleoside carriers, we here report the functional expression and characterization of the main adenosine transporters of T. vivax (TvxNT3) and T. congolense (TcoAT1/NT10), in a Leishmania mexicana cell line ('SUPKO') lacking adenosine uptake. Both carriers were similar to the T. brucei P1-type transporters and bind adenosine mostly through interactions with N3, N7 and 3'-OH. Expression of TvxNT3 and TcoAT1 sensitized SUPKO cells to various 7-substituted tubercidins and other nucleoside analogs although tubercidin itself is a poor substrate for P1-type transporters. Individual nucleoside EC50s were similar for T. b. brucei, T. congolense, T. evansi and T. equiperdum but correlated less well with T. vivax. However, multiple nucleosides including 7-halogentubercidines displayed pEC50>7 for all species and, based on transporter and anti-parasite SAR analyses, we conclude that nucleoside chemotherapy for AAT is viable.

Blood serum acute phase proteins and iron dynamics during acute phase response of Salmonella enterica serotype Dublin experimentally infected buffalo calves.

The study aimed to evaluate clinical signs, blood serum acute phase proteins (APP) and iron dynamics during the acute phase response (APR) of Salmonella Dublin experimentally infected Murrah buffalo calves. Six buffalo calves constituted the control group (CNT) and six were orally inoculate with 108 CFU of S. Dublin (INF). Clinical evaluation was performed, rectal swabs to detect S. Dublin strains were collected and venous blood was sampled before and throughout seven days after inoculation. The APP fractions ß-haptoglobin, α-haptoglobin, ceruloplasmin and transferrin were analyzed by 1-D and 2-D electrophoresis. Proteins were identified using LC/ESI-MS/MS and NCBI database. Plasma fibrinogen, serum iron and serum haptoglobin concentrations were measured. The inoculation of 108 CFU of S. Dublin was effective in inducing clinical signs of Salmonellosis, such as hyperthermia and diarrhea. 1-DE showed that ß and α-haptoglobin increased 204% (p = 0.008) and 184% (p = 0.022) 48 h after inoculation (HAI), respectively, with highest concentrations 120 HAI (498% increased, p = 0.012; 431% increased, p = 0.011) and 168 HAI (492% increased, p = 0.019; 523% increased, p = 0.028). 2-DE showed that the expression of two spots, identified as ß-haptoglobin, were increased 693% (p = 0.0006) and 580% (p = 0.0003) 168 HAI, respectively, while one spot, identified as α-haptoglobin, increased 714% (p = 0.040). Haptoglobin concentrations increased 1339% (p < 0.0001) 168 HAI. 1-DE showed that ceruloplasmin increased 42% (p = 0.034) 48 HAI, with highest concentration 120 HAI (133% increased, p = 0.022). 2-DE showed that the expression of two spots, identified as ceruloplasmin, were increased 218% (p = 0.0153) and 85% (p = 0.0143) 168 HAI, respectively. Fibrinogen increased 78% (p = 0.012) 96 HAI, with highest concentration 120 HAI (increased 114%, p = 0.002). Iron decreased 33% 24 HAI (p = 0.015) and 37% 72 HAI (p = 0.029), and began to be restored 96 HAI. 1-DE showed that transferrin decreased 23% 120 HAI (p = 0.047), and that values were restored 168 HAI. 2-DE showed that expression patterns of transferrin comparing 0 h and 168 HAI were similar, evidencing that values were restored 168 HAI. In conclusion, the inoculation of 108 CFU was effective in inducing hyperthermia and diahrrea. ß and α-haptoglobin, ceruloplasmin and fibrinogen worked as positive APP during the APR to S. Dublin infection and are potential biomarker candidates. Concentrations of iron and transferrin decreased during the infection, highlighting the fact that mechanisms for restricting iron availability are part of the APR triggered against S. Dublin infection in buffalo calves.

PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast.

The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His(99) and Cys(136)), and an Asp (Asp(134)) in the potential S1 binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.

Agarose gel serum protein electrophoresis in cats with and without lymphoma and preliminary results of tandem mass fingerprinting analysis.

BACKGROUND: Serum electrophoretic profiles in cats are poorly characterized with respect to the proteins that comprise the globulin fractions, and interpretation of the electrophoretograms is routinely done in the absence of information about identity of the proteins found within each fraction. OBJECTIVES: The aims of this study were to compare protein fractions separated by serum protein electrophoresis (SPE) in healthy cats and in cats with lymphoma and to confirm some component proteins in the major fractions following SPE using tandem mass fingerprinting analysis (TMFA). METHODS: Total protein concentration was measured and agarose gel SPE performed on serum from 14 healthy cats and 14 cats with lymphoma. The absolute protein concentration within each fraction was compared between the 2 groups. Bands corresponding to the SPE fractions were excised from the gels of 2 control cats and 1 cat with lymphoma and analyzed by liquid chromatography coupled to mass spectrometry. Results were compared with sequences in the National Center for Biotechnology Information protein database. RESULTS: Median albumin concentrations were significantly decreased and median ß-globulin concentrations were significantly increased in cats with lymphoma. Narrow electrophoretic spikes were present in the ß/γ-globulin fraction in 3 cats with lymphoma. Following TMFA, multiple proteins were identified in each fraction, and their mobility agreed with results from previous studies generated using alternative techniques. Inter-α (globulin) inhibitor 4 was identified in feline serum for the first time. CONCLUSIONS: Cats with lymphoma had lower albumin and higher ß-globulin concentrations than did healthy cats. Despite limitations of one-dimensional agarose gel SPE, TMFA provided preliminary data to confirm the protein components of the various fractions.

Trypanocidal furamidine analogues: influence of pyridine nitrogens on trypanocidal activity, transport kinetics, and resistance patterns.

Current therapies for human African trypanosomiasis (HAT) are unsatisfactory and under threat from emerging drug resistance linked to the loss of transporters, e.g., the P2 aminopurine transporter (TbAT1). Here we compare the uptake and trypanocidal properties of furamidine (DB75), recently evaluated in clinical trials against stage 1 (haemolymphatic) HAT, and two aza analogues, DB820 and CPD0801 (DB829), which are candidate compounds for treatment of stage 2 (neurological) disease. Values of 50% inhibitory concentrations (IC50s) determined in vitro against both wild-type and transporter mutant parasites were submicromolar, with DB75 trypanotoxicity shown to be better than and DB820 trypanotoxicity similar to that of the widely used veterinary trypanocide diminazene, while CPD0801 was less active. Activity correlated with uptake and with the minimum drug exposure time necessary to kill trypanosomes: DB75 accumulated at double and 10-fold the rates of DB820 and CPD0801, respectively. All three compounds inhibited P2-mediated adenosine transport with similar Ki values, indicating affinity values for this permease in the low to submicromolar range. Uptake of DB75, DB820, and CPD0801 was significantly reduced in tbat1-/- parasites and was sensitive to inhibition by adenine, showing that all three compounds are substrates for the P2 transporter. Uptake in vitro was significantly less than that seen with parasites freshly isolated from infected rats, correlating with a downregulation of P2 activity in vitro. We conclude that DB75, DB820, and CPD0801 are actively accumulated by Trypanosoma brucei brucei, with P2 as the main transport route. The aza analogues of DB75 accumulate more slowly than furamidine itself and reveal less trypanocidal activity in standard in vitro drug sensitivity assays.

Multiple genetic mechanisms lead to loss of functional TbAT1 expression in drug-resistant trypanosomes.

The P2 aminopurine transporter, encoded by TbAT1 in African trypanosomes in the Trypanosoma brucei group, carries melaminophenyl arsenical and diamidine drugs into these parasites. Loss of this transporter contributes to drug resistance. We identified the genomic location of TbAT1 to be in the subtelomeric region of chromosome 5 and determined the status of the TbAT1 gene in two trypanosome lines selected for resistance to the melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), and in a Trypanosoma equiperdum clone selected for resistance to the diamidine, diminazene aceturate. In the Trypanosoma brucei gambiense STIB 386 melarsamine hydrochloride-resistant line, TbAT1 is deleted, while in the Trypanosoma brucei brucei STIB 247 melarsamine hydrochloride-resistant and T. equiperdum diminazene-resistant lines, TbAT1 is present, but expression at the RNA level is no longer detectable. Further characterization of TbAT1 in T. equiperdum revealed that a loss of heterozygosity at the TbAT1 locus accompanied loss of expression and that P2-mediated uptake of [(3)H]diminazene is lost in drug-resistant T. equiperdum. Adenine-inhibitable adenosine uptake is still detectable in a DeltaTbat1 T. b. brucei mutant, although at a greatly reduced capacity compared to that of the wild type, indicating that an additional adenine-inhibitable adenosine permease, distinct from P2, is present in these cells.

The trypanocide diminazene aceturate is accumulated predominantly through the TbAT1 purine transporter: additional insights on diamidine resistance in african trypanosomes.

Resistance to diminazene aceturate (Berenil) is a severe problem in the control of African trypanosomiasis in domestic animals. It has been speculated that resistance may be the result of reduced diminazene uptake by the parasite. We describe here the mechanisms by which [(3)H]diminazene is transported by Trypanosoma brucei brucei bloodstream forms. Diminazene was rapidly accumulated through a single transporter, with a K(m) of 0.45 +/- 0.11 micro M, which was dose dependently inhibited by pentamidine and adenosine. The K(i) values for these inhibitors were consistent with this transporter being the P2/TbAT1 adenosine transporter. Yeast expressing TbAT1 acquired the ability to take up [(3)H]diminazene and [(3)H]pentamidine. TbAT1-null mutants had lost almost all capacity for [(3)H]diminazene transport. However, this cell line still displayed a small but detectable rate of [(3)H]diminazene accumulation, in a nonsaturable manner. We conclude that TbAT1 mediates [(3)H]diminazene transport almost exclusively and that this explains the observed diminazene resistance phenotypes of TbAT1-null mutants and field isolates.

Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei.

Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.