Oligopeptide Signaling through TbGPR89 Drives Trypanosome Quorum Sensing.

Trypanosome parasites control their virulence and spread by using quorum sensing (QS) to generate transmissible "stumpy forms" in their host bloodstream. However, the QS signal "stumpy induction factor" (SIF) and its reception mechanism are unknown. Although trypanosomes lack G protein-coupled receptor signaling, we have identified a surface GPR89-family protein that regulates stumpy formation. TbGPR89 is expressed on bloodstream "slender form" trypanosomes, which receive the SIF signal, and when ectopically expressed, TbGPR89 drives stumpy formation in a SIF-pathway-dependent process. Structural modeling of TbGPR89 predicts unexpected similarity to oligopeptide transporters (POT), and when expressed in bacteria, TbGPR89 transports oligopeptides. Conversely, expression of an E. coli POT in trypanosomes drives parasite differentiation, and oligopeptides promote stumpy formation in vitro. Furthermore, the expression of secreted trypanosome oligopeptidases generates a paracrine signal that accelerates stumpy formation in vivo. Peptidase-generated oligopeptide QS signals being received through TbGPR89 provides a mechanism for both trypanosome SIF production and reception.

Synthesis and evaluation of analogs of the phenylpyridazinone NPD-001 as potent trypanosomal TbrPDEB1 phosphodiesterase inhibitors and in vitro trypanocidals.

Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of Trypanosoma brucei, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. Knock down of both enzymes leads to cell cycle arrest and is lethal to the parasite. Recently, we reported the phenylpyridazinone, NPD-001, with low nanomolar IC50 values on both TbrPDEB1 (IC50: 4nM) and TbrPDEB2 (IC50: 3nM) (J. Infect. Dis.2012, 206, 229). In this study, we now report on the first structure activity relationships of a series of phenylpyridazinone analogs as TbrPDEB1 inhibitors. A selection of compounds was also shown to be anti-parasitic. Importantly, a good correlation between TbrPDEB1 IC50 and EC50 against the whole parasite was observed. Preliminary analysis of the SAR of selected compounds on TbrPDEB1 and human PDEs shows large differences which shows the potential for obtaining parasite selective PDE inhibitors. The results of these studies support the pharmacological validation of the Trypanosome PDEB family as novel therapeutic approach for HAT and provide as well valuable information for the design of potent TbrPDEB1 inhibitors that could be used for the treatment of this disease.

PUF Proteins as Critical RNA-Binding Proteins in TriTryp Parasites: A Review Article.

In eukaryotes, translation is a fundamental step in the long pathway of protein synthesis within the cell. In this process, several proteins and factors have involved directly or indirectly, individually or in association with other elements to contact mRNA. For perfect translation, many essential modifications should be done, such as cis-splicing to remove introns and two main events for capping and poly A polymerization in 5' and 3' end of mRNA, respectively. Gene expression is then regulated at both translation and stability of the target mRNA molecule levels. Pumilio/FBFs (PUFs) are the main group of RNA-binding proteins which bind to the 3'-UTR of target RNA and thereby regulate the fate, stability and subcellular localization of mRNAs and adjust the translated protein level. PUF proteins have been found both in nucleus where that bind to precursor mRNA, for processing and maturation of rRNA, and in cytoplasm where that bind to mRNA, stall the ribosomes, suppress the translation and localization of the mRNA. They can regulate the expression of mRNAs through activation or suppression of translation. Therefore, these proteins have recently garnered much attention as new generation of therapeutic targets against diseases such as cancer and neurological disorders. In comparison to other eukaryotes, trypanosomatids have a high number of PUF proteins, which function not only as gene expression regulatory factors but also in several biological processes such as differentiation and life-cycle progression of the cells. Here, we review the molecular and biological roles of known PUF proteins in TriTryp parasites (Trypanosome brucei, T. cruzi and Leishmania) beside some other parasites.

Unwrap RAP1's Mystery at Kinetoplastid Telomeres.

Although located at the chromosome end, telomeres are an essential chromosome component that helps maintain genome integrity and chromosome stability from protozoa to mammals. The role of telomere proteins in chromosome end protection is conserved, where they suppress various DNA damage response machineries and block nucleolytic degradation of the natural chromosome ends, although the detailed underlying mechanisms are not identical. In addition, the specialized telomere structure exerts a repressive epigenetic effect on expression of genes located at subtelomeres in a number of eukaryotic organisms. This so-called telomeric silencing also affects virulence of a number of microbial pathogens that undergo antigenic variation/phenotypic switching. Telomere proteins, particularly the RAP1 homologs, have been shown to be a key player for telomeric silencing. RAP1 homologs also suppress the expression of Telomere Repeat-containing RNA (TERRA), which is linked to their roles in telomere stability maintenance. The functions of RAP1s in suppressing telomere recombination are largely conserved from kinetoplastids to mammals. However, the underlying mechanisms of RAP1-mediated telomeric silencing have many species-specific features. In this review, I will focus on Trypanosoma brucei RAP1's functions in suppressing telomeric/subtelomeric DNA recombination and in the regulation of monoallelic expression of subtelomere-located major surface antigen genes. Common and unique mechanisms will be compared among RAP1 homologs, and their implications will be discussed.

Molecular detection and therapeutic study of Trypanosoma brucei evansi from naturally infected horses in Punjab, Pakistan.

Trypanosomiasis is one of the severe pathogenic infections, caused by several Trypanosoma species, affecting both animals and humans, causing substantial economic losses and severe illness. The objective of this study was to determine the molecular diagnosis and the risk factors associated with trypanosomiasis in District Jhang, Punjab, Pakistan. For this purpose, blood samples were randomly collected from 200 horses. A predesigned questionnaire was used to collect data on risk factors before the sample collection. The microscopy examination through Giemsa staining, formol gel test and PCR techniques were used to find the prevalence. The prevalence was recorded as 22.5% with microscopy examination, 21% through formol gel test and 15.5% with PCR based results. Analysis of risk factors associated with Trypanosoma brucei evansi occurrence was carried out using Chi-square test. It showed the prevalence of Trypanosoma brucei evansi was significantly (p⟨0.05) associated with sex, age, rearing purpose and body condition whereas non-significantly (p⟩0.05) with insects control practices. This study supports the idea that PCR is a sensitive, robust and more reliable technique to diagnose trypanosomiasis. It was concluded that Trypanosoma brucei evansi is widely prevalent in Jhang (Pakistan), highlighting a dire need to develop control strategies and education programmes to control this disease in developing countries.

Synthesis of proline derived benzenesulfonamides: A potent anti-Trypanosoma brucei gambiense agent.

Thousands of death in Africa and other developing nations are still attributed to trypanosomiasis. Excessive sleep has been associated with increased inflammation. We report herein, the synthesis, antitrypanosomal and anti-inflammatory activities of eight new carboxamide derivatives bearing substituted benzenesulfonamides. The base promoted reactions of l-proline and L-4-hydroxyproline with substituted benzenesulfonyl chlorides gave the benzenesulfonamides (11a-h) in excellent yields. Boric acid mediated amidation of the benzenesulfonamides (11a-h) and p-aminobenzoic acid (12) gave the new carboxamides (13a-h) in excellent yields. The new carboxamides were tested for their antitrypanosomal and anti-inflammatory activities against Trypanosome brucei gambiense and inhibition of carrageenan-induced rat paw edema. Compound 13f was the most potent antitrypanosomal agent with an IC50 value of 2 nM as against 5 nM for melarsoprol; whereas compound 13a was the most potent anti-inflammatory agent with percentage inhibition of carrageenan-induced rat paw edema of 58, 60, 67 and 84% after 0.5 h, 1 h, 2 h and 3 h administration respectively. The structure-activity relationship study revealed that substitution at the para position in the benzenesulfonamide ring increased both the antitrypanosomal and anti-inflammatory activities. The 4-hydroxyprolines (13a-d) showed higher anti-inflammatory activity than the prolines (13e-h). In contrast, the prolines (13e-h) had higher antitrypanosomal activities than the 4-hydroxyprolines. The link between excessive sleep and inflammation makes the report of this class of compounds possessing both antitrypanosomal and anti-inflammatory activity worthwhile. The pharmacokinetic studies showed that the compounds would not pose oral bioavailability, transport and permeability problems.

Codon choice directs constitutive mRNA levels in trypanosomes.

Selective transcription of individual protein coding genes does not occur in trypanosomes and the cellular copy number of each mRNA must be determined post-transcriptionally. Here, we provide evidence that codon choice directs the levels of constitutively expressed mRNAs. First, a novel codon usage metric, the gene expression codon adaptation index (geCAI), was developed that maximised the relationship between codon choice and the measured abundance for a transcriptome. Second, geCAI predictions of mRNA levels were tested using differently coded GFP transgenes and were successful over a 25-fold range, similar to the variation in endogenous mRNAs. Third, translation was necessary for the accelerated mRNA turnover resulting from codon choice. Thus, in trypanosomes, the information determining the levels of most mRNAs resides in the open reading frame and translation is required to access this information.

Relief of autoinhibition by conformational switch explains enzyme activation by a catalytically dead paralog.

Catalytically inactive enzyme paralogs occur in many genomes. Some regulate their active counterparts but the structural principles of this regulation remain largely unknown. We report X-ray structures of Trypanosoma brucei S-adenosylmethionine decarboxylase alone and in functional complex with its catalytically dead paralogous partner, prozyme. We show monomeric TbAdoMetDC is inactive because of autoinhibition by its N-terminal sequence. Heterodimerization with prozyme displaces this sequence from the active site through a complex mechanism involving a cis-to-trans proline isomerization, reorganization of a ß-sheet, and insertion of the N-terminal α-helix into the heterodimer interface, leading to enzyme activation. We propose that the evolution of this intricate regulatory mechanism was facilitated by the acquisition of the dimerization domain, a single step that can in principle account for the divergence of regulatory schemes in the AdoMetDC enzyme family. These studies elucidate an allosteric mechanism in an enzyme and a plausible scheme by which such complex cooperativity evolved.

Bioassay-guided isolation of a sesquiterpene lactone of deoxyelephantopin from Elephantopus scaber Linn. active on Trypanosome brucei rhodesience.

Methanolic extracts of 70 Malaysia plants were screened for their in vitro antitrypanosomal activity using Trypanosome brucei rhodesience, strain STIB 900 and mouse skeletal cell (L-6) in cytotoxicity activity assay. Results indicated that methanol extract from Elephantopus scaber Linn. (E. scaber) possessed the highest value of antitrypanosomal activity with good selectivity index (antitrypanosomal IC50 of 0.22±0.02 µg/ml, SI value of 204.55). Based on these results, E. scaber was chosen for further study by applying bioassay guided fractionation to isolate its antiprotozoal principle. The antiprotozoal principle was isolated from the ethyl acetate partition through solvent fractionation and crystallization process. The isolated active compound 1 was identified as deoxyelephantopin on the basis of its spectral analysis (FTIR, MS, 1D and 2D NMR).