Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains.

Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 µM at the promastigote stage and 32 and 195 µM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.

Molecular cloning and localization of a calpain-like protease from the abdominal muscle of Norway lobster Nephrops norvegicus.

Calpains are ubiquitous cysteine-proteases found in many, if not all, living organisms and their roles within these organisms are diverse, ranging from the mediation of cytoskeletal remodeling to the regulation of gene expression. In crustaceans calpains have so far been shown to be important mainly during moulting and growth. In the present study we report the expression of a calpain in the abdominal muscle of Norway lobster (Nephrops norvegicus) using degenerate primer, rapid amplification of cDNA ends (5'-3'-RACE), reverse transcriptase-PCR and RNA in situ hybridization approaches. The full-length mRNA sequence (2,774 bp) was found to include an open reading frame (bp 225-1,940) encoding a 572 amino acid polypeptide with a predicted mass of 65.9 kDa and a predicted pI of 5.17. The calpain was found to be an arthropod M-class calpain homologue to Homarus americanus Calpain M (Ha-CalpM) and has thus been termed Nephrops norvegicus calpain M (Nn-CalpM). When its expression pattern in abdominal muscle of adult intermoult Nephrops norvegicus was investigated an exclusive expression in a thin layer of connective tissue cells surrounding muscle fibres was found. This localization suggests a role in tenderizing connective tissue networks during growth and moulting.

Differential inhibition of high and low Mr thioredoxin reductases of parasites by organotelluriums supports the concept that low Mr thioredoxin reductases are good drug targets.

Thioredoxin reductase (TrxR), a NADPH-dependent disulfide oxidoreductase, is vital in numerous cellular processes including defence against reactive oxygen species, cell proliferation and signal transduction. TrxRs occur in 2 forms, a high Mr enzyme characterized by those of mammals, the malaria parasite Plasmodium falciparum and some worms, and a low Mr form is present in bacteria, fungi, plants and some protozoan parasites. Our hypothesis is that the differences between the forms can be exploited in the development of selective inhibitors. In this study, cyclodextrin- and sulfonic acid-derived organotelluriums known to inhibit mammalian TrxR were investigated for their relative efficacy against P. falciparum TrxR (PfTrxR), a high Mr enzyme, and Trichomonas vaginalis TrxR (TvTrxR), a low Mr form of TrxR. The results suggest that selective inhibition of low Mr TrxRs is a feasible goal.

Functional conservation of a natural cysteine peptidase inhibitor in protozoan and bacterial pathogens.

Cysteine peptidase inhibitor genes (ICP) of the chagasin family have been identified in protozoan (Leishmania mexicana and Trypanosoma brucei) and bacterial (Pseudomonas aeruginosa) pathogens. The encoded proteins have low sequence identities with each other and no significant identity with cystatins or other known cysteine peptidase inhibitors. Recombinant forms of each ICP inhibit protozoan and mammalian clan CA, family C1 cysteine peptidases but do not inhibit the clan CD cysteine peptidase caspase 3, the serine peptidase trypsin or the aspartic peptidases pepsin and thrombin. The functional homology between ICPs implies a common evolutionary origin for these bacterial and protozoal proteins.

The stage-regulated expression of Leishmania mexicana CPB cysteine proteases is mediated by an intercistronic sequence element.

The tandemly arranged CPB genes of Leishmania mexicana are polycistronically transcribed and encode cysteine proteases that are differentially stage-specific; CPB1 and CPB2 are expressed predominantly in metacyclics, whereas CPB3-CPB18 are expressed mainly in amastigotes. The mechanisms responsible for this differential expression have been studied via gene analysis and re-integration of individual CPB genes, and variants thereof, into a CPB-deficient parasite mutant. Comparison of the nucleotide sequences of the repeat units of CPB1 and CPB2 with CPB2.8 (typical of CPB3-CPB18) revealed two major regions of divergence as follows: one of 258 base pairs (bp) corresponding to the C-terminal extension of CPB2.8; another, designated InS, of 120 bp, with insertions totaling 57 bp, localized to the intercistronic region downstream of CPB1 and CPB2. Cell lines expressing CPB2.8 or CPB2 with the 3'-untranslated region and intercistronic sequence of CPB2.8 showed up-regulation in amastigotes. Conversely, metacyclic-specific expression occurred with CPB2 or CPB2.8 with the 3'-untranslated region and intercistronic sequence of CPB2. Moreover, the InS down-regulated expression in amastigotes of a reporter gene integrated into the CPB locus. It is proposed that the InS mediates metacyclic-specific stage-regulated expression of CPB by affecting the maturation of polycistronic pre-mRNA. This is the first well defined cis-regulatory element implicated in post-transcriptional stage-specific gene expression in Leishmania.

Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro.

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC. The role of the cyaC gene product in the acylation reaction has not been determined. We have developed an efficient T7 RNA polymerase system for over-expression of cyaA and cyaC separately in Escherichia coli. Each protein accumulated intracellularly in an insoluble form and could be collected by centrifugation of lysed cells. A single-step purification was achieved by extraction of the aggregated material with 8 M urea. Active cell-invasive CyaA was produced in vitro when the proCyaA and CyaC proteins were mixed with a cytosolic extract of either E. coli or B. pertussis. Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cytosolic factor required for toxin activation was lost if the S100 extract was dialysed before use and the cytosolic factor could be replaced in the in vitro reaction by ACP charged separately in vitro with palmitic acid, as reported previously for activation of the homologous E. coli haemolysin (HlyA). The in vitro activation system may be used to investigate the mechanism of the CyaC-dependent acylation of proCyaA and the effect of variation of the modifying fatty acyl group on target cell specificity and toxic activity of CyaA.

A new assay for the invasive adenylate cyclase toxin of Bordetella pertussis based on its morphological effects on the fibronectin-stimulated spreading of BHK21 cells.

When baby hamster kidney (BHK) cells are allowed to spread on fibronectin-coated substrata in the absence of serum and the presence of agents which elevate intracellular 3':5'-cyclic AMP (cAMP) levels they adopt an abnormal, stellated morphology. To determine whether the invasive adenylate cyclase (AC) toxin of Bordetella pertussis induced the same response, cell extracts were prepared from several B. pertussis strains. They were characterized for AC toxin production by enzymic assay and by immunoblotting with an AC-toxin-specific monoclonal antibody. Extracts of strains producing AC toxin induced elevated levels of intracellular cAMP in BHK cells and promoted a stellation response during cell spreading. Extracts prepared from strains defective in AC toxin production showed no effect. Using image analysis to quantify the morphological change, we have demonstrated that the effect of AC toxin on cell spreading is dose dependent. This technique is a rapid and sensitive assay for the invasive AC toxin.

Genetic basis of attenuation of the Sabin type 3 oral poliovirus vaccine.

The poliovirus type 3 Sabin oral poliovirus vaccine strain P3/Leon/12a1b differs in nucleotide sequence from its neurovirulent progenitor P3/Leon/37 by just 10 point mutations. The contribution of each mutation to the attenuation phenotype of the vaccine strain was determined by the construction of a series of recombinant viruses from infectious cDNA clones. The neurovirulence testing of recombinant viruses indicated that the attenuation phenotype is determined by just two point mutations: a C to U in the noncoding region at position 472 and a C to U at nucleotide 2034 which results in a serine-to-phenylalanine amino acid substitution in the structural protein VP3.

Studies on the attenuation of the Sabin type 3 oral polio vaccine.

The genetic basis of attenuation of the poliovirus type 3 vaccine strain P3/Leon 12a1b has been investigated by comparing the nucleotide sequence of this strain with that of its neurovirulent progenitor P3/Leon/37 and by constructing recombinants between these two viruses using infectious cDNAs. Preliminary results suggest that attenuation is caused by just two point mutations, one occurring in the 5' non-coding region and the other causing an amino acid change in coat protein VP3.

Construction of poliovirus intertypic recombinants by use of cDNA.

We investigated the use of infectious cDNA for the production of poliovirus type 1-type 3 recombinants. One such recombinant virus was produced, but a second construct involving the transfer of part of the capsid protein region was not infectious. Our results suggest that the approach may prove valuable but that not all cDNA constructs will give rise to viable viruses.

New poliovirus vaccines: a molecular approach.

This article summarizes recent work on the determinants of antigenicity in poliovirus type 3 and reports on experiments in progress aimed at understanding the molecular basis of attenuation in Sabin's type 3 vaccines. Ways in which this new information might be used to produce alternative, safe, inexpensive, multivalent vaccines against polio and other enteroviruses are discussed.