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G H Coombs

Publications and source records attributed to G H Coombs.

At least 55 records · Page 3Linked to original sources
PubMed1998

Roles of cysteine proteinases of trypanosomes and Leishmania in host-parasite interactions.

Trypanosomes and Leishmania contain an abundance of stage-regulated cysteine proteinases encoded by several gene families. Analysis of parasites rendered defective in cysteine proteinase function, either through genetic manipulation or through the use of specific inhibitors, has revealed roles for the enzymes in parasite virulence, in modulation of the host's immune response and in parasite differentiation.

Animals↗
PubMed1997

The multiple cpb cysteine proteinase genes of Leishmania mexicana encode isoenzymes that differ in their stage regulation and substrate preferences.

The cpb genes of Leishmania mexicana encode stage-regulated, cathepsin L-like cysteine proteinases that are leishmanial virulence factors. Field inversion gel electrophoresis and genomic mapping indicate that there are 19 cpb genes arranged in a tandem array. Five genes from the array have been sequenced and their expression analyzed. The first two genes, cpb1 and cpb2, differ significantly from the remaining 17 copies (cpb3-cpb19) in that: 1) they are expressed predominantly in metacyclic promastigotes (the form in the insect vector which is infective to mammalian macrophages) rather than amastigotes (the form that parasitizes mammals); 2) they encode enzymes with a truncation in the COOH-terminal extension, an unusual feature of these cysteine proteinases of trypanosomatids. Transfection of cpb1 into a cpb null mutant resulted in expression of an active enzyme that was shown by immunogold labeling with anti-CPB antibodies to be targeted to large lysosomes. This demonstrates that the 100-amino acid COOH-terminal extension is not essential for the activation or activity of the enzyme or for its correct intracellular trafficking. Transfection into the cpb null mutant of different copies of cpb and analysis of the phenotype of the lines showed that individual isoenzymes differ in their substrate preferences and ability to restore the loss of virulence associated with the null mutant. Comparison of the predicted amino acid sequences of the isoenzymes implicates five residues located in the mature domain (Asn18, Asp60, Asn61, Ser64, and Tyr84) with differences in the activities of the encoded isoenzymes. The results suggest that the individual isoenzymes have distinct roles in the parasite's interaction with its host. This complexity reflects the adaptation of cathepsin L-like cysteine proteinases to diverse functions in parasitic protozoa.

Amino Acid Sequence↗
PubMed1997

Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis.

Designing cysteine proteinase inhibitors as antitrichomonal drugs requires knowledge of which cysteine proteinases are essential to the parasite. In an attempt to obtain such information, the effects of a number of cysteine proteinase inhibitors on trichomonad growth in vitro and proteinase activity were investigated. The broad specificity inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (known as E-64) had little effect on growth of Trichomonas vaginalis (27% inhibition at 280 microM, none at 28 microM) even though the addition of 2.8 microM E-64 to growth medium resulted in inhibition of all but two (apparent molecular masses: 35 k and 49 k) of the parasite's proteinases detected by gelatin SDS-PAGE. This shows that many of the parasite's cysteine proteinases are not essential for growth in axenic culture. In contrast, a peptidyl acyloxymethyl ketone, N-benzoyloxycarbonyl-Phe-Ala-CH2OCO-(2,6,-(CF3)2)Ph, at 16 microM killed T. vaginalis and severely inhibited growth of Tritrichomonas foetus. Exposure of Trichomonas vaginalis to 16 microM of this compound for 1 h resulted in both the 35 kDa and 49 kDa proteinases being inhibited, whereas some other proteinases were unaffected. Similar distinctions between the inhibitor sensitivity of the parasite's cysteine proteinases were apparent when a biotinylated peptidyl diazomethyl ketone was used to detect active proteinases. These data suggest that the growth inhibitory effects of the peptidyl acyloxymethyl ketone are through inhibition of cysteine proteinases that are not affected when the parasites are grown in the presence of E-64. At least one of these enzymes, which include the 35 kDa and 49 kDa cysteine proteinases, must be essential and so a suitable target for chemotherapeutic attack.

Animals↗
PubMed1997

Cathepsin B-like cysteine proteinase-deficient mutants of Leishmania mexicana.

Mutants null for the cathepsin B-like cysteine proteinase gene (cpc) of Leishmania mexicana have been generated by targeted gene disruption. The gene deletion was confirmed using a polymerase chain reaction (PCR) method with cpc-specific primers and genomic DNA isolated from the mutants. cpc was re-expressed in the null mutants from an episomal vector. Re-expression of the enzyme (CPC) was detected by Western blotting with a specific anti-peptide antiserum. The cpc null mutants grew apparently normally as promastigotes and amastigotes in axenic cultures, but they showed greatly reduced infectivity to macrophages in vitro with only a low percentage of the cells being infected. Re-expression of cpc in the null mutant increased the parasite's infectivity in vitro. The null mutant parasites formed lesions in mice at a similar rate as wild type parasites, although somewhat smaller lesions were produced. The results suggest that although the cysteine proteinase encoded by cpc plays a role in the parasite's interaction with macrophages it alone is not crucial for infectivity or virulence.

Amino Acid Sequence↗
PubMed1996

The use of a new viability assay to determine the susceptibility of Cryptosporidium and Eimeria sporozoites to respiratory inhibitors and extremes of pH.

A new viability assay for Cryptosporidium and Eimeria sporozoites is described. It involves the use of both acridine orange and bis-benzimide and is more rapid, easier and less subjective than procedures used previously. The assay has been used to investigate the effects of respiratory inhibitors and pH on the sporozoites of C. parvum, C. muris and E. tenella. Neither cyanide nor azide reduced the viability of C. parvum or E. tenella, whereas they had some effect on C. muris. This latter organism, an intracellular parasite of stomach epithelial cells, also differed from the other two in being able to survive pH 2 for as long as 1 h.

Acridine Orange↗
PubMed1996

Evidence from disruption of the lmcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors.

The mammalian form of the protozoan parasite Leishmania mexicana contains high activity of a cysteine proteinase (LmCPb) encoded on a tandem array of 19 genes (lmcpb). Homozygous null mutants for lmcpb have been produced by targeted gene disruption. All life-cycle stages of the mutant can be cultured in vitro, demonstrating that the gene is not essential for growth or differentiation of the parasite. However, the mutant exhibits a marked phenotype affecting virulence-- its infectivity to macrophages is reduced by 80%. The mutants are as efficient as wild-type parasites in invading macrophages but they only survive in a small proportion of the cells. However, those parasites that successfully infect these macrophages grow normally. Despite their reduced virulence, the mutants are still able to produce subcutaneous lesions in mice, albeit at a slower rate than wild-type parasites. The product of a single copy of lmcpb re-expressed in the null mutant was enzymatically active and restored infectivity toward macrophages to wild-type levels. Double null mutants created for lmcpb and lmcpa (another cathepsin L-like cysteine proteinase) have a similar phenotype to the lmcpb null mutant, showing that LmCPa does not compensate for the loss of LmCPb.

Animals↗
PubMed1996

Enzymes of energy metabolism in the bradyzoites and tachyzoites of Toxoplasma gondii.

The bradyzoite and tachyzoite forms of Toxoplasma gondii, purified from infected animals, were analysed for their activities of phosphofructokinase, pyruvate kinase, lactate dehydrogenase, NAD(+)- and NADH-linked isocitrate dehydrogenases, and succinic dehydrogenase. Both developmental stages contained high activities of phosphofructokinase (specific for pyrophosphate rather than ATP), pyruvate kinase and lactate dehydrogenase, suggesting that energy metabolism in both forms may centre around a high glycolytic flux linked to lactate production. The markedly higher activity of the latter two enzymes in bradyzoites suggests that lactate production is particularly important in this developmental form. NAD(+)-specific isocitrate dehydrogenase was not detectable in either stage of the parasite (and proved useful as a measure of the purity of the bradyzoite preparation), whereas both parasite forms contained low activities of NADP(+)-linked isocitrate dehydrogenase. The results are consistent with the bradyzoites lacking a functional TCA cycle and respiratory chain and are suggestive of a lack of susceptibility of this developmental stage to atovaquone.

Animals↗
PubMed1995

Amino acid consumption by the parasitic, amoeboid protists Entamoeba histolytica and E. invadens.

Amino acid consumption by Entamoeba histolytica and E. invadens has been measured in order to assess the possible roles of amino acids as energy substrates. Mixtures of amino acids enhanced the growth of the parasites in complex medium and their survival in simple medium. The consumption of several amino acids by the parasites suspended in simple media was greater when glucose was absent, suggesting that they may act as alternative energy sources. Under these conditions, asparagine was consumed extremely rapidly by E. histolytica in particular, and arginine, leucine and threonine were used greatly by both species. There was also a marked consumption of aspartate, but this occurred even when glucose was present. These five amino acids and phenylalanine were the ones consumed in greatest amounts during growth of E. histolytica in complex medium. Under the same growth conditions, E. invadens also used asparagine, arginine, leucine and threonine and in addition there was a large consumption of serine and especially glutamate. In contrast, the aspartate concentration in the complex medium increased and there was also a net increase in the concentration of some other amino acids. Alanine was produced by both species when the parasites were incubated in simple medium with glucose, and in greater amounts during growth in complex media, suggesting that it is an end product of energy metabolism. The findings provide support for the suggestion that energy generation through amino acid catabolism may be a characteristic feature of anaerobic parasitic protists.

Amino Acids↗
PubMed1995

Multiple protease activities in Giardia intestinalis trophozoites.

Azocasein and a wide range of chromogenic and fluorogenic peptidyl substrates, representing 21 different peptide sequences, were degraded by the intracellular proteases present in lysates of trophozoites of Giardia intestinalis (syn lamblia) Portland 1 strain. Total protease activity differed considerably with the substrate with the p-nitroanilide derivatives Bz-pro-phe-arg-pNA and Bz-phe-val-arg-pNA being most rapidly hydrolysed (specific activities 4.2 +/- 0.4 and 1.1 +/- 0.6 U/mg protein, respectively). Activities were increased (16-72%) by addition of 1 mM dithiothreitol and were maximal, for the substrates monitored, in the range pH 5.5-7.0. These data and the inhibitor susceptibilities of the trophozoite proteases confirmed that the activity was predominantly due to cysteine proteases, although the presence of some serine protease, aspartic protease and aminopeptidase activity in the lysates was also indicated. The multiplicity of the protease activities was confirmed by gelatin-polyacrylamide electrophoretic analysis. Eighteen proteolytic activities with Mr values in the range 30,000 to > 211,000 were detected. These gelatinase activities were enhanced by dithiothreitol, were maximal at or close to pH 6, and were inhibited by cysteine proteinase and some serine proteinase inhibitors. Four of the proteases present in the gels exhibited activity towards fluorogenic amidomethylcoumarin peptides, but with different substrate preferences. The results show that G. intestinalis contains multiple proteases, many of which are of the cysteine type.

Amino Acid Sequence↗
PubMed1995

The pathway of secretion of proteinases in Trichomonas vaginalis.

Trichomonads secrete large amounts of hydrolytic enzymes into liquid growth medium. Proteinase release by Trichomonas vaginalis has been quantified after resuspension of the parasite in a simple buffered maltose medium. After 6 h incubation, 70-90% of each of two cysteine proteinase activities, one towards benzyloxycarbonyl-arginyl-arginine 4-nitroanilide (Z-RR-Nan) and the other active towards N-benzoyl-prolyl-phenylalanyl-arginine 4-nitroanilide (Bz-PFR-Nan), was extracellular. This release was insensitive to changes in pH within the range from 5.5 to 8.6 but was partially inhibited by chloride ions. The secretion of activity towards Bz-PFR-Nan was temperature-sensitive but was still detectable down to 14 degrees C. Neither this nor other cysteine proteinase activities were detectable on the surface of parasites. Release was stimulated by various amines and monensin, suggesting that secretion was from or via acidic compartments. The intracellular activity towards Bz-PFR-Nan could be totally and irreversibly inhibited by treating the parasites with benzyloxycarbonyl-phenylalanyl-alanine diazomethylketone (Z-FA-DMK), without otherwise harming the cells. Regeneration and routing of the proteinases responsible for this activity was followed after removal of the inhibitor. There was a significant rise in the intracellular level of activity before it became detectable in the medium. The release of this activity was accelerated by amines and monensin, but the build-up of enzyme activity within cells was not prevented. Organelles containing cysteine proteinases banded as a single peak in Percoll density gradients. The density of these increased when cells were treated with dextran. The activity towards Bz-PFR-Nan which reappeared after Z-FA-DMK treatment has a similar distribution. The proteinase-containing fraction could be distinguished from an early (5 min) endosome fraction, suggesting that it was composed of late endosomes/lysosomes. Thus these results imply that the secretion pathway for proteinases necessarily involves lysosomes/late endosomes.

Amines↗
PubMed1995

Uptake of amino acids by the parasitic, flagellated protist Trichomonas vaginalis.

HPLC techniques have been applied to study amino acid uptake and release by Trichomonas vaginalis under a variety of conditions. Studies on the growth of T. vaginalis in complex media and the survival of the parasite in simple media, with and without amino acids and/or maltose, have shown that the growth or survival of T. vaginalis is better in the presence of maltose than when it is absent, and that greater amounts of amino acids are consumed by T. vaginalis in the absence of maltose. The results are consistent with several amino acids, notably arginine, threonine, leucine and methionine, being used by T. vaginalis as energy substrates. T. vaginalis released alanine and glycine into the culture media, the excretion being greater in the presence of maltose. These studies have provided new data on the uptake and release of amino acids by T. vaginalis and pave the way for detailed analysis of key enzymes and the regulation of the pathways involved.

Amino Acids↗
PubMed1995

Multiple cysteine proteinases of the pathogenic protozoon Tritrichomonas foetus: identification of seven diverse and differentially expressed genes.

The cattle protozoan parasite Tritrichomonas foetus has multiple forms of cysteine proteinases. To investigate their diversity, PCR and reverse transcriptase PCR were used to isolate genomic DNA and cDNA fragments, respectively, encoding different cysteine proteinases. Seven genes have been identified, TFCP3-6 from amplification of genomic DNA and TFCP7-9 from amplification of cDNA. Comparison of the predicted amino acid sequences indicates that the T. foetus enzymes are cathepsin-L-like rather than cathepsin-B-like in structure. However, there is considerable diversity among the proteinases. TFCP7 and TFCP8 are most similar to one another (78% identity), while TFCP3 and TFCP9 are the least closely related (30% identity). All but one of the genes are single-copy, the exception being TFCP3, which was present in multiple copies in one of the three isolates examined. Single transcripts were detected for each of the seven genes. TFCP8 was expressed at the highest levels, while transcripts for TFCP4 were only just detectable. In T. foetus F2, the strain from which the genomic DNA and mRNA were isolated, transcripts of the five other genes were present at intermediate levels. When two other isolates were compared with F2, differences in the expression of individual genes were apparent, with either one or two of them not expressed. In spite of these differences the major cysteine proteinases detected in the three isolates using substrate-SDS-PAGE appeared identical. The data show that the multiplicity of cysteine proteinases in T. foetus is due, in part at least, to the presence of multiple genes and that some of the genes encode cysteine proteinases which are not among the high-activity enzymes detected previously.

Amino Acid Sequence↗
PubMed1994

Analysis of the cysteine proteinases of Leishmania mexicana and Trypanosoma cruzi using specific antisera.

Antisera raised against papain and cysteine proteinases (CPs) purified from Leishmania mexicana and Trypanosoma cruzi have been used to study the proteins in the two parasites. The antisera against the major CP of T. cruzi (cruzipain) not only cross-reacted with known CPs of L. mexicana but also detected stage-specific molecules that may represent previously unrecognised CPs. The binding of the same antisera to extracts of different life cycle stages of T. cruzi suggested that the stages possess different isoforms of cruzipain. The lack of cross-reactivity of anti-papain antiserum against cruzipain suggests that the major immunogenic epitopes of these CPs are different, whereas the detection of the major CPs of L. mexicana with both heterologous antisera shows that the parasite's enzymes share epitopes with the other CPs.

Animals↗