Juan Venegas

Identification and characterization of potentially algal-lytic marine bacteria strongly associated with the toxic dinoflagellate Alexandrium catenella.

Abstract. The toxic dinoflagellate Alexandrium catenella isolated from fjords in Southern Chile produces several analogues of saxitoxin and has been associated with outbreaks of paralytic shellfish poisoning. Three bacterial strains, which remained in close association with this dinoflagellate in culture, were isolated by inoculating the dinoflagellate onto marine agar. The phenotypically different cultivable bacterial colonies were purified. Their genetic identification was done by polymerase chain reaction amplification of the 16S rRNA genes. Partial sequence analysis suggested that the most probable affiliations were to two bacterial phyla: Proteobacteria and the Cytophaga group. The molecular identification was complemented by morphological data and biochemical profiling. The three bacterial species, when grown separately from phytoplankton cells in high‐nutrient media, released algal‐lytic compounds together with aminopeptidase, lipase, glucosaminidase, and alkaline phosphatase. When the same bacteria, free of organic nutrients, were added back to the algal culture they displayed no detrimental effects on the dinoflagellate cells and recovered their symbiotic characteristics. This observation is consistent with phylogenetic analysis that reveals that these bacteria correspond to species distinct from other bacterial strains previously classified as algicidal bacteria. Thus, bacterial‐derived lytic activities are expressed only in the presence of high‐nutrient culture media and it is likely that in situ environmental conditions may modulate their expression.

Differential distribution of Trypanosoma cruzi clones in human chronic chagasic cardiopathic and non-cardiopathic individuals.

PCR and Southern blot hybridization were used to determine the distribution of Trypanosoma cruzi clones in 37 chronic chagasic cardiopathic and non-cardiopathic patients. Parasite DNA amplified from peripheral blood or dejections of Triatoma infestans fed on patient blood was hybridized with probes containing hypervariable minicircle nucleotide sequences capable of detecting three sublineages of T. cruzi. Probes Z-I and Z-IIb detect unique sequences in lineages TcI and TcIIb, respectively. Probe Z-hybrid detects sequences of lineages TcIId and TcIIe. T. cruzi clones of the Z-I sublineage were detected in 62.2% of T. infestans dejections and 5.4% of peripheral blood samples. Clones of Z-IIb and Z-hybrid sublineages had similar distribution in blood and dejection samples. Interestingly, clones of the Z-IIb sublineage were significantly lower in cardiopathic than in non-cardiopathic patients (23.5% versus 75%; P=0.0006). Clones of the Z-hybrid sublineage were found in 29.4% of cardiopathic and 75% of non-cardiopathic patients, respectively (P=0.0051). By contrast, clones of sublineage Z-I were similarly distributed in both groups of patients. The low frequency of Z-IIb and Z-hybrid sublineage clones detected in cardiopathic patients suggests that the immunological mechanisms involved in controlling and eliminating these T. cruzi parasites may be detrimental to the host, leading to the development of chagasic cardiomyopathy.

Phylogenetic analysis of microsatellite markers further supports the two hybridization events hypothesis as the origin of the Trypanosoma cruzi lineages

To better understand the evolution of the etiologic agent of Chagas disease, we cloned and sequenced 25 alleles from five Tripanosoma cruzi microsatellite markers. The study of the sequences showed highly conserved alleles present in T. cruzi clones belonging to TCI, TCIIc, and TCIIe. This result was also confirmed by the phylogenetic analysis of MCLE01 allele sequences. The examination by capillary electrophoresis of six microsatellite markers from 19 T. cruzi clones showed a high proportion of the alleles found both in the TCI and TCII sublineages. The phylogenetic reconstruction of these 19 clones produced a tree with two major clusters with bootstrap support of 100% and 95%. The first cluster includes T. cruzi clones belonging to the TCI and TCIIa lineages. The second cluster is composed of TCI, TCIIc, TCIId, and TCIIe T. cruzi clones. The analysis of five microsatellite markers in the CLBrener genome showed that almost all the microsatellite markers are synteny; non-Esmeraldo and Esmeraldo haplotypes probably come from the TCIIc and TCIIb lineages. Taken together, our results are in agreement with the two hybridization events hypothesis as the origin of current T. cruzi lineages.

Two DNA polymerases from Trypanosoma cruzi: biochemical characterization and effects of inhibitors

1. Two DNA polymerases have been partially purified from Trypanosoma cruzi epimastigotes by DEAE-cellulose, phosphocellulose, and DNA agarose chromatography. 2. Both enzyme activities were characterized by several biochemical criteria. 3. They showed different sensitivity to KCl and displayed characteristic Mg2+ and Mn2+ requirements, although they exhibited almost identical primer-template utilization. 4. The preferred substrates were poly dC-oligo dG, activated calf thymus DNA, and poly dT-oligo rA. 5. Both enzyme fractions are not inhibited by aphidicolin while N-ethylmaleimide and phosphonacetic acid inhibited them to different extents. 6. ButylphenyldGTP strongly inhibited T. cruzi enzyme fraction I while it had no effect on enzyme fraction III. 7. This dGTP analog also inhibited the poly dT-directed polymerization of dAMP as described for other mammalian DNA polymerases. Kinetic studies indicated that butylphenyldGTP inhibited enzyme fraction I in a non-competitive fashion.

Detection of the G3 genotype of Echinococcus granulosus from hydatid cysts of Chilean cattle using COX1and ND1mitochondrial markers

For a deeper understanding of the phylogenetic relationships of Echinococcus genotypes and species in different intermediate hosts, we analyzed samples from human and bovine hydatid cysts. For this, segments of the cytochrome oxidase (COX1) and NADH dehydrogenase (ND1) mitochondrial genes were used. To obtain sufficient amounts of the ND1 marker to be sequenced properly, a new variant of the PCR assay was implemented. Phylogenetic analysis with both markers showed that most of the analyzed samples correspond to genotype G1. However, a sample from cysts of a bovine lung (Q21), with the COX1 marker, was grouped in a node together with a sample belonging to genotype G3. In the phylogenetic tree obtained with the ND1 marker, this sample was grouped with sequences of genotypes G3, G2, and G4. Analyzing the single nucleotide polymorphic (SNP) sites of both markers, it was observed that the Q21 sequence is almost identical to the G3 sequence and differ in only one SNP from the G2 sequence, and is completely different from G4. These results are noteworthy, since neither G2 nor G3 genotypes have been described previously in Chile, raising the possibility that the G3 genotype is present in these latitudes. This information is highly relevant; it can be employed to uncover additional unknown details of transmission cycles of this important parasite.

Purification and characterization of a β-like DNA polymerase from Trypanosoma cruzi

A DNA polymerase was purified to near homogeneity from Trypanosoma cruzi epimastigotes. This preparation had a major polypeptide of 50 kDa and a minor band of 45 kDa. SDS-PAGE studies and a novel colorimetric activity gel technique demonstrated that the 50-kDa polypeptide chain is the catalytic subunit of this T. cruzi DNA polymerase. Western blot analysis of different purification stage fractions strongly suggests that this 50-kDa protein is the intact catalytic subunit and does not correspond to a degradation product from a larger one. This T. cruzi DNA polymerase is insensitive to aphidicolin, butylphenyldeoxyguanosine triphosphate, berenil, ethidium bromide and N-ethylmaleimide, but is markedly inhibited by the dideoxythymidine triphosphate analogue. Studies with different DNA templates showed that the DNA polymerase prefers activated DNA as substrate and that it cannot elongate oligoriboadenylate primers. The data presented in this paper are consistent with the hypothesis that this enzyme corresponds to a beta-like DNA polymerase present in the parasitic protozoon T. cruzi.

Microsatellite marker analysis shows differentiation among Trypanosoma cruzi populations of peripheral blood and dejections of Triatoma infestans fed on the same chronic chagasic patients: Microsatellite marker analysis and T. cruzi

To investigate whether Trypanosoma cruzi populations found in chagasic cardiopathic and non-cardiopathic patients are genetically differentiated, three molecular microsatellite markers were analysed. This analysis was also applied to compare T. cruzi samples from peripheral blood or dejections of Triatoma infestans fed on the blood of the same patients. In order to obtain the first objective, analyses of predominant T. cruzi genotypes were conducted using three approaches: a locus-by-locus analysis; a Fisher method across three loci; and analysis of molecular variance by Genepop and Arlequin programs. Only with one locus and on the blood samples was a significant differentiation detected among non-cardiopathic and cardiopathic groups, which was not confirmed by the other two methods. On the contrary, with the three approaches, it was found that T. cruzi clones present in the blood of patients are genetically differentiated from those detected in dejections of T. infestans fed on the same patients. Our results showed that the most frequent lineage both in blood as well as in triatomine dejection samples was TcI. No significant difference in T. cruzi lineage distribution was observed among chagasic cardiopathic and non-cardiopathic patients. The majority of the samples (50–60%) had only one T. cruzi clone (uniclonal) either in blood or dejection samples.

Geographical structuring of Trypanosoma cruzi populations from Chilean Triatoma infestans triatomines and their genetic relationship with other Latino American counterparts.

Abstract In order to obtain more information about the population structure of Chilean Trypanosoma cruzi, and their genetic relationship with other Latino American counterparts, we performed the study of T. cruzi samples detected in the midgut content of Triatoma infestans insects from three endemic regions of Chile. The genetic characteristics of these samples were analysed using microsatellite markers and PCR conditions that allow the detection of predominant T. cruzi clones directly in triatomine midgut content. Population genetic analyses using the Fisher’s exact method, analysis of molecular variance (AMOVA) and the determination of FST showed that the northern T. cruzi population sample was genetically differentiated from the two southern population counterparts. Further analysis showed that the cause of this genetic differentiation was the asymmetrical distribution of TcIII T. cruzi predominant clones. Considering all triatomines from the three regions, the most frequent predominant lineages were TcIII (38%), followed by TcI (34%) and hybrid (8%). No TcII lineage was observed along the predominant T. cruzi clones. The best phylogenetic reconstruction using the shared allelic genetic distance was concordant with the population genetic analysis and tree topology previously described studying foreign samples. The correlation studies showed that the lineage TcIII from the III region was genetically differentiated from the other two, and this differentiation was correlated with geographical distance including Chilean and mainly Brazilian samples. It will be interesting to investigate whether this geographical structure may be related with different clinical manifestation of Chagas disease.

Isolation and partial characterization of three DNA polymerases from Trypanosoma cruzi

Three distinct DNA polymerase fractions (A, B and C), were isolated from Trypanosoma cruzi epimastigote forms. Fraction A is a low molecular mass enzyme corresponding to beta-like DNA polymerase of T. cruzi. Fraction B co-purified along several purification steps with fraction A, but in the last step it was clearly separated by a phosphocellulose chromatography. Fraction C was separated from fractions A and B by binding to DEAE-cellulose column, since the other two fractions were eluted in the flowthrough. This enzyme has an apparent native molecular mass of 100 kDa and showed a high preference for poly(dC)-oligo(dG) among different template-primers tested as substrate. Western-blot and biochemical analysis strongly suggest that the three DNA polymerase fractions correspond to different molecular entities. These results are in agreement with the idea that fraction C is a new DNA polymerase of T. cruzi, not described before.

Microsatellite loci-based distribution of Trypanosoma cruzi genotypes from Chilean chronic Chagas disease patients and Triatoma infestans is concordant with a specific host-parasite association hypothesis.

The objective of this study was to investigate if there is specific host-parasite association in Chilean populations of Trypanosoma cruzi. For this purpose, two groups of parasites were analyzed, one from chronic chagasic patients, and the other from Triatoma infestans triatomines in three regions of the country. The first group consisted of four types of samples: parasites from peripheral blood of non-cardiopathic T. cruzi infected patients (NB); parasites from their corresponding xenodiagnosis (NX); parasites from peripheral blood of T. cruzi infected cardiopathic patients (CB) and parasites from their xenodiagnostics (CX). The T. infestans sample in turn was from three regions: III, V and M (Metropolitan). The genetic differentiation by the Fisher exact method, the lineage distribution of the samples, the molecular phylogeny and the frequency of multiclonality were analysed. The results show that not only are the groups of T. cruzi clones from Chagas disease patients and vectors genetically differentiated, but also all the sub-groups (NB, NX, CB and CX) from the III, V and M regions. The analysis of lineage distribution was concordant with the above results, because significant differences among the percentages of TcI, TcIII and hybrids (TcV or TcVI) were observed. The phylogenetic reconstruction with these Chilean T. cruzi samples was coherent with the above results because the four chagasic samples clustered together in a node with high bootstrap support, whereas the three triatomine samples (III, V and M) were located apart from that node. The topology of the tree including published T. cruzi clones and isolates was concordant with the known topology, which confirmed that the results presented here are correct and are not biased by experimental error. Taken together the results presented here are concordant with a specific host-parasite association between some Chilean T. cruzi populations.