Expanded repertoire of kinetoplast associated proteins and unique mitochondrial DNA arrangement of symbiont-bearing trypanosomatids.

In trypanosomatids, the kinetoplast is the portion of the single mitochondrion that is connected to the basal body and contains the kDNA, a network composed by circular and interlocked DNA. The kDNA packing is conducted by Kinetoplast Associated Proteins (KAPs), which are similar to eukaryotic histone H1. In symbiont-harboring trypanosomatids (SHTs) such as Angomonas deanei and Strigomonas culicis, a ß-proteobacterium co-evolves with the host in a mutualistic relationship. The prokaryote confers nutritional benefits to the host and affects its cell structure. Atomic force microscopy showed that the topology of isolated kDNA networks is quite similar in the two SHT species. Ultrastructural analysis using high-resolution microscopy techniques revealed that the DNA fibrils are more compact in the kinetoplast region that faces the basal body and that the presence of the symbiotic bacterium does not interfere with kDNA topology. However, RT-PCR data revealed differences in the expression of KAPs in wild-type protozoa as compared to aposymbiotic cells. Immunolocalization showed that different KAPs present distinct distributions that are coincident in symbiont-bearing and in symbiont-free cells. Although KAP4 and KAP7 are shared by all trypanosomatid species, the expanded repertoire of KAPs in SHTs can be used as phylogenetic markers to distinguish different genera.

Effects of miltefosine on the proliferation, ultrastructure, and phospholipid composition of Angomonas deanei, a trypanosomatid protozoan that harbors a symbiotic bacterium.

Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.

The effect of topoisomerase II inhibitors on the kinetoplast ultrastructure.

Topoisomerases from trypanosomatids play key functions in the replication and organization of kinetoplast DNA (kDNA). Hence, they are considered as potential targets for anti-parasite drugs. In this paper, the effect of topoisomerase II inhibitors, such as nalidixic acid, novobiocin and etoposide, on the ultrastructure of trypanosomatids that present distinct kDNA arrangements was evaluated. Prokaryotic topoisomerase II inhibitors were more effective on growth arrest and ultrastructure changes than etoposide, a eukaryotic topoisomerase II inhibitor. With the exception of novobiocin, drug concentrations which inhibited cell proliferation also promoted kinetoplast ultrastructure alterations, including the redistribution of topoisomerase II. The data reinforce the concept that prokaryotic topoisomerase II inhibitors may offer greater selectivity in drug therapy of trypanosomatid infections.