Trypanosoma cruzi modulates lipid metabolism and highjacks phospholipids from the midgut of Rhodnius prolixus.

Chagas disease is potentially life-threatening and caused by the protozoan parasite Trypanosoma cruzi. The parasite cannot synthesize some lipids and depends on the uptake of these lipids from its vertebrate and invertebrate hosts. To achieve this, T. cruzi may need to modify the physiology of the insect host for its own benefit. In this study, we investigated the interaction of T. cruzi (Y strain) with its insect vector Rhodnius prolixus and how it manipulates the vector lipid metabolism. We observed a physiological change in lipid flux in of infected insects. In the fat body of infected insects, triacylglycerol levels decreased by 80.6% and lipid storage droplet-1(LSD-1) mRNA levels were lower, when compared to controls. Lipid sequestration by infected midguts led to increased levels of 5' AMP-activated protein kinase (AMPK) phosphorylation and activation in the fat body, inhibiting the synthesis of fatty acids and stimulating their oxidation. This led to reduced lipid levels in the fat body of infected insets, despite the fact that T. cruzi does not colonize this tissue. There was a 3-fold increase, in lipid uptake and synthesis in the midgut of infected insects. Finally, our results suggest that the parasite modifies the lipid flux and metabolism of its vector R. prolixus through the increase in lipid delivery from the fat body to midgut that are then scavenge by T cruzi.

Fluorescent membrane markers elucidate the association of Borrelia burgdorferi with tick cell lines.

This study aimed to describe the association of Borrelia burgdorferi s.s. with ixodid tick cell lines by flow cytometry and fluorescence and confocal microscopy. Spirochetes were stained with a fluorescent membrane marker (PKH67 or PKH26), inoculated into 8 different tick cell lines and incubated at 30°C for 24 h. PKH efficiently stained B. burgdorferi without affecting bacterial viability or motility. Among the tick cell lines tested, the Rhipicephalus appendiculatus cell line RA243 achieved the highest percentage of association/internalization, with both high (90%) and low (10%) concentrations of BSK-H medium in tick cell culture medium. Treatment with cytochalasin D dramatically reduced the average percentage of cells with internalized spirochetes, which passed through a dramatic morphological change during their internalization by the host cell as observed in time-lapse photography. Almost all of the fluorescent bacteria were seen to be inside the tick cells. PKH labeling of borreliae proved to be a reliable and valuable tool to analyze the association of spirochetes with host cells by flow cytometry, confocal and fluorescence microscopy.

Fluorescent membrane markers elucidate the association of Borrelia burgdorferi with tick cell lines

This study aimed to describe the association of Borrelia burgdorferi s.s. with ixodid tick cell lines by flow cytometry and fluorescence and confocal microscopy. Spirochetes were stained with a fluorescent membrane marker (PKH67 or PKH26), inoculated into 8 different tick cell lines and incubated at 30°C for 24 h. PKH efficiently stained B. burgdorferi without affecting bacterial viability or motility. Among the tick cell lines tested, the Rhipicephalus appendiculatus cell line RA243 achieved the highest percentage of association/internalization, with both high (90%) and low (10%) concentrations of BSK-H medium in tick cell culture medium. Treatment with cytochalasin D dramatically reduced the average percentage of cells with internalized spirochetes, which passed through a dramatic morphological change during their internalization by the host cell as observed in time-lapse photography. Almost all of the fluorescent bacteria were seen to be inside the tick cells. PKH labeling of borreliae proved to be a reliable and valuable tool to analyze the association of spirochetes with host cells by flow cytometry, confocal and fluorescence microscopy.

The role of lipoxygenase products on the endocytosis of yolk proteins in insects: participation of cAMP.

The participation of eicosanoids and second messengers in the regulation of endocytosis by the ovaries was investigated using the uptake of Rhodnius heme binding protein (RHBP) as an experimental model. The rate of RHBP uptake decreased up to 40% in the presence of BWA4C and NDGA, 5 and 12-lipoxygenase inhibitors, respectively, suggesting the involvement of lipoxygenase products in endocytosis regulation. Addition of Leukotriene B4 (LTB(4); one product of the 5 lipoxygenase pathway) increased in vitro the uptake of RHBP by 30%. The content of cAMP in the Rhodnius' ovaries were monitored after treatment with different eicosanoids and inhibitors of eicosanoids synthesis. The amount of cAMP decreased in the presence of indomethacin (by 50%), while treatment with PGE(2) induced an increase of 85% of this messenger in the ovaries. The presence of LTB(4) in the medium inhibited in 60% the content of cAMP in the ovaries, while BWA4C induced a 100% increase of this messenger in the ovaries. Addition of 1 microM DBcAMP in the medium resulted in a 30% decrease in the rate of RHBP uptake. Taken together, these data show that cyclooxygenase and lipoxygenase products participate in the control of protein internalization by modulation of cAMP levels.

HeLp, a heme lipoprotein from the hemolymph of the cattle tick, Boophilus microplus.

The main protein of the hemolymph of the cattle tick Boophilus microplus has been isolated and shown to be a heme lipoprotein (HeLp). HeLp has an apparent molecular mass of 354,000 and contains two apoproteins (103 and 92 kDa) found in equal amounts. HeLp presents a pI of 5.8 and a density of 1.28 g/ml and contains 33% lipids, containing both neutral lipids and phospholipids, and 3% of sugars. A remarkable feature of HeLp is the abundance of cholesterol ester (35% of total lipids), a lipid not previously reported in invertebrate lipoproteins. Western blot analysis showed HeLp in hemolymph from adult females and males, but not in eggs. Although HeLp contains 2 heme molecules, it is capable of binding 6 additional molecules of heme. Boophilus feeds large amount of blood, and we recently showed that this tick is unable to perform de novo synthesis of heme (Braz, G. R. C., Coelho, H. S. L., Masuda, H., and Oliveira, P. L. (1999) Curr. Biol. 9, 703-706). Injection of tick females with (55)Fe-labeled heme-HeLp indicated that this protein transports heme from hemolymph to tissues. HeLp is suggested to be an essential adaptation to the loss of the heme synthesis pathway.