"Urate and NOX5 Control Blood Digestion in the Hematophagous Insect Rhodnius prolixus".

Low levels of reactive oxygen species (ROS) are now recognized as essential players in cell signaling. Here, we studied the role of two conserved enzymes involved in redox regulation that play a critical role in the control of ROS in the digestive physiology of a blood-sucking insect, the kissing bug Rhodnius prolixus. RNAi-mediated silencing of RpNOX5 and RpXDH induced early mortality in adult females after a blood meal. Recently, a role for RpNOX5 in gut motility was reported, and here, we show that midgut peristalsis is also under the control of RpXDH. Together with impaired peristalsis, silencing either genes impaired egg production and hemoglobin digestion, and decreased hemolymph urate titers. Ultrastructurally, the silencing of RpNOX5 or RpXDH affected midgut cells, changing the cells of blood-fed insects to a phenotype resembling the cells of unfed insects, suggesting that these genes work together in the control of blood digestion. Injection of either allopurinol (an XDH inhibitor) or uricase recapitulated the gene silencing effects, suggesting that urate itself is involved in the control of blood digestion. The silencing of each of these genes influenced the expression of the other gene in a complex way both in the unfed state and after a blood meal, revealing signaling crosstalk between them that influences redox metabolism and nitrogen excretion and plays a central role in the control of digestive physiology.

Starvation and pH stress conditions induced mitochondrial dysfunction, ROS production and autophagy in Trypanosoma cruzi epimastigotes.

Chagas disease is a neglected illness endemic in Latin America that mainly affects rural populations. The etiological agent of Chagas disease is the protozoan Trypanosoma cruzi, which has three different parasite stages and a dixenous life cycle that includes colonization of the vertebrate and invertebrate hosts. During its life cycle, T. cruzi is subjected to stress conditions, including variations in nutrient availability and pH, which impact parasite survival and differentiation. The plasticity of mitochondrial function in trypanosomatids has been defined as mitochondrial activity related to substrate availability. Thus, mitochondrial remodeling and autophagy, which is a constitutive cellular process of turnover and recycling of cellular components, may constitute a response to the nutritional and pH stress in the host. To assess these processes, epimastigotes were subjected to acidic, alkaline, and nutritional stress conditions, and mitochondrial function and its influence on the autophagic process were evaluated. Our data demonstrated that the three stress conditions affected the mitochondrial structure, inducing organelle swelling and impaired oxidative phosphorylation. Stressed epimastigotes produced increased ROS levels and overexpressed antioxidant enzymes. The stress conditions resulted in an increase in the number of autophagosomes and exacerbated the expression of different autophagy-related genes (Atgs). A correlation between mitochondrial dysfunction and autophagic phenotypes was also observed. After 24 h, acid stress and nutritional deprivation induced metacyclogenesis phenotypes (mitochondrial remodeling and autophagy). On the other hand, alkaline stress was transient due to insect blood feeding and culminated in an increase in autophagic flux as a survival mechanism.

Heme crystallization in a Chagas disease vector acts as a redox-protective mechanism to allow insect reproduction and parasite infection.

Heme crystallization as hemozoin represents the dominant mechanism of heme disposal in blood feeding triatomine insect vectors of the Chagas disease. The absence of drugs or vaccine for the Chagas disease causative agent, the parasite Trypanosoma cruzi, makes the control of vector population the best available strategy to limit disease spread. Although heme and redox homeostasis regulation is critical for both triatomine insects and T. cruzi, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization in vivo by the antimalarial drug quinidine, caused systemic heme overload and redox imbalance in distinct insect tissues, assessed by spectrophotometry and fluorescence microscopy. Quinidine treatment activated compensatory defensive heme-scavenging mechanisms to cope with excessive heme, as revealed by biochemical hemolymph analyses, and fat body gene expression. Importantly, egg production, oviposition, and total T. cruzi parasite counts in R. prolixus were significantly reduced by quinidine treatment. These effects were reverted by oral supplementation with the major insect antioxidant urate. Altogether, these data underscore the importance of heme crystallization as the main redox regulator for triatomine vectors, indicating the dual role of hemozoin as a protective mechanism to allow insect fertility, and T. cruzi life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and T. cruzi transmission.

Detergent-Mediated Formation of ß-Hematin: Heme Crystallization Promoted by Detergents Implicates Nanostructure Formation for Use as a Biological Mimic.

Hemozoin is a unique biomineral that results from the sequestration of toxic free heme liberated as a consequence of hemoglobin degradation in the malaria parasite. Synthetic neutral lipid droplets (SNLDs) and phospholipids were previously shown to support the rapid formation of ß-hematin, abiological hemozoin, under physiologically relevant pH and temperature, though the mechanism by which heme crystallization occurs remains unclear. Detergents are particularly interesting as a template because they are amphiphilic molecules that spontaneously organize into nanostructures and have been previously shown to mediate ß-hematin formation. Here, 11 detergents were investigated to elucidate the physicochemical properties that best recapitulate crystal formation in the parasite. A strong correlation between the detergent's molecular structure and the corresponding kinetics of ß-hematin formation was observed, where higher molecular weight polar chains promoted faster reactions. The larger hydrophilic chains correlated to the detergent's ability to rapidly sequester heme into the lipophilic core, allowing for crystal nucleation to occur. The data presented here suggest that detergent nanostructures promote ß-hematin formation in a similar manner to SNLDs and phospholipids. Through understanding mediator properties that promote optimal crystal formation, we are able to establish an in vitro assay to probe this drug target pathway.

Unsaturated glycerophospholipids mediate heme crystallization: biological implications for hemozoin formation in the kissing bug Rhodnius prolixus.

Hemozoin (Hz) is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM). Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML) in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient ß-hematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE) and phosphatidylcholine (uPC), with half-lives of 0.04 and 0.7 minutes, respectively. ß-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to ß-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9-17.7 minutes) than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, ß-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.

On the mechanisms involved in biological heme crystallization.

Blood-feeding organisms digest hemoglobin, releasing large quantities of heme inside their digestive tracts. Free heme is very toxic, and these organisms have evolved several mechanisms to protect against its deleterious effects. One of these adaptations is the crystallization of heme into the dark-brown pigment hemozoin (Hz). Here we review the process of Hz formation, focusing on organisms other than Plasmodium that have contributed to a better understanding of heme crystallization. Hemozoin has been found in several distinct classes of organisms including protozoa, helminths and insects and Hz formation is the predominant form of heme detoxification. The available evidence indicates that amphiphilic structures such as phospholipid membranes and lipid droplets accompanied by specific proteins play a major role in heme crystallization. Because this process is specific to a number of blood-feeding organisms and absent in their hosts, Hz formation is an attractive target for the development of novel drugs to control illnesses associated with these hematophagous organisms.

Increase on the initial soluble heme levels in acidic conditions is an important mechanism for spontaneous heme crystallization in vitro.

BACKGROUND: Hemozoin (Hz) is a heme crystal that represents a vital pathway for heme disposal in several blood-feeding organisms. Recent evidence demonstrated that ß-hematin (ßH) (the synthetic counterpart of Hz) formation occurs under physiological conditions near synthetic or biological hydrophilic-hydrophobic interfaces. This seems to require a heme dimer acting as a precursor of Hz crystals that would be formed spontaneously in the absence of the competing water molecules bound to the heme iron. Here, we aimed to investigate the role of medium polarity on spontaneous ßH formation in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the effect of water content on spontaneous ßH formation by using the aprotic solvent dimethylsulfoxide (DMSO) and a series of polyethyleneglycols (PEGs). We observed that both DMSO and PEGs (3.350, 6.000, 8.000, and 22.000) increased the levels of soluble heme under acidic conditions. These compounds were able to stimulate the production of ßH crystals in the absence of any biological sample. Interestingly, the effects of DMSO and PEGs on ßH formation were positively correlated with their capacity to promote previous heme solubilization in acidic conditions. Curiously, a short chain polyethyleneglycol (PEG 300) caused a significant reduction in both soluble heme levels and ßH formation. Finally, both heme solubilization and ßH formation strongly correlated with reduced medium water activity provided by increased DMSO concentrations. CONCLUSIONS: The data presented here support the notion that reduction of the water activity is an important mechanism to support spontaneous heme crystallization, which depends on the previous increase of soluble heme levels.

On the physico-chemical and physiological requirements of hemozoin formation promoted by perimicrovillar membranes in Rhodnius prolixus midgut.

Triatomine insects are obligatory blood-feeders that detoxify most of the hemoglobin-derived heme through its crystallization into hemozoin (Hz). Previous evidence demonstrates the key role of midgut perimicrovillar membranes (PMVM) on heme crystallization in triatomines. Here, we investigated some of the physico-chemical and physiological aspects of heme crystallization induced by Rhodnius prolixus PMVM. Hz formation in vitro proceeded optimally at pH 4.8 and 28 degrees C, apparently involving three kinetically distinct mechanisms along this process. Furthermore, the insect feeding status and age affected PMVM-induced heme crystallization whereas pharmacological blockage of PMVM formation by azadirachtin, reduced hemoglobin digestion and Hz formation in vivo. Mössbauer spectrometry analyses of R. prolixus midgut showed that Hz represents the only measurable iron species found four days after a blood meal. Autocatalytic heme crystallization to Hz is revealed to be an inefficient process and this conversion is further reduced as the Hz concentration increases. Also, PMVM-derived lipids were able to induce rapid Hz formation, regardless of the diet composition. These results indicate that PMVM-driven Hz formation in R. prolixus midgut occurs at physiologically relevant physico-chemical conditions and that lipids derived from this structure play an important role in heme crystallization.

Effects of retinoids and juvenoids on moult and on phenoloxidase activity in the blood-sucking insect Rhodnius prolixus.

Retinoic acid and insect juvenile hormone (JH) are structurally related terpenoids which are widespread in nature and are involved in many biological events such as morphogenesis, embryogenesis and cellular differentiation. Here, we investigated the effects of the retinoids 9-cis retinoic acid (9cisRA), all trans retinol (atROH), all trans retinoic acid (atRA) and the juvenoids methoprene (Met) and JH injection on moult and on phenoloxidase activity in the blood-sucking insect Rhodnius prolixus. Overall, we observed that injection of retinoids or juvenoids (120 pmols) in the hemocoel of 4th instar nymphs reduced the percentage of insects which appeared normal in morphology upon moult. Noteworthy, insects exposed to 9cisRA or JH underwent profound morphological changes upon moult, generating abnormal 5th instar nymphs and also markedly increased the death of insects during the moulting process. In addition, reduction in the percentage of insects that moult without any morphological alteration, induced by retinoids or juvenoids treatment, was negatively correlated with insects that both display abnormal moult and those that die during moult. Hemolymphatic phenoloxidase activity in adult male insects injected with 9cisRA, Met and JH were significantly reduced after a bacterial challenge. Together, these results indicate that not only juvenoids but also retinoids play an important role on morphogenesis and on immune response in R. prolixus, suggesting that the molecular mechanisms involved in these events recognize the terpenoid backbone as an important structural determinant in insects.