Fernando A. Genta

Interactions between intestinal compounds of triatomines and Trypanosoma cruzi

Triatomine bugs are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease, a devastating disease that disables and leads to the death of many people in Latin America. In this review, factors from the insect vector are described, including digestive enzymes, hemolysins, agglutinins, microbiota and especially antimicrobial factors, which are potentially involved in regulating the development of T. cruzi in the gut. Differential regulation of parasite populations shows that some triatomine defense reactions discriminate not only between molecular signals specific for trypanosome infections but also between different strains of T. cruzi.

Action pattern, specificity, lytic activities, and physiological role of five digestive β-glucanases isolated from Periplaneta americana

Three laminarinases (LAM, LIC 1, and LIC 2) and two cellulases (CEL 1 and CEL 2) were purified to homogeneity from Periplaneta americana midguts. These beta-glucanases are secreted by salivary glands, stabilized by calcium ions, and have pH optima around 6. LAM (46 kDa) is active only on laminarin, native or with oxidized ends, and so it is an endo-beta-1,3-glucanase (EC 3.2.1.39). It processively releases mainly glucose from laminarin and shows lytic activity on fungal cells. LIC 1 (25 kDa) is an endo-beta-1,3(4)-glucanase (EC 3.2.1.6.), because it cleaves internal bonds on both laminarin and lichenin. LIC 1 lyses fungal cells and apparently have high affinity for sequences of cellotetraoses linked by beta-1,3 links, releasing cellotetraose from lichenin. The reaction catalyzed by LIC 1 is not in rapid equilibrium, as suggested by activity-pH data. These data also showed that a group in LIC 1 with pK=4.9 is necessary for substrate binding. LIC 2 (23 kDa) seems to be similar to LIC 1. The laminarinases are inactivated by carbodiimide, suggesting the presence of a carboxyl group involved in catalysis. LAM and LIC 2 are inhibited by excess laminarin as substrate. CEL 1 (72 kDa) and CEL 2 (73 kDa) quickly decrease the molecular weight of lichenin used as substrate. Therefore, they are endo-beta-1,4-glucanases (EC 3.2.1.4). Both CEL 1 and CEL 2 are also active on crystalline cellulose. The specificities of P. americana beta-glucanases agree with the omnivorous detritus-feeding habit of this insect, as they are able to attack plant (CEL 1, CEL 2, LIC 1 and LIC 2) and fungal (LIC 1 and LAM) cell walls.

Reactive Oxygen Species-mediated Immunity against Leishmania mexicana and Serratia marcescens in the Phlebotomine Sand Fly Lutzomyia longipalpis

Background: Reactive oxygen species are part of the sand fly innate immune system. Results: ROS production in the gut increases in response to a bacterial pathogen but not to Leishmania. Conclusion: Sand flies tolerate the presence of Leishmania by differential response of the ROS system. Significance: The successful use of sand flies as vehicles for Leishmania transmission relies partially on the parasite circumventing the ROS immune response. Phlebotomine sand flies are the vectors of medically important Leishmania. The Leishmania protozoa reside in the sand fly gut, but the nature of the immune response to the presence of Leishmania is unknown. Reactive oxygen species (ROS) are a major component of insect innate immune pathways regulating gut-microbe homeostasis. Here we show that the concentration of ROS increased in sand fly midguts after they fed on the insect pathogen Serratia marcescens but not after feeding on the Leishmania that uses the sand fly as a vector. Moreover, the Leishmania is sensitive to ROS either by oral administration of ROS to the infected fly or by silencing a gene that expresses a sand fly ROS-scavenging enzyme. Finally, the treatment of sand flies with an exogenous ROS scavenger (uric acid) altered the gut microbial homeostasis, led to an increased commensal gut microbiota, and reduced insect survival after oral infection with S. marcescens. Our study demonstrates a differential response of the sand fly ROS system to gut microbiota, an insect pathogen, and the Leishmania that utilize the sand fly as a vehicle for transmission between mammalian hosts.

Identification, molecular cloning and functional characterization of an octaprenyl pyrophosphate synthase in intra-erythrocytic stages of Plasmodium falciparum

Isoprenoids play important roles in all living organisms as components of structural cholesterol, steroid hormones in mammals, carotenoids in plants, and ubiquinones. Significant differences occur in the length of the isoprenic side chains of ubiquinone between different organisms, suggesting that different enzymes are involved in the synthesis of these side chains. Whereas in Plasmodium falciparum the isoprenic side chains of ubiquinone contain 7-9 isoprenic units, 10-unit side chains are found in humans. In a search for the P. falciparum enzyme responsible for the biosynthesis of isoprenic side chains attached to the benzoquinone ring of ubiquinones, we cloned and expressed a putative polyprenyl synthase. Polyclonal antibodies raised against the corresponding recombinant protein confirmed the presence of the native protein in trophozoite and schizont stages of P. falciparum. The recombinant protein, as well as P. falciparum extracts, showed an octaprenyl pyrophosphate synthase activity, with the formation of a polyisoprenoid with eight isoprenic units, as detected by reverse-phase HPLC and reverse-phase TLC, and confirmed by electrospray ionization and tandem MS analysis. The recombinant and native versions of the enzyme had similar Michaelis constants with the substrates isopentenyl pyrophosphate and farnesyl pyrophosphate. The recombinant enzyme could be competitively inhibited in the presence of the terpene nerolidol. This is the first report that directly demonstrates an octaprenyl pyrophosphate synthase activity in parasitic protozoa. Given the rather low similarity of the P. falciparum enzyme to its human counterpart, decaprenyl pyrophosphate synthase, we suggest that the identified enzyme and its recombinant version could be exploited in the screening of novel drugs.

Colonisation resistance in the sand fly gut: Leishmania protects Lutzomyia longipalpis from bacterial infection

BackgroundPhlebotomine sand flies transmit the haemoflagellate Leishmania, the causative agent of human leishmaniasis. The Leishmania promastigotes are confined to the gut lumen and are exposed to the gut microbiota within female sand flies. Here we study the colonisation resistance of yeast and bacteria in preventing the establishment of a Leishmania population in sand flies and the ability of Leishmania to provide colonisation resistance towards the insect bacterial pathogen Serratia marcescens that is also pathogenic towards Leishmania.MethodsWe isolated microorganisms from wild-caught and laboratory-reared female Lutzomyia longipalpis, identified as Pseudozyma sp. Asaia sp. and Ochrobactrum intermedium. We fed the females with a sugar meal containing the microorganisms and then subsequently fed them with a bloodmeal containing Leishmania mexicana and recorded the development of the Leishmania population. Further experiments examined the effect of first colonising the sand fly gut with L. mexicana followed by feeding with, Serratia marcescens, an insect bacterial pathogen. The mortality of the flies due to S. marcescens was recorded in the presence and absence of Leishmania.ResultsThere was a reduction in the number of flies harbouring a Leishmania population that had been pre-fed with Pseudozyma sp. and Asaia sp. or O. intermedium. Experiments in which L. mexicana colonised the sand fly gut prior to being fed an insect bacterial pathogen, Serratia marcescens, showed that the survival of flies with a Leishmania infection was significantly higher compared to flies without Leishmania infection.ConclusionsThe yeast and bacterial colonisation experiments show that the presence of sand fly gut microorganisms reduce the potential for Leishmania to establish within the sand fly vector. Sand flies infected with Leishmania were able to survive an attack by the bacterial pathogen that would have killed the insect and we concluded that Leishmania may benefit its insect host whilst increasing the potential to establish itself in the sand fly vector. We suggest that the increased ability of the sand fly to withstand a bacterial entomopathogen, due to the presence of the Leishmania, may provide an evolutionary pressure for the maintenance of the Leishmania-vector association.

Reactive Oxygen Species Scavenging by Catalase Is Important for Female Lutzomyia longipalpis Fecundity and Mortality

The phlebotomine sand fly Lutzomyia longipalpis is the most important vector of American visceral leishmaniasis (AVL), the disseminated and most serious form of the disease in Central and South America. In the natural environment, most female L. longipalpis are thought to survive for less than 10 days and will feed on blood only once or twice during their lifetime. Successful transmission of parasites occurs when a Leishmania-infected female sand fly feeds on a new host. Knowledge of factors affecting sand fly longevity that lead to a reduction in lifespan could result in a decrease in parasite transmission. Catalase has been found to play a major role in survival and fecundity in many insect species. It is a strong antioxidant enzyme that breaks down toxic reactive oxygen species (ROS). Ovarian catalase was found to accumulate in the developing sand fly oocyte from 12 to 48 hours after blood feeding. Catalase expression in ovaries as well as oocyte numbers was found to decrease with age. This reduction was not found in flies when fed on the antioxidant ascorbic acid in the sugar meal, a condition that increased mortality and activation of the prophenoloxidase cascade. RNA interference was used to silence catalase gene expression in female Lu. longipalpis. Depletion of catalase led to a significant increase of mortality and a reduction in the number of developing oocytes produced after blood feeding. These results demonstrate the central role that catalase and ROS play in the longevity and fecundity of phlebotomine sand flies.

Trypanosoma rangeli: a new perspective for studying the modulation of immune reactions of Rhodnius prolixus

Insects are exposed to a wide range of microorganisms (bacteria, fungi, parasites and viruses) and have interconnected powerful immune reactions. Although insects lack an acquired immune system they have well-developed innate immune defences that allow a general and rapid response to infectious agents.Over the last few decades we have observed a dramatic increase in the knowledge of insect innate immunity, which relies on both humoral and cellular responses. However, innate reactions to natural insect pathogens and insect-transmitted pathogens, such as parasites, still remain poorly understood.In this review, we briefly introduce the general immune system of insects and highlight our current knowledge of these reactions focusing on the interactions of Trypanosoma rangeli with Rhodnius prolixus, an important model for innate immunity investigation.

Purification, characterization and sequencing of the major β-1,3-glucanase from the midgut of Tenebrio molitor larvae

The major beta-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an endoglucanase that hydrolyzes beta-1,3-glucans as laminarin and yeast beta-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched beta-1,3-glucans or mixed beta-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections.

Lipid mediators and vector infection: Trypanosoma rangeli inhibits Rhodnius prolixus hemocyte phagocytosis by modulation of phospholipase A2 and PAF-acetylhydrolase activities

In this work we investigated the effects of Trypanosoma rangeli infection through a blood meal on the hemocyte phagocytosis in experiments using the 5th instar larvae of Rhodnius prolixus. Hemocyte phagocytic activity was strongly blocked by oral infection with the parasites. In contrast, hemocyte phagocytosis inhibition caused by T. rangeli infection was rescued by exogenous arachidonic acid (20 microg/insect) or platelet activating factor (PAF; 1 microg/insect) applied by hemocelic injection. Following the oral infection with the protozoan we observed significant attenuation of phospholipase A2 (PLA2) activities in R. prolixus hemocytes (cytosolic PLA2: cPLA2, secreted PLA2: sPLA2 and Ca+2-independent PLA2: iPLA2) and enhancement of sPLA2 activities in cell-free hemolymph. At the same time, the PAF-acetyl hydrolase (PAF-AH) activity in the cell-free hemolymph increased considerably. Our results suggest that T. rangeli infection depresses eicosanoid and insect PAF analogous (iPAF) pathways giving support to the role of PLA2 in the regulation of arachidonic acid and iPAF biosynthesis and of PAF-AH by reducing the concentration of iPAF in R. prolixus. This illustrates the ability of T. rangeli to modulate the immune responses of R. prolixus to favor its own multiplication in the hemolymph.

High throughput screening of hydrolytic enzymes from termites using a natural substrate derived from sugarcane bagasse

BackgroundThe description of new hydrolytic enzymes is an important step in the development of techniques which use lignocellulosic materials as a starting point for fuel production. Sugarcane bagasse, which is subjected to pre-treatment, hydrolysis and fermentation for the production of ethanol in several test refineries, is the most promising source of raw material for the production of second generation renewable fuels in Brazil. One problem when screening hydrolytic activities is that the activity against commercial substrates, such as carboxymethylcellulose, does not always correspond to the activity against the natural lignocellulosic material. Besides that, the macroscopic characteristics of the raw material, such as insolubility and heterogeneity, hinder its use for high throughput screenings.ResultsIn this paper, we present the preparation of a colloidal suspension of particles obtained from sugarcane bagasse, with minimal chemical change in the lignocellulosic material, and demonstrate its use for high throughput assays of hydrolases using Brazilian termites as the screened organisms.ConclusionsImportant differences between the use of the natural substrate and commercial cellulase substrates, such as carboxymethylcellulose or crystalline cellulose, were observed. This suggests that wood feeding termites, in contrast to litter feeding termites, might not be the best source for enzymes that degrade sugarcane biomass.