3-phenacylideneoxindoles as a new class of antifungal compounds against Paracoccidioides spp.

Aims: Considering the need to identify new compounds with antifungal action, the activity of five 3-phenacylideneoxindoles compounds was evaluated. Materials & methods: The compounds were synthesized, and their antifungal activity was elucidated through minimum inhibitory concentration tests and interaction assay with other antifungals. Potential targets of compounds were predicted in silico. Results: 3-phenacylideneoxindoles compounds inhibited fungal growth with minimum inhibitory concentration and minimum fungicidal concentration ranging from 3.05 to 12.26 µM. The compounds demonstrated high selectivity index and presented a synergistic effect with itraconazole. In silico prediction revealed the pentafunctional AROM polypeptide, enolase, superoxide dismutase, catalase and kinases as proteins targets of the compound 4a. Conclusion: The results demonstrate that 3-phenacylideneoxindoles is a potential new class of antifungal compounds for paracoccidioidomycosis treatment.

Patients affected by paracoccidioidomycosis (PCM) require long-term treatment, which commonly influences their adherence. In addition, only three drugs are in clinical use, which indicates the relevance of research in identifying new drugs for treating PCM. Thus, five drugs were tested in the laboratory to verify whether they could prevent the growth of the fungus without being toxic to humans. In addition, whether these compounds in combination with drugs used to treat PCM could be even more potent was evaluated. All compounds tested efficiently inhibited the growth of Paracoccidioides, the fungus that causes PCM. One drug was identified that, combined with itraconazole, decreased the required dose of both the discovered compound and itraconazole needed to inhibit fungal growth. Using computational tools, this work suggests how the new drug could act against the fungus. The results demonstrate a potential new treatment option, but more studies are needed to confirm the safety of these drugs.

Setting New Routes for Antifungal Drug Discovery Against Pathogenic Fungi.

Fungal diseases are life-threatening to human health and responsible for millions of deaths around the world. Fungal pathogens lead to a high number of morbidity and mortality. Current antifungal treatment comprises drugs, such as azoles, echinocandins, and polyenes and the cure is not guaranteed. In addition, such drugs are related to severe side effects and the treatment lasts for an extended period. Thus, setting new routes for the discovery of effective and safe antifungal drugs should be a priority within the health care system. The discovery of alternative and efficient antifungal drugs showing fewer side effects is time-consuming and remains a challenge. Natural products can be a source of antifungals and used in combinatorial therapy. The most important natural products are antifungal peptides, antifungal lectins, antifungal plants, and fungi secondary metabolites. Several proteins, enzymes, and metabolic pathways could be targets for the discovery of efficient inhibitor compounds and recently, heat shock proteins, calcineurin, salinomycin, the trehalose biosynthetic pathway, and the glyoxylate cycle have been investigated in several fungal species. HSP protein inhibitors and echinocandins have been shown to have a fungicidal effect against azole-resistant fungi strains. Transcriptomic and proteomic approaches have advanced antifungal drug discovery and pointed to new important specific-pathogen targets. Certain enzymes, such as those from the glyoxylate cycle, have been a target of antifungal compounds in several fungi species. Natural and synthetic compounds inhibited the activity of such enzymes and reduced the ability of fungal cells to transit from mycelium to yeast, proving to be promisor antifungal agents. Finally, computational biology has developed effective approaches, setting new routes for early antifungal drug discovery since normal approaches take several years from discovery to clinical use. Thus, the development of new antifungal strategies might reduce the therapeutic time and increase the quality of life of patients.

Quantitative proteomic analysis of A549 cells infected with human adenovirus type 2.

Human adenovirus (HAdV-2) is considered a common agent of respiratory tract infection in the human, especially in children. Virus infection is believed to modify host cell expression necessary for its replication and therefore cell proteome can reflect the changes of specific cellular pathways during infection. This study aims to identify differentially expressed proteins of A549 cells in response to HAdV-2 infection using a label-free liquid chromatography-high-resolution tandem mass spectrometry strategy (LC-MS/MS) at 24 and 48 hpi. A total of 248 and 216 proteins were deregulated by 1.35-fold at 24 and 48 hpi, respectively. Among them, 155 were upregulated at 24 hpi and 86 at 48 hpi, whereas 93 and 130 were downregulated at 24 and 48 hpi, respectively. The identified proteins were involved in different pathways as energy, transcription, protein synthesis, cytoskeleton, rescue and defense, cell cycle, DNA processing, transportation, and metabolism. Glycolytic pathway and histone deregulated proteins were further confirmed by chemical testing and immunofluorescence, respectively. The results suggest that the identified proteins influenced HAdV-2 infection in the context of viral replication and propagation. This study complement proteomic data obtained from previous studies and reinforce the understanding of the relationship between HAdV and host cell.

Response of Paracoccidioides lutzii to the antifungal camphene thiosemicarbazide determined by proteomic analysis.

AIM: To perform the proteomic profile of Paracoccidioides lutzii after treatment with the compound camphene thiosemicarbazide (TSC-C) in order to study its mode of action. METHODS: Proteomic analysis was carried out after cells were incubated with TSC-C in a subinhibitory concentration. Validation of the proteomic results comprised the azocasein assay, western blot and determination of the susceptibility of a mutant to the compound. RESULTS: Proteins related to metabolism, energy and protein fate were regulated after treatment. In addition, TSC-C reduces the proteolytic activity of the protein extract similarly to different types of protease inhibitors. CONCLUSION: TSC-C showed encouraging antifungal activity, working as a protease inhibitor and downregulating important pathways impairing the ability of the fungi cells to produce important precursors.

The second International Symposium on Fungal Stress: ISFUS.

The topic of 'fungal stress' is central to many important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The second ISFUS was held in May 8-11 2017 in Goiania, Goiás, Brazil and hosted by the Instituto de Patologia Tropical e Saúde Pública at the Universidade Federal de Goiás. It was supported by grants from CAPES and FAPEG. Twenty-seven speakers from 15 countries presented their research related to fungal stress biology. The Symposium was divided into seven topics: 1. Fungal biology in extreme environments; 2. Stress mechanisms and responses in fungi: molecular biology, biochemistry, biophysics, and cellular biology; 3. Fungal photobiology in the context of stress; 4. Role of stress in fungal pathogenesis; 5. Fungal stress and bioremediation; 6. Fungal stress in agriculture and forestry; and 7. Fungal stress in industrial applications. This article provides an overview of the science presented and discussed at ISFUS-2017.

Inhibition of protein kinase A affects Paracoccidioides lutzii dimorphism.

A critical step in the lifecycle of many fungal pathogens is the ability to switch between filamentous and yeast growth, a process known as dimorphism. cAMP-dependent protein kinase (PKA) controls morphological changes and the pathogenicity of several animal and plant pathogenic fungi. In this work, we report the analysis of PKA activity during the mycelium to yeast transition in the pathogenic fungus Paracoccidioides lutzii. This fungus, as well as the closely related species Paracoccidioides brasiliensis, causes paracoccidioidomycosis, a systemic mycosis that affects thousands of people in Latin America. Infection occurs when hypha fragments or spores released from mycelium are inhaled by the host, an event that triggers the morphological switch. We show here that PKA activity is regulated in the fungus phase, increasing during the mycelium to yeast transition. Also, morphological transition from mycelium to yeast is blocked by the compound H89, a specific PKA inhibitor. Nevertheless, the fungus recovers its ability to change morphology when H89 is removed from the culture media. This recovery is accompanied by a significant increase in PKA activity. Our results strongly indicate that PKA directly affects phase transition in P. lutzii.

Differential Metabolism of a Two-Carbon Substrate by Members of the Paracoccidioides Genus.

The genus Paracoccidioides comprises known fungal pathogens of humans and can be isolated from different infection sites. Metabolic peculiarities in different members of the Paracoccidioides led us to perform proteomic studies in the presence of the two-carbon molecule acetate, which predominates in the nutrient-poor environment of the phagosome. To investigate the expression rates of proteins of different members of Paracoccidioides, including one isolate of P. lutzii (Pb01) and three isolates of P. brasiliensis (Pb03, Pb339, and PbEPM83), using sodium acetate as a carbon source, proteins were quantified using label-free and data-independent liquid chromatography-mass spectrometry. Protein profiles of the isolates were statistically analyzed, revealing proteins that were differentially expressed when the fungus was cultivated in a non-preferential carbon source rather than glucose. A total of 1,160, 1,211, 1,280, and 1,462 proteins were reproducibly identified and relatively quantified in P. lutzii and the P. brasiliensis isolates Pb03, Pb339, and PbEPM83, respectively. Notably, 526, 435, 744, and 747 proteins were differentially expressed among P. lutzii and the P. brasiliensis isolates Pb03, Pb339, and PbEPM83, respectively, with a fold-change equal to or higher than 1.5. This analysis revealed that reorganization of metabolism occurred through the induction of proteins related to gluconeogenesis, glyoxylic/glyoxylate cycle, response to stress, and degradation of amino acids in the four isolates. The following differences were observed among the isolates: higher increases in the expression levels of proteins belonging to the TCA and respiratory chain in PbEPM83 and Pb01; increase in ethanol production in Pb01; utilization of cell wall components for gluconeogenesis in Pb03 and PbEPM83; and increased ß-oxidation and methylcitrate cycle proteins in Pb01and PbEPM83. Proteomic profiles indicated that the four isolates reorganized their metabolism in different manners to use acetate as a carbon source.

Identification and Analysis of the Role of Superoxide Dismutases Isoforms in the Pathogenesis of Paracoccidioides spp.

The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms.

Proteomic profile response of Paracoccidioides lutzii to the antifungal argentilactone.

The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a mycosis of high incidence in Brazil. The toxicity of drug treatment and the emergence of resistant organisms have led to research for new candidates for drugs. In this study, we demonstrate that the natural product argentilactone was not cytotoxic or genotoxic to MRC5 cells at the IC50 concentration to the fungus. We also verified the proteomic profile of Paracoccidioides lutzii after incubation with argentilactone using a label free quantitative proteome nanoUPLC-MS(E). The results of this study indicated that the fungus has a global metabolic adaptation in the presence of argentilactone. Enzymes of important pathways, such as glycolysis, the Krebs cycle and the glyoxylate cycle, were repressed, which drove the metabolism to the methylcytrate cycle and beta-oxidation. Proteins involved in cell rescue, defense and stress response were induced. In this study, alternative metabolic pathways adopted by the fungi were elucidated, helping to elucidate the course of action of the compound studied.

ß-Carboline alkaloids from Galianthe ramosa inhibit malate synthase from Paracoccidioides spp.

As part of our continuing chemical and biological analyses of Rubiaceae species from Cerrado, we isolated novel alkaloids 1 and 2, along with known compounds epicatechin, ursolic acid, and oleanolic acid, from Galianthe ramosa. Alkaloid 2 inhibited malate synthase from the pathogenic fungus Paracoccidioides spp. This enzyme is considered an important molecular target because it is not found in humans. Molecular docking simulations were used to describe the interactions between the alkaloids and malate synthase.

New developments of RNAi in Paracoccidioides brasiliensis: prospects for high-throughput, genome-wide, functional genomics.

BACKGROUND: The Fungal Genome Initiative of the Broad Institute, in partnership with the Paracoccidioides research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1), Pb03 (PS2) and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for P. brasiliensis. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×106 viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the Streptoalloteichus hindustanus bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in P. brasiliensis. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA) from a Gateway-adapted cassette (cALf) which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach. CONCLUSIONS/SIGNIFICANCE: We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future Paracoccidioides spp. functional genomics research.

Efferent pathways in sodium overload-induced renal vasodilation in rats.

Hypernatremia stimulates the secretion of oxytocin (OT), but the physiological role of OT remains unclear. The present study sought to determine the involvement of OT and renal nerves in the renal responses to an intravenous infusion of hypertonic saline. Male Wistar rats (280-350 g) were anesthetized with sodium thiopental (40 mg. kg(-1), i.v.). A bladder cannula was implanted for collection of urine. Animals were also instrumented for measurement of mean arterial pressure (MAP) and renal blood flow (RBF). Renal vascular conductance (RVC) was calculated as the ratio of RBF by MAP. In anesthetized rats (n = 6), OT infusion (0.03 µg • kg(-1), i.v.) induced renal vasodilation. Consistent with this result, ex vivo experiments demonstrated that OT caused renal artery relaxation. Blockade of OT receptors (OXTR) reduced these responses to OT, indicating a direct effect of this peptide on OXTR on this artery. Hypertonic saline (3 M NaCl, 1.8 ml • kg(-1) b.wt., i.v.) was infused over 60 s. In sham rats (n = 6), hypertonic saline induced renal vasodilation. The OXTR antagonist (AT; atosiban, 40 µg • kg(-1) • h(-1), i.v.; n = 7) and renal denervation (RX) reduced the renal vasodilation induced by hypernatremia. The combination of atosiban and renal denervation (RX+AT; n = 7) completely abolished the renal vasodilation induced by sodium overload. Intact rats excreted 51% of the injected sodium within 90 min. Natriuresis was slightly blunted by atosiban and renal denervation (42% and 39% of load, respectively), whereas atosiban with renal denervation reduced sodium excretion to 16% of the load. These results suggest that OT and renal nerves are involved in renal vasodilation and natriuresis induced by acute plasma hypernatremia.

The endothelin system has a significant role in the pathogenesis and progression of Mycobacterium tuberculosis infection.

Tuberculosis (TB) remains a major global health problem, and although multiple studies have addressed the relationship between Mycobacterium tuberculosis and the host on an immunological level, few studies have addressed the impact of host physiological responses. Proteases produced by bacteria have been associated with important alterations in the host tissues, and a limited number of these enzymes have been characterized in mycobacterial species. M. tuberculosis produces a protease called Zmp1, which appears to be associated with virulence and has a putative action as an endothelin-converting enzyme. Endothelins are a family of vasoactive peptides, of which 3 distinct isoforms exist, and endothelin 1 (ET-1) is the most abundant and the best-characterized isoform. The aim of this work was to characterize the Zmp1 protease and evaluate its role in pathogenicity. Here, we have shown that M. tuberculosis produces and secretes an enzyme with ET-1 cleavage activity. These data demonstrate a possible role of Zmp1 for mycobacterium-host interactions and highlights its potential as a drug target. Moreover, the results suggest that endothelin pathways have a role in the pathogenesis of M. tuberculosis infections, and ETA or ETB receptor signaling can modulate the host response to the infection. We hypothesize that a balance between Zmp1 control of ET-1 levels and ETA/ETB signaling can allow M. tuberculosis adaptation and survival in the lung tissues.

Predicting copper-, iron-, and zinc-binding proteins in pathogenic species of the Paracoccidioides genus.

Approximately one-third of all proteins have been estimated to contain at least one metal cofactor, and these proteins are referred to as metalloproteins. These represent one of the most diverse classes of proteins, containing metal ions that bind to specific sites to perform catalytic, regulatory and structural functions. Bioinformatic tools have been developed to predict metalloproteins encoded by an organism based only on its genome sequence. Its function and the type of metal binder can also be predicted via a bioinformatics approach. Paracoccidioides complex includes termodimorphic pathogenic fungi that are found as saprobic mycelia in the environment and as yeast, the parasitic form, in host tissues. They are the etiologic agents of Paracoccidioidomycosis, a prevalent systemic mycosis in Latin America. Many metalloproteins are important for the virulence of several pathogenic microorganisms. Accordingly, the present work aimed to predict the copper, iron and zinc proteins encoded by the genomes of three phylogenetic species of Paracoccidioides (Pb01, Pb03, and Pb18). The metalloproteins were identified using bioinformatics approaches based on structure, annotation and domains. Cu-, Fe-, and Zn-binding proteins represent 7% of the total proteins encoded by Paracoccidioides spp. genomes. Zinc proteins were the most abundant metalloproteins, representing 5.7% of the fungus proteome, whereas copper and iron proteins represent 0.3 and 1.2%, respectively. Functional classification revealed that metalloproteins are related to many cellular processes. Furthermore, it was observed that many of these metalloproteins serve as virulence factors in the biology of the fungus. Thus, it is concluded that the Cu, Fe, and Zn metalloproteomes of the Paracoccidioides spp. are of the utmost importance for the biology and virulence of these particular human pathogens.

Cell survival and altered gene expression following photodynamic inactivation of Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides brasiliensis. Currently, the treatment approach involves the use of antifungal drugs and requires years of medical therapy, which can induce nephrotoxicity and lead to resistance in yeast strains. Photodynamic inactivation (PDI) is a new therapy capable of killing microorganisms via the combination of a nontoxic dye with visible light to generate toxic reactive oxygen species (ROS). We investigated the phototoxic effect of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin (TMPyP), a cationic porphyrin, on the survival of P. brasiliensis following exposure to light. Phototoxicity was found to depend on both the fluence and concentration of the photosensitizer (PS). Although the biological effects of PDI are known, the molecular mechanisms underlying the resultant damage to cells are poorly defined. Therefore, we evaluated the molecular response to PDI-induced oxidative stress by gene transcription analysis. We selected genes associated with the high-osmolarity glycerol (HOG)-mitogen-activated protein kinase (MAPK) pathway and antioxidant enzymes. The genes analyzed were all overexpressed after PDI treatment, suggesting that the oxidative stress generated in our experimental conditions induces antioxidant activity. In addition to PDI-induced gene expression, there was high cell mortality, suggesting that the antioxidant response was not sufficient to avoid fungal mortality.

A quantitative view of the morphological phases of Paracoccidioides brasiliensis using proteomics.

Paracoccidioides brasiliensis is a fungal pathogen with a broad distribution in Latin American countries. The mycelia-to-yeast morphological transition of P. brasiliensis is involved in the virulence of this pathogen, and this event is essential to the establishment of infection. Here, we report the first proteomic comparison between the mycelia, the mycelia-to-yeast transition and the yeast cells. Changes in the relative abundance of the components of the proteome during phase conversion of P. brasiliensis were analyzed by two-dimensional gel electrophoresis coupled to mass spectrometry. Using MALDI-TOF-MS, we identified 100 total proteins/isoforms. We show that 18, 30 and 33 proteins/isoforms in our map are overexpressed in the mycelia, the mycelia-to-yeast transition and in yeast cells, respectively. Nineteen proteins/isoforms did not present significant differences in the volume spots in the three analyzed conditions. The differential expression was confirmed for six different proteins by Western blot analysis. The quantitative differences observed by the proteomic analysis were correlated with the transcript levels, as determined by quantitative RT-PCR of the analyzed conditions, including conidial formation and the transition from conidia-to-yeast cells. The analysis of the functional categories to which these proteins belong provided an integrated view of the metabolic reorganization during the morphogenesis of P. brasiliensis.

Proteomic analysis reveals that iron availability alters the metabolic status of the pathogenic fungus Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a thermodimorphic fungus and the causative agent of paracoccidioidomycosis (PCM). The ability of P. brasiliensis to uptake nutrients is fundamental for growth, but a reduction in the availability of iron and other nutrients is a host defense mechanism many pathogenic fungi must overcome. Thus, fungal mechanisms that scavenge iron from host may contribute to P. brasiliensis virulence. In order to better understand how P. brasiliensis adapts to iron starvation in the host we compared the two-dimensional (2D) gel protein profile of yeast cells during iron starvation to that of iron rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity as determined by image analysis. A total of 1752 protein spots were selected for comparison, and a total of 274 out of the 1752 protein spots were determined to have changed significantly in abundance due to iron depletion. Ninety six of the 274 proteins were grouped into the following functional categories; energy, metabolism, cell rescue, virulence, cell cycle, protein synthesis, protein fate, transcription, cellular communication, and cell fate. A correlation between protein and transcript levels was also discovered using quantitative RT-PCR analysis from RNA obtained from P. brasiliensis under iron restricting conditions and from yeast cells isolated from infected mouse spleens. In addition, western blot analysis and enzyme activity assays validated the differential regulation of proteins identified by 2-D gel analysis. We observed an increase in glycolytic pathway protein regulation while tricarboxylic acid cycle, glyoxylate and methylcitrate cycles, and electron transport chain proteins decreased in abundance under iron limiting conditions. These data suggest a remodeling of P. brasiliensis metabolism by prioritizing iron independent pathways.

Diversity of anti-haemostatic proteins in the salivary glands of Rhodnius species transmitters of Chagas disease in the greater Amazon.

The triatomines in the tribe Rhodniini are the main vectors of the Trypanosoma cruzi to humans in recent outbreaks of acute Chagas disease in the Amazon. These insects dwelling in palm trees do not colonize the human domicile. Their success to transmit the infection relies partially on the efficacy of their salivary gland apparatuses. Here we show the transcriptome of the Rhodnius brethesi and Rhodnius robustus salivary glands, comprising 56 and 122 clusters, respectively. Approximately one third of these clusters are described for the first time. The LC-MS/MS analysis identified 123 and 111 proteins in R. brethesi and R. robustus sialome, respectively. Noteworthy, lipocalin platelet aggregation inhibitors, inositol polyphosphate 5-phosphatases, and Kazal domain proteins, which are essential for the insect's successful acquisition of blood meals, were found in our analysis. Moreover, glutathione S transferase and antigen-5, which play roles in the insect's defense and resistance against insecticide, were also observed.

Phosphorylation is the major mechanism regulating isocitrate lyase activity in Paracoccidioides brasiliensis yeast cells.

The glyoxylate cycle plays an essential role for anaplerosis of oxaloacetate during growth of microorganisms on carbon sources such as acetate or fatty acids and has been shown to contribute to virulence of several pathogens. Here, we investigated the transcriptional and post-translational regulation of the glyoxylate cycle key enzyme isocitrate lyase (PbICL) in the human pathogenic fungus Paracoccidioides brasiliensis. Although sequence analyses on fungal isocitrate lyases revealed a high phylogenetic conservation, their regulation seems to differ significantly. Closely related Aspergillus species regulate the glyoxylate cycle at the transcriptional level, whereas Pbicl was constitutively expressed in yeast cells. However, only low PbICL activity was detected when cells were grown in the presence of glucose. Two-dimensional gel analyses with subsequent antibody hybridization revealed constitutive production of PbICL, but low PbICL activity on glucose coincided with extensive protein phosphorylation. Since an in vitro dephosphorylation of PbICL from glucose grown cells strongly increased ICL activity and resembled the phosphorylation pattern of highly active acetate grown cells, post-translational modification seems the main mechanism regulating PbICL activity in yeast cells. In agreement, a transfer of yeast cells from glucose to acetate medium increased PbICL activity without requirement of de novo protein synthesis. Thus, inactivation of PbICL by phosphorylation is reversible, denoting a new strategy for the rapid adaptation to changing environmental conditions.

Redundancy of proteins in the salivary glands of Panstrongylus megistus secures prolonged procurement for blood meals.

Panstrongylus megistus, a vector for the Chagas disease parasite Trypanosoma cruzi, is a hematophagous bug widely distributed in South America. This ubiquitous triatomine is known to colonize different wild life habitats. Additionally, P. megistus synanthropy, preying upon mammals, birds, reptiles, and eventually being predators upon insect's hemolymph probably increases its ability to survive after prolonged fasting. It was suspected that the P. megistus mechanisms of adaptation to survival might include a salivary gland complex tool-box with a diversity of pharmacologically active proteins for obtaining blood meals. Herein we describe comprehensive proteome and transcriptome of the P. megistus salivary gland. The proteomic analysis led to the identification of 159 proteins, and the transcriptome revealed 47 complete cDNAs. A diversity of protein functions associated to blood feeding was identified. The most prevalent proteins were related to blood clotting, anti-platelet aggregation and anti-vasoconstriction activities, which correlate with the insect's ability to obtain meals from different sources. Moreover, a gene of resistance to insecticides was identified. These features augments the comprehension towards P. megistus enormous capacity to survive in adverse wild life-changing habitats.