Sirtuin E deacetylase is required for full virulence of Aspergillus fumigatus.

Aspergillus fumigatus represents a public health problem due to the high mortality rate in immunosuppressed patients and the emergence of antifungal-resistant isolates. Protein acetylation is a crucial post-translational modification that controls gene expression and biological processes. The strategic manipulation of enzymes involved in protein acetylation has emerged as a promising therapeutic approach for addressing fungal infections. Sirtuins, NAD+-dependent lysine deacetylases, regulate protein acetylation and gene expression in eukaryotes. However, their role in the human pathogenic fungus A. fumigatus remains unclear. This study constructs six single knockout strains of A. fumigatus and a strain lacking all predicted sirtuins (SIRTKO). The mutant strains are viable under laboratory conditions, indicating that sirtuins are not essential genes. Phenotypic assays suggest sirtuins' involvement in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. Deletion of sirE attenuates virulence in murine and Galleria mellonella infection models. The absence of SirE alters the acetylation status of proteins, including histones and non-histones, and triggers significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

Deacetylation by sirtuins is important for Aspergillus fumigatus pathogenesis and virulence.

Protein acetylation is a crucial post-translational modification that controls gene expression and a variety of biological processes. Sirtuins, a prominent class of NAD + -dependent lysine deacetylases, serve as key regulators of protein acetylation and gene expression in eukaryotes. In this study, six single knockout strains of fungal pathogen Aspergillus fumigatus were constructed, in addition to a strain lacking all predicted sirtuins (SIRTKO). Phenotypic assays suggest that sirtuins are involved in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. AfsirE deletion resulted in attenuation of virulence, as demonstrated in murine and Galleria infection models. The absence of AfSirE leads to altered acetylation status of proteins, including histones and non-histones, resulting in significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

Sirtuins: Key pieces in the host response to pathogens' puzzle.

Global warming is changing the distribution of different pathogens around the globe, and humans are more susceptible to new or re-emerging infections. The human response to microbes is complex and involves different mechanisms of the immune system. Regulation of gene expression of immunity genes and of metabolism of immune cells are essential in this process. Both mechanisms could be regulated by protein lysine acetylation that will control chromatin structure affecting gene expression or key enzyme activity involved in cellular processes. Protein acetylation is crucial for the immunity and involves two families of enzymes: lysine acetyltransferases (KATs), which will promote protein acetylation, and lysine deacetylases (KDACs) that will reduce this modification. Lysine deacetylases are divided into Zinc-dependent or HDACs and NAD+ -dependent, or Sirtuins. These enzymes are in the nucleus, cytosol, and mitochondria of mammalian cells affecting different cellular pathways, such as metabolism, gene expression, DNA repair, cell proliferation, and apoptosis, opening the opportunity to explore these proteins as drug targets in different diseases, including cancer and neurodegenerative illness. Although widely explored in chronic diseases, very little is known about the role of Sirtuins during host response against microbes' infection. In this review we aim to explore the most recent literature evidencing a role for these enzymes during host responses to viruses, bacterial and protozoan infections, pointing out how these proteins can be manipulated by these pathogens to progress in the infection. Moreover, we will uncover the potential of host KDACs as therapeutic targets to prevent infections by activating effector immune functions.

Antileishmanial metallodrugs and the elucidation of new drug targets linked to post-translational modifications machinery: pitfalls and progress.

Despite the increasing number of manuscripts describing potential alternative antileishmanial compounds, little is advancing on translating these knowledges to new products to treat leishmaniasis. This is in part due to the lack of standardisations during pre-clinical drug discovery stage and also depends on the alignment of goals among universities/research centers, government and pharmaceutical industry. Inspired or not by drug repurposing, metal-based antileishmanial drugs represent a class that deserves more attention on its use for leishmaniasis chemotherapy. Together with new chemical entities, progresses have been made on the knowledge of parasite-specific drug targets specially after using CRISPR/Cas system for functional studies. In this regard, Leishmania parasites undergoe post-translational modification as key regulators in several cellular processes, which represents an entire new field for drug target elucidation, once this is poorly explored. This perspective review describes the advances on antileishmanial metallodrugs and the elucidation of drug targets based on post-translational modifications, highlighting the limitations on the drug discovery/development process and suggesting standardisations focused on products addressed to who need it most.

Sex under pressure: stress facilitates Leishmania in vitro hybridization.

The selection of Leishmania hybrids in axenic culture was considered rare until recently, when Louradour and Ferreira et al., demonstrated that induced DNA damage facilitates genetic exchange, resulting in full genome tetraploid progenies in vitro. Meiosis-related gene homologues HAP2, GEX1, and RAD51 were found to be involved, opening new avenues for functional genomic studies.

Disruption of Active Trans-Sialidase Genes Impairs Egress from Mammalian Host Cells and Generates Highly Attenuated Trypanosoma cruzi Parasites.

Trans-sialidases (TS) are unusual enzymes present on the surface of Trypanosoma cruzi, the causative agent of Chagas disease. Encoded by the largest gene family in the T. cruzi genome, only few members of the TS family have catalytic activity. Active trans-sialidases (aTS) are responsible for transferring sialic acid from host glycoconjugates to mucins, also present on the parasite surface. The existence of several copies of TS genes has impaired the use of reverse genetics to study this highly polymorphic gene family. Using CRISPR-Cas9, we generated aTS knockout cell lines displaying undetectable levels of TS activity, as shown by sialylation assays and labeling with antibodies that recognize sialic acid-containing mucins. In vitro infection assays showed that disruption of aTS genes does not affect the parasite's capacity to invade cells or to escape from the parasitophorous vacuole but resulted in impaired differentiation of amastigotes into trypomastigotes and parasite egress from the cell. When inoculated into mice, aTS mutants were unable to establish infection even in the highly susceptible gamma interferon (IFN-γ) knockout mice. Mice immunized with aTS mutants were fully protected against a challenge infection with the virulent T. cruzi Y strain. Altogether, our results confirmed the role of aTS as a T. cruzi virulence factor and indicated that aTS play a major role during the late stages of intracellular development and parasite egress. Notably, mutants lacking TS activity are completely avirulent in animal models of infection and may be used as a live attenuated vaccine against Chagas disease. IMPORTANCE Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects approximately 6 to 8 million people and for which there is no effective treatment or vaccine. The parasite expresses a family of surface proteins, named trans-sialidases, responsible for transferring sialic acid from host glycoconjugates to parasite mucins. Although recognized as a main virulence factor, the multiple roles of these proteins during infection have not yet been fully characterized, mainly because the presence of several copies of aTS genes has impaired their study using reverse genetics. By applying CRISPR-Cas9, we generated aTS knockout parasites and showed that, although aTS parasite mutants were able to infect cells in vitro, they have an impaired capacity to egress from the infected cell. Importantly, aTS mutants lost the ability to cause infection in vivo but provided full protection against a challenge infection with a virulent strain.

Panacea within a Pandora's box: the antiparasitic effects of phospholipases A2 (PLA2s) from snake venoms.

Parasitic diseases affect millions of individuals worldwide, mainly in low-income regions. There is no cure for most of these diseases, and the treatment relies on drugs that have side effects and lead to drug resistance, emphasizing the urgency to find new treatments. Snake venom has been gaining prominence as a rich source of molecules with antiparasitic potentials, such as phospholipases A2 (PLA2s). Here, we compile the findings involving PLA2s with antiparasitic activities against helminths, Plasmodium, Toxoplasma, and trypanosomatids. We indicate their molecular features, highlighting the possible antiparasitic mechanisms of action of these proteins. We also demonstrate interactions between PLA2s and some parasite membrane components, shedding light on potential targets for drug design that may provide better treatment for the illnesses caused by parasites.

The current drug discovery landscape for trypanosomiasis and leishmaniasis: Challenges and strategies to identify drug targets.

Human trypanosomiasis and leishmaniasis are vector-borne neglected tropical diseases caused by infection with the protozoan parasites Trypanosoma spp. and Leishmania spp., respectively. Once restricted to endemic areas, these diseases are now distributed worldwide due to human migration, climate change, and anthropogenic disturbance, causing significant health and economic burden globally. The current chemotherapy used to treat these diseases has limited efficacy, and drug resistance is spreading. Hence, new drugs are urgently needed. Phenotypic compound screenings have prevailed as the leading method to discover new drug candidates against these diseases. However, the publication of the complete genome sequences of multiple strains, advances in the application of CRISPR/Cas9 technology, and in vivo bioluminescence-based imaging have set the stage for advancing target-based drug discovery. This review analyses the limitations of the narrow pool of available drugs presently used for treating these diseases. It describes the current drug-based clinical trials highlighting the most promising leads. Furthermore, the review presents a focused discussion on the most important biological and pharmacological challenges that target-based drug discovery programs must overcome to advance drug candidates. Finally, it examines the advantages and limitations of modern research tools designed to identify and validate essential genes as drug targets, including genomic editing applications and in vivo imaging.

Antileishmanial metallodrugs and the elucidation of new drug targets linked to post-translational modifications machinery: pitfalls and progress

Despite the increasing number of manuscripts describing potential alternative antileishmanial compounds, little is advancing on translating these knowledges to new products to treat leishmaniasis. This is in part due to the lack of standardisations during pre-clinical drug discovery stage and also depends on the alignment of goals among universities/research centers, government and pharmaceutical industry. Inspired or not by drug repurposing, metal-based antileishmanial drugs represent a class that deserves more attention on its use for leishmaniasis chemotherapy. Together with new chemical entities, progresses have been made on the knowledge of parasite-specific drug targets specially after using CRISPR/Cas system for functional studies. In this regard, Leishmania parasites undergoe post-translational modification as key regulators in several cellular processes, which represents an entire new field for drug target elucidation, once this is poorly explored. This perspective review describes the advances on antileishmanial metallodrugs and the elucidation of drug targets based on post-translational modifications, highlighting the limitations on the drug discovery/development process and suggesting standardisations focused on products addressed to who need it most.

Mitochondrial Sirtuin TcSir2rp3 Affects TcSODA Activity and Oxidative Stress Response in Trypanosoma cruzi.

Trypanosoma cruzi faces a variety of environmental scenarios during its life cycle, which include changes in the redox environment that requires a fine regulation of a complex antioxidant arsenal of enzymes. Reversible posttranslational modifications, as lysine acetylation, are a fast and economical way for cells to react to environmental conditions. Recently, we found that the main antioxidant enzymes, including the mitochondrial superoxide dismutase A (TcSODA) are acetylated in T. cruzi, suggesting that protein acetylation could participate in the oxidative stress response in T. cruzi. Therefore, we investigated whether mitochondrial lysine deacetylase TcSir2rp3 was involved in the activity control of TcSODA. We observed an increased resistance to hydrogen peroxide and menadione in parasites overexpressing TcSir2rp3. Increased resistance was also found for benznidazole and nifurtimox, known to induce reactive oxidative and nitrosactive species in the parasite, associated to that a reduction in the ROS levels was observed. To better understand the way TcSir2rp3 could contributes to oxidative stress response, we analyzed the expression of TcSODA in the TcSir2rp3 overexpressing parasites and did not detect any increase in protein levels of this enzyme. However, we found that these parasites presented higher levels of superoxide dismutase activity, and also that TcSir2rp3 and TcSODA interacts in vivo. Knowing that TcSODA is acetylated at lysine residues K44 and K97, and that K97 is located at a similar region in the protein structure as K68 in human manganese superoxide dismutase (MnSOD), responsible for regulating MnSOD activity, we generated mutated versions of TcSODA at K44 and K97 and found that replacing K97 by glutamine, which mimics an acetylated lysine, negatively affects the enzyme activity in vitro. By using molecular dynamics approaches, we revealed that acetylation of K97 induces specific conformational changes in TcSODA with respect to hydrogen-bonding pattern to neighbor residues, suggesting a key participation of this residue to modulate the affinity to O2- . Taken together, our results showed for the first time the involvement of lysine acetylation in the maintenance of homeostatic redox state in trypanosomatids, contributing to the understanding of mechanisms used by T. cruzi to progress during the infection.

Protein acetylation in the critical biological processes in protozoan parasites.

Protein lysine acetylation has emerged as a major regulatory post-translational modification in different organisms, present not only on histone proteins affecting chromatin structure and gene expression but also on nonhistone proteins involved in several cellular processes. The same scenario was observed in protozoan parasites after the description of their acetylomes, indicating that acetylation might regulate crucial biological processes in these parasites. The demonstration that glycolytic enzymes are regulated by acetylation in protozoans shows that this modification might regulate several other processes implicated in parasite survival and adaptation during the life cycle, opening the chance to explore the regulatory acetylation machinery of these parasites as drug targets for new treatment development.

Raising the Bar(-seq) in Leishmania Genetic Screens.

Our understanding of regulatory factors in Leishmania differentiation has long been restricted by the available genetic tools, but the availability of CRISPR/Cas9 has changed the landscape forever. Recently, Baker and Catta-Preta et al. applied Cas9 editing and kinome-wide bar-seq to dissect the function of 204 kinases in the Leishmania mexicana life cycle.

Epitranscriptome machinery in Trypanosomatids: New players on the table?

Trypanosoma and Leishmania parasites cause devastating tropical diseases resulting in serious global health consequences. These organisms have complex life cycles with mammalian hosts and insect vectors. The parasites must, therefore, survive in different environments, demanding rapid physiological and metabolic changes. These responses depend upon regulation of gene expression, which primarily occurs posttranscriptionally. Altering the composition or conformation of RNA through nucleotide modifications is one posttranscriptional mechanism of regulating RNA fate and function, and modifications including N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytidine (m5C), N4-acetylcytidine (ac4C), and pseudouridine (Ψ), dynamically regulate RNA stability and translation in diverse organisms. Little is known about RNA modifications and their machinery in Trypanosomatids, but we hypothesize that they regulate parasite gene expression and are vital for survival. Here, we identified Trypanosomatid homologs for writers of m1A, m5C, ac4C, and Ψ and analyze their evolutionary relationships. We systematically review the evidence for their functions and assess their potential use as therapeutic targets. This work provides new insights into the roles of these proteins in Trypanosomatid parasite biology and treatment of the diseases they cause and illustrates that Trypanosomatids provide an excellent model system to study RNA modifications, their molecular, cellular, and biological consequences, and their regulation and interplay.

Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi.

The flagellum of Trypanosomatids is an organelle that contributes to multiple functions, including motility, cell division, and host-pathogen interaction. Trypanin was first described in Trypanosoma brucei and is part of the dynein regulatory complex. TbTrypanin knockdown parasites showed motility defects in procyclic forms; however, silencing in bloodstream forms was lethal. Since TbTrypanin mutants show drastic phenotypic changes in mammalian stages, we decided to evaluate if the Trypanosoma cruzi ortholog plays a similar role by using the CRISPR-Cas9 system to generate null mutants. A ribonucleoprotein complex of SaCas9 and sgRNA plus donor oligonucleotide were used to edit both alleles of TcTrypanin without any selectable marker. TcTrypanin -/- epimastigotes showed a lower growth rate, partially detached flagella, normal numbers of nuclei and kinetoplasts, and motility defects such as reduced displacement and speed and increased tumbling propensity. The epimastigote mutant also showed decreased efficiency of in-vitro metacyclogenesis. Mutant parasites were able to complete the entire life cycle in vitro; however, they showed a reduction in their infection capacity compared with WT and addback cultures. Our data show that T. cruzi life cycle stages have differing sensitivities to TcTrypanin deletion. In conclusion, additional work is needed to dissect the motility components of T. cruzi and to identify essential molecules for mammalian stages.

Cyclophilin 19 secreted in the host cell cytosol by Trypanosoma cruzi promotes ROS production required for parasite growth.

Infection by Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, depends on reactive oxygen species (ROS), which has been described to induce parasite proliferation in mammalian host cells. It is unknown how the parasite manages to increase host ROS levels. Here, we found that intracellular T. cruzi forms release in the host cytosol its major cyclophilin of 19 kDa (TcCyp19). Parasites depleted of TcCyp19 by using CRISPR/Cas9 gene replacement proliferate inefficiently and fail to increase ROS, compared to wild type parasites or parasites with restored TcCyp19 gene expression. Expression of TcCyp19 in L6 rat myoblast increased ROS levels and restored the proliferation of TcCyp19 depleted parasites. These events could also be inhibited by cyclosporin A, (a cyclophilin inhibitor), and by polyethylene glycol-linked to antioxidant enzymes. TcCyp19 was found more concentrated in the membrane leading edges of the host cells in regions that also accumulate phosphorylated p47phox , as observed to the endogenous cyclophilin A, suggesting some mechanisms involved with the translocation process of the regulatory subunit p47phox in the activation of the NADPH oxidase enzymatic complex. We concluded that cyclophilin released in the host cell cytosol by T. cruzi mediates the increase of ROS, required to boost parasite proliferation in mammalian hosts.

Identification of Inhibitors to Trypanosoma cruzi Sirtuins Based on Compounds Developed to Human Enzymes.

Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi, affecting more than 7 million people in the world. Benznidazole and nifurtimox are the only drugs available for treatment and in addition to causing several side effects, are only satisfactory in the acute phase of the disease. Sirtuins are NAD+-dependent deacetylases involved in several biological processes, which have become drug target candidates in various disease settings. T. cruzi presents two sirtuins, one cytosolic (TcSir2rp1) and the latter mitochondrial (TcSir2rp3). Here, we characterized the effects of human sirtuin inhibitors against T. cruzi sirtuins as an initial approach to develop specific parasite inhibitors. We found that, of 33 compounds tested, two inhibited TcSir2rp1 (15 and 17), while other five inhibited TcSir2rp3 (8, 12, 13, 30, and 32), indicating that specific inhibitors can be devised for each one of the enzymes. Furthermore, all inhibiting compounds prevented parasite proliferation in cultured mammalian cells. When combining the most effective inhibitors with benznidazole at least two compounds, 17 and 32, demonstrated synergistic effects. Altogether, these results support the importance of exploring T. cruzi sirtuins as drug targets and provide key elements to develop specific inhibitors for these enzymes as potential targets for Chagas disease treatment.

Preclinical Gold Complexes as Oral Drug Candidates to Treat Leishmaniasis Are Potent Trypanothione Reductase Inhibitors.

The drugs currently used to treat leishmaniases have limitations concerning cost, efficacy, and safety, making the search for new therapeutic approaches urgent. We found that the gold(I)-derived complexes were active against L. infantum and L. braziliensis intracellular amastigotes with IC50 values ranging from 0.5 to 5.5 µM. All gold(I) complexes were potent inhibitors of trypanothione reductase (TR), with enzyme IC50 values ranging from 1 to 7.8 µM. Triethylphosphine-derived complexes enhanced reactive oxygen species (ROS) production and decreased mitochondrial respiration after 2 h of exposure, indicating that gold(I) complexes cause oxidative stress by direct ROS production, by causing mitochondrial damage or by impairing TR activity and thus accumulating ROS. There was no cross-resistance to antimony; in fact, SbR (antimony-resistant mutants) strains were hypersensitive to some of the complexes. BALB/c mice infected with luciferase-expressing L. braziliensis or L. amazonensis and treated orally with 12.5 mg/kg/day of AdT Et (3) or AdO Et (4) presented reduced lesion size and parasite burden, as revealed by bioimaging. The combination of (3) and miltefosine allowed for a 50% reduction in miltefosine treatment time. Complexes 3 and 4 presented favorable pharmacokinetic and toxicity profiles that encourage further drug development studies. Gold(I) complexes are promising antileishmanial agents, with a potential for therapeutic use, including in leishmaniasis caused by antimony-resistant parasites.

Effect of lysine acetylation on the regulation of Trypanosoma brucei glycosomal aldolase activity.

Post-translational modifications provide suitable mechanisms for cellular adaptation to environmental changes. Lysine acetylation is one of these modifications and occurs with the addition of an acetyl group to Nε-amino chain of this residue, eliminating its positive charge. Recently, we found distinct acetylation profiles of procyclic and bloodstream forms of Trypanosoma brucei, the agent of African Trypanosomiasis. Interestingly, glycolytic enzymes were more acetylated in the procyclic, which develops in insects and uses oxidative phosphorylation to obtain energy, compared with the bloodstream form, whose main source of energy is glycolysis. Here, we investigated whether acetylation regulates the T. brucei fructose 1,6-bisphosphate aldolase. We found that aldolase activity was reduced in procyclic parasites cultivated in the absence of glucose and partial recovered by in vitro deacetylation. Similarly, acetylation of protein extracts from procyclics cultivated in glucose-rich medium, caused a reduction in the aldolase activity. In addition, aldolase acetylation levels were higher in procyclics cultivated in the absence of glucose compared with those cultivated in the presence of glucose. To further confirm the role of acetylation, lysine residues near the catalytic site were substituted by glutamine in recombinant T. brucei aldolase. These replacements, especially K157, inhibited enzymatic activity, changed the electrostatic surface potential, decrease substrate binding and modify the catalytic pocket structure of the enzyme, as predicted by in silico analysis. Taken together, these data confirm the role of acetylation in regulating the activity of an enzyme from the glycolytic pathway of T. brucei, expanding the factors responsible for regulating important pathways in this parasite.