Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila.

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

Plasmodium berghei-Released Factor, PbTIP, Modulates the Host Innate Immune Responses.

The Plasmodium parasite has to cross various immunological barriers for successful infection. Parasites have evolved mechanisms to evade host immune responses, which hugely contributes to the successful infection and transmission by parasites. One way in which a parasite evades immune surveillance is by expressing molecular mimics of the host molecules in order to manipulate the host responses. In this study, we report a Plasmodium berghei hypothetical protein, PbTIP (PbANKA_124360.0), which is a Plasmodium homolog of the human T-cell immunomodulatory protein (TIP). The latter possesses immunomodulatory activities and suppressed the host immune responses in a mouse acute graft-versus-host disease (GvHD) model. The Plasmodium berghei protein, PbTIP, is expressed on the merozoite surface and exported to the host erythrocyte surface upon infection. It is shed in the blood circulation by the activity of an uncharacterized membrane protease(s). The shed PbTIP could be detected in the host serum during infection. Our results demonstrate that the shed PbTIP exhibits binding on the surface of macrophages and reduces their inflammatory cytokine response while upregulating the anti-inflammatory cytokines such as TGF-ß and IL-10. Such manipulated immune responses are observed in the later stage of malaria infection. PbTIP induced Th2-type gene transcript changes in macrophages, hinting toward its potential to regulate the host immune responses against the parasite. Therefore, this study highlights the role of a Plasmodium-released protein, PbTIP, in immune evasion using macrophages, which may represent the critical strategy of the parasite to successfully survive and thrive in its host. This study also indicates the human malaria parasite TIP as a potential diagnostic molecule that could be exploited in lateral flow-based immunochromatographic tests for malaria disease diagnosis.

Role of inflammasomes in innate host defense against Entamoeba histolytica.

Intestinal amebiasis is the disease caused by the extracellular protozoan parasite Entamoeba histolytica (Eh) that induces a dynamic and heterogeneous interaction profile with the host immune system during disease pathogenesis. In 90% of asymptomatic infection, Eh resides with indigenous microbiota in the outer mucus layer of the colon without prompting an immune response. However, for reasons that remain unclear, in a minority of the Eh-infected individuals, this fine tolerated relationship is switched to a pathogenic phenotype and advanced to an increasingly complex host-parasite interaction. Eh disease susceptibility depends on parasite virulence factors and their interactions with indigenous bacteria, disruption of the mucus bilayers, and adherence to the epithelium provoking host immune cells to evoke a robust pro-inflammatory response mediated by inflammatory caspases and inflammasome activation. To understand Eh pathogenicity and innate host immune responses, this review highlights recent advances in our understanding of how Eh induces outside-in signaling via Mϕs to activate inflammatory caspases and inflammasome to regulate pro-inflammatory responses.

Leishmania dual-specificity tyrosine-regulated kinase 1 (DYRK1) is required for sustaining Leishmania stationary phase phenotype.

Although the multiplicative and growth-arrested states play key roles in Leishmania development, the regulators of these transitions are largely unknown. In an attempt to gain a better understanding of these processes, we characterised one member of a family of protein kinases with dual specificity, LinDYRK1, which acts as a stasis regulator in other organisms. LinDYRK1 overexpressing parasites displayed a decrease in proliferation and in cell cycle re-entry of arrested cells. Parasites lacking LinDYRK1 displayed distinct fitness phenotypes in logarithmic and stationary growth phases. In logarithmic growth phase, LinDYRK1-/- parasites proliferated better than control lines, supporting a role of this kinase in stasis, while in stationary growth phase, LinDYRK1-/- parasites had important defects as they rounded up, accumulated vacuoles and lipid bodies and displayed subtle but consistent differences in lipid composition. Moreover, they expressed less metacyclic-enriched transcripts, displayed increased sensitivity to complement lysis and a significant reduction in survival within peritoneal macrophages. The distinct LinDYRK1-/- growth phase phenotypes were mirrored by the distinct LinDYRK1 localisations in logarithmic (mainly in flagellar pocket area and endosomes) and late stationary phase (mitochondrion). Overall, this work provides first evidence for the role of a DYRK family member in sustaining promastigote stationary phase phenotype and infectivity.

Functional analyses of an axonemal inner-arm dynein complex in the bloodstream form of Trypanosoma brucei uncover its essential role in cytokinesis initiation.

The flagellated eukaryote Trypanosoma brucei alternates between the insect vector and the mammalian host and proliferates through an unusual mode of cell division. Cell division requires flagellum motility-generated forces, but flagellum motility exerts distinct effects between different life cycle forms. Motility is required for the final cell abscission of the procyclic form in the insect vector, but is necessary for the initiation of cell division of the bloodstream form in the mammalian host. The underlying mechanisms remain elusive. Here we carried out functional analyses of a flagellar axonemal inner-arm dynein complex in the bloodstream form and investigated its mechanistic role in cytokinesis initiation. We showed that the axonemal inner-arm dynein heavy chain TbIAD5-1 and TbCentrin3 form a complex, localize to the flagellum, and are required for viability in the bloodstream form. We further demonstrated the interdependence between TbIAD5-1 and TbCentrin3 for maintenance of protein stability. Finally, we showed that depletion of TbIAD5-1 and TbCentrin3 arrested cytokinesis initiation and disrupted the localization of multiple cytokinesis initiation regulators. These findings identified the essential role of an axonemal inner-arm dynein complex in cell division, and provided molecular insights into the flagellum motility-mediated cytokinesis initiation in the bloodstream form of T. brucei.

Evaluation of clan CD C11 peptidase PNT1 and other Leishmania mexicana cysteine peptidases as potential drug targets.

Leishmania mexicana is one of the causative agents of cutaneous leishmaniasis in humans. There is an urgent need to identify new drug targets to combat the disease. Cysteine peptidases play crucial role in pathogenicity and virulence in Leishmania spp. and are promising targets for developing new anti-leishmanial drugs. Genetic drug target validation has been performed on a number of cysteine peptidases, but others have yet to be characterized. We targeted 16 L. mexicana cysteine peptidases for gene deletion and tagging using CRISPR-Cas9 in order to identify essential genes and ascertain their cellular localization. Our analysis indicates that two clan CA, family C2 calpains (LmCAL27.1, LmCAL31.6) and clan CD, family C11 PNT1 are essential for survival in the promastigote stage. The other peptidases analysed, namely calpains LmCAL4.1, LmCAL25.1, and members of clan CA C51, C78, C85 and clan CP C97 were found to be non-essential. We generated a gene deletion mutant (Δpnt1) which was severely compromised in its cell growth and a conditional gene deletion mutant of PNT1 (Δpnt1: PNT1flox/Δ pnt1:HYG [SSU DiCRE]). PNT1 localizes to distinct foci on the flagellum and on the surface of the parasite. The conditional gene deletion of PNT1 induced blebs and pits on the cell surface and eventual cell death. Over-expression of PNT1, but not an active site mutant PNT1C134A, was lethal, suggesting that active PNT1 peptidase is required for parasite survival. Overall, our data suggests that PNT1 is an essential gene and one of a number of cysteine peptidases that are potential drug targets in Leishmania.

Neutrophil elastase promotes Leishmania donovani infection via interferon-ß.

Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major, named inhibitor of serine peptidases (ISP) 2, inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF-α and IFN-ß production, and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knockout mice for NE (ela-/-), TLR4, or TLR2. NE and TLR4 colocalized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela-/- mice were reduced and accompanied by increased NO and decreased TGF-ß production. Expression of ISP2 was not detected in L. donovani, and a transgenic line constitutively expressing ISP2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela-/- macrophages displayed significantly lower IFN-ß mRNA than background mice macrophages, and the intracellular growth was fully restored by exogenous IFN-ß. We propose that L. donovani utilizes the host NE-TLR machinery to induce IFN-ß necessary for parasite survival and growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE-TLR pathway.-Dias, B. T., Dias-Teixeira, K. L., Godinho, J. P., Faria, M. S., Calegari-Silva, T., Mukhtar, M. M., Lopes, U. G., Mottram, J. C., Lima, A. P. C. A. Neutrophil elastase promotes Leishmania donovani infection via interferon-ß.

Nuclear Factor Y (NF-Y) Modulates Encystation in Entamoeba via Stage-Specific Expression of the NF-YB and NF-YC Subunits.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor composed of three subunits, namely, NF-YA, NF-YB, and NF-YC, which are conserved throughout evolution. In higher eukaryotes, NF-Y plays important roles in several cellular processes (development, cell cycle regulation, apoptosis, and response to growth, stress, and DNA damage) by controlling gene expression through binding to a CCAAT promoter motif. We demonstrated that NF-Y subunits in the protist Entamoeba, while significantly divergent from those of higher eukaryotes, have well-conserved domains important for subunit interactions and DNA binding and that NF-YB and NF-YC are developmentally expressed during encystation. Electrophoretic mobility shift assays confirmed that the NF-Y protein(s) from Entamoeba cysts binds to a CCAAT motif. Consistent with a role as a transcription factor, the NF-Y proteins show nuclear localization during development. Additionally, we demonstrated that NF-YC localizes to the chromatoid body (an RNA processing center) during development, indicating that it may have a role in RNA processing. Finally, silencing of the NF-YC subunit resulted in reduced stability of the NF-Y complex and decreased encystation efficiency. We demonstrated that the NF-Y complex functions at a time point subsequent to the NAD+ flux and expression of the transcription factor encystation regulatory motif-binding protein, both of which are early regulators of Entamoeba development. Taken together, our results demonstrate that the NF-Y complex plays an important role in regulating encystation in Entamoeba and add to our understanding of the transcriptional networks and signals that control this essential developmental pathway in an important human pathogen.IMPORTANCE The human parasite Entamoeba histolytica is an important pathogen with significant global impact and is a leading cause of parasitic death in humans. Since only the cyst form can be transmitted, blocking encystation would prevent new infections, making the encystation pathway an attractive target for the development of new drugs. Identification of the genetic signals and transcriptional regulatory networks that control encystation would be an important advance in understanding the developmental cascade. We show that the Entamoeba NF-Y complex plays a crucial role in regulating the encystation process in Entamoeba.

Oscillatory cAMP cell-cell signalling persists during multicellular Dictyostelium development.

Propagating waves of cAMP, periodically initiated in the aggregation centre, are known to guide the chemotactic aggregation of hundreds of thousands of starving individual Dictyostelium discoideum cells into multicellular aggregates. Propagating optical density waves, reflecting cell periodic movement, have previously been shown to exist in streaming aggregates, mounds and migrating slugs. Using a highly sensitive cAMP-FRET reporter, we have now been able to measure periodically propagating cAMP waves directly in these multicellular structures. In slugs cAMP waves are periodically initiated in the tip and propagate backward through the prespore zone. Altered cAMP signalling dynamics in mutants with developmental defects strongly support a key functional role for cAMP waves in multicellular Dictyostelium morphogenesis. These findings thus show that propagating cAMP not only control the initial aggregation process but continue to be the long range cell-cell communication mechanism guiding cell movement during multicellular Dictyostelium morphogenesis at the mound and slugs stages.

A post-transcriptional respiratome regulon in trypanosomes.

Post-transcriptional regulons coordinate the expression of groups of genes in eukaryotic cells, yet relatively few have been characterized. Parasitic trypanosomatids are particularly good models for studies on such mechanisms because they exhibit almost exclusive polycistronic, and unregulated, transcription. Here, we identify the Trypanosoma brucei ZC3H39/40 RNA-binding proteins as regulators of the respiratome; the mitochondrial electron transport chain (complexes I-IV) and the FoF1-ATP synthase (complex V). A high-throughput RNAi screen initially implicated both ZC3H proteins in variant surface glycoprotein (VSG) gene silencing. This link was confirmed and both proteins were shown to form a cytoplasmic ZC3H39/40 complex. Transcriptome and mRNA-interactome analyses indicated that the impact on VSG silencing was indirect, while the ZC3H39/40 complex specifically bound and stabilized transcripts encoding respiratome-complexes. Quantitative proteomic analyses revealed specific positive control of >20 components from complexes I, II and V. Our findings establish a link between the mitochondrial respiratome and VSG gene silencing in bloodstream form T. brucei. They also reveal a major respiratome regulon controlled by the conserved trypanosomatid ZC3H39/40 RNA-binding proteins.

Poly(ADP-ribose) polymerase-1 (PARP-1) regulates developmental morphogenesis and chemotaxis in Dictyostelium discoideum.

BACKGROUND INFORMATION: Poly(ADP-ribose) polymerase-1 (PARP-1) has been attributed to varied roles in DNA repair, cell cycle, cell death, etc. Our previous reports demonstrate the role of PARP-1 during Dictyostelium discoideum development by its constitutive downregulation as well as by PARP-1 ortholog, ADP ribosyl transferase 1 A (ADPRT1A) overexpression. The current study analyses and strengthens the function of ADPRT1A in multicellular morphogenesis of D. discoideum. ADPRT1A was knocked out, and its effect was studied on cAMP signalling, chemotaxis and development of D. discoideum. RESULTS: We report that ADPRT1A is essential in multicellular development of D. discoideum, particularly at the aggregation stage. Genetic alterations of ADPRT1A and chemical inhibition of its activity affects the intracellular and extracellular cAMP levels during aggregation along with chemotaxis. Exogenous cAMP pulses could rescue this defect in the ADPRT1A knockout (ADPRT1A KO). Expression analysis of genes involved in cAMP signalling reveals altered transcript levels of four essential genes (PDSA, REGA, ACAA and CARA). Moreover, ADPRT1A KO affects prespore- and prestalk-specific gene expression and prestalk tendency is favoured in the ADPRT1A KO. CONCLUSION: ADPRT1A plays a definite role in regulating developmental morphogenesis via cAMP signalling. SIGNIFICANCE: This study helps in understanding the role of PARP-1 in multicellular development and differentiation in higher complex organisms.

Tissue self-organization based on collective cell migration by contact activation of locomotion and chemotaxis.

Despite their central role in multicellular organization, navigation rules that dictate cell rearrangement remain largely undefined. Contact between neighboring cells and diffusive attractant molecules are two of the major determinants of tissue-level patterning; however, in most cases, molecular and developmental complexity hinders one from decoding the exact governing rules of individual cell movement. A primordial example of tissue patterning by cell rearrangement is found in the social amoeba Dictyostelium discoideum where the organizing center or the "tip" self-organizes as a result of sorting of differentiating prestalk and prespore cells. By employing microfluidics and microsphere-based manipulation of navigational cues at the single-cell level, here we uncovered a previously overlooked mode of Dictyostelium cell migration that is strictly directed by cell-cell contact. The cell-cell contact signal is mediated by E-set Ig-like domain-containing heterophilic adhesion molecules TgrB1/TgrC1 that act in trans to induce plasma membrane recruitment of the SCAR complex and formation of dendritic actin networks, and the resulting cell protrusion competes with those induced by chemoattractant cAMP. Furthermore, we demonstrate that both prestalk and prespore cells can protrude toward the contact signal as well as to chemotax toward cAMP; however, when given both signals, prestalk cells orient toward the chemoattractant, whereas prespore cells choose the contact signal. These data suggest a model of cell sorting by competing juxtacrine and diffusive cues, each with potential to drive its own mode of collective cell migration.

Insight from the maximal activation of the signal transduction excitable network in Dictyostelium discoideum.

Cell migration requires the coordination of an excitable signal transduction network involving Ras and PI3K pathways with cytoskeletal activity. We show that expressing activated Ras GTPase-family proteins in cells lacking PTEN or other mutations which increase cellular protrusiveness transforms cells into a persistently activated state. Leading- and trailing-edge markers were found exclusively at the cell perimeter and the cytosol, respectively, of the dramatically flattened cells. In addition, the lifetimes of dynamic actin puncta were increased where they overlapped with actin waves, suggesting a mechanism for the coupling between these two networks. All of these phenotypes could be reversed by inhibiting signal transduction. Strikingly, maintaining cells in this state of constant activation led to a form of cell death by catastrophic fragmentation. These findings provide insight into the feedback loops that control excitability of the signal transduction network, which drives migration.

Catalytic Mechanism of Cruzain from Trypanosoma cruzi As Determined from Solvent Kinetic Isotope Effects of Steady-State and Pre-Steady-State Kinetics.

Cruzain, an important drug target for Chagas disease, is a member of clan CA of the cysteine proteases. Understanding the catalytic mechanism of cruzain is vital to the design of new inhibitors. To this end, we have determined pH-rate profiles for substrates and affinity agents and solvent kinetic isotope effects in pre-steady-state and steady-state modes using three substrates: Cbz-Phe-Arg-AMC, Cbz-Arg-Arg-AMC, and Cbz-Arg-Ala-AMC. The pH-rate profile of kcat/ Km for Cbz-Arg-Arg-AMC indicated p K1 = 6.6 (unprotonated) and p K2 ∼ 9.6 (protonated) groups were required for catalysis. The temperature dependence of the p K = 6.2-6.6 group exhibited a Δ Hion value of 8.4 kcal/mol, typical of histidine. The pH-rate profile of inactivation by iodoacetamide confirmed that the catalytic cysteine possesses a p Ka of 9.8. Normal solvent kinetic isotope effects were observed for both D2O kcat (1.6-2.1) and D2O kcat/ Km (1.1-1.4) for all three substrates. Pre-steady-state kinetics revealed exponential bursts of AMC production for Cbz-Phe-Arg-AMC and Cbz-Arg-Arg-AMC, but not for Cbz-Arg-Ala-AMC. The overall solvent isotope effect on kcat can be attributed to the solvent isotope effect on the deacylation step. Our results suggest that cruzain is unique among papain-like cysteine proteases in that the catalytic cysteine and histidine have neutral charges in the free enzyme. The generation of the active thiolate of the catalytic cysteine is likely preceded (and possibly triggered) by a ligand-induced conformational change, which could bring the catalytic dyad into the proximity to effect proton transfer.

Binding synergy as an essential step for tRNA editing and modification enzyme codependence in Trypanosoma brucei.

Transfer RNAs acquire a variety of naturally occurring chemical modifications during their maturation; these fine-tune their structure and decoding properties in a manner critical for protein synthesis. We recently reported that in the eukaryotic parasite, Trypanosoma brucei, a methylation and deamination event are unexpectedly interconnected, whereby the tRNA adenosine deaminase (TbADAT2/3) and the 3-methylcytosine methyltransferase (TbTrm140) strictly rely on each other for activity, leading to formation of m3C and m3U at position 32 in several tRNAs. Still however, it is not clear why these two enzymes, which work independently in other systems, are strictly codependent in T. brucei Here, we show that these enzymes exhibit binding synergism, or a mutual increase in binding affinity, that is more than the sum of the parts, when added together in a reaction. Although these enzymes interact directly with each other, tRNA binding assays using enzyme variants mutated in critical binding and catalytic sites indicate that the observed binding synergy stems from contributions from tRNA-binding domains distal to their active sites. These results provide a rationale for the known interactions of these proteins, while also speaking to the modulation of substrate specificity between seemingly unrelated enzymes. This information should be of value in furthering our understanding of how tRNA modification enzymes act together to regulate gene expression at the post-transcriptional level and provide a basis for the interdependence of such activities.

Loss of FrmB results in increased size of developmental structures during the multicellular development of Dictyostelium cells.

FERM domain-containing proteins are involved in diverse biological and pathological processes, including cell-substrate adhesion, cell-cell adhesion, multicellular development, and cancer metastasis. In this study, we determined the functions of FrmB, a FERM domain-containing protein, in the cell morphology, cell adhesion, and multicellular development of Dictyostelium cells. Our results show that FrmB appears to play an important role in regulating the size of developmental structures. frmB null cells showed prolonged aggregation during development, resulting in increased size of developmental structures, such as mounds and fruiting bodies, compared to those of wild-type cells, whereas FrmB overexpressing cells exhibited decreased size of developmental structures. These results suggest that FrmB may be necessary for limiting the sizes of developmental structures. Loss of FrmB also resulted in decreased cell-substrate adhesion and slightly increased cell area, suggesting that FrmB had important roles in the regulation of cell adhesion and cell morphology. These studies would contribute to our understanding of the intertwined and overlapped functions of FERM domain-containing proteins.

Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ-mediated host defenses.

An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii-targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ-stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1(+) inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism.

HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.

HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics.

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.

Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis.

Palmitoylation is the post-translational reversible addition of the acyl moiety, palmitate, to cysteine residues of proteins and is involved in regulating protein trafficking, localization, stability and function. The Aspartate-Histidine-Histidine-Cysteine (DHHC) protein family, named for their highly conserved DHHC signature motif, is thought to be responsible for catalysing protein palmitoylation. Palmitoylation is widespread in all eukaryotes, including the malaria parasite, Plasmodium falciparum, where over 400 palmitoylated proteins are present in the asexual intraerythrocytic schizont stage parasites, including proteins involved in key aspects of parasite maturation and development. The P. falciparum genome includes 12 proteins containing the conserved DHHC motif. In this study, we adapted a palmitoyl-transferase activity assay for use with P. falciparum proteins and demonstrated for the first time that P. falciparum DHHC proteins are responsible for the palmitoylation of P. falciparum substrates. This assay also reveals that multiple DHHCs are capable of palmitoylating the same substrate, indicating functional redundancy at least in vitro. To test whether functional redundancy also exists in vivo, we investigated the endogenous localization and essentiality of a subset of schizont-expressed PfDHHC proteins. Individual PfDHHC proteins localized to distinct organelles, including parasite-specific organelles such as the rhoptries and inner membrane complex. Knock-out studies identified individual DHHCs that may be essential for blood-stage growth and others that were functionally redundant in the blood stages but may have functions in other stages of parasite development. Supporting this hypothesis, disruption of PfDHHC9 had no effect on blood-stage growth but reduced the formation of gametocytes, suggesting that this protein could be exploited as a transmission-blocking target. The localization and stage-specific expression of the DHHC proteins may be important for regulating their substrate specificity and thus may provide a path for inhibitor development.