Juliana Alves-Silva

The Ancestry of Brazilian mtDNA Lineages

We have analyzed 247 Brazilian mtDNAs for hypervariable segment (HVS)-I and selected restriction fragment-length-polymorphism sites, to assess their ancestry in different continents. The total sample showed nearly equal amounts of Native American, African, and European matrilineal genetic contribution but with regional differences within Brazil. The mtDNA pool of present-day Brazilians clearly reflects the imprints of the early Portuguese colonization process (involving directional mating), as well as the recent immigrant waves (from Europe) of the last century. The subset of 99 mtDNAs from the southeastern region encompasses nearly all mtDNA haplogroups observed in the total Brazilian sample; for this regional subset, HVS-II was analyzed, providing, in particular, some novel details of the African mtDNA phylogeny.

The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles.

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1‐HA in HEK 293 cells establishes Na+‐dependent, hemicholinium‐3 sensitive high‐affinity choline transport activity. Confocal microscopy reveals that CHT1‐HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1‐HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin‐mediated endocytic pathway. In a neuronal cell line, CHT1‐HA colocalizes with the early endocytic marker green fluorescent protein (GFP)‐Rab 5 and with two markers of synaptic‐like vesicles, VAMP‐myc and GFP‐VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.

Trypanosomes Modify the Behavior of Their Insect Hosts: Effects on Locomotion and on the Expression of a Related Gene

Background As a result of evolution, the biology of triatomines must have been significantly adapted to accommodate trypanosome infection in a complex network of vector-vertebrate-parasite interactions. Arthropod-borne parasites have probably developed mechanisms, largely still unknown, to exploit the vector-vertebrate host interactions to ensure their transmission to suitable hosts. Triatomines exhibit a strong negative phototaxis and nocturnal activity, believed to be important for insect survival against its predators. Methodology/Principal Findings In this study we quantified phototaxis and locomotion in starved fifth instar nymphs of Rhodnius prolixus infected with Trypanosoma cruzi or Trypanosoma rangeli. T. cruzi infection did not alter insect phototaxis, but induced an overall 20% decrease in the number of bug locomotory events. Furthermore, the significant differences induced by this parasite were concentrated at the beginning of the scotophase. Conversely, T. rangeli modified both behaviors, as it significantly decreased bug negative phototaxis, while it induced a 23% increase in the number of locomotory events in infected bugs. In this case, the significant effects were observed during the photophase. We also investigated the expression of Rpfor, the triatomine ortholog of the foraging gene known to modulate locomotion in other insects, and found a 4.8 fold increase for T. rangeli infected insects. Conclusions/Significance We demonstrated for the first time that trypanosome infection modulates the locomotory activity of the invertebrate host. T. rangeli infection seems to be more broadly effective, as besides affecting the intensity of locomotion this parasite also diminished negative phototaxis and the expression of a behavior-associated gene in the triatomine vector.

Zika Virus Promotes Neuronal Cell Death in a Non-Cell Autonomous Manner by Triggering the Release of Neurotoxic Factors

Zika virus (ZIKV) has recently caused a worldwide outbreak of infections associated with severe neurological complications, including microcephaly in infants born from infected mothers. ZIKV exhibits high neurotropism and promotes neuroinflammation and neuronal cell death. We have recently demonstrated that N-methyl-d-aspartate receptor (NMDAR) blockade by memantine prevents ZIKV-induced neuronal cell death. Here, we show that ZIKV induces apoptosis in a non-cell autonomous manner, triggering cell death of uninfected neurons by releasing cytotoxic factors. Neuronal cultures infected with ZIKV exhibit increased levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and glutamate. Moreover, infected neurons exhibit increased expression of GluN2B and augmented intracellular Ca2+ concentration. Blockade of GluN2B-containing NMDAR by ifenprodil normalizes Ca2+ levels and rescues neuronal cell death. Notably, TNF-α and IL-1β blockade decreases ZIKV-induced Ca2+ flux through GluN2B-containing NMDARs and reduces neuronal cell death, indicating that these cytokines might contribute to NMDAR sensitization and neurotoxicity. In addition, ZIKV-infected cultures treated with ifenprodil exhibits increased activation of the neuroprotective pathway including extracellular signal-regulated kinase and cAMP response element-binding protein, which may underlie ifenprodil-mediated neuroprotection. Together, our data shed some light on the neurotoxic mechanisms triggered by ZIKV and begin to elucidate how GluN2B-containing NMDAR blockade can prevent neurotoxicity.

Sm10.3, a Member of the Micro-Exon Gene 4 (MEG-4) Family, Induces Erythrocyte Agglutination In Vitro and Partially Protects Vaccinated Mice against Schistosoma mansoni Infection

Background The parasitic flatworm Schistosoma mansoni is a blood fluke that causes schistosomiasis. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Numerous antigens that are expressed at the interface between the parasite and the mammalian host have been assessed. Among the most promising molecules are the proteins present in the tegument and digestive tract of the parasite. Methodology/Principal Findings In this study, we evaluated the potential of Sm10.3, a member of the micro-exon gene 4 (MEG-4) family, for use as part of a recombinant vaccine. We confirmed by real-time PCR that Sm10.3 was expressed at all stages of the parasite life cycle. The localization of Sm10.3 on the surface and lumen of the esophageal and intestinal tract in adult worms and lung-stage schistosomula was confirmed by confocal microscopy. We also show preliminary evidence that rSm10.3 induces erythrocyte agglutination in vitro. Immunization of mice with rSm10.3 induced a mixed Th1/Th2-type response, as IFN-γ, TNF-α, and low levels of IL-5 were detected in the supernatant of cultured splenocytes. The protective effect conferred by vaccination with rSm10.3 was demonstrated by 25.5–32% reduction in the worm burden, 32.9–43.6% reduction in the number of eggs per gram of hepatic tissue, a 23.8% reduction in the number of granulomas, an 11.8% reduction in the area of the granulomas and a 39.8% reduction in granuloma fibrosis. Conclusions/Significance Our data suggest that Sm10.3 is a potential candidate for use in developing a multi-antigen vaccine to control schistosomiasis and provide the first evidence for a possible role for Sm10.3 in the blood feeding process.

Identification in Portugal and Brazil of a mtDNA Lineage Containing a 9-bp Triplication of the Intergenic COII/tRNALys Region

Although the deletion of one of the 9-bp repeats in region V of mitochondrial DNA is very common in Asians, Asian-derived populations and Africans, the triplication of the 9-bp segment was described only a few times, mostly on individuals from Asian origin. Here, we report for the first time the presence of the 9-bp triplication in Europeans. The triplication was initially found in one Brazilian individual. Sequencing of the hypervariable segments I (HVSI) and II (HVS2) of the control region and RFLP analysis of the coding region classified the mtDNA as belonging to the European haplogroup H. Since white Brazilians are predominantly of Portuguese descent, we screened 96 unrelated Northern Portuguese for the 9-bp triplication and found its presence in two of them (2.1%). One of these had an mtDNA haplotype identical to that of the Brazilian individual, while the other differed in a single base change in HVS2. The fact that the 9-bp triplication has reached polymorphic frequencies in Northern Portugal and that it has apparently differentiated into at least two lineages defined by the mutuation in HVS2 suggests that it probably occurred a long time ago.

Mutant Brucella abortus Membrane Fusogenic Protein Induces Protection against Challenge Infection in Mice

ABSTRACT Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies.

Brucella abortus Triggers a cGAS-Independent STING Pathway to Induce Host Protection That Involves Guanylate-Binding Proteins and Inflammasome Activation

Immunity against microbes depends on recognition of pathogen-associated molecular patterns by innate receptors. Signaling pathways triggered by Brucella abortus DNA involves TLR9, AIM2, and stimulator of IFN genes (STING). In this study, we observed by microarray analysis that several type I IFN–associated genes, such as IFN-β and guanylate-binding proteins (GBPs), are downregulated in STING knockout (KO) macrophages infected with Brucella or transfected with DNA. Additionally, we determined that STING and cyclic GMP–AMP synthase (cGAS) are important to engage the type I IFN pathway, but only STING is required to induce IL-1β secretion, caspase-1 activation, and GBP2 and GBP3 expression. Furthermore, we determined that STING but not cGAS is critical for host protection against Brucella infection in macrophages and in vivo. This study provides evidence of a cGAS-independent mechanism of STING-mediated protection against an intracellular bacterial infection. Additionally, infected IFN regulatory factor-1 and IFNAR KO macrophages had reduced GBP2 and GBP3 expression and these cells were more permissive to Brucella replication compared with wild-type control macrophages. Because GBPs are critical to target vacuolar bacteria, we determined whether GBP2 and GBPchr3 affect Brucella control in vivo. GBPchr3 but not GBP2 KO mice were more susceptible to bacterial infection, and small interfering RNA treated–macrophages showed reduction in IL-1β secretion and caspase-1 activation. Finally, we also demonstrated that Brucella DNA colocalizes with AIM2, and AIM2 KO mice are less resistant to B. abortus infection. In conclusion, these findings suggest that the STING-dependent type I IFN pathway is critical for the GBP-mediated release of Brucella DNA into the cytosol and subsequent activation of AIM2.

Modulation of Microtubule Dynamics Affects Brucella abortus Intracellular Survival, Pathogen-Containing Vacuole Maturation, and Pro-inflammatory Cytokine Production in Infected Macrophages

The microtubule (MT) cytoskeleton regulates several cellular processes related to the immune system. For instance, an intricate intracellular transport mediated by MTs is responsible for the proper localization of vesicular receptors of innate immunity and its adaptor proteins. In the present study, we used nocodazole to induce MTs depolymerization and paclitaxel or recombinant (r) TIR (Toll/interleukin-1 receptor) domain containing protein (TcpB) to induce MT stabilization in bone marrow-derived macrophages infected with Brucella abortus. Following treatment of the cells, we evaluated their effects on pathogen intracellular replication and survival, and in pro-inflammatory cytokine production. First, we observed that intracellular trafficking and maturation of Brucella-containing vesicles (BCVs) is affected by partial destabilization or stabilization of the MTs network. A typical marker of early BCVs, LAMP-1, is retained in late BCVs even 24 h after infection in the presence of low doses of nocodazole or paclitaxel and in the presence of different amounts of rTcpB. Second, microscopy and colony forming unit analysis revealed that bacterial load was increased in infected macrophages treated with lower doses of nocodazole or paclitaxel and with rTcpB compared to untreated cells. Third, innate immune responses were also affected by disturbing MT dynamics. MT depolymerization by nocodazole reduced IL-12 production in infected macrophages. Conversely, rTcpB-treated cells augmented IL-12 and IL-1β secretion in infected cells. In summary, these findings demonstrate that modulation of MTs affects several crucial steps of B. abortus pathogenesis, including BCV maturation, intracellular survival and IL-12 secretion in infected macrophages.

Orchestrated activation of mGluR5 and CB1 promotes neuroprotection.

The metabotropic glutamate receptor 5 (mGluR5) and the cannabinoid receptor 1 (CB1) exhibit a functional interaction, as CB1 regulates pre-synaptic glutamate release and mGluR5 activation increases endocannabinoid synthesis at the post-synaptic site. Since both mGluR5 and CB1 promote neuroprotection, we delineated experiments to investigate a possible link between CB1 and mGluR5 activation in the induction of neuroprotection using primary cultured corticostriatal neurons. We find that either the pharmacological blockade or the genetic ablation of either mGluR5 or CB1 can abrogate both CB1- and mGluR5-mediated neuroprotection against glutamate insult. Interestingly, decreased glutamate release and diminished intracellular Ca2+ do not appear to play a role in CB1 and mGluR5-mediated neuroprotection. Rather, these two receptors work cooperatively to trigger the activation of cell signaling pathways to promote neuronal survival, which involves MEK/ERK1/2 and PI3K/AKT activation. Interestingly, although mGluR5 activation protects postsynaptic terminals and CB1 the presynaptic site, intact signaling of both receptors is required to effectively promote neuronal survival. In conclusion, mGluR5 and CB1 act in concert to activate neuroprotective cell signaling pathways and promote neuronal survival.