Application of crispr/cas9-based reverse genetics in leishmania braziliensis: Conserved roles for hsp100 and hsp23

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23-and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. major HSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.Alexander von Humboldt-StiftungRevisión por pare

Altered Cytokine Response of Human Brain Endothelial Cells after Stimulation with Malaria Patient Plasma

Infections with the deadliest malaria parasite, Plasmodium falciparum, are accompanied by a strong immunological response of the human host. To date, more than 30 cytokines have been detected in elevated levels in plasma of malaria patients compared to healthy controls. Endothelial cells (ECs) are a potential source of these cytokines, but so far it is not known if their cytokine secretion depends on the direct contact of the P. falciparum-infected erythrocytes (IEs) with ECs in terms of cytoadhesion. Culturing ECs with plasma from malaria patients (27 returning travellers) resulted in significantly increased secretion of IL-11, CXCL5, CXCL8, CXCL10, vascular endothelial growth factor (VEGF) and angiopoietin-like protein 4 (ANGPTL4) if compared to matching controls (22 healthy individuals). The accompanying transcriptome study of the ECs identified 43 genes that were significantly increased in expression (≥1.7 fold) after co-incubation with malaria patient plasma, including cxcl5 and angptl4. Further bioinformatic analyses revealed that biological processes such as cell migration, cell proliferation and tube development were particularly affected in these ECs. It can thus be postulated that not only the cytoadhesion of IEs, but also molecules in the plasma of malaria patients exerts an influence on ECs, and that not only the immunological response but also other processes, such as angiogenesis, are altered

Male offspring born to mildly ZIKV-infected mice are at risk of developing neurocognitive disorders in adulthood

Congenital Zika virus (ZIKV) syndrome may cause fetal microcephaly in -1% of affected newborns. Here, we investigate whether the majority of clinically inapparent newborns might suffer from long-term health impairments not readily visible at birth. Infection of immunocompetent pregnant mice with high-dose ZIKV caused severe offspring phenotypes, such as fetal death, as expected. By contrast, low-dose (LD) maternal ZIKV infection resulted in reduced fetal birth weight but no other obvious phenotypes. Male offspring born to LD ZIKV-infected mothers had increased testosterone (TST) levels and were less likely to survive in utero infection compared to their female littermates. Males also presented an increased number of immature neurons in apical and basal hippocampal dendrites, while female offspring had immature neurons in basal dendrites only. Moreover, male offspring with high but not very high (storm) TST levels were more likely to suffer from learning and memory impairments compared to females. Future studies are required to understand the impact of TST on neuropathological and neurocognitive impairments in later life. In summary, increased sex-specific vigilance is required in countries with high ZIKV prevalence, where impaired neurodevelopment may be camouflaged by a healthy appearance at birth.Peer reviewe

Monocyte dysregulation: consequences for hepatic infections

Liver disorders due to infections are a substantial health concern in underdeveloped and industrialized countries. This includes not only hepatotropic viruses (e.g., hepatitis B, hepatitis C) but also bacterial and parasitic infections such as amebiasis, leishmaniasis, schistosomiasis, or echinococcosis. Recent studies of the immune mechanisms underlying liver disease show that monocytes play an essential role in determining patient outcomes. Monocytes are derived from the mononuclear phagocyte lineage in the bone marrow and are present in nearly all tissues of the body; these cells function as part of the early innate immune response that reacts to challenge by external pathogens. Due to their special ability to develop into tissue macrophages and dendritic cells and to change from an inflammatory to an anti-inflammatory phenotype, monocytes play a pivotal role in infectious and non-infectious liver diseases: they can maintain inflammation and support resolution of inflammation. Therefore, tight regulation of monocyte recruitment and termination of monocyte-driven immune responses in the liver is prerequisite to appropriate healing of organ damage. In this review, we discuss monocyte-dependent immune mechanisms underlying hepatic infectious disorders. Better understanding of these immune mechanisms may lead to development of new interventions to treat acute liver disease and prevent progression to organ failure

Myeloperoxidase Attenuates Pathogen Clearance during Plasmodium yoelii Nonlethal Infection

Myeloperoxidase (MPO), a leukocyte-derived enzyme mainly secreted by activated neutrophils, is known to be involved in the immune response during bacterial and fungal infection and inflammatory diseases. Nevertheless, the role of MPO in a parasitic disease like malaria is unknown. We hypothesized that MPO contributes to parasite clearance. To address this hypothesis, we used Plasmodium yoelii nonlethal infection in wild-type and MPO-deficient mice as a murine malaria model. We detected high MPO plasma levels in wild-type mice with Plasmodium yoelii infection. Unexpectedly, infected MPO-deficient mice did not show increased parasite loads but were able to clear the infection more rapidly than wild-type mice. Additionally, the presence of neutrophils at the onset of infection seemed not to be essential for the control of the parasitemia. The effect of decreased parasite levels in MPO-deficient mice was absent from animals lacking mature T and B cells, indicating that this effect is most likely dependent on adaptive immune response mechanisms. Indeed, we observed increased gamma interferon and tumor necrosis factor alpha production by T cells in infected MPO-deficient mice. Together, these results suggest that MPO modulates the adaptive immune response during malaria infection, leading to an attenuated parasite clearance

Data for: Persistent hepatitis B virus and HIV coinfections in dually humanized mice engrafted with human liver and immune system

Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), remains a major medical problem. HBV has a high propensity for progressing to chronicity and can result in severe liver disease, including fibrosis, cirrhosis and hepatocellular carcinoma. CHB patients frequently present with viral coinfection, including HIV and hepatitis delta virus. About 10% of chronic HIV carriers are also persistently infected with HBV which can result in more exacerbated liver disease. Mechanistic studies of HBV-induced immune responses and pathogenesis, which could be significantly influenced by HIV infection, have been hampered by the scarcity of immunocompetent animal models. Here, we demonstrate that humanized mice dually engrafted with components of a human immune system and a human liver supported HBV infection, which was partially controlled by human immune cells, as evidenced by lower levels of serum viremia and HBV replication intermediates in the liver. HBV infection resulted in priming and expansion of human HLA-restricted CD8+ T cells, which acquired an activated phenotype. Notably, our dually humanized mice support persistent coinfections with HBV and HIV which opens opportunities for analyzing immune dysregulation during HBV and HIV coinfection and preclinical testing of novel immunotherapeutics.This study was supported by grants from the National Institutes of Health (R01 AI138797 to A.P. and L.S., R01 AI153236, R01 AI146917, R01 AI168048 all to A.P), a Research Scholar Award from the American Cancer Society (RSG-15-048-01-MPC to A.P.), a Burroughs Wellcome Fund Award for Investigators in Pathogenesis (to A.P.) a Graduate fellowship from the Health Grand Challenge from the Global Health Fund of Princeton University (to B.Y.W.). The NYU Experimental Pathology Immunohistochemistry Core Laboratory is supported in part by the Laura and Isaac Perlmutter Cancer Center Support Grant; NIH /NCI P30CA016087” and the National Institutes of Health S10 Grants; NIH/ORIP S10OD01058 and S10OD018338. B.Y.W. was a recipient of F31 NIH/NRSA Ruth L. Kirschstein Predoctoral awarded from the NIAID and a graduate fellowship from the New Jersey Commission on Cancer Research and was supported by an NIH training grant (T32GM007388). J.S. was a recipient of a postdoctoral fellowships from the German Research Foundation.Excel spreadsheets with all the raw data for the main figures 1-7 and supplementary figures 1&2; readme fil

Application of CRISPR/Cas9-based reverse genetics in Leishmania braziliensis : conserved roles for HSP100 and HSP23

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. majorHSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis

High Content Analysis of Macrophage-Targeting <i>Eh</i>PIb-Compounds against Cutaneous and Visceral <i>Leishmania</i> Species

An immunostimulatory glycolipid molecule from the intestinal protozoan parasite Entamoeba histolytica (Eh) and its synthetic analogs derived from its phosphatidylinositol-b-anchor (EhPIb) previously showed considerable immunotherapeutic effects against Leishmania major infection in vitro and in vivo. Here, we describe a high content screening assay, based on primary murine macrophages. Parasites detection is based on a 90 kDA heat shock protein-specific staining, enabling the detection of several Leishmania species. We validated the assay using L. major, L. braziliensis, L. donovani, and L. infantum as well as investigated the anti-leishmanial activity of six immunostimulatory EhPIb-compounds (Eh-1 to Eh-6). Macrophages infected with dermotropic species were more sensitive towards treatment with the compounds as their viability showed a stronger reduction compared to macrophages infected with viscerotropic species. Most compounds caused a significant reduction of the infection rates and the parasite burdens depending on the infecting species. Only compound Eh-6 was found to have activity against all Leishmania species. Considering the challenges in anti-leishmanial drug discovery, we developed a multi-species screening assay capable of utilizing non-recombinant parasite strains, and demonstrated its usefulness by screening macrophage-targeting EhPIb-compounds showing their potential for the treatment of cutaneous and visceral leishmaniasis

Identification of Plasmodium falciparum proteoforms from liver stage models.

Immunization with attenuated malaria sporozoites protects humans from experimental malaria challenge by mosquito bite. Protection in humans is strongly correlated with the production of T cells targeting a heterogeneous population of pre-erythrocyte antigen proteoforms, including liver stage antigens. Currently, few T cell epitopes derived from Plasmodium falciparum, the major aetiologic agent of malaria in humans are known. In this study both in vitro and in vivo malaria liver stage models were used to sequence host and pathogen proteoforms. Proteoforms from these diverse models were subjected to mild acid elution (of soluble forms), multi-dimensional fractionation, tandem mass spectrometry, and top-down bioinformatics analysis to identify proteoforms in their intact state. These results identify a group of host and malaria liver stage proteoforms that meet a 5% false discovery rate threshold. This work provides proof-of-concept for the validity of this mass spectrometry/bioinformatic approach for future studies seeking to reveal malaria liver stage antigens towards vaccine development

Androgens predispose males to monocyte-mediated immunopathology by inducing the expression of leukocyte recruitment factor CXCL1

Altered monocyte responses and testosterone levels correlate, individually, with the pathogenesis of hepatic amebiasis in mice. Here the authors show that testosterone induces enhanced TNF/CXCL1 expression and stronger proinflammatory responses in both human and mouse monocytes to support an androgen-monocyte axis of inflammation regulation