KCC1 Activation protects Mice from the Development of Experimental Cerebral Malaria

Plasmodium falciparum malaria causes half a million deaths per year, with up to 9% of this mortality caused by cerebral malaria (CM). One of the major processes contributing to the development of CM is an excess of host inflammatory cytokines. Recently K+ signaling has emerged as an important mediator of the inflammatory response to infection; we therefore investigated whether mice carrying an ENU induced activation of the electroneutral K+ channel KCC1 had an altered response to Plasmodium berghei. Here we show that KCC1(M935K/M935K )mice are protected from the development of experimental cerebral malaria, and that this protection is associated with an increased CD4+ and TNFa response. This is the first description of a K+ channel affecting the development of experimental cerebral malaria.We would like to acknowledge Shelley Lampkin and Australian Phenomics Facility (APF) for the maintenance of the mouse colonies. Tis study was funded by the National Health and Medical Research Council of Australia (Program Grant 490037, and Project Grants 605524 and APP1047090), the National Collaborative Research Infrastructure Strategy (NCRIS), the Education Investment Fund from the Department of Education and Training, the Australian Phenomics Network, Howard Hughes Medical Institute and the Bill and Melinda Gates Foundation

Bone marrow transplantation corrects haemolytic anaemia in a novel ENU mutagenesis mouse model of TPI deficiency

In this study, we performed a genome-wide N-ethyl-N-mtrosourea (ENU) mutagenesis screen in mice to identify novel genes or alleles that regulate erythropoiesis. Here, we describe a recessive mouse strain, called RBC19, harbouring a point mutation within the housekeeping gene, Tpi1, which encodes the glycolysis enzyme, triosephosphate isomerase (TPI). A serine in place of a phenylalanine at amino acid 57 severely diminishes enzyme activity in red blood cells and other tissues, resulting in a macrocytic haemolytic phenotype in homozygous mice, which closely resembles human TPI deficiency. A rescue study was performed using bone marrow transplantation of wild-type donor cells, which restored all haematological parameters and increased red blood cell enzyme function to wild-type levels after 7 weeks. This is the first study performed in a mammalian model of TPI deficiency, demonstrating that the haematological phenotype can be rescued.d the Sylvia and Charles Viertel Charitable Foundation (Senior Medical Research Fellowship to D.J.C.

Rough and smooth variants of Mycobacterium abscessus are differentially controlled by host immunity during chronic infection of adult zebrafish.

Prevalence of Mycobacterium abscessus infections is increasing in patients with respiratory comorbidities. After initial colonisation, M. abscessus smooth colony (S) variants can undergo an irreversible genetic switch into highly inflammatory, rough colony (R) variants, often associated with a decline in pulmonary function. Here, we use an adult zebrafish model of chronic infection with R and S variants to study M. abscessus pathogenesis in the context of fully functioning host immunity. We show that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signalling, mediated by the tnfa, tnfr1 and tnfr2 gene products. T cell-dependent immunity is stronger against the R variant early in infection, and regulatory T cells associate with R variant granulomas and limit bacterial growth. In comparison, an S variant proliferates to high burdens but appears to be controlled by TNF-dependent innate immunity early during infection, resulting in delayed granuloma formation. Thus, our work demonstrates the applicability of adult zebrafish to model persistent M. abscessus infection, and illustrates differences in the immunopathogenesis induced by R and S variants during granulomatous infection

ENU mutagenesis and the quest for a malaria host-directed therapy

Empirical thesis.Bibliography: pages 129-152.Chapter 1. Literature review -- Chapter 2. Experimental procedures -- Chapter 3. MR149372 blood phenotype -- Chapter 4. MR149372 malaria -- Chapter 5. RBC10 -- Chapter 6. Final discussion and conclusions.Malaria is a disease of global concern, which causes over 800,000 deaths every year. Due to the rapid emergence of parasite drug resistance, ongoing control of malaria requires the urgent development of new anti-malarials that will have a prolonged life span. Genetic polymorphisms that provide natural resistance to malaria have existed in human populations for thousands of years without losing their efficacy. This suggests a possible new treatment strategy; drugs can be used to mimic the protective effects of such polymorphisms, in what is known as host-directed therapy (HDT). It is hoped that HDT will be able to effectively treat malaria without driving drug resistance. An ENU mutagenesis screen was established in mice to identify novel genes mediating host resistance to malaria, and to thereby uncover potential drug targets for an anti-malarial HDT. Two mutant mouse lines were investigated: MR149372, in which an over-activation of the enzyme AMPD3 causes striking resemblance to P. Chabaudi parasitaemia through high RBC turnover; and RBC10, in which an over-activation of the transporter KCC1 causes resistance to the development of cerebral malaria and modulation of the inflammatory response during P. berghei infection.Through the work in this thesis, novel insight has been gained into the role of AMPD3 in determining red blood cell half-­‐life, the effect of altered purine balance on intra-­erythrocytic parasite growth, and the host factors affecting the immune response to malaria infection. Moreover, potential novel drug targets have been identified both for lengthening the life span of blood stored for transfusion, and for treating cerebral malaria.Mode of access: World wide web1 online resource (xiv, 163 pages) illustrations (some colour

A zebrafish model of tuberculosis comorbidity and the effects of HIF‐activating intervention

Comorbidities are an important factor in tuberculosis pathophysiology and treatment but are understudied in animal models. Schild et al. present a zebrafish model of Mycobacterium marinum infection and wound comorbidity that retains responsiveness to protective hypoxia-inducible factor-1α activation as an example of a host-directed therapy. This platform is a new paradigm for the zebrafish-M. marinum infection model and provides a blueprint to test therapeutic interventions on infection and comorbid pathologies. Comment on: https://doi.org/10.1111/febs.15433

Directional surface plasmon coupled chemiluminescence from nickel thin films : fixed angle observation

Directional surface plasmon coupled chemiluminescence (SPCC) from nickel thin films is demonstrated. Free-space and angular-dependent SPCC emission from blue, green and turquoise chemiluminescent solutions placed onto nickel thin films attached to a hemispherical prism were measured. SPCC emission was found to be highly directional and preferentially p-polarized, in contrast to the unpolarized and isotropic chemiluminescence emission. The largest SPCC emission for all chemiluminescence solutions was observed at a fixed observation angle of 60°, which was also predicted by theoretical Fresnel calculations. It was found that nickel thin films did not have a catalytic effect on chemiluminescence emission.6 page(s

Surface plasmon coupled chemiluminescence from iron thin films : directional and approaching fixed angle observation

We report the observation of surface plasmon coupled chemiluminescence (SPCC) from ironthin films. Theoretical Fresnel calculations were employed to determine the optimum thickness of ironthin films to be 15 nm, which is assessed by the value of minimum reflectivity curves for incident light upon the thin film. An overlayer 10 nm thick SiO₂ was used to protect the ironthin films from oxidation by air or physical changes induced by chemiluminescent solutions. SPCC emission from blue, green, and chartreuse chemiluminescent solutions on ironfilms can be observed at a fixed angle of 60° for all the chemiluminescent solutions, while free-space emission was isotropic. Ironthin films do not have a catalytic effect on chemiluminescence emission.5 page(s

Fixed-angle observation of surface plasmon coupled chemiluminescence from palladium thin films

In this letter, the fixed-angle observation of surface plasmon coupled chemiluminescence (SPCC) from palladiumthin films is reported. Fresnel calculations predict that light 492–549 nm spectral range can efficiently induce surface plasmon modes in 15 nm palladiumthin films. Free-space emission from blue, green, and chartreuse chemiluminescent solutions on palladiumthin films was isotropic, while the SPCC emission was highly directional and predominantly p-polarized in accordance with the predictions of the Fresnel calculations. In addition, the decay rates of the SPCC and free-space emission were similar, which suggests that palladiumthin films have no catalytic effect on chemiluminescence emission.3 page(s

Mycobacterium marinum infection drives foam cell differentiation in zebrafish infection models

Host lipid metabolism is an important target for subversion by pathogenic mycobacteria such as Mycobacterium tuberculosis. The appearance of foam cells within the granuloma are well-characterised effects of chronic tuberculosis. The zebrafish-Mycobacterium marinum infection model recapitulates many aspects of human-M. tuberculosis infection and is used as a model to investigate the structural components of the mycobacterial granuloma. Here, we demonstrate that the zebrafish-M. marinum granuloma contains foam cells and that the transdifferentiation of macrophages into foam cells is driven by the mycobacterial ESX1 pathogenicity locus. This report demonstrates conservation of an important aspect of mycobacterial infection across species.This work was supported by the Australian National Health and Medical Research Council (APP1099912 and APP1053407 to S.H.O.); Meat and Livestock Australia (P.PSH. 0813 to A.C.P.); the Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney (grant to S.H.O. and A.C.P.); University of Sydney Fellowship (grant to S.H.O.); and the Kenyon Family Inflammation Award (grant to S.H.O.)

The cyclic nitroxide antioxidant 4-methoxy-TEMPO decreases mycobacterial burden in vivo through host and bacterial targets

Tuberculosis is a chronic inflammatory disease caused by persistent infection with Mycobacterium tuberculosis. The rise of antibiotic resistant strains necessitates the design of novel treatments. Recent evidence shows that not only is M. tuberculosis highly resistant to oxidative killing, it also co-opts host oxidant production to induce phagocyte death facilitating bacterial dissemination. We have targeted this redox environment with the cyclic nitroxide derivative 4-methoxy-TEMPO (MetT) in the zebrafish-M. marinum infection model. MetT inhibited the production of mitochondrial ROS and decreased infection-induced cell death to aid containment of infection. We identify a second mechanism of action whereby stress conditions, including hypoxia, found in the infection microenvironment appear to sensitise M. marinum to killing by MetT both in vitro and in vivo. Together, our study demonstrates MetT inhibited the growth and dissemination of M. marinum through host and bacterial targets.This work was supported by the Australian National Health and Medical Research Council grants APP1099912 and APP1053407; The University of Sydney, Australia Fellowship G197581; NSW Ministry of Health, Australia under the NSW Health Early-Mid Career Fellowships Scheme H18/31086; and the Kenyon Family Inflammation Award (S.H.O.)