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Proteasomal Control of Cytokinin Synthesis Protects Mycobacterium tuberculosis against Nitric Oxide
Samanovic, Marie I; Tu, Shengjiang; Novak, Ondrej; Iyer, Lakshminarayan M; McAllister, Fiona E; Aravind, L; Gygi, Steven P; Hubbard, Stevan R; Strnad, Miroslav; Darwin, K Heran
One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homolog of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO.
PMCID:4369403
PMID: 25728768
ISSN: 1097-4164
CID: 1520722
The copper-responsive RicR regulon contributes to Mycobacterium tuberculosis virulence
Shi, Xiaoshan; Festa, Richard A; Ioerger, Thomas R; Butler-Wu, Susan; Sacchettini, James C; Darwin, K Heran; Samanovic, Marie I
As with most life on Earth, the transition metal copper (Cu) is essential for the viability of the human pathogen Mycobacterium tuberculosis. However, infected hosts can also use Cu to control microbial growth. Several Cu-responsive pathways are present in M. tuberculosis, including the regulated in copper repressor (RicR) regulon, which is unique to pathogenic mycobacteria. In this work, we describe the contribution of each RicR-regulated gene to Cu resistance in vitro and to virulence in animals. We found that the deletion or disruption of individual RicR-regulated genes had no impact on virulence in mice, although several mutants had Cu hypersensitivity. In contrast, a mutant unable to activate the RicR regulon was not only highly susceptible to Cu but also attenuated in mice. Thus, these data suggest that several genes of the RicR regulon are required simultaneously to combat Cu toxicity in vivo or that this regulon is also important for resistance against Cu-independent mechanisms of host defense. IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, killing millions of people every year. Therefore, understanding the biology of M. tuberculosis is crucial for the development of new therapies to treat this devastating disease. Our studies reveal that although host-supplied Cu can suppress bacterial growth, M. tuberculosis has a unique pathway, the RicR regulon, to defend against Cu toxicity. These findings suggest that Cu homeostasis pathways in both the host and the pathogen could be exploited for the treatment of tuberculosis.
PMCID:3944814
PMID: 24549843
ISSN: 2150-7511
CID: 953442
Activity of trypanosome lytic factor: a novel component of innate immunity
Thomson, Russell; Samanovic, Marie; Raper, Jayne
Trypanosome lytic factors (TLFs) are high-density lipoproteins and components of primate innate immunity. TLFs are characterized by their ability to kill extracellular protozoon parasites of the genus Trypanosoma. Two subspecies of Trypanosoma brucei have evolved resistance to TLFs and can consequently infect humans, resulting in the disease African sleeping sickness. The unique protein components of TLFs are a hemoglobin-binding protein, haptoglobin-related protein and a pore-forming protein, apoL-I. The recent advances in our understanding of the roles that these proteins play in the mechanism of TLF-mediated lysis are highlighted in this article. In light of recent data, which demonstrate that TLFs can ameliorate infection by the intracellular pathogen Leishmania, we also discuss the broader function of TLFs as components of innate immunity
PMCID:2777647
PMID: 19722834
ISSN: 1746-0921
CID: 101964
Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection
Samanovic, Marie; Molina-Portela, Maria Pilar; Chessler, Anne-Danielle C; Burleigh, Barbara A; Raper, Jayne
Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system
PMCID:2622765
PMID: 19165337
ISSN: 1553-7374
CID: 96765
Identification of Three Classes of Heteroaromatic Compounds with Activity against Intracellular Trypanosoma cruzi by Chemical Library Screening
Bettiol, Esther; Samanovic, Marie; Murkin, Andrew S; Raper, Jayne; Buckner, Frederick; Rodriguez, Ana
The development of new drugs against Chagas disease is a priority since the currently available medicines have toxic effects, partial efficacy and are targeted against the acute phase of disease. At present, there is no drug to treat the chronic stage. In this study, we have optimized a whole cell-based assay for high throughput screening of compounds that inhibit infection of mammalian cells by Trypanosoma cruzi trypomastigotes. A 2000-compound chemical library was screened using a recombinant T. cruzi (Tulahuen strain) expressing beta-galactosidase. Three hits were selected for their high activity against T. cruzi and low toxicity to host cells in vitro: PCH1, NT1 and CX1 (IC(50): 54, 190 and 23 nM, respectively). Each of these three compounds presents a different mechanism of action on intracellular proliferation of T. cruzi amastigotes. CX1 shows strong trypanocidal activity, an essential characteristic for the development of drugs against the chronic stage of Chagas disease where parasites are found intracellular in a quiescent stage. NT1 has a trypanostatic effect, while PCH1 affects parasite division. The three compounds also show high activity against intracellular T. cruzi from the Y strain and against the related kinetoplastid species Leishmania major and L. amazonensis. Characterization of the anti-T. cruzi activity of molecules chemically related to the three library hits allowed the selection of two compounds with IC(50) values of 2 nM (PCH6 and CX2). These values are approximately 100 times lower than those of the medicines used in patients against T. cruzi. These results provide new candidate molecules for the development of treatments against Chagas disease and leishmaniasis
PMCID:2639639
PMID: 19238193
ISSN: 1935-2735
CID: 96764
Distinct roles of apolipoprotein components within the trypanosome lytic factor complex revealed in a novel transgenic mouse model
Molina-Portela, Maria Pilar; Samanovic, Marie; Raper, Jayne
Humans express a unique subset of high-density lipoproteins (HDLs) called trypanosome lytic factors (TLFs) that kill many Trypanosoma parasite species. The proteins apolipoprotein (apo) A-I, apoL-I, and haptoglobin-related protein, which are involved in TLF structure and function, were expressed through the introduction of transgenes in mice to explore their physiological roles in vivo. Transgenic expression of human apolipoprotein L-I alone conferred trypanolytic activity in vivo. Coexpression of human apolipoprotein A-I and haptoglobin-related protein (Hpr) had an effect on the integration of apolipoprotein L-I into HDL, and both proteins were required to increase the specific activity of TLF, which was measurable in vitro. Unexpectedly, truncated apolipoprotein L-I devoid of the serum resistance gene interacting domain, which was previously shown to kill human infective trypanosomes, was not trypanolytic in transgenic mice despite being coexpressed with human apolipoprotein A-I and Hpr and incorporated into HDLs. We conclude that all three human apolipoproteins act cooperatively to achieve maximal killing capacity and that truncated apolipoprotein L-I does not function in transgenic animals
PMCID:2525602
PMID: 18606856
ISSN: 1540-9538
CID: 96766