Methods in molecular biology (Clifton, N.J.), 2012
The complexity of the metabolic networks in even the simplest organisms has raised new challenges... more The complexity of the metabolic networks in even the simplest organisms has raised new challenges in organizing metabolic information. To address this, specialized computer frameworks have been developed to capture, manage, and visualize metabolic knowledge. The leading databases of metabolic information are those organized under the umbrella of the BioCyc project, which consists of the reference database MetaCyc, and a number of pathway/genome databases (PGDBs) each focussed on a specific organism. A number of PGDBs have been developed for bacterial, fungal, and protozoan pathogens, greatly facilitating dissection of the metabolic potential of these organisms and the identification of new drug targets. Leishmania are protozoan parasites belonging to the family Trypanosomatidae that cause a broad spectrum of diseases in humans. In this work we use the LeishCyc database, the BioCyc database for Leishmania major, to describe how to build a BioCyc database from genomic sequences and as...
Parasitic protozoa belonging to the genus Leishmania are the cause of a spectrum of diseases in h... more Parasitic protozoa belonging to the genus Leishmania are the cause of a spectrum of diseases in humans, as well as chronic long-term infections. These parasites exhibit a remarkable capacity to survive and proliferate within the phagolysosome compartment of host macrophages. Studies with defined Leishmania mutants in mouse models of infection have highlighted processes that are required for parasite survival in macrophages. Parasite mutants have been identified that (i) are poorly virulent when the insect (promastigote) stage is used to initiate infection, but retain wild-type virulence following transformation to the obligate intracellular amastigote stage, (ii) are highly attenuated when either promastigotes or amastigotes are used, and (iii) are unable to induce characteristic lesion granulomas, but can persist within macrophages in other tissues. From these analyses it can be concluded that promastigote stages of some species require the surface expression of lipophosphoglycan, ...
Protozoan parasites belonging to the genus Leishmania exhibit a pronounced tropism for macrophage... more Protozoan parasites belonging to the genus Leishmania exhibit a pronounced tropism for macrophages although they have the capacity to infect a variety of other phagocytic and non-phagocytic mammalian cells. Unlike most other intramacrophage pathogens, the major proliferative stage of Leishmania resides in the mature phagolysosomes of these host cells. In this review we highlight some of the strategies utilized by the intracellular amastigote stage of Leishmania to survive in this compartment. Remarkably, and in contrast to many other intracellular pathogens, Leishmania amastigotes have a minimalist surface glycocalyx which may facilitate uptake of essential lipids and promote exposure of phospholipids required for phagocytosis via macrophage apoptotic cell receptors. Leishmania amastigotes also differ from many other intracellular pathogens in having complex nutritional requirements which must be scavenged from the host cell. Amino acids and polyamines appear to be important carbon ...
The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the m... more The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular beta1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold ...
Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial ce... more Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a c...
Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2006
Leishmania are protozoan parasites that replicate within mature phagolysosomes of mammalian macro... more Leishmania are protozoan parasites that replicate within mature phagolysosomes of mammalian macrophages. To define the biochemical composition of the phagosome and carbon source requirements of intracellular stages of L. major, we investigated the role and requirement for the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP). L. major FBP was constitutively expressed in both extracellular and intracellular stages and was primarily targeted to glycosomes, modified peroxisomes that also contain glycolytic enzymes. A L. major FBP-null mutant was unable to grow in the absence of hexose, and suspension in glycerol-containing medium resulted in rapid depletion of internal carbohydrate reserves. L. major Deltafbp promastigotes were internalized by macrophages and differentiated into amastigotes but were unable to replicate in the macrophage phagolysosome. Similarly, the mutant persisted in mice but failed to generate normal lesions. The data suggest that Leishmania amastigotes reside ...
Page 223. CHAPTER Glycosylated compounds of parasitic protozoa Joanne Heng, Thomas Naderer, Stuar... more Page 223. CHAPTER Glycosylated compounds of parasitic protozoa Joanne Heng, Thomas Naderer, Stuart A. Ralph and MalcolmJ. McConville SUMMARY Parasitic protists belong to a range of deeply diverging eukaryotic ...
Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and se... more Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri-and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host. Citation: Sansom FM, Ralton JE, Sernee MF, Cohen AM, Hooker DJ, et al. (2014) Golgi-Located NTPDase1 of Leishmania major Is Required for Lipophosphoglycan Elongation and Normal Lesion Development whereas Secreted NTPDase2 Is Dispensable for Virulence. PLoS Negl Trop Dis 8(12): e3402.
The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that tr... more The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.
Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the ho... more Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Dgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Dgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Dgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.
Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophag... more Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA) that could have a role in reducing oxidized free and proteinogenic methionine residues. A GFP-fusion of L. major MsrA was shown to have a cytoplasmic localization by immunofluorescence microscopy and subcellular fractionation. An L. major msrA null mutant, generated by targeted replacement of both chromosomal allelles, was viable in rich medium but was unable to reduce exogenous methionine sulfoxide when cultivated in the presence of this amino acid, indicating that msrA encodes a functional MsrA. The DmsrA mutant exhibited increased sensitivity to H 2 O 2 compared to wild type parasites and was unable to proliferate normally in macrophages. Wild type sensitivity to H 2 O 2 and infectivity in macrophages was restored by complementation of the mutant with a plasmid encoding MsrA. Unexpectedly, the DmsrA mutant was able to induce normal lesions in susceptible BALB/c indicating that this protein is not essential for pathogenesis in vivo. Our results suggest that Leishmania MsrA contributes to the anti-oxidative defences of these parasites, but that complementary oxidative defence mechansims are up-regulated in lesion amastigotes.
Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial ce... more Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a characteristic feature of autotransporters-was found to be composed from seven conserved sequence segments that can be arranged in multiple ways in the tertiary structure of the assembled autotransporter. One of these conserved motifs overlays the targeting information required for autotransporters to reach the outer membrane. Another conserved and diagnostic motif maps to the linker region between the passenger domain and barrel-domain, indicating it as an important feature in the assembly of autotransporters.
Leishmania spp. are sandfly-transmitted protozoa parasites that cause a spectrum of diseases in h... more Leishmania spp. are sandfly-transmitted protozoa parasites that cause a spectrum of diseases in humans. Many enzymes involved in Leishmania central carbon metabolism differ from their equivalents in the mammalian host and are potential drug targets. In this review we summarize recent advances in our understanding of Leishmania central carbon metabolism, focusing on pathways of carbon utilization that are required for growth and pathogenesis in the mammalian host. While Leishmania central carbon metabolism shares many features in common with other pathogenic trypanosomatids, significant differences are also apparent. Leishmania parasites are also unusual in constitutively expressing most core metabolic pathways throughout their life cycle, a feature that may allow these parasites to exploit a range of different carbon sources (primarily sugars and amino acids) rapidly in both the insect vector and vertebrate host. Indeed, recent gene deletion studies suggest that mammal-infective stages are dependent on multiple carbon sources in vivo. The application of metabolomic approaches, outlined here, are likely to be important in defining aspects of central carbon metabolism that are essential at different stages of mammalian host infection.
Leishmania parasites incorporate N-acetylglucosamine (GlcNAc) into surface-expressed glycosylphos... more Leishmania parasites incorporate N-acetylglucosamine (GlcNAc) into surface-expressed glycosylphosphatidylinositol (GPI) glycolipids and N-linked glycans. To investigate whether these glycoconjugates are required for infectivity of promastigote and intracellular amastigote stages, we generated a Leishmania major mutant lacking the gene encoding glutamine : fructose-6-phosphate amidotransferase (GFAT). The L. majorDeltagfat mutant is unable to synthesize GlcN-6-phosphate de novo and is auxotrophic for GlcN or GlcNAc. GlcN starvation leads to the rapid depletion of dolichol-linked oligosaccharides and GPI precursors, hypersensitivity to elevated temperatures encountered in the mammalian host and eventual parasite death. Short-term tunicamycin treatment induces a similar hypersensitivity to temperature, indicating that N-linked glycans are required for thermotolerance and viability. L. majorDeltagfat promastigotes are unable to proliferate in ex vivo infected macrophages, demonstrating that GlcN(Ac) levels in the phagolysosome are low. In contrast, Deltagfat amastigotes grow as well as wild-type amastigotes in macrophages and induce lesions in susceptible mice. These stages still require GlcN(Ac) for viability but can apparently scavenge all of their glucosamine requirements from the macrophage phagolysosome. These results highlight significant differences in the nutrient requirements of promastigote and amastigote stages and suggest that enzymes involved in UDP-GlcNAc biosynthesis are essential for pathogenesis in the mammalian host.
Leishmania parasites must adapt to elevated temperatures and other environmental stresses during ... more Leishmania parasites must adapt to elevated temperatures and other environmental stresses during infection of their mammalian hosts. How these environmental cues are sensed is poorly understood. In this study we show that calcium uptake is required for parasite thermotolerance at 34-37°C. To identify potential downstream targets of calcium influx, a Leishmania major mutant lacking the essential regulatory subunit (CnB) of the Ca 2+ /calmodulin-dependent serine/threonine-specific phosphatase, calcineurin, was generated. The Dcnb mutant grew as well as wild-type parasites at 27°C and differentiated normally to infective metacyclic promastigotes. However, Dcnb parasites lost viability when exposed to increased temperature (34°C) and were hypersensitive to endoplasmic reticulum and membrane stress, induced by tunicamycin and inhibitors of sterol and sphingolipid biosynthesis respectively. Dcnb promastigotes were internalized by macrophages, but their differentiation to the heat adapted amastigote stage was delayed and the resulting parasites failed to proliferate. Strikingly, the Dcnb parasites were completely cleared by susceptible BALB/c mice. Complementation of Dcnb parasites with CnB restored thermotolerance and infectivity in both macrophages and animal models. Our results suggest that Ca 2+ influx and calcineurin signalling are required for both early and long-term adaptive parasite responses to environmental stresses encountered in the mammalian host.
The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia ar... more The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.
Methods in molecular biology (Clifton, N.J.), 2012
The complexity of the metabolic networks in even the simplest organisms has raised new challenges... more The complexity of the metabolic networks in even the simplest organisms has raised new challenges in organizing metabolic information. To address this, specialized computer frameworks have been developed to capture, manage, and visualize metabolic knowledge. The leading databases of metabolic information are those organized under the umbrella of the BioCyc project, which consists of the reference database MetaCyc, and a number of pathway/genome databases (PGDBs) each focussed on a specific organism. A number of PGDBs have been developed for bacterial, fungal, and protozoan pathogens, greatly facilitating dissection of the metabolic potential of these organisms and the identification of new drug targets. Leishmania are protozoan parasites belonging to the family Trypanosomatidae that cause a broad spectrum of diseases in humans. In this work we use the LeishCyc database, the BioCyc database for Leishmania major, to describe how to build a BioCyc database from genomic sequences and as...
Parasitic protozoa belonging to the genus Leishmania are the cause of a spectrum of diseases in h... more Parasitic protozoa belonging to the genus Leishmania are the cause of a spectrum of diseases in humans, as well as chronic long-term infections. These parasites exhibit a remarkable capacity to survive and proliferate within the phagolysosome compartment of host macrophages. Studies with defined Leishmania mutants in mouse models of infection have highlighted processes that are required for parasite survival in macrophages. Parasite mutants have been identified that (i) are poorly virulent when the insect (promastigote) stage is used to initiate infection, but retain wild-type virulence following transformation to the obligate intracellular amastigote stage, (ii) are highly attenuated when either promastigotes or amastigotes are used, and (iii) are unable to induce characteristic lesion granulomas, but can persist within macrophages in other tissues. From these analyses it can be concluded that promastigote stages of some species require the surface expression of lipophosphoglycan, ...
Protozoan parasites belonging to the genus Leishmania exhibit a pronounced tropism for macrophage... more Protozoan parasites belonging to the genus Leishmania exhibit a pronounced tropism for macrophages although they have the capacity to infect a variety of other phagocytic and non-phagocytic mammalian cells. Unlike most other intramacrophage pathogens, the major proliferative stage of Leishmania resides in the mature phagolysosomes of these host cells. In this review we highlight some of the strategies utilized by the intracellular amastigote stage of Leishmania to survive in this compartment. Remarkably, and in contrast to many other intracellular pathogens, Leishmania amastigotes have a minimalist surface glycocalyx which may facilitate uptake of essential lipids and promote exposure of phospholipids required for phagocytosis via macrophage apoptotic cell receptors. Leishmania amastigotes also differ from many other intracellular pathogens in having complex nutritional requirements which must be scavenged from the host cell. Amino acids and polyamines appear to be important carbon ...
The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the m... more The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular beta1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold ...
Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial ce... more Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a c...
Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2006
Leishmania are protozoan parasites that replicate within mature phagolysosomes of mammalian macro... more Leishmania are protozoan parasites that replicate within mature phagolysosomes of mammalian macrophages. To define the biochemical composition of the phagosome and carbon source requirements of intracellular stages of L. major, we investigated the role and requirement for the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP). L. major FBP was constitutively expressed in both extracellular and intracellular stages and was primarily targeted to glycosomes, modified peroxisomes that also contain glycolytic enzymes. A L. major FBP-null mutant was unable to grow in the absence of hexose, and suspension in glycerol-containing medium resulted in rapid depletion of internal carbohydrate reserves. L. major Deltafbp promastigotes were internalized by macrophages and differentiated into amastigotes but were unable to replicate in the macrophage phagolysosome. Similarly, the mutant persisted in mice but failed to generate normal lesions. The data suggest that Leishmania amastigotes reside ...
Page 223. CHAPTER Glycosylated compounds of parasitic protozoa Joanne Heng, Thomas Naderer, Stuar... more Page 223. CHAPTER Glycosylated compounds of parasitic protozoa Joanne Heng, Thomas Naderer, Stuart A. Ralph and MalcolmJ. McConville SUMMARY Parasitic protists belong to a range of deeply diverging eukaryotic ...
Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and se... more Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri-and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host. Citation: Sansom FM, Ralton JE, Sernee MF, Cohen AM, Hooker DJ, et al. (2014) Golgi-Located NTPDase1 of Leishmania major Is Required for Lipophosphoglycan Elongation and Normal Lesion Development whereas Secreted NTPDase2 Is Dispensable for Virulence. PLoS Negl Trop Dis 8(12): e3402.
The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that tr... more The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.
Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the ho... more Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Dgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Dgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Dgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.
Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophag... more Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA) that could have a role in reducing oxidized free and proteinogenic methionine residues. A GFP-fusion of L. major MsrA was shown to have a cytoplasmic localization by immunofluorescence microscopy and subcellular fractionation. An L. major msrA null mutant, generated by targeted replacement of both chromosomal allelles, was viable in rich medium but was unable to reduce exogenous methionine sulfoxide when cultivated in the presence of this amino acid, indicating that msrA encodes a functional MsrA. The DmsrA mutant exhibited increased sensitivity to H 2 O 2 compared to wild type parasites and was unable to proliferate normally in macrophages. Wild type sensitivity to H 2 O 2 and infectivity in macrophages was restored by complementation of the mutant with a plasmid encoding MsrA. Unexpectedly, the DmsrA mutant was able to induce normal lesions in susceptible BALB/c indicating that this protein is not essential for pathogenesis in vivo. Our results suggest that Leishmania MsrA contributes to the anti-oxidative defences of these parasites, but that complementary oxidative defence mechansims are up-regulated in lesion amastigotes.
Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial ce... more Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a characteristic feature of autotransporters-was found to be composed from seven conserved sequence segments that can be arranged in multiple ways in the tertiary structure of the assembled autotransporter. One of these conserved motifs overlays the targeting information required for autotransporters to reach the outer membrane. Another conserved and diagnostic motif maps to the linker region between the passenger domain and barrel-domain, indicating it as an important feature in the assembly of autotransporters.
Leishmania spp. are sandfly-transmitted protozoa parasites that cause a spectrum of diseases in h... more Leishmania spp. are sandfly-transmitted protozoa parasites that cause a spectrum of diseases in humans. Many enzymes involved in Leishmania central carbon metabolism differ from their equivalents in the mammalian host and are potential drug targets. In this review we summarize recent advances in our understanding of Leishmania central carbon metabolism, focusing on pathways of carbon utilization that are required for growth and pathogenesis in the mammalian host. While Leishmania central carbon metabolism shares many features in common with other pathogenic trypanosomatids, significant differences are also apparent. Leishmania parasites are also unusual in constitutively expressing most core metabolic pathways throughout their life cycle, a feature that may allow these parasites to exploit a range of different carbon sources (primarily sugars and amino acids) rapidly in both the insect vector and vertebrate host. Indeed, recent gene deletion studies suggest that mammal-infective stages are dependent on multiple carbon sources in vivo. The application of metabolomic approaches, outlined here, are likely to be important in defining aspects of central carbon metabolism that are essential at different stages of mammalian host infection.
Leishmania parasites incorporate N-acetylglucosamine (GlcNAc) into surface-expressed glycosylphos... more Leishmania parasites incorporate N-acetylglucosamine (GlcNAc) into surface-expressed glycosylphosphatidylinositol (GPI) glycolipids and N-linked glycans. To investigate whether these glycoconjugates are required for infectivity of promastigote and intracellular amastigote stages, we generated a Leishmania major mutant lacking the gene encoding glutamine : fructose-6-phosphate amidotransferase (GFAT). The L. majorDeltagfat mutant is unable to synthesize GlcN-6-phosphate de novo and is auxotrophic for GlcN or GlcNAc. GlcN starvation leads to the rapid depletion of dolichol-linked oligosaccharides and GPI precursors, hypersensitivity to elevated temperatures encountered in the mammalian host and eventual parasite death. Short-term tunicamycin treatment induces a similar hypersensitivity to temperature, indicating that N-linked glycans are required for thermotolerance and viability. L. majorDeltagfat promastigotes are unable to proliferate in ex vivo infected macrophages, demonstrating that GlcN(Ac) levels in the phagolysosome are low. In contrast, Deltagfat amastigotes grow as well as wild-type amastigotes in macrophages and induce lesions in susceptible mice. These stages still require GlcN(Ac) for viability but can apparently scavenge all of their glucosamine requirements from the macrophage phagolysosome. These results highlight significant differences in the nutrient requirements of promastigote and amastigote stages and suggest that enzymes involved in UDP-GlcNAc biosynthesis are essential for pathogenesis in the mammalian host.
Leishmania parasites must adapt to elevated temperatures and other environmental stresses during ... more Leishmania parasites must adapt to elevated temperatures and other environmental stresses during infection of their mammalian hosts. How these environmental cues are sensed is poorly understood. In this study we show that calcium uptake is required for parasite thermotolerance at 34-37°C. To identify potential downstream targets of calcium influx, a Leishmania major mutant lacking the essential regulatory subunit (CnB) of the Ca 2+ /calmodulin-dependent serine/threonine-specific phosphatase, calcineurin, was generated. The Dcnb mutant grew as well as wild-type parasites at 27°C and differentiated normally to infective metacyclic promastigotes. However, Dcnb parasites lost viability when exposed to increased temperature (34°C) and were hypersensitive to endoplasmic reticulum and membrane stress, induced by tunicamycin and inhibitors of sterol and sphingolipid biosynthesis respectively. Dcnb promastigotes were internalized by macrophages, but their differentiation to the heat adapted amastigote stage was delayed and the resulting parasites failed to proliferate. Strikingly, the Dcnb parasites were completely cleared by susceptible BALB/c mice. Complementation of Dcnb parasites with CnB restored thermotolerance and infectivity in both macrophages and animal models. Our results suggest that Ca 2+ influx and calcineurin signalling are required for both early and long-term adaptive parasite responses to environmental stresses encountered in the mammalian host.
The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia ar... more The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.
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Papers by Thomas Naderer