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PubMed2005

Downregulation of the human Lon protease impairs mitochondrial structure and function and causes cell death.

Lon now emerges as a major regulator of multiple mitochondrial functions in human beings. Lon catalyzes the degradation of oxidatively modified matrix proteins, chaperones the assembly of inner membrane complexes, and participates in the regulation of mitochondrial gene expression and genome integrity. An early result of Lon downregulation in WI-38 VA-13 human lung fibroblasts is massive caspase 3 activation and extensive (although not universal) apoptotic death. At a later stage, the surviving cells fail to divide, display highly abnormal mitochondrial function and morphology, and rely almost exclusively on anaerobic metabolism. In a selected subpopulation of cells, the mitochondrial mass decreases probably as a result of mitochondrial inability to divide. At this final point the Lon-deficient cells are not engaged anymore in apoptosis, and are lost by necrosis or "mitoptosis." Our results indicate that mitochondrial Lon is required for normal survival and proliferation; a clear impetus for Lon's evolutionary conservation.

Apoptosis↗
PubMed2005

Ultrastructural aspects of Fallisia effusa (Haemosporina: Garniidae) in thrombocytes of the lizard Neusticurus bicarinatus (Reptilia: Teiidae).

The fine structure of the different stages of the Fallisia effusa (Haemosporina: Garniidae), infecting the thrombocytes of the semi-aquatic Amazonian lizard Neusticurus bicarinatus (Reptilia: Teiidae) is described. Gametocytes, meronts, and merozoites of Fallisia effusa were found within a parasitophorous vacuole (PV). Multiple infections of micro- and macrogametocytes were observed. A circumferential coil of microtubules was seen in the cytoplasm of the infected host cell and this microtubule array was pronounced in cells harboring gametocytes. A deep invagination of the inner membrane complex of gametocytes may be involved in nutrition. The non-pigmented parasites underwent both merogony and gametogony in thrombocytes of the peripheral blood. No infection of the erythrocytes was observed. These observations confirm that Fallisia effusa displays characteristic features distinguishing it from other members of the Haemosporidian families, and that it has the ability to modulate microtubule assembly.

Animals↗
PubMed2006

Ultrastructural study of the gametocytes and merogonic stages of Fallisia audaciosa (Haemosporina: Garniidae) that infect neutrophils of the lizard Plica umbra (Reptilia: Iguanidae).

Little is known regarding the ultrastructure of the genus Fallisia (Apicomplexa: Haemosporina: Garniidae). This report describes the fine structure of some developmental stages of Fallisia audaciosa that infect neutrophils in the peripheral blood of the Amazonian lizard Plica umbra (Reptilia: Iguanidae). The parasites lie within a parasitophorous vacuole and exhibit the basic structures of members of the Apicomplexa, such as the pellicle and the cytostome. Invaginations of the inner membrane complex were seen in the gametocytes and may be concerned with nutrition. The meronts were irregularly shaped before division, a feature unusual among members of the Apicomplexa. The unusual presence of a parasitic protozoan within neutrophils, in some way interfering with or modulating the microbicidal activity of such cells, is discussed.

Animals↗
PubMed1997

Enrichment and biochemical characterization of Toxoplasma gondii tachyzoite plasmalemma.

The protozoan parasite Toxoplasma gondii possesses a triple surface membrane called the pellicle. This is made of an outer plasmalemma and an inner membrane complex lying under the plasmalemma. Using a high salt glycerol treatment followed by sonication, we have obtained a partial dissociation of the pellicle. A plasmalemma-enriched fraction was isolated on 0.7 M sucrose. It was identified by immunodetection of the tachyzoite major surface antigens. Protein content, resolved by SDS-PAGE, revealed that the surface protein SAG1 is the major component of the plasmalemma. The plasmalemma fraction is made of small vesicles (20-100 nm) which possess a low density (1.085-1.090 g/cm3 in sucrose) contrasting with other eukaryotic plasma membranes (1.12-1.16 g/cm3).

Animals↗
PubMed2006

Importance of the adaptor (membrane fusion) protein hairpin domain for the functionality of multidrug efflux pumps.

Drug efflux pumps of Gram-negative bacteria are tripartite export machineries located in the bacterial envelopes contributing to multidrug resistance. Protein structures of all three components have been determined, but the exact interaction sites are still unknown. We could confirm that the hybrid system composed of Pseudomonas aeruginosa channel tunnel OprM and the Escherichia coli inner membrane complex, formed by adaptor protein (membrane fusion protein) AcrA and transporter AcrB of the resistance nodulation cell division (RND) family, is not functional. However, cross-linking experiments show that the hybrid exporter assembles. Exchange of the hairpin domain of AcrA with the corresponding hairpin from adaptor protein MexA of P. aeruginosa restored the functionality. This shows the importance of the MexA hairpin domain for the functional interaction with the OprM channel tunnel. On the basis of these results, we have modeled the interaction of the hairpin domain and the channel tunnel on a molecular level for AcrA and TolC as well as MexA and OprM, respectively. The model of two hairpin docking sites per TolC protomer corresponding with hexameric adaptor proteins was confirmed by disulfide cross-linking experiments. The role of this interaction for functional efflux pumps is discussed.

Bacterial Outer Membrane Proteins↗
PubMed2004

Lactoferrin receptors in gram-negative bacteria: insights into the iron acquisition process.

One component of the anti-microbial function of lactoferrin (Lf) is its ability to sequester iron from potential pathogens. To overcome this iron limitation, a number of gram-negative bacterial pathogens have developed a mechanism for acquiring iron directly from this host glycoprotein. This mechanism involves surface receptors capable of specifically binding Lf from the host, removing iron and transporting it across the outer membrane. The iron is then bound by a periplasmic iron-binding protein, FbpA, and transported into the cell via an inner membrane complex comprised of FbpB and FbpC. The receptor has been shown to consist of two proteins, LbpA and LbpB. LbpB is bilobed lipoprotein anchored to the outer membrane via fatty acyl groups attached to the N-terminal cysteine. LbpA is a homologue of siderophore receptors, which consist of an N-terminal plug and a C-terminal beta-barrel region. We propose that the receptor proteins, LbpA and LbpB, induce conformational changes in human Lf (hLf) that lower its affinity for iron that binding by FbpA can drive the transport across the outer membrane, a mechanism shared with transferrin (Tf) receptors. The interaction between the receptor proteins and Lf is quite extensive and has been previously studied by using chimeric proteins comprised of Lf & Tf. In an attempt to evaluate the role of FbpA in the transport process, a series of site-directed mutants of FbpA were prepared and used to replace the wild-type protein in the iron acquisition pathway. The mutations were made in the iron-binding and anion-binding ligands of FbpA and were designed to result in altered binding properties. Protein crystallography of the iron-bound form of the Q58L mutant protein revealed that it was in the open conformation with iron coordinated by Y195 and Y196 from the C-terminal domain but not by the other iron-liganding amino acids from the N-terminal domain, H9 and E57. Replacement of the native FbpA in Neisseria meningitidis with wild-type or mutant Haemophilus influenzae FbpAs resulted in a defect in growth on Tf or Lf, suggesting that there may be a barrier to functional expression of H. influenzae FbpAs in Neisseria meningitidis. Thus mutants of the N. meningitidis FbpA are being prepared to replace wild-type protein in order to test their ability to mediate transport from hLf.

Anti-Infective Agents↗
PubMed1999

Crystal structure of the outer membrane active transporter FepA from Escherichia coli.

Integral outer membrane receptors for iron chelates and vitamin B12 carry out specific ligand transport against a concentration gradient. Energy for active transport is obtained from the proton-motive force of the inner membrane through physical interaction with TonB-ExbB-ExbD, an inner membrane complex. Here we report the crystal structure of an active transport, outer membrane receptor at 2.4 A resolution. Two distinct functional domains are revealed: (i) a 22-stranded beta-barrel that spans the outer membrane and contains large extracellular loops which appear to function in ligand binding; and (ii) a globular N-terminal domain that folds into the barrel pore, inhibiting access to the periplasm and contributing two additional loops for potential ligand binding. These loops could provide a signaling pathway between the processes of ligand recognition and TonB-mediated transport. The blockage of the pore suggests that the N-terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm.

Amino Acid Sequence↗
PubMed2005

Biochemical dysfunction in heart mitochondria exposed to ischaemia and reperfusion.

Heart tissue is remarkably sensitive to oxygen deprivation. Although heart cells, like those of most tissues, rapidly adapt to anoxic conditions, relatively short periods of ischaemia and subsequent reperfusion lead to extensive tissue death during cardiac infarction. Heart tissue is not readily regenerated, and permanent heart damage is the result. Although mitochondria maintain normal heart function by providing virtually all of the heart's ATP, they are also implicated in the development of ischaemic damage. While mitochondria do provide some mechanisms that protect against ischaemic damage (such as an endogenous inhibitor of the F1Fo-ATPase and antioxidant enzymes), they also possess a range of elements that exacerbate it, including ROS (reactive oxygen species) generators, the mitochondrial permeability transition pore, and their ability to release apoptotic factors. This review considers the process of ischaemic damage from a mitochondrial viewpoint. It considers ischaemic changes in the inner membrane complexes I-V, and how this might affect formation of ROS and high-energy phosphate production/degradation. We discuss the contribution of various mitochondrial cation channels to ionic imbalances which seem to be a major cause of reperfusion injury. The different roles of the H+, Ca2+ and the various K+ channel transporters are considered, particularly the K+(ATP) (ATP-dependent K+) channels. A possible role for the mitochondrial permeability transition pore in ischaemic damage is assessed. Finally, we summarize the metabolic and pharmacological interventions that have been used to alleviate the effects of ischaemic injury, highlighting the value of these or related interventions in possible therapeutics.

Animals↗
PubMed2002

Transition to the open state of the TolC periplasmic tunnel entrance.

The TolC channel-tunnel spans the bacterial outer membrane and periplasm, providing a large exit duct for protein export and multidrug efflux when recruited by substrate-engaged inner membrane complexes. The sole constriction in the single pore of the homotrimeric TolC is the periplasmic tunnel entrance, which in its resting configuration is closed by dense packing of the 12 tunnel-forming alpha-helices. Recruitment of TolC must trigger opening for substrate transit to occur, but the mechanism underlying transition from the closed to the open state is not known. The high resolution structure of TolC indicates that the tunnel helices are constrained at the entrance by a circular network of intra- and intermonomer hydrogen bonds and salt bridges. To assess how opening is achieved, we disrupted these connections and monitored changes in the aperture size by measuring the single channel conductance of TolC derivatives in black lipid bilayers. Elimination of individual connections caused incremental weakening of the circular network, accompanied by gradual relaxation from the closed state and increased flexibility of the entrance. Simultaneous abolition of the key links caused a substantial increase in conductance, generating an aperture that corresponds to the modeled open state, with the capacity to allow access and passage of diverse substrates. The results support a model in which transition to the open state of TolC is achieved by an iris-like realignment of the tunnel entrance helices.

Amino Acid Substitution↗
PubMed2005

Differential localization of alternatively spliced hypoxanthine-xanthine-guanine phosphoribosyltransferase isoforms in Toxoplasma gondii.

A unique feature of the Toxoplasma gondii purine salvage pathway is the expression of two isoforms of the hypoxanthine-xanthine-guanine phosophoribosyltransferase (HXGPRT) of the parasite encoded by a single genetic locus. These isoforms differ in the presence or absence of a 49-amino acid insertion (which is specified by a single differentially spliced exon) but exhibit similar substrate specificity, kinetic characteristics, and temporal expression patterns. To examine possible functional differences between the two HXGPRT isoforms, fluorescent protein fusions were expressed in parasites lacking the endogenous hxgprt gene. Immunoblot analysis of fractionated cell extracts and fluorescence microscopy indicated that HXGPRT-I (which lacks the 49-amino acid insertion) is found in the cytosol, whereas HXGPRT-II (which contains the insertion) localizes to the inner membrane complex (IMC) of the parasite. Simultaneous expression of both isoforms resulted in the formation of hetero-oligomers, which distributed between the cytosol and IMC. Chimeric constructs expressing N-terminal peptides from either isoform I (11 amino acids) or isoform II (60 amino acids) fused to a chloramphenicol acetyl transferase (CAT) reporter demonstrated that the N-terminal domain of isoform II is both necessary and sufficient for membrane association. Metabolic labeling experiments with transgenic parasites showed that isoform II or an isoform II-CAT fusion protein (but not isoform I or isoform I-CAT) incorporate [(3)H]palmitate. Mutation of three adjacent cysteine residues within the isoform II-targeting domain to serines blocked both palmitate incorporation and IMC attachment without affecting enzyme activity, demonstrating that acylation of N-terminal isoform II cysteine residues is responsible for the association of HXGPRT-II with the IMC.

Alternative Splicing↗
PubMed2005

A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites.

Apicomplexan parasites constitute one of the most significant groups of pathogens infecting humans and animals. The liver stage sporozoites of Plasmodium spp. and tachyzoites of Toxoplasma gondii, the causative agents of malaria and toxoplasmosis, respectively, use a unique mode of locomotion termed gliding motility to invade host cells and cross cell substrates. This amoeboid-like movement uses a parasite adhesin from the thrombospondin-related anonymous protein (TRAP) family and a set of proteins linking the extracellular adhesin, via an actin-myosin motor, to the inner membrane complex. The Plasmodium blood stage merozoite, however, does not exhibit gliding motility. Here we show that homologues of the key proteins that make up the motor complex, including the recently identified glideosome-associated proteins 45 and 50 (GAP40 and GAP50), are present in P. falciparum merozoites and appear to function in erythrocyte invasion. Furthermore, we identify a merozoite TRAP homologue, termed MTRAP, a micronemal protein that shares key features with TRAP, including a thrombospondin repeat domain, a putative rhomboid-protease cleavage site, and a cytoplasmic tail that, in vitro, binds the actin-binding protein aldolase. Analysis of other parasite genomes shows that the components of this motor complex are conserved across diverse Apicomplexan genera. Conservation of the motor complex suggests that a common molecular mechanism underlies all Apicomplexan motility, which, given its unique properties, highlights a number of novel targets for drug intervention to treat major diseases of humans and livestock.

Actins↗
PubMed2001

Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division.

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

Alternative Splicing↗
PubMed2004

A malaria membrane skeletal protein is essential for normal morphogenesis, motility, and infectivity of sporozoites.

Membrane skeletons are structural elements that provide mechanical support to the plasma membrane and define cell shape. Here, we identify and characterize a putative protein component of the membrane skeleton of the malaria parasite. The protein, named PbIMC1a, is the structural orthologue of the Toxoplasma gondii inner membrane complex protein 1 (TgIMC1), a component of the membrane skeleton in tachyzoites. Using targeted gene disruption in the rodent malaria species Plasmodium berghei, we show that PbIMC1a is involved in sporozoite development, is necessary for providing normal sporozoite cell shape and mechanical stability, and is essential for sporozoite infectivity in insect and vertebrate hosts. Knockout of PbIMC1a protein expression reduces, but does not abolish, sporozoite gliding locomotion. We identify a family of proteins related to PbIMC1a in Plasmodium and other apicomplexan parasites. These results provide new functional insight in the role of membrane skeletons in apicomplexan parasite biology.

Amino Acid Sequence↗
PubMed2000

Chunnel vision. Export and efflux through bacterial channel-tunnels.

The Escherichia coli TolC protein is central to toxin export and drug efflux across the inner and outer cell membranes and the intervening periplasmic space. The crystal structure has revealed that TolC assembles into a remarkable alpha-helical trans-periplasmic cylinder (tunnel) embedded in the outer membrane by a contiguous beta-barrel (channel), so providing a large duct open to the outside environment. The channel-tunnel structure is conserved in TolC homologues throughout Gram-negative bacteria, and it is envisaged that they are recruited and opened, through a common mechanism, by substrate-specific inner-membrane complexes.

Bacterial Outer Membrane Proteins↗
PubMedDate not supplied

Characterization of the hrpJ and hrpU operons of Pseudomonas syringae pv. syringae Pss61: similarity with components of enteric bacteria involved in flagellar biogenesis and demonstration of their role in HarpinPss secretion.

The hrp/hrmA gene cluster of Pseudomonas syringae pv. syringae Pss61 has been shown to form a minimum genetic unit sufficient to enable nonpathogenic bacteria, such as Escherichia coli, to elicit the hypersensitive response associated with disease resistance. The biochemical functions of most of these genes have not been established. The nucleotide sequence of a 4.3-kb SstI-BglII fragment carrying hrp apparent translational units V, VI, and VII revealed one partial open reading frame (ORF) and five complete ORFs producing 35,126-, 48,866-, 17,308-, 20,482-, and 26,364-Da gene products (hrpJ3, J4, J5, U1, U2, respectively). The production of these proteins was confirmed by using T7 RNA polymerase-directed expression. The partial ORF was found to be identical to the C terminus of HrpJ2. The absence of apparent transcriptional terminators and promoters between hrpI (hrpJ2), hrpJ3, hrpJ4, and hrpJ5 together with the observation that the HrpL-dependent hrpJ promoter directs expression of hrpJ3-J5 indicates that these genes form a single operon controlled by the HrpL-dependent hrpJ promoter. A second HrpL-dependent promoter consensus sequence was also identified upstream of hrpU1 and demonstrated to function as a HrpL-dependent promoter, thus indicating that hrpU1, hrpU2, and additional downstream genes may be part of a second operon. The deduced product of hrpJ3 exhibits similarity to FliG of Salmonella typhimurium, a cytoplasmic protein that regulates flagellar rotation and biogenesis. HrpJ4 shares extensive similarity with the FliI family of ATPase-like proteins and retains the known functional domains conserved among this family of proteins. HrpJ5 has properties similar to the S. typhimurium FliJ. Neither HrpU1 nor HrpU2 exhibit significant similarity to known proteins. Secretion of HarpinPss by E. coli MC4100 transformants carrying pHIR11::TnphoA derivatives was blocked in hrpJ4, J5, and U2 mutants. In view of the previously reported similarity of HrpJ2 to the LcrD super-family that includes FlhA, these results predict that the gene products of the hrpJ and hrpU operons form an inner membrane complex for translocation of proteins similar to that used by the flagellar biogenesis system of S. typhimurium.

Amino Acid Sequence↗
PubMed1975

[Genesis of merozoites in the coccidia, Eimeria necatrix. Ultrastructural study].

Second generation schizonts of Eimeria necatrix were studied with the aid of the electron microscope. Differentiation of daughter merozoites is associated with the last mitosis, which is not significantly different from the earlier ones. The merozoites develop at the periphery of the schizont. The conoid and 22 subpellicular microtubules, probably induced by centrioles, and the inner membranes complex and the rhoptry anlagen which seem to be produced by the Golgi apparatus, appear close to each nuclear pole, just near the schizont membrane. These organelles are the first to appear in the merozoite anlagen. Then, nucleus, dictyosome and multimembranous vescles enter the budding merozoites. Micronemes, probably originating from Golgi apparatus, and amylopectin granules are produced later, when daughter merozoites separate from the residuum. The genesis of these various organelles and the relation between the last mitosis and differentiation are discussed.

Animals↗
PubMed1986

Ultrastructural studies of microgametogenesis and macrogametogenesis of Eimeria truncata of the lesser snow goose.

Microgamonts and macrogamonts of Eimeria truncata were observed in renal epithelial cells of collecting tubules and ducts and occasionally in macrophages of experimentally infected lesser snow geese (Anser c. caerulescens) beginning 8.5 days post inoculation. Intraparasitophorous vesicles in parasitophorous vacuoles of both types of gamonts appeared to originate in host cell cytoplasm and enter gamonts through micropores by budding of plasmalemma or by pinocytosis. Within the parasite's cytoplasm, the vesicles were broken down in Golgi-associated vacuoles. The surfaces of microgamonts were highly invaginated to facilitate extrusion of numerous microgametes. Formation and maturation of microgametes were similar to those of other eimerian species. Each microgamete had two flagella, a mitochondrion, and a peculiarly shaped electron-dense nucleus that was oval anteriorly in cross section and somewhat dumbbell-shaped posteriorly. A longitudinally arranged inner membrane complex lay between a portion of the mitochondrion and the plasmalemma. About five subpellicular microtubules extended the length of the microgamete body. Macrogametogony differed little from that described in other eimerian species. Type 1 wall-forming bodies (WFB) formed in Golgi complexes early in macrogametogony, and type 2 WFB formed in cisternae of endoplasmic reticulum in intermediate stages of macrogamont development.

Animals↗
PubMed2006

Identification of PhIL1, a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide.

The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[(125)I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [(125)I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.

Amino Acid Sequence↗