Host Cell Mitochondria Research Articles

Toxoplasma gondiiinfection confers resistance against BimS-induced apoptosis by preventing the activation and mitochondrial targeting of pro-apoptotic Bax

In order to accomplish their life style, intracellular pathogens, including the apicomplexan Toxoplasma gondii, subvert the innate apoptotic response of infected host cells. However, the precise mechanisms of parasite interference with the mitochondrial apoptotic pathway remain unknown. Here, we used the conditional expression of the BH3-only protein Bim(S) to pinpoint the interaction of T. gondii with the intrinsic pathway of apoptosis. Infection of epithelial cells with T. gondii dose-dependently abrogated Bim(S)-triggered release of cytochrome c from host-cell mitochondria into the cytosol, induction of activity of caspases 3, 7 and 9, and chromatin condensation. Furthermore, inhibition of apoptosis in parasite-infected lymphocytes counteracted death of Toxoplasma-infected host cells. Although total cellular levels and mitochondrial targeting of Bim(S) was not altered by the infection, the activation of pro-apoptotic effector proteins Bax and Bak was strongly impaired. Inhibition of Bax and Bak activation by T. gondii was seen with regard to their conformational changes, the cytosol-to-mitochondria targeting and the oligomerization of Bax but not their cellular protein levels. Blockade of Bax and Bak activation was not mediated by the upregulation of anti-apoptotic Bcl-2-like proteins following infection. Further, the BH3-mimetic ABT-737 failed to overcome the Toxoplasma-imposed inhibition of Bim(S)-triggered apoptosis. These results indicate that T. gondii targets activation of pro-apoptotic Bax and Bak to inhibit the apoptogenic function of mitochondria and to increase host-cell viability.

Mitochondrial localization of non‐histone protein HMGB1 during human endothelial cell–Toxoplasma gondii infection

Toxoplasma gondii is an obligate intracellular pathogen, replicating only within a specialized membrane-bounded cytoplasmic vacuole, the parasitophorous vacuole (PV), which interacts with host cell mitochondria. High mobility group box 1 (HMGB1), a known nuclear transcription factor, also may be involved in pathological conditions, whose function is to signal tissue damage. Using confocal microscopy, we have investigated the localization of HMGB1 and the mitochondria performance during interaction between human umbilical vein endothelial cells (HUVEC) and Toxoplasma. Immunofluorescence showed HMGB1 localization in HUVEC tubular mitochondria stained with Mito Tracker (MT). At 2h post-infection, MT labeled spherical structures scattered throughout the cytoplasm and HMGB1 were still present. After 24h of infection, long and tubular structures were localized around PVs and were double labeled by MT and HMGB1, suggesting a structural reorganization of the mitochondria over a long period of infection. For the first time, these results show there is HMGB1 in HUVEC mitochondria and that this protein could be playing a part in mitochondrial DNA events which are important for fission and fusion processes reported here during HUVEC-T. gondii infection.

Shigella flexneri Induces Cell Death by Damage to and Autophagy of Host Cell Mitochondria: An Electron-Microscopic Study of Infected Human Monocyte Derived Macrophages

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

Multinucleate host cells induced by Vittaforma corneae (Microsporidia)

The microsporidium Vittaforma corneae develops within the target cell cytoplasm. In the present study, green monkey kidney (E6) cells infected at 30 degrees C, 35 degrees C or 37 degrees C with V. corneae developed enlarged multinucleate structures of up to 200 microm in any horizontal dimension made up either of a single cell or of multiple fused cells. A number of epithelial cell types (SW-480, HT-29, Caco-2 and HCT-8) were infected with V. corneae but did not induce the same highly organized structures, suggesting that for the structure to develop, the host cell must be capable of continued mitosis, and not be differentiated or be detaching from the surface matrix. Live cell imaging of infected E6 cells revealed large, multinucleate infected cells characterized by a central focus from which radiated parasite stages and host cell mitochondria. Immunocytochemistry identifying gamma and alpha tubulin suggested that a single centrally-located microtubule organizing centre governed the distribution of parasite stages and host cell organelles, with mitochondria and parasites being eventually transported towards the periphery of the structure. Whole cell patch clamp analysis of infected cells indicated an average five-fold increase in total membrane capacitance, consistent with an enlarged single cell. Scanning electron microscopy revealed cell-like protrusions around the periphery of the structure with the intervening space being made up of parasites and cell debris. Clearly in the case of V. corneae-infected E6 cells the parasite-host cell relationship involves subverting the host cell cytoskeleton and cell volume control, providing the parasite with the same protected niche as does a xenoma.

Relationship between the host cell mitochondria and the parasitophorous vacuole in cells infected with Encephalitozoon microsporidia.

Encephalitozoon microsporidia proliferate and differentiate within a parasitophorous vacuole. Using the fluorescent probe, calcein, and the mitochondrial probe, MitoTracker-CMXRos, a vital method was developed that confirmed ultrastructural reports that the host cell mitochondria frequently lie in immediate proximity to the parasitophorous vacuole. Morphometry failed to demonstrate any infection-induced increase in host cell mitochondria as there was no correlation between the mitochondrial volume and the extent of infection as judged by the parasitophorous vacuole volume. The total ATP concentration of infected cells did not differ from that of uninfected cells in spite of the increased metabolic demands of the infection. Treatment with 10(-6) M albendazole, more than ten times the antiparasitic IC50 dose, and demecolcine had no subjective effect on the proximity of mitochondria to the parasitophorous vacuole membrane when studied by either transmission electron microscopy or by confocal microscopy even though these drug concentrations affected microtubule structure. Thus, once the association between mitochondria and the parasitophorous vacuole has been established, host cell microtubule integrity is probably not required for its maintenance. It is unlikely that the antimicrosporidial action of albendazole involves physically uncoupling developing parasite stages from host cell organelle metabolic support.

VDAC and the bacterial porin PorB of Neisseria gonorrhoeae share mitochondrial import pathways.

The human pathogen Neisseria gonorrhoeae induces host cell apoptosis during infection by delivering the outer membrane protein PorB to the host cell's mitochondria. PorB is a pore-forming beta-barrel protein sharing several features with the mitochondrial voltage-dependent anion channel (VDAC), which is involved in the regulation of apoptosis. Here we show that PorB of pathogenic Neisseria species produced by host cells is efficiently targeted to mitochondria. Imported PorB resides in the mitochondrial outer membrane and forms multimers with similar sizes as in the outer bacterial membrane. The mitochondria completely lose their membrane potential, a characteristic previously observed in cells infected with gonococci or treated with purified PorB. Closely related bacterial porins of non-pathogenic Neisseria mucosa or Escherichia coli remain in the cytosol. Import of PorB into mitochondria in vivo is independent of a linear signal sequence. Insertion of PorB into the mitochondrial outer membrane in vitro depends on the activity of Tom5, Tom20 and Tom40, but is independent of Tom70. Our data show that human VDAC and bacterial PorB are imported into mitochondria by a similar mechanism.

Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole.

Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry-derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein-rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite-containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre-existing, parasite-containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two-step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by TOXOPLASMA: The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.

Molecular cloning, organellar targeting and developmental expression of mitochondrial chaperone HSP60 in Toxoplasma gondii

The obligate intracellular protozoan parasite Toxoplasma gondii has a single tubular mitochondrion. During infection, it recruits the host cell's mitochondria abutting to the intracellular vacuole, that contains the parasites. The respective contribution of host and parasitic mitochondria in the intracellular growth of T. gondii remains unknown. Heat shock protein, HSP60 has been reported in all eukaryotes examined, as an essential chaperone required for the folding and multimeric complex assembly of mitochondrial proteins. Here, we report the isolation and molecular characterization of two cDNAs corresponding to a single T. gondii gene coding for HSP60. Using a model fusion protein, preHSP60-chloramphenicol acetyl transferase (CAT), we demonstrate that the classical 22 amino acid mitochondrial presequence and the adjacent 32 amino acids of the mature protein are both required for the in vivo import into T. gondii mitochondria. The T. gondii HSP60 gene composed of five introns and six exons is transcribed into two related but differently spliced transcripts. Whereas the two transcripts can be detected in both developmental stages within the intermediate host, their levels are significantly increased in bradyzoites when compared to tachyzoites. By immunoblot analysis, the predicted 60-kDa protien corresponding to HSP60 was detected in both tachyzoite and bradyzoite forms. Using immunofluorescence assays, the polyclonal antibodies specific to T. gondii HSP60 recognized the mitochondrion in tachyzoites, as expected. In contrast, these antibodies reacted against two unknown vesicular bodies which are distinct from the classical mitochondrial pattern in bradyzoites. Taken together, these expression patterns of mitochondrial chaperone HSP60 suggests stage-specific induction of the respiratory pathway in the protozoan parasite T. gondii.

Ultrastructural differentiation of the genogroups in the genus Ehrlichia.

Ultrastructural characteristics of 15 strains and isolates of ehrlichiae belonging to three genogroups, or clades of genetically related organisms united in the genera Ehrlichia, Cowdria, Anaplasma, Neorickettsia and a strain of Wolbachia pipientis which represents a fourth genogroup in this cluster of species, were studied in continuous cell culture or in vivo: E. canis (Oklahoma strain and VHE isolate), E. muris (AS 145), E. chaffeensis (Arkansas, 91HE17 and Sapulpa), human granulocytic ehrlichiae (HGE)(BDS, 96HE27, 96HE37, #54, #55 and #72), E. equi (MRK), E. sennetsu (Miyayama), E. risticii (HRC-IL). Wolbachia pipientis was studied in the naturally infected Aedes albopictus mosquito cell line Aa23. All organisms were similar in the normal ultrastructure of individual cells and in the ability to form abnormal, pathological ehrlichial cells of the same type irrespective of the species. Normally all ehrlichiae studied in cell culture existed in two morphological forms - reticulate and dense-cored cells, both of which could divide by binary fission. Most alterations were related to their membranes, especially the cell wall. Differences in the structure of intravacuolar microcolonies (morulae) of ehrlichiae and their inter-relations with the host cells allowed differentiation of the genogroups: the E. canis-E. chaffeensis-E. muris genogroup formed large morulae, with many ehrlichiae, often suspended in a fibrillar matrix, and the host cell mitochondria and endoplasmic reticulum usually aggregated near the morulae and were in contact with the morula membrane; the E. phagocytophila-E. equi-HGE group morulae had no fibrillar matrix, no contacts with host cell mitochodria, and they did not aggregate around the morulae; E. sennetsu-E. risticii group usually developed in small individual vacuoles that did not fuse with each other and divided along with the ehrlichiae.

Ultrastructure of the endophyte and localization of nifH transcripts in root nodules of Coriaria nepalensis Wall, by in situ hybridization

SUMMARYStudies on the root nodules of Coriaria nepalensis Wall. using light and electron microscopy revealed that infected cortical cells are enlarged and form a compact kidney‐shaped region with an acentric stele. Within a single cell, the actinomycetous endophyte has branched hyphae and elongate vesicles. Hyphae penetrate the host cell wall in different directions. Inside the host ceil, the hyphae are located in the peripheral region of the cytoplasm near the host cell whilst the vesicles are positioned towards the centre of the cell around the central vacuole. No septa formation was observed in the microsymbiont vesicles. Host cell mitochondria were aggregated near the hyphal/vesicular junction of the endophyte. In situ hybridization studies demonstrated that the nifH transcripts are unequally distributed over the infected cortical cells. In infected cells, nifH mRNA was located in the region occupied by the elongate vesicles of the endophyte near the host cell central vacuole. The results indicate that the elongate vesicles of the nodule endophyte of C. nepalensis are functionally identical to the spherical vesicles of the nodule endophyte of Alnus.

Development of a rickettsia isolated from an aborted bovine fetus

An obligate intracellular rickettsial organism isolated from an aborted bovine fetus was studied in bovine turbinate and mouse macrophage cell cultures with light and electron microscopy. Development of the organism was similar in both cell types. The organism replicated within cytoplasmic vacuoles in a developmental cycle that resembled that of both the ehrlichiae and chlamydiae. The inoculum contained only electron-dense forms, which infected cells within 2 h postinoculation by adhering to cell membranes at thickened areas that appeared to be coated pits and then being endocytosed. A striking feature occurred next as the organisms became surrounded by host cell mitochondria and, by light microscopy, appeared to have halos. During this intimate association with mitochondria, the electron-dense organisms changed into large reticulated forms that began to divide by binary fission. These large forms were often in direct contact with mitochondrial membranes. The organisms continued to divide by binary fission, and host cells contained large cytoplasmic inclusions of reticulated organisms. The reticulated organisms gradually changed into electron-dense forms that were released from degenerated host cells.

O 2 consumption and superoxide dismutase content in purified mitochondria from soybean root nodules

Soybean nodule mitochondria have been separated from cotnaminating organella on discontinuous Percoll gradients. The preparations appeared highly purified and at least 80% of the mitochondria were estimated to be derived from infected cells. Percoll-purified mitochondria showed important respiratory activity; in the case of succinate, the rate of O 2 consumption was 185 nmol O 2 min −1 mg −1 and the respiratory control and ADP/O ratio reached 2.72 and 1.16, respectively. These organelles also exhibited an active manganese containing superoxide dismutase (9.7 U mg −1), whose purification is reported. These results are consistent with a significant O 2 consumption by host cell mitochondria in vivo and the possibility of a competition for O 2 supply with the bacteroids is discussed.

Untersuchungen zur Anfälligkeit und Resistenz von Kopfsalat (Lactuca sativa) gegen falschen Mehltau (Bremia lactucae) III. Peroxydase‐, peroxydatische Katalase‐und Polyphenoloxydase‐Aktivitäten

Abstract Investigations on the susceptibility of head lettuce (Lactuca sativa) to downy mildew (Bremia lactucae) III. Activities of peroxidase, catalase and polyphenoloxidase Host cell walls in contact with intercellular hyphae of Bremia lactucae stain electron positively in susceptible and incompletely resistant varieties of lettuce after appropriate electron microscopy preparation for peroxidase activity. The outer membranes of the mitochondria of the parasite also stained darkly in susceptible varieties whereas in incompletely resistant plants Bremia innermost mitochondrial membranes and host cell mitochondria were darkly stained. This latter observation suggests increased respiration and could be explained as a resistance reaction. Catalase activity was observed in the microbodies of susceptible, in incompletely resistant and healthy varieties. There were no differences in stain intensity in the three kinds of varieties suggesting that catalase activity is not involved in resistance reactions.Polyphenoloxidase activity was infrequently observed on the host cell wall in susceptible and healthy plants, whereas strong activity in incompletely resistant varieties was observed in vesicles in the haustorial sheath. These vesicles were not surrounded by unit membranes and therefore could not have originated from the unit membranes of the extrahaustorial matrix or from the host plasmalemma. They may have been derived from the host protoplast and involved in inactivation of parasite produced toxins thereby contributing to resistance.

Morphological effects of arprinocid on developmental stages of Eimeria tenella and E. brunetti.

Medication of chicks with 70 p.p.m. arprinocid in the food, starting 2 days prior to inoculation with Eimeria tenella, resulted in decreased oocyst production and oocyst sporulation. The main morphological alteration seen during the development of the second-generation schizont was a reduction in the number of second-generation merozoites due to incomplete merogony, resulting in large masses of vacuolated residual cytoplasm. In places around the developing macrogamete the integrity of the parasitophorous vacuole was lost by either the host cell limiting membrane making contact with the parasite pellicle, or the disruption of one or both of these surfaces apparently brought about by the intervention of the host cell mitochondria. Thirty p.p.m. arprinocid reduced sporulation of the oocysts of E. brunetti and caused enlargement of the perinuclear space in the late merozoites and swelling of the endoplasmic reticulum around wall-forming bodies II.

Visualization of the mitochondria of Toxoplasma gondii-infected mouse fibroblasts by the cationic permeant fluorescent dye rhodamine 123.

The mitochondria of living mouse fibroblasts infected with Toxoplasma gondii were monitored with the cationic permeant fluorescent dye rhodamine 123. Fluorescence microscopy revealed that host cell mitochondria accumulated at the cytoplasmic surface of parasitophorous vacuoles and increased the dye uptake in the periparasitophorous vacuole as T. gondii multiplied.

Reduction in the mitochondrial membrane potential of Toxoplasma gondii after invasion of host cells.

The membrane potential of Toxoplasma gondii, an obligatory intracellular protozoan parasite, was monitored with the cationic permeant fluorescent dye rhodamine 123 (R123). Fluorescence microscopy revealed R123 to be partitioned predominantly in a restricted part of the parasite, which consisted of twisted or branched tubules, or of granular bodies. These structures were frequently connected to each other. The dye retention by these structures was markedly reduced by treating R123-labelled parasites with the proton ionophore, carbonylcyanide m-chlorophenylhydrazone, the potassium ionophore, valinomycin and the inhibitor of electron transport, antimycin A. Thus, these structures are regarded as the parasite mitochondria. Another cationic fluorescent dye, rhodamine 6G, stained the parasite mitochondria, whereas a negatively charged fluorescent dye, fluorescein, and the neutral compounds, rhodamine 110 and rhodamine B, did not. This fact indicates that R123 monitored the parasite mitochondrial membrane potential. T. gondii-infected 3T3 cells were also stained with R123. In contrast to the mitochondria of extracellular parasites, those of intracellular parasites failed to take up the dye. The absence of fluorescence in intracellular parasites persisted until the infected host cells ruptured and liberated daughter parasites 1 day after infection. Parasites, liberated from the host cells, either spontaneously or artificially by passing the infected cells through a 27G needle, regained the ability to take up the dye. After direct microinjection of R123 into the vacuole in which the parasite grows and multiples, the dye appeared in the host-cell mitochondria but not in the parasite's mitochondria. Thus, we conclude that the mitochondrial membrane potential of T. gondii was reduced after invasion of host cells by the parasite.

Ultrastructure of the Peripheral Zone of a Glugea‐Induced Xenoma*

The Glugea stephani-induced xenoma in the winter flounder, Pseudopleuronectes americanus, is a large spherical host-parasite complex, up to 4.0 mm in diameter, with the host and parasite components of the xenoma being most active in the peripheral zone. The xenoma has an extensive periodic acid-silver methenamine-positive surface coat covering the plasma membrane. The surface of this membrane is amplified by the presence of numerous folds and fine tubular extensions. The peripheral zone of the xenoma contains many host-cell mitochondria in addition to numerous microsporidan parasites. At the ultrastructural level, the peripheral zone of the host-cell cytoplasm appears normal. Inside the peripheral region of the 0.4-1.0 mm xenoma, the host-cell component largely disintegrates in the presence of microsporidan parasites undergoing sporogenesis.

In vitro development from sporozoites to first-generation merozoites in Eimeria contorta Haberkorn, 1971. A fine structural study.

The asexual development of Eimeria contorta from sporozoites to first-generation merozoites in tissue culture was investigated with the electron microscope. Sporozoites with a three-layered pellicle, 26 subpellicular microtubules, a conoid, 4-7 rhoptries, and an abundance of micronemes actively entered host cells and showed direct contact to the host cell's cytoplasm. Shortly after penetration, small vacuoles surrounding the parasite merged into a parasitophorous vacuole. Inside this vacuole, sporozoites assumed a definite U-shape before transformation into schizonts took place. This process was characterised by the occurrence of subpellicular microtubules exclusively in the anterior half of the sporozoite, by a degeneration of the 2 inner pellicular membranes, by an outpocketing of the parasite's surface, and by the arrangement of microtubules in clusters. About 25 merozoites were formed at the surface of mature schizonts, to which they remained attached at their posterior pole. A polar ring was present at that area. Anterior and posterior refractile bodies were conspicuous in merozoites and showed close association with mitochondria. The significance of a fibrillar substructure in rhoptries and micronemes is discussed, and special attention is drawn to the pathway of nutrient transport from host cell mitochondria and dictyosomes through intravacuolar folds, parasitophorous vacuole and crescent body into the parasite's food vacuoles.

Isolation and biochemical activities of trehalose-6-monomycolate of Mycobacterium tuberculosis.

A monoester of trehalose linked at the 6-position with mycolic acids (trehalose-6-monomycolate) was isolated from the wax D fraction of virulent human Mycobacterium tuberculosis, and its biochemical action on host-cell mitochondria was studied. Trehalose-6-monomycolate showed a delayed toxicity for mice. The 50% lethal dose at 2 weeks was 452 mug. It induced in vitro a swelling of mouse liver mitochondria and uncoupled respiration and phosphorylation in the nicotinamide adenine dinucleotide pathway of the electron transport chain. The site of functional damage was located specifically at coupling site II. Mitochondrial adenosine triphosphatase was slightly stimulated by trehalose-6-monomycolate. These findings indicate that trehalose-6-monomycolate affects mitochondrial oxidative phosphorylation in a similar manner to, but to a lesser extent than, trehalose-6, 6'-dimycolate (cord factor) of M. tuberculosis.

Action of a toxic glycolipid of Corynebacterium diphtheriae on mitochondrial structure and function.

The toxicity for mice of the cord factor of Cornebacterium diphtheriae, trehalose-6,6'-dicorynomycolate, was studied. The diphtherial cord factor showed a delayed lethal toxicity for mice; the median lethal dose was 120 mug. Mouse liver mitochondria were disrupted in vivo under the toxic action of the diphtherial cord factor into fragments deficient in both respiration and phosphorylation. The site of metabolic defect in the mitochondrial electron transport system was located at the region prior to the level of cytochrome c in the livers of cord factor-intoxicated mice. These effects of the diphtherial cord factor on the host-cell mitochondria were essentially similar to those of the cord factor of Mycobacterium tuberculosis, trehalose-6,6'-dimycolate.