Aberrant Bodies Research Articles

Differential expression of chlamydial signal transduction genes in normal and interferon gamma-induced persistent Chlamydophila pneumoniae infections

Characteristic features of the persistent chlamydial developmental cycle, associated with chronic infections in both humans and animals, include the generation of non-replicative, morphologically aberrant bodies which are distinct from normal propagating reticulate bodies. Previous studies have correlated these morphological and metabolic changes with differential expression of diverse functional subsets of chlamydial genes. To further investigate these correlations, we compared mRNA expression of predicted chlamydial signal transduction genes between normal Chlamydophila pneumoniae A-03 infections in HEp-2 cells and those treated with gamma interferon (IFN-γ) by using real-time RT-PCR. Inspection of the Cp. pneumoniae genome revealed at least 39 candidate signal transduction genes, of which 30 were differentially expressed in Cp. pneumoniae mediated persistence. Functional sub-groups of differentially expressed signal transduction genes include chlamydial GTPases ( hflX, ychF, yhbZ and yphC), linked to bacterial cellular processes such as cell cycle control and ribosome assembly and stability. Other up-regulated signal transduction genes sharing similarity to bacterial stress response genes ( htrA, surE, lytB and hrcA) were also detected. The transcriptional changes observed for the majority of signal transduction genes appear to be unique for Cp. pneumoniae, as similar changes were not observed in recent whole genomic analysis of C. trachomatis IFN-γ mediated persistence. These results suggest that chlamydial signal transduction genes play potentially important roles in the establishment and maintenance of Cp. pneumoniae persistence, likely as part of the IFN-γ response stimulon as described for C. trachomatis, but with considerable differences in the transcriptional profile.

Tobacco smoke induces persistent infection of Chlamydophila pneumoniae in HEp-2 cells

We examined tobacco smoke exposure and its effect on the life cycle of Chlamydophila pneumoniae ( C. pneumoniae) in HEp-2, a human respiratory epithelial cell line. Using noncytotoxic concentrations of smoke medium, chlamydiae were grown in tissue culture and infectious particles were quantitated indirectly by immunocytometry of infected indicator cells. Chlamydial genome copy number was assessed with real-time polymerase chain reaction, and ultrastructure was examined by transmission electron microscopy. There was a significant reduction (56–64%; p<0.05) in the number of infectious elementary bodies following smoke exposure compared to untreated cultures. Under the same conditions, at late time points, smoke-exposed cultures showed significantly fewer chlamydial DNA copies ( p<0.04). Moreover, smoke exposure induced large aberrant bodies that predominated within the inclusion. Following in vitro smoke exposure, alterations in the developmental cycle of C. pneumoniae included: inhibition of productive infection, reduced bacterial cell division, and formation of aberrant bodies. Thus, using this novel system, we were able to induce chlamydial persistence. Tobacco smoke exposure may represent a risk for establishment of a chronic reservoir of C. pneumoniae infection within respiratory epithelium.

A HEX-1 crystal lattice required for Woronin body function in Neurospora crassa.

The Woronin body is a dense-core vesicle specific to filamentous ascomycetes (Euascomycetes), where it functions to seal the septal pore in response to cellular damage. The HEX-1 protein self-assembles to form this solid core of the vesicle. Here, we solve the crystal structure of HEX-1 at 1.8 A, which provides the structural basis of its self-assembly. The structure reveals the existence of three intermolecular interfaces that promote the formation of a three-dimensional protein lattice. Consistent with these data, self-assembly is disrupted by mutations in intermolecular contact residues and expression of an assembly-defective HEX-1 mutant results in the production of aberrant Woronin bodies, which possess a soluble noncrystalline core. This mutant also fails to complement a hex-1 deletion in Neurospora crassa, demonstrating that the HEX-1 protein lattice is required for Woronin body function. Although both the sequence and the tertiary structure of HEX-1 are similar to those of eukaryotic initiation factor 5A (eIF-5A), the amino acids required for HEX-1 self-assembly and peroxisomal targeting are absent in eIF-5A. Thus, we propose that a new function has evolved following duplication of an ancestral eIF-5A gene and that this may define an important step in fungal evolution.

ForC, a novel type of formin family protein lacking an FH1 domain, is involved in multicellular development in Dictyostelium discoideum.

Formins are highly conserved regulators of cytoskeletal organization and share three regions of homology: the FH1, FH2 and FH3 domains. Of the nine known formin genes or pseudogenes carried by Dictyostelium, forC is novel in that it lacks an FH1 domain. Mutant Dictyostelium lacking forC (DeltaforC) grew normally during the vegetative phase and, when starved, migrated normally and formed tight aggregates. Subsequently, however, DeltaforC cells made aberrant fruiting bodies with short stalks and sori that remained unlifted. DeltaforC aggregates were also unable to migrate as slugs, suggesting forC is involved in mediating cell movement during multicellular stages of Dictyostelium development. Consistent with this idea, expression of forC was increased significantly in aggregates of wild-type cells. GFP-ForC expressed in DeltaforC cells was localized at the crowns, which are macropinocytotic structures rich in F-actin, suggesting that, like other formin isoforms, ForC functions in close relation with the actin cytoskeleton. Truncation analysis of GFP-ForC revealed that the FH3 domain is required for ForC localization; moreover, localization of a truncated GFP-ForC mutant at the site of contacts between cells on substrates and along the cortex of cells within a multicellular culminant suggests that ForC is involved in the local actin cytoskeletal reorganization mediating cell-cell adhesion.

Giant electron dense chains, clusters and granules in megakaryocytes and platelets with normal dense bodies: an inherited thrombocytopenic disorder IV. Ultrastructural cytochemistry and analytical electron microscopy

Platelets from a mother and son with prolonged thrombocytopenia were shown in previous studies to contain giant organelles that developed in megakaryocytes and continued to evolve in circulating cells. Whole mount platelet preparations revealed that the large organelles were electron opaque like the serotonin-rich dense bodies in normal and patient platelets, and analytical electron microscopy revealed they contained large amounts of calcium and phosphorous in a ratio close to that found in normal platelet dense bodies. However, differences in physiology, biochemistry and morphology indicated the large opaque bodies and target-organelles in patient platelets were not aberrant dense bodies. The present study has shown that the giant organelles contain peroxidase activity like primary lysosomes in polymorphonuclear (PMN) leukocytes. Further, the giant organelles in patient platelets contain acid phosphatase activity. Analytical electron microscopy demonstrated that cerium, the capture ion for the acid phosphatase reaction product, was present in the opaque organelles with calcium and phosphorous, but not present in their normal dense bodies. Since normal sized lysosomes appeared to be reduced in patient platelets, it was concluded that the large structures were abnormal lysosomes, or fused with normal platelet lysosomes during their development. Similar giant lysosomes were not present in other patient blood cells. As a result the disorder can be considered a unique lysosomal disease of platelets.

The intracellular life of chlamydiae

The ability to cause persistent infection is one of the major characteristics of all chlamydial species in their appropriate hosts. Persistent infection with Chlamydia trachomatis and Chlamydia pneumoniae has been implicated in the pathogenesis of many chronic diseases, some initially not thought to be infectious, including pelvic inflammatory disease, arthritis, asthma, and atherosclerosis. Chlamydiae have a unique developmental cycle with morphologically distinct infectious and reproductive forms: elementary (EB) and reticulate bodies (RB). Chlamydiae appear to circumvent the host endocytic pathway and inhabit a nonacidic vacuole that is dissociated from late endosomes and lysosomes. Chlamydiae also have been demonstrated to enter a persistent state after treatment with cytokines such as interferon-γ (IFN-γ), treatment with antibiotics, or restriction of certain nutrients, or to enter this state spontaneously under certain culture conditions. While the organism is in the persistent state, metabolic activity is reduced, and the organism is often refractory to antibiotic treatment. Ultrastructural analysis of IFN-γ–treated C pneumoniae demonstrates atypical inclusions containing large reticulate-like aberrant bodies with no evidence of redifferentiation into EBs. Persistent C pneumoniae infection appears to be associated with continued expression of genes associated with DNA replication but not with those genes involved with bacterial cell division. The latter observation may explain the appearance of the large abnormal RBs seen in ultrastructural studies. Studies of the association of chlamydiae with chronic disease have been hampered by difficulties in diagnosing chronic, persistent infection with the organism, which, in turn, render determining the efficacy of antibiotic therapy very difficult. Copyright 2002, Elsevier Science (USA). All rights reserved.

Characterization of Chlamydia pneumoniae persistence in HEp-2 cells treated with gamma interferon.

Infection with Chlamydia pneumoniae has been implicated as a potential risk factor for atherosclerosis. This study demonstrated the effects of gamma interferon (IFN-gamma)-mediated indoleamine 2,3-dioxygenase activity on C. pneumoniae persistence in HEp-2 cells, inclusion morphology, and ultrastructure. C. pneumoniae replication showed a dose-dependent decrease when treated with increasing concentrations of IFN-gamma and a phenotypic switch resulting in a decrease in typical inclusions with an increase in smaller, less-dense atypical inclusions. Ultrastructural analysis of IFN-gamma-treated C. pneumoniae revealed atypical inclusions containing large reticulatate-like aberrant bodies with no evidence of redifferentiation into elementary bodies.

Chlamydia pneumoniae expresses genes required for DNA replication but not cytokinesis during persistent infection of HEp-2 cells.

Chlamydia pneumoniae causes community-acquired pneumonia and is associated with several chronic diseases, including asthma and atherosclerosis. The intracellular growth rate of C. pneumoniae slows dramatically during chronic infection, and such persistence leads to attenuated production of new elementary bodies, appearance of morphologically aberrant reticulate bodies, and altered expression of several chlamydial genes. We used an in vitro system to further characterize persistent C. pneumoniae infection, employing both ultrastructural and transcriptional activity measurements. HEp-2 cells were infected with C. pneumoniae (TW-183) at a multiplicity of infection of 3:1, and at 2 h postinfection gamma interferon (IFN-gamma) was added to the medium at 0.15 or 0.50 ng/ml. Treated and untreated cultures were harvested at several times postinfection. RNA was isolated and reverse transcribed, and reverse transcription (RT)-PCR analyses targeting primary transcripts from chlamydial rRNA operons as well as dnaA, polA, mutS, minD, ftsK, and ftsW mRNA were done. Some cultures were fixed and stained for electron microscopic analysis, and a real-time PCR assay was used to assess relative chlamydial chromosome accumulation under each culture condition. The latter assays showed that bacterial chromosome copies accumulated severalfold during IFN-gamma treatment of infected HEp-2 cells, although less accumulation was observed in cells treated with the higher dose. Electron microscopy demonstrated that high-dose IFN-gamma treatment elicited aberrant forms of the bacterium. RT-PCR showed that chlamydial primary rRNA transcripts were present in all IFN-gamma-treated and untreated cell cultures, indicating bacterial metabolic activity. Transcripts from dnaA, polA, mutS, and minD, all of which encode products for bacterial chromosome replication and partition, were expressed in IFN-gamma-treated and untreated cells. In contrast, ftsK and ftsW, encoding products for bacterial cell division, were expressed in untreated cells, but expression was attenuated in cells treated with low-dose IFN-gamma and absent in cells given the high dose of cytokine. Thus, the development of persistence included production of transcripts for DNA replication-related, but not cell division-related, genes. These results provide new insight regarding molecular activities that accompany persistence of C. pneumoniae, as well as suggesting requirements for reactivation from persistent to productive growth.

Pten is essential for embryonic development and tumour suppression.

The PTEN gene encodes a dual-specificity phosphatase mutated in a variety of human cancers. PTEN germline mutations are found in three related human autosomal dominant disorders, Cowden disease (CD), Lhermitte-Duclos disease (LDD) and Bannayan-Zonana syndrome (BZS), characterized by tumour susceptibility and developmental defects. To examine the role of PTEN in ontogenesis and tumour suppression, we disrupted mouse Pten by homologous recombination. Pten inactivation resulted in early embryonic lethality. Pten-/- ES cells formed aberrant embryoid bodies and displayed an altered ability to differentiate into endodermal, ectodermal and mesodermal derivatives. Pten+/- mice and chimaeric mice derived from Pten+/- ES cells showed hyperplastic-dysplastic changes in the prostate, skin and colon, which are characteristic of CD, LDD and BZS. They also spontaneously developed germ cell, gonadostromal, thyroid and colon tumours. In addition, Pten inactivation enhanced the ability of ES cells to generate tumours in nude and syngeneic mice, due to increased anchorage-independent growth and aberrant differentiation. These results support the notion that PTEN haploinsufficiency plays a causal role in CD, LDD and BZS pathogenesis, and demonstrate that Pten is a tumour suppressor essential for embryonic development.

A Baculovirus Mutant Defective in PKIP, a Protein Which Interacts with a Virus-Encoded Protein Kinase

We have found that a temperature-sensitive mutant of the baculovirus AcMNPV, tsB97, is defective in PKIP, the product of ORF24 which was previously found to interact with and stimulate the activity of a virus-encoded protein kinase, PK-1. The mutant lacks the ability to form plaques and occlusion bodies at the nonpermissive temperature. The mutant displays several properties which suggest a defect in the latter half of the late phase of infection; these properties include a delay in the shutoff of host protein synthesis, the presence of aberrant electron-dense bodies associated with the virogenic stroma, and the production of few, if any, progeny budded virus. A study of the expression of selected late genes showed no difference in the timing or level of transcription or translation of most late genes. However, elevated levels of the late 6.9K protein, a protamine-like protein, were observed in mutant-infected cells at 24 h postinfection, suggesting a defect in the regulation of this protein. Two polypeptides, 40 and 6 kDa, exhibited considerably higher levels of steady-state phosphorylation in wt-infected cells versus tsB97-infected cells at 24 h p.i. and could be candidates for PK-1/PKIP-mediated phosphorylation. The tsB97 mutant also displayed a severe defect in very late gene transcription which accounts for its inability to form occlusion bodies. The effect of PKIP on very late gene transcription may be a secondary effect of the block in the late phase of infection. PKIP showed no ability to transactivate expression from a very late promoter in transient expression assays.

Evidence that the RdeA protein is a component of a multistep phosphorelay modulating rate of development in Dictyostelium.

We have isolated an insertional mutant of Dictyostelium discoideum that aggregated rapidly and formed spores and stalk cells within 14 h of development instead of the normal 24 h. We have shown by parasexual genetics that the insertion is in the rdeA locus and have cloned the gene. It encodes a predicted 28 kDa protein (RdeA) that is enriched in charged residues and is very hydrophilic. Constructs with the DNA for the c-Myc epitope or for the green fluorescent protein indicate that RdeA is not compartmentalized. RdeA displays homology around a histidine residue at amino acid 65 with members of the H2 module family of phosphotransferases that participate in multistep phosphoryl relays. Replacement of this histidine rendered the protein inactive. The mutant is complemented by transformation with the Ypd1 gene of Saccharomyces cerevisiae, itself an H2 module protein. We propose that RdeA is part of a multistep phosphorelay system that modulates the rate of development.

Induction of terminal differentiation of Dictyostelium by cAMP-dependent protein kinase and opposing effects of intracellulr and extracellular cAMP on stalk cell differentiation.

Expression of the catalytic (C) subunit of the cAMP-dependent protein kinase (PKA) of Dictyostelium under the control of heterologous, cell-type-specific promoters causes ectopic terminal differentiation. When expressed under the control of a prespore-specific promoter, development is accelerated, to yield highly aberrant fruiting bodies that contain a basal mass of spore cells surrounding a central stalk-like structure. When expressed under the control of a prestalk-specific promoter, development arrests much earlier, at the tight mound stage. Prestalk cells move to the apices of these mounds, apparently normally, but no tip is formed. Most of the prestalk cells remain arrested in their development but there are a few isolated stalk cells scattered within such mounds. We show that extracellular cAMP represses stalk cell-specific gene expression in cells where the kinase is constitutively active, suggesting that inhibition of stalk cell differentiation by cAMP in normal cells (Berks and Kay, 1988) occurs because of an effect of extracellular cAMP on an intracellular signalling pathway independent of PKA. We propose a scheme whereby two separate events, a rise in intracellular cAMP levels and a fall in extracellular cAMP concentration, are required to induce stalk cell differentiation.

Analysis of a spatially regulated phosphotyrosine phosphatase identifies tyrosine phosphorylation as a key regulatory pathway in Dictyostelium

We have cloned a Dictyostelium phosphotyrosine phosphatase ( PTP1) with a catalytic domain showing ∼38%–50% amino acid identity to those of other PTPs. PTP1 contains an ∼99 amino acid insert and bacterially produced PTP1 possesses PTP activity. PTP1 is expressed at a very low level in vegetative cells, induced by 4 hr, and maximally expressed at the tight aggregate stage. PTP1-lacZ studies indicate that PTP1 is spatially localized to prestalk and anterior-like cell types. PTP1 gene disruptants show accelerated development, whereas strains overexpressing PTP1 to a high level fail to aggregate. Strains overexpressing moderate levels exhibit severe morphological defects following aggregation, including multiply tipped aggregates and morphologically aberrant fruiting bodies. Western blot analysis using anti-phosphotyrosine antibodies shows specific changes in the mutant strains when compared with wild-type cells. The results indicate that reversible protein-tyrosine phosphorylation and PTP1 play important regulatory roles during Dictyostelium development.

Implications for persistent chlamydial infections of phagocyte-microorganism interplay

In vitro models of Chlamydia trachomatis inhibition by cytokines, human-monocyte derived macrophages (HMDM) and human polymorphonuclear leukocytes (HPMN) are discussed in an attempt to delineate the molecular basis of parasite-host cell interplay in persistent and chronic chlamydial infection. Interferon gamma (IFN) has been found to reversibly inhibit chlamydial growth at an early stage in the replicative cycle, while tumor necrosis factor (TNF) has a more profound effect on chlamydial growth resulting in production of aberrant reticulate bodies and enhancement of production of prostaglandin E2 (PGE2). Chlamydia trachomatis (serovar L2) replicate in HMDM while serovar K has been found to be restricted in these cells. Chlamydiae are killed by HPMN but the cell walls persist undegraded, inducing production of oxygen radicals which can be demonstrated to induce DNA strand scissions in HeLa target cells. Evidence is accumulating that chlamydia specific serum IgA antibodies may serve as a noninvasive serological marker for diagnosis of a number of acute and persistent Chlamydia trachomatis infections.

The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene

Mutations in the fission yeast cut1 +, cut2 +, and cut10 + genes uncouple normally coordinated mitotic events and deregulate, rather than arrest, mitosis. DNA synthesis continues, making polyploid nuclei with several spindles. Multiple, aberrant spindle pole bodies (SPBs) are produced in cut1 mutant cells. The cut1 + and cut2 + genes are cloned by transformation. High gene dosage of cut1 + also complements cut2 and cut10 mutants. The cut2 + gene, however, complements only cut2. The 210 kd cut1 + gene product contains putative ATP binding and helical coll regions followed by a COOH-terminal domain homologous to the S. cerevislae gene ESP1. Mutations in the ESP1 gene also result in many SPBs. The cut1 + product is shown by anti-cut1 antibody to be a rare component of the insoluble nuclear fraction. It may play a key role in coupling chromosome disjunction with other cell cycle events and is potentially a component, regulator, or motor for the SPB and/or kinetochores.

Microtubules and lithocyst morphogenesis in Pilea cadierei

Lithocysts in Pilea cadierei are initiated by differential divisions, the plane of which is predicted by typical preprophase microtubule bands. They soon become polarized and undergo a unique differentiation. The external periclinal wall thickens considerably in the absence of microtubules. In incipient lithocysts, a periclinal band of microtubules lines the external ends of the anticlinal walls. A distinct local thickening, possessing periclinal cellulose microfibrils, underlies the microtubule band. The cystolith stalk originates as a cylindrical wall ingrowth of a limited central region of the external periclinal wall. This grows inwards in the absence of microtubules in a preformed cytoplasmic diaphragm, approaching the internal periclinal wall. Stalk formation as well as the external periclinal wall thickening are not affected by colchicine and isopropyl n(3-chlorophenyl) carbamate. A cystolith body is formed within a cytoplasmic diaphragm in which most of the lithocyst organelles are located; this body is rich in cellulose microfibrils and is formed by the deposition of large amounts of wall material at the free end of the stalk, at right angles to its axis. This morphogenetic shift is preceded by the formation of a system of microtubules that converge in cortical sites close to the stalk. They proliferate and form an axial sheath around the cystolith body. The cellulose microfibrils are aligned in a parallel fashion with microtubules. Microtubule destruction by colchicine results in malformation of both the lithocyst and the cystolith body. Aberrant cystolith bodies are also formed in the presence of isopropyl n(3-chlorophenyl) carbamate, which seems to interfere with microtubule formation. These observations suggest that the lithocyst – cystolith body morphogenesis is controlled by microtubules.

Inhibition of growth of Chlamydia trachomatis by human gamma interferon

Treatment of HEp-2 cell cultures with highly purified human gamma interferon before infection resulted in the reduction of Chlamydia trachomatis (L2/434/Bu) infectious particle yield. Electron microscope studies showed that interferon did not affect chlamydial conversion to reticulate bodies but influenced the extent of maturation to elementary bodies. High interferon concentrations (greater than 350 IU/ml) inhibited inclusion body formation and resulted in the appearance of aberrant reticulate bodies.

Disruption of microtubules and retardation of development ofDictyostelium with ethylN-phenylcarbamate and thiabendazole

The effects of ethylN-phenylcarbamate (EPC) and thiabendazole (TB) onDictyostelium discoideum andD. mucoroides cells were examined as a step toward purifying tubulin and clarifying the function of microtubules in cellular slime molds. EPC (1.5 × 10−3M) or TB (5 × 10−5M) inhibited the development ofDictyostelium, inducing the formation of aberrant fruiting bodies with stalks irregular in shape and sori containing spores of various sizes and shapes.

FINE STRUCTURE OF CHLOROPLASTS IN “GREEN ISLANDS” AND IN SURROUNDING CHLOROTIC AREAS OF BARLEY LEAVES INFECTED BY POWDERY MILDEW

The fine structure of mesophyll chloroplasts in green islands and in adjacent chlorotic areas of barley leaves infected with Erysiphe graminis (DC.) was compared with healthy non‐inoculated tissue. Chloroplasts in green islands were persistent. Green‐island chloroplast grana were enlarged and fewer in number than in healthy tissue. In contrast, cells in chlorotic areas had fewer chloroplasts and their lamellae showed progressive degeneration and fragmentation. The lamellae often resembled aberrant prolamellar bodies. As lamellar degeneration progressed there was a marked increase in the amount of osmophilic material within the chloroplasts.