Peculiarities of metabolism of the microsporidia Nosema grylli during the intracellular development

A long adaptation of Microsporidia to intracellular development supposes the host-derived ATP dependence of merogony and sporogony stages. To prove this assumption the activities of ten carbohydrate and energy metabolism enzymes were compared in the microsporidia Nosema grylli intracellular stages and mature spores. This species infects the fat body of crickets Gryllus bimaculatus. We have demonstrated lower activities of glycolytic enzymes, phosphoglucomutase and glucose-6-PhDH in the metabolically active meronts and sporonts than in the dormant mature spores. Low glycolysis level indicates that carbohydrate catabolism is not a principal mechanism of ATP supply in the N. grylli intracellular stages. Furthermore, we have not revealed a preferable expenditure of glycogen in comparison with triglycerides in infected cricket fat bodies. The N. grylli infection causes an equal reduction of glycogen and lipid content approximately in 2-3 times. Microsporidia have not mitochondria, Krebs cycle and electron-transport chain. Therefore they are not able to utilise fat reserves for ATP production. It seems to be proposed that microsporidia consume exogenous ATP which is produced by host cell metabolic system. The N. grylli infection provokes an increase of ATP content and ratio of ATP/ADP concentrations in cricket fat bodies approximately in 4 times. These data indicates a rise of host cell energy metabolism rate during the infection.     

Augmentation of microsporidia adherence and host cell infection by divalent cations

The infection process of intracellular opportunistic microsporidia involves the forcible eversion of a coiled hollow polar filament that pierces the host cell membrane, allowing the passage of infectious sporoplasm into the host cell cytoplasm. Although the exact mechanism of spore activation leading to polar filament discharge is unknown, we have shown that spore adherence to host cells, which is mediated by sulfated glycosaminoglycans, may play a vital role. When adherence is inhibited, host cell infection decreases, indicating a direct link between adherence and infection. The goal of this study was to evaluate the effects of exogenous divalent cations on microsporidia spore adherence and infection. Data generated using an in vitro spore adherence assay show that spore adherence is augmented by manganese (Mn2+) and magnesium (Mg2+), but not by calcium (Ca2+). However, each of the three divalent cations contributed to increased host cell infection when included in the assay. Finally, we show that Mn2+ and Mg2+ may activate a constituent on the microsporidia spore, not on the host cell, leading to higher infection efficiency. This report further supports recent evidence that spore adherence to the host cell surface is an important aspect of the microsporidial infection process.     

Hydroxyurea inhibits intracellular Toxoplasma gondii multiplication

Tachyzoites of Toxoplasma gondii multiply within the parasitophorous vacuole (PV) until the lysis of the host cell. This study was undertaken to evaluate the effect of hydroxyurea (a specific drug that arrests cell division at G1/S phase) on the multiplication of T. gondii tachyzoites in infected Vero cells. Infected host cells were treated with hydroxyurea for periods varying from 5 to 48 h, and the survival and morphology of the parasite were determined. Hydroxyurea arrested intracellular T. gondii multiplication in all periods tested. After 48 h of incubation with hydroxyurea, intracellular parasites were not easily observed in Vero cells. Ultrastructural observations showed that infected host cells treated with hydroxyurea for 24 h or more presented disrupted intracellular parasites within the PV. However, the host cells exhibited a normal morphology. Our observations suggest that hydroxyurea was able to interfere with the cycle of the intracellular parasite, leading to the complete destruction of the T. gondii without affecting the host cells.     

Unique physiology of host–parasite interactions in microsporidia infections

Microsporidia are intracellular parasites of all major animal lineages and have a described diversity of over 1200 species and an actual diversity that is estimated to be much higher. They are important pathogens of mammals, and are now one of the most common infections among immunocompromised humans. Although related to fungi, microsporidia are atypical in genomic biology, cell structure and infection mechanism. Host cell infection involves the rapid expulsion of a polar tube from a dormant spore to pierce the host cell membrane and allow the direct transfer of the spore contents into the host cell cytoplasm. This intimate relationship between parasite and host is unique. It allows the microsporidia to be highly exploitative of the host cell environment and cause such diverse effects as the induction of hypertrophied cells to harbour prolific spore development, host sex ratio distortion and host cell organelle and microtubule reorganization. Genome sequencing has revealed that microsporidia have achieved this high level of parasite sophistication with radically reduced proteomes and with many typical eukaryotic pathways pared-down to what appear to be minimal functional units. These traits make microsporidia intriguing model systems for understanding the extremes of reductive parasite evolution and host cell manipulation.     

Inhibition of Trypanosoma cruzi egress from infected fibroblasts is mediated by CD4+ and mu+ immune cells

Trypanosoma cruzi, the causative agent of Chagas disease, is able to reproduce intracellularly in many host cell types while in the mammalian host. Although cellular immunity is known to be important in resistance to infection, the ability of immune cells to interfere with the completion of the intracellular growth cycle of T. cruzi has not been described. Using a tissue culture system to study the parasite growth cycle, we have found that spleen cells from infected mice are able to decrease the number of parasites released from infected fibroblasts. Spleen cells from mice infected for as few as 14 days and as long as 300 days display this inhibitory ability. Parasite egress from infected cells is inhibited by factor(s) released by immune cells during coculture with infected fibroblasts. Immune cell depletion studies indicate that the inhibitory activity requires the presence of both CD4+ T cells and mu+ B cells. These results suggest a direct ability of immune cells to somehow interfere with the completion of the intracellular cycle, and this ability may play a role in control of this parasite.     

Infection by microsporidia disrupts the host cell cycle

Microsporidia of the genus Encephalitozoon infect mammalian cells and have become a source of morbidity and mortality in immunocompromised humans. Encephalitozoon microsporidia develop and mature within parasitophorous vacuoles, enlarging the vacuole over time until it eventually occupies most of the cytoplasm of the host cell. The ability of the host cell to accommodate such a large burden for several days suggests that the parasite subverts normal host cell processes to ensure optimal environmental conditions for its growth and development. Since this environment would be threatened if cell division of the host cell occurred, we have formulated the hypothesis that infection with Encephalitozoon microsporidia induces an arrest in the cell cycle of the host cell. In support of this hypothesis, we have found that mitotic index and DNA duplication are reduced in infected cells as compared to uninfected cells. The number of host cell nuclei in S phase is increased. The levels of cyclin D1 and the percentage of cells in G1 are reduced; however, the levels of cyclin B1 are elevated even though the percentage of cells in G2/M is decreased. These results suggest that host cells infected with Encephalitozoon microsporidia are blocked at multiple points in the cell cycle.     

The ins and outs of host‐microsporidia interactions during invasion,proliferation and exit

Microsporidia are a large group of fungal‐related obligate intracellular parasites. They are responsible for infections in humans as well as in agriculturally and environmentally important animals. Although microsporidia are abundant in nature, many of the molecular mechanisms employed during infection have remained enigmatic. In this review, we highlight recent work showing how microsporidia invade, proliferate and exit from host cells. During invasion, microsporidia use spore wall and polar tube proteins to interact with host receptors and adhere to the host cell surface. In turn, the host has multiple defence mechanisms to prevent and eliminate these infections. Microsporidia encode numerous transporters and steal host nutrients to facilitate proliferation within host cells. They also encode many secreted proteins which may modulate host metabolism and inhibit host cell defence mechanisms. Spores exit the host in a non‐lytic manner that is dependent on host actin and endocytic recycling proteins. Together, this work provides a fuller picture of the mechanisms that these fascinating organisms use to infect their hosts.     

Kinetics of Encephalitozoon spp. infection of human macrophages

Microsporidia are obligate intracellular, eukaryotic parasites that are known to infect a variety of invertebrate and vertebrate species and have been reported to include a broad range of host specificities for various cell types. Although it is clear that some species of microsporidia have the ability to disseminate, causing multiorgan infections, it is not understood how dissemination occurs. One hypothesis suggests that mononuclear phagocytes engulf the pathogen and migrate to various organs while the parasite persists and proliferates. This implies that microsporidia have developed methods by which to escape intracellular degradation and can, instead, use the host as a source of nourishment and a vehicle for dissemination. In our study, we investigated the infection kinetics of 2 Encephalitozoon spp. known to cause disseminated disease in humans. Using fluorescence and scanning electron microscopy, it was determined that spore adherence to the host was rapid (3-6 hr), as was the uptake and organization of internal parasitophorous vacuoles (24 hr). Furthermore, replication was shown to occur within macrophages at 72 hr, as measured by the bromodeoxyuridine proliferation assay, and the production of mature spores occurred in host cells at 120 hr. Parasitic replication could be reduced by pretreatment of macrophages with interferon-gamma and bacterial lipopolysaccharide.     

Perturbation of cell cycle progression and cellular gene expression as a function of herpes simplex virus ICP0.

Herpes simplex virus type 1 is capable of inhibiting host cell DNA synthesis following lytic infection. However, the mechanism and nature of potential effects on cell cycle progression have not been described. In this report, we characterize the dysregulation of the cell cycle following infection with the replication-incompetent virus d106, where immediate-early gene expression is restricted to infected-cell polypeptide 0 (ICP0) and the expression of all other viral genes is dramatically reduced or is not observed. Infection with d106 resulted in the accumulation of cells in both the G(1)/S and G(2)/M compartments, consistent with cell cycle arrest at both checkpoints. The isogenic variant d109, which does not express any viral proteins, failed to induce this phenotype, suggesting that the expression of ICP0 is crucial for cell cycle arrest. Analysis of global cellular gene expression patterns following infection with d106 and d109 revealed that a relatively small subset of cellular genes were induced as a consequence of ICP0 expression. A number of these genes induced in the presence of ICP0 are classically considered p53-responsive genes, including p21, gadd45, and mdm-2. However, infection with d106 of cells with both alleles of p53 deleted resulted in the same cell cycle arrest phenotype and similar cellular gene expression patterns, suggesting that the expression of ICP0 results in cell cycle arrest potentially via p53-dependent and p53-independent mechanisms. In addition, it was found that the effects of infection with d106 on viral and cellular gene expression were similar to the effects observed following treatment of cells with the histone deacetylase inhibitor trichostatin A.     

Inhibition of African swine fever virus binding and infectivity by purified recombinant virus attachment protein p12.

The African swine fever virus protein p12, involved in virus attachment to the host cell, has an apparent molecular mass of 17 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We have also identified 12- and 10-kDa forms of the p12 protein in infected Vero cells and found that the mature 17-kDa protein is the only form present in virus particles. The p12 protein has been produced in large amounts in Spodoptera frugiperda insect cells infected with a recombinant baculovirus. A 17-kDa protein that possessed the biological properties of the viral protein was produced, since it bound to susceptible Vero cells and not to receptor-negative L cells, which do not support virus replication. The binding of the baculovirus-expressed protein p12 to Vero cells was specifically blocked by virus particles. In addition, the recombinant protein purified by immunoaffinity chromatography blocked the specific binding of virus particles to susceptible cells and prevented infection, demonstrating that the p12 protein mediates the attachment of virions to specific receptors and indicating that blocking the p12-mediated interaction between African swine fever virus and receptors in Vero cells can inhibit infection. However, although antibodies specific for protein p12 are induced in natural infections and in animals inoculated with inactivated virus or recombinant protein p12, these antisera did not inhibit virus binding to the host cell or neutralize virus infectivity.     

Inhibition of p53-Dependent,but Not p53-Independent,Cell Death by U19 Protein from Human Herpesvirus 6B

Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein.     

Toxoplasma gondii exploits UHRF1 and induces host cell cycle arrest at G2 to enable its proliferation

Toxoplasma gondii is an obligate intracellular parasite that causes severe disease in humans. It is able to infect all nucleated mammalian cells leading to lifelong persistence of the parasite in the host. Here, we studied the effect of T. gondii infection on host cell proliferation and explored the molecular mechanisms involved in host cell cycle progression. We found that T. gondii induced G1/S transition in host cells in the presence of UHRF1, followed by G2 arrest after cyclin B1 downregulation which is probably the major cause of the arrest. Other molecules at the G2/M checkpoint including p53, p21 and Cdk1 were normally regulated. Interestingly, while parasite proliferation was normal in cells that were in the G2 phase, it was suppressed in G1-arrested cells induced by UHRF1-siRNA, indicating the importance of the G2 phase via UHRF1-induced G1/S transition for T. gondii growth.     

Coexistence of three microsporidia parasites in populations of the freshwater amphipod Gammarus roeseli: evidence for vertical transmission and positive effect on reproduction

We investigated the prevalence, transmission mode and fitness effects of infections by obligatory intracellular, microsporidian parasites in the freshwater amphipod Gammarus roeseli. We found three different microsporidia species in this host, all using transovarial (vertical) transmission. All three coexist at different prevalences in two host populations, but bi-infected individuals were rarely found, suggesting no (or very little) horizontal transmission. It is predicted that vertically-transmitted parasites may exhibit sex-specific virulence in their hosts, or they may have either positive or neutral effects on host fitness. All three species differed in their transmission efficiency and infection intensity and our data suggest that these microsporidia exert sex-specific virulence by feminising male hosts. The patterns of infection we found exhibit convergent evolution with those of another amphipod host, Gammarus duebeni. Interestingly, we found that infected females breed earlier in the reproductive season than uninfected females. This is the first study, to our knowledge, to report a positive effect of microsporidian infection on female host reproduction.     

Mitotic catastrophe occurs in the absence of apoptosis in p53-null cells with a defective G1 checkpoint

Cell death occurring during mitosis, or mitotic catastrophe, often takes place in conjunction with apoptosis, but the conditions in which mitotic catastrophe may exhibit features of programmed cell death are still unclear. In the work presented here, we studied mitotic cell death by making use of a UV-inactivated parvovirus (adeno-associated virus; AAV) that has been shown to induce a DNA damage response and subsequent death of p53-defective cells in mitosis, without affecting the integrity of the host genome. Osteosarcoma cells (U2OSp53DD) that are deficient in p53 and lack the G1 cell cycle checkpoint respond to AAV infection through a transient G2 arrest. We found that the infected U2OSp53DD cells died through mitotic catastrophe with no signs of chromosome condensation or DNA fragmentation. Moreover, cell death was independent of caspases, apoptosis-inducing factor (AIF), autophagy and necroptosis. These findings were confirmed by time-lapse microscopy of cellular morphology following AAV infection. The assays used readily revealed apoptosis in other cell types when it was indeed occurring. Taken together the results indicate that in the absence of the G1 checkpoint, mitotic catastrophe occurs in these p53-null cells predominantly as a result of mechanical disruption induced by centrosome overduplication, and not as a consequence of a suicide signal.     

Induction of p53-dependent and mitochondria-mediated cell death pathway by dengue virus infection of human and animal cells

Previous studies have suggested that cells undergo apoptosis in response to dengue virus infection. However, the potential significance of dengue virus-induced apoptosis and the pathways are still not clearly defined. In this study, comparative analysis of dengue virus-induced apoptosis in BHK, H1299, HUH-7 and Vero cell lines was carried out. We show here that infection of BHK, HUH-7 and Vero cell lines with dengue type 1 virus (DEN1V) induces cell death typical of apoptosis. Virus-induced cell death was assayed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, detection of oligonucleosomal DNA fragmentation, DNA content analysis and assay for the externalization of phosphatidylserine residues. Detailed study of dengue virus infection in HUH-7 cells showed activation of cell death via the mitochondrial pathway causing lowering of mitochondrial transmembrane potential (DeltaPsim) in HUH-7 cells. Interestingly, in the p53-deficient cell line, H1299, apoptosis was largely undetectable compared with the other cell lines used; suggesting that a p53- and mitochondria-mediated cell death pathway may play an important role in dengue virus-induced apoptosis.     

Trichosanthin suppresses the elevation of p38 MAPK, and Bcl-2 induced by HSV-1 infection in Vero cells

Trichosanthin (TCS) is a type 1 ribosome-inactivating protein (RIP) effective against HIV-1 and HSV-1 replication. The mechanism of its antiviral activity is not clear. Many believe that it is related to ribosome inactivation. Some RIPs and viral infection affect the phosphorylation of MAPK and Bcl-2 and these proteins may be the common element linking RIP and viral infection. This study investigated the effect of HSV-1 infection on p38 MAPK and Bcl-2 as well as possible interference by TCS. Results showed that HSV-1 infection induced an elevation of phosphorylated p38 and Bcl-2 in Vero cells, which could be partially blocked by TCS. At the same time, both viral replication and host cells viability were lowered. Viral replication, Vero cell viability, p38 MAPK and Bcl-2 were further reduced with the addition of a p38 MAPK inhibitor (SB203580). This suggested that TCS may interfere with MAPK and Bcl-2 signals generated by infection leading to inhibition of viral replication. In summary, our results demonstrated that HSV-1 infection in Vero cells induced an elevation of p38 MAPK and Bcl-2. TCS suppressed this rise and reduced viral replication. The MAPK family may play a role in the antiviral mechanism of TCS.     

ORF73-null murine gammaherpesvirus 68 reveals roles for mLANA and p53 in virus replication

Gammaherpesviruses establish lifelong, latent infections in host lymphocytes, during which a limited subset of viral gene products facilitates maintenance of the viral episome. Among the gamma-2-herpesvirus (rhadinovirus) subfamily, this includes expression of the conserved ORF73-encoded LANA proteins. We previously demonstrated by loss-of-function mutagenesis that the murine gammaherpesvirus 68 (MHV68) ORF73 gene product, mLANA, is required for the establishment of latency following intranasal inoculation of mice (N. J. Moorman, D. O. Willer, and S. H. Speck, J. Virol. 77:10295-10303, 2003). mLANA-deficient viruses also exhibited a defect in acute virus replication in the lungs of infected mice. The latter observation led us to examine the role of mLANA in productive viral replication. We assessed the capacity of mLANA-deficient virus (73.Stop) to replicate in cell culture at low multiplicities of infection (MOIs) and found that 73.Stop growth was impaired in murine fibroblasts but not in Vero cells. A recombinant virus expressing an mLANA-green fluorescent protein (GFP) fusion revealed that mLANA is expressed throughout the virus replication cycle. In addition, 73.Stop infection of murine fibroblasts at high MOIs was substantially more cytotoxic than infection with a genetically repaired marker rescue virus (73.MR), a phenotype that correlated with enhanced kinetics of viral gene expression and increased activation of p53. Notably, augmented cell death, viral gene expression, and p53 induction were independent of viral DNA replication. Expression of a mLANA-GFP fusion protein in fibroblasts correlated with both reduced p53 stabilization and reduced cell death following treatment with p53-inducing agonists. In agreement, accentuated cell death associated with 73.Stop infection was reduced in p53-deficient murine embryonic fibroblasts. Additionally, replication of 73.Stop in p53-deficient cells was restored to levels comparable to those of 73.MR. More remarkably, the absence of p53 led to an overall delay in replication for both 73.Stop and 73.MR viruses, which correlated with delayed viral gene expression, indicating a role for p53 in MHV68 replication. Consistent with these findings, the expression of replication-promoting viral genes was positively influenced by p53 overexpression or treatment with the p53 agonist etoposide. Overall, these data demonstrate the importance of mLANA in MHV68 replication and suggest that LANA proteins limit the induction of cellular stress responses to regulate the viral gene expression cascade and limit host cell injury.     

Liberation of functional p53 by proteasome inhibition in human papilloma virus-positive head and neck squamous cell carcinoma cells promotes apoptosis and cell cycle arrest

Human papilloma virus (HPV) infection represents an emerging risk factor in head and neck squamous cell carcinoma (HNSCC). In contrast to HPV-negative HNSCC, most cases of HPV-positive HNSCC encode wild-type p53, although the p53 protein in these cells is rapidly degraded via HPV E6-mediated ubiquitination and subsequent proteasomal degradation. This unique feature of HPV-positive HNSCC has raised hope that liberation of wild-type p53 from the E6 protein may have therapeutic benefit in this disease. Indeed, suppression of E6 expression promotes apoptosis in HPV-positive HNSCC cell lines. However, the role of p53 in mediating this cell death has not been determined. Here, we demonstrate that siRNAs targeting the E6/E7 RNA, or treatment with the proteasome inhibitor bortezomib, resulted in upregulation of functional p53 and p53 gene targets in three HPV-positive HNSCC cell lines, but not in HPV-negative HNSCC cells. Apoptosis induced by E6/E7 siRNA in HPV-positive cells was found to be dependent on p53, while bortezomib-induced cell death was modestly p53-dependent. Treatment with subtoxic doses of bortezomib led to cell cycle arrest in HPV-positive, but not HPV-negative HNSCC cells. Moreover, this cell cycle arrest was mediated by p53 and the cell cycle inhibitor p21, the product of a p53 target gene. Collectively, these findings establish that wild-type p53 encoded by HPV-positive HNSCC cells, once liberated from HPV E6, can play important roles in promoting apoptosis and cell cycle arrest.