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• W2048288056 abstract "Cells infected with the intracellular protozoan parasite Toxoplasma gondii undergo up-regulation of pro-inflammatory cytokines, organelle redistribution, and protection from apoptosis. To examine the molecular basis of these and other changes, gene expression profiles of human foreskin fibroblasts infected with Toxoplasma were studied using human cDNA microarrays consisting of ∼22,000 known genes and uncharacterized expressed sequence tags. Early during infection (1–2 h), <1% of all genes show a significant change in the abundance of their transcripts. Of the 63 known genes in this group, 27 encode proteins associated with the immune response. These genes are also up-regulated by secreted, soluble factors from extracellular parasites indicating that the early response does not require parasite invasion. Later during infection, genes involved in numerous host cell processes, including glucose and mevalonate metabolism, are modulated. Many of these late genes are dependent on the direct presence of the parasite;i.e. secreted products from either the parasite or infected cells are insufficient to induce these changes. These results reveal several previously unknown effects on the host cell and lay the foundation for detailed analysis of their role in the host-pathogen interaction. Cells infected with the intracellular protozoan parasite Toxoplasma gondii undergo up-regulation of pro-inflammatory cytokines, organelle redistribution, and protection from apoptosis. To examine the molecular basis of these and other changes, gene expression profiles of human foreskin fibroblasts infected with Toxoplasma were studied using human cDNA microarrays consisting of ∼22,000 known genes and uncharacterized expressed sequence tags. Early during infection (1–2 h), <1% of all genes show a significant change in the abundance of their transcripts. Of the 63 known genes in this group, 27 encode proteins associated with the immune response. These genes are also up-regulated by secreted, soluble factors from extracellular parasites indicating that the early response does not require parasite invasion. Later during infection, genes involved in numerous host cell processes, including glucose and mevalonate metabolism, are modulated. Many of these late genes are dependent on the direct presence of the parasite;i.e. secreted products from either the parasite or infected cells are insufficient to induce these changes. These results reveal several previously unknown effects on the host cell and lay the foundation for detailed analysis of their role in the host-pathogen interaction. interferon γ human foreskin fibroblasts hours post-infection tricarboxylic acid Dulbecco's modified Eagle's medium enzyme-linked immunosorbent assay interleukin-1 tumor necrosis factor α expressed sequence tag low density lipoprotein 3-hydroxy-3-methylglutaryl-Coenzyme A Toxoplasma gondii, which is an obligate intracellular apicomplexan parasite that can infect most nucleated cells, causes devastating disease in humans and is related to Plasmodium,the causative agent of malaria (1Luft B.J. Remington J.S. Clin. Infect. Dis. 1992; 15: 211-222Crossref PubMed Scopus (1071) Google Scholar). Immediately following invasion,T. gondii resides and replicates within a parasitophorous vacuole that, although free of host membrane proteins, is surrounded by host mitochondria and endoplasmic reticulum (2Sinai A.P. Webster P. Joiner K.A. J. Cell Sci. 1997; 110: 2117-2128Crossref PubMed Google Scholar). Once the parasite begins growing and dividing within the parasitophorous vacuole, it must acquire nutrients such as purine nucleosides and cholesterol from the host cell (3Schwartzman J.D. Pfefferkorn E.R. Exp. Parasitol. 1982; 53: 77-86Crossref PubMed Scopus (101) Google Scholar). The mechanisms by which the morphological and metabolic changes to the host cell occur are unknown. T. gondii infection also causes the induction of numerous immune modulators that activate both the innate and adaptive immune responses (4Li Z.Y. Manthey C.L. Perera P.Y. Sher A. Vogel S.N. Infect. Immun. 1994; 62: 3434-3440Crossref PubMed Google Scholar, 5Brenier-Pinchart M.P. Pelloux H. Simon J. Ricard J. Bosson J.L. Ambroise-Thomas P. Mol. Cell Biochem. 2000; 209: 79-87Crossref PubMed Google Scholar, 6Yap G.S. Sher A. Immunobiology. 1999; 201: 240-247Crossref PubMed Scopus (181) Google Scholar, 7Denney C.F. Eckmann L. Reed S.L. Infect. Immun. 1999; 67: 1547-1552Crossref PubMed Google Scholar). Both live parasites and parasite extracts stimulate IFN-γ1 synthesis, which is required for protection, primarily via dendritic cell activation (6Yap G.S. Sher A. Immunobiology. 1999; 201: 240-247Crossref PubMed Scopus (181) Google Scholar, 8Alexander J. Scharton-Kersten T.M. Yap G. Roberts C.W. Liew F.Y. Sher A. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1997; 352: 1355-1359Crossref PubMed Scopus (55) Google Scholar,9Yap G. Pesin M. Sher A. J. Immunol. 2000; 165: 628-631Crossref PubMed Scopus (247) Google Scholar). However, the mechanism by which infected cells activate and recruit dendritic cells remain unclear but presumably requires synthesis of C-C chemokines that bind and activate the C-C chemokine receptor, CCR5 (10Aliberti J. Sousa C.R.E. Schito M. Hieny S. Wells T. Huffnagel G.B. Sher A. Nat. Immunol. 2000; 1: 83-87Crossref PubMed Scopus (308) Google Scholar). Although previous data demonstrated that T. gondii-infected cells produce these chemokines, the molecular mechanisms that regulate their expression are still unknown (4Li Z.Y. Manthey C.L. Perera P.Y. Sher A. Vogel S.N. Infect. Immun. 1994; 62: 3434-3440Crossref PubMed Google Scholar, 5Brenier-Pinchart M.P. Pelloux H. Simon J. Ricard J. Bosson J.L. Ambroise-Thomas P. Mol. Cell Biochem. 2000; 209: 79-87Crossref PubMed Google Scholar, 7Denney C.F. Eckmann L. Reed S.L. Infect. Immun. 1999; 67: 1547-1552Crossref PubMed Google Scholar). Moreover, the full repertoire of host genes whose expression is modulated by T. gondii as well as the signal transduction cascades underlying these changes are also unknown. Recently, the study of host-parasite interactions has been greatly aided by large-scale gene expression analysis using DNA microarrays (11Manger I.D. Relman D.A. Curr. Opin. Immunol. 2000; 12: 215-218Crossref PubMed Scopus (114) Google Scholar). Spotted with grids of highly dense and organized spots of DNA, cDNA microarrays are hybridized to two different cDNA samples each labeled with a different fluorophore thus allowing the simultaneous monitoring of differential gene expression for thousands of genes (12Eisen M.B. Brown P.O. Methods Enzymol. 1999; 303: 179-205Crossref PubMed Scopus (901) Google Scholar). Recent studies using microarrays have enhanced our knowledge of the host response to viruses (e.g. human immunodeficiency virus, cytomegalovirus, and coxsackievirus) and bacteria (e.g. Salmonella typhimurium,Pseudomonas aeruginosa, and Listeria monocytogenes) (13Zhu H. Cong J.P. Mamtora G. Gingeras T. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14470-14475Crossref PubMed Scopus (412) Google Scholar, 14Rosenberger C.M. Scott M.G. Gold M.R. Hancock R.E. Finlay B.B. J. Immunol. 2000; 164: 5894-5904Crossref PubMed Scopus (186) Google Scholar, 15Cohen P. Bouaboula M. Bellis M. Baron V. Jbilo O. Poinot-Chazel C. Galiegue S. Hadibi E.H. Casellas P. J. Biol. Chem. 2000; 275: 11181-11190Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16Taylor L.A. Carthy C.M. Yang D. Saad K. Wong D. Schreiner G. Stanton L.W. McManus B.M. Circ. Res. 2000; 87: 328-334Crossref PubMed Scopus (93) Google Scholar, 17Ichikawa J.K. Norris A. Bangera M.G. Geiss G.K. van't Wout A.B. Bumgarner R.E. Lory S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9659-9664Crossref PubMed Scopus (167) Google Scholar, 18Geiss G.K. Bumgarner R.E. An M.C. Agy M.B. van't Wout A.B. Hammersmark E. Carter V.S. Upchurch D. Mullins J.I. Katze M.G. Virology. 2000; 266: 8-16Crossref PubMed Scopus (229) Google Scholar). Microarrays demonstrated that the transcriptional response of macrophages to lipopolysaccharide andSalmonella are largely overlapping, suggesting that lipopolysaccharide may be largely responsible for the inflammatory response to Salmonella (14Rosenberger C.M. Scott M.G. Gold M.R. Hancock R.E. Finlay B.B. J. Immunol. 2000; 164: 5894-5904Crossref PubMed Scopus (186) Google Scholar). The goal of our study was to analyze the global changes that occur when a protozoan intracellular parasite infects a mammalian host cell. Thus, we examined the transcriptional profile of HFFs in response to T. gondii infection. Intact parasites, as well as soluble parasite-derived factors, rapidly increase the abundance of transcripts associated with the immune response. Later following infection, numerous host genes were modulated that are involved in diverse cellular processes, including metabolism, transcription, protein targeting, and apoptosis. Finally, we were able to discriminate between genes that are modulated due to either parasite- or host cell-derived secreted factors and those that are modulated only in the presence of intracellular parasites. HFFs (passage 10–16 post-isolation) were used for all assays. The PDS strain of T. gondii, cloned from ME49 (19Boothroyd J.C. Black M. Bonnefoy S. Hehl A. Knoll L.J. Manger I.D. Ortega-Barria E. Tomavo S. Philos. Trans. R. Soc. Lond-Biol. Sci. 1997; 352: 1347-1354Crossref PubMed Scopus (92) Google Scholar), was routinely passaged in HFFs using standard T. gondii culture conditions (19Boothroyd J.C. Black M. Bonnefoy S. Hehl A. Knoll L.J. Manger I.D. Ortega-Barria E. Tomavo S. Philos. Trans. R. Soc. Lond-Biol. Sci. 1997; 352: 1347-1354Crossref PubMed Scopus (92) Google Scholar) with DMEM supplemented with 10% fetal bovine serum, 2 mm glutamine, and gentamicin (Life Technologies; Rockville, MD). HFF and parasite strains were regularly inspected for mycoplasma, and found to be negative, using a Mycoplasma PCR ELISA detection kit (Roche Molecular Biochemicals; Indianapolis, IN). Parasites were harvested from infected HFF monolayers when lysis by infection was almost completed and few, if any, intact host cells remained. The flasks were then scraped, and the entire material harvested and passed twice through a 27-gauge needle to rupture any remaining HFFs and release the parasites within. The resulting material was pelleted at 600 × g, washed three times in serum-free DMEM, and parasites counted by light microscopy. No intact host cells could be detected in this preparation. Parasites were added to HFF monolayers (4–7 days old) at an multiplicity of infection of 5–10. Infection assays were routinely performed with HFF monolayers in 75-cm2 flasks grown in 15 ml of media or a proportionate volume for different size flasks for the indicated times. For the Transwell experiments, HFFs were plated in the lower chamber of a 10-cm Transwell tissue culture insert (final volume 10 ml; Corning-Costar, Corning, NY) with 5 ml of media in the upper chamber. Parasites, in serum free media, were added to the upper chamber, and the plates were incubated for 4 h. Total RNA was prepared using the RNAeasy Midi kit (Qiagen, Valencia, CA). The amount of T. gondiitotal RNA co-purified 24 hpi was estimated to be ∼20% by comparing ribosomal RNA band intensities of total RNA separated by agarose gel electrophoresis (not shown). cDNA microarrays were synthesized at Stanford University using standard protocols (12Eisen M.B. Brown P.O. Methods Enzymol. 1999; 303: 179-205Crossref PubMed Scopus (901) Google Scholar). The microarrays were spotted with 18,000–27,000 sequence-verified clones that are available from Research Genetics (Huntsville, AL). cDNA probe preparation was performed essentially as described previously (12Eisen M.B. Brown P.O. Methods Enzymol. 1999; 303: 179-205Crossref PubMed Scopus (901) Google Scholar). Briefly, total RNA (20–25 μg) was converted to first-strand cDNA using Superscript II (Life Technologies, Rockville, MD) with oligo-dT18 (New England BioLabs, Bedford, MA). The cDNA was labeled with Cy3-dUTP (infected) or Cy5-dUTP (uninfected) (Amersham Pharmacia Biotech, Uppsala, Sweden) via a random prime reaction using random nanomer. We found that labeling cDNA with random nanomer versus direct dye incorporation during the first-stand cDNA synthesis reaction (12Eisen M.B. Brown P.O. Methods Enzymol. 1999; 303: 179-205Crossref PubMed Scopus (901) Google Scholar) did not indicate any significant difference between the two protocols (not shown). Microarrays were hybridized overnight at 65 °C, washed as described (12Eisen M.B. Brown P.O. Methods Enzymol. 1999; 303: 179-205Crossref PubMed Scopus (901) Google Scholar) and scanned with a GenePix 4000 microarray scanner (Axon Instruments, South San Francisco, CA). Each time point and condition was repeated typically three-five times. Microarrays were analyzed with the Scanalyze program (written by Mike Eisen and available on the Web) to determine the fluorescent intensities of the two dyes for each spot. The fluorescence intensities were normalized by applying a scaling factor so that the median fluorescence ratio of all spots with detectable signals above background on each microarray was 1.0 (20Alizadeh A.A. Eisen M.B. Davis R.E. Ma C. Lossos I.S. Rosenwald A. Boldrick J.C. Sabet H. Tran T., Yu, X. Powell J.I. Yang L. Marti G.E. Moore T. Hudson Jr., J. Lu L. Lewis D.B. Tibshirani R. Sherlock G. Chan W.C. Greiner T.C. Weisenburger D.D. Armitage J.O. Warnke R. Staudt L.M. et al.Nature. 2000; 403: 503-511Crossref PubMed Scopus (8087) Google Scholar). The data were then filtered such that only spots with intensities that were three times greater than background in either channel were used in the analysis. Only those spots that displayed a 2-fold or greater difference in fluorescence intensities between the two dyes were used to generate gene clusters. For all such clusters, each spot was manually examined to assess its quality and those that exhibited poor quality throughout the analysis were removed. Spots of poor quality in individual microarrays were discarded from the dataset and are represented in the gene clusters as gray bars. Poor quality spots were removed if they were 1) very small, 2) irregularly shaped, or 3) with pixels that were not uniformly distributed throughout the spot. In the gene clusters, black bars represent genes that displayed -fold changes of 2 or less. Data clustering was performed using the Cluster program and figures generated using Tree View (both available on the Web) (21Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13268) Google Scholar). Total RNA (5 μg) was separated by agarose gel electrophoresis using standard molecular biological techniques.GRO1 cDNA was prepared by RT-PCR from total RNA fromT. gondii-infected HFFs using the following oligonucleotides: GRO1-F 5′-CGGAAAGCTTGCCTCAATCCT-3′ and GRO1-R 5′-GATCCGCCAGCCTCTATCACA-3′. β-actin (IMAGE: 867606) and DKK1 (IMAGE: 669375) cDNAs were purchased from Research Genetics (Huntsville, AL) and were sequenced-verified. Probes were labeled with [α-32P]dGTP with the Random Primed DNA labeling kit (Roche Molecular Biochemicals, Germany) and hybridized with Express-Hyb (CLONTECH, Palo Alto, CA) according to the manufacturer's instructions. Blots were exposed to film, and the autoradiographs were scanned and analyzed using the ImageQuaNT analysis program (Molecular Dynamics, Sunnyvale, CA) Secreted GRO1 protein was detected by ELISA using the GRO1 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. To characterize the host cell response to T. gondii infection, we employed cDNA microarray analysis, which is a powerful tool for analyzing global changes in gene expression. Because this technique can be subject to variability due to a combination of RNA preparation and handling, spot quality, and data analysis (15Cohen P. Bouaboula M. Bellis M. Baron V. Jbilo O. Poinot-Chazel C. Galiegue S. Hadibi E.H. Casellas P. J. Biol. Chem. 2000; 275: 11181-11190Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), it is necessary first to determine the experimental error for the conditions being used. Once determined, these variables allow thresholds to be set below which changes are not regarded as significant. Thus, HFF cultures were mock-infected or infected with T. gondii tachyzoites for 14 h in duplicate, and RNA was isolated from infected and mock-infected cells. HFFs were used in this study, because they are a well characterized T. gondii cell culture model. Moreover, they are non-transformed and do not require pretreatment with differentiation factors, such as growth factors or phorbol esters, which limit the extent of variability between cell preparations. Microarrays were then hybridized with cDNA probes synthesized using RNA from the infected and the mock-infected samples. We compared the difference between infected and uninfected cells for each spot whose signal intensity in either channel was at least 500 fluorescence units, which corresponds to three times greater than background. Using this filter, 11,976 spots (45% of the total number of spots) were included in the analysis. To assess assay reproducibility, the fold difference for each spot was plotted for the two duplicate experiments (Fig.1 A). The data showed a linear regression of 0.88, indicating a generally high level of correlation. Next, the 11,976 spots were filtered to include only spots of high quality that displayed a fluorescence ratio between infected and uninfected cells of >2 in either microarray. Using these criteria, a total of 1503 spots (∼6% of the original 11,976 spots) were analyzed with a regression correlation of r = 0.95 (Fig.1 B). Similar analysis demonstrated that no significant bias was observed when the Cy3-dUTP and Cy5-dUTP labels were reversed (not shown). Because T. gondii is an intracellular parasite, the RNA preparation protocol used resulted in the purification of not only HFF RNA but also parasite RNA. Thus, the apparent gene modulation observed could have conceivably been an artifact due to cross-hybridization between T. gondii cDNA and spots on the human microarray. To control for this, a human cDNA microarray was hybridized with Cy5-dUTP-labeled cDNA probe made from 20 μg of uninfected HFF RNA and Cy3-dUTP-labeled cDNA probe made from 16 μg of uninfected HFF RNA to which 4 μg (20%) total parasite RNA, prepared from washed and filtered tachyzoites, was added. Data analysis indicated that T. gondii cDNA does not cross-hybridize to any measurable extent with any spot on the human microarray (not shown). Because the parasites were cultured in vitro in HFF monolayers and purified by syringe-lysis, host cell debris remaining in the parasite preparation could have modulated gene expression in uninfected monolayers. Thus, the response of HFFs infected for 2 h with T. gondii were compared with HFFs treated with an equivalent preparation of uninfected HFFs, which were scraped and prepared using conditions identical to those used to prepare the parasites. Two different microarrays were hybridized with either cDNA from the parasite-infected or debris-treated cells against cDNA from untreated HFFs. The microarray from the cell debris-treated sample showed no significant changes in gene expression whereas the infected cells displayed the usual profile of gene induction (not shown). These data indicate that the changes in gene expression detected by microarrays are real, reproducible, and the result of T. gondii infection. To dissect the temporal response of HFFs infected withT. gondii, a time course was performed during which RNA was purified from HFFs that were mock- or parasite-infected for 1, 2, 4, 6, and 24 hpi with T. gondii tachyzoites. To minimize host cell lysis following parasite egress, the time course was limited to 24 h and we used the PDS strain, a ME49 clonal derivative, that grows more slowly than the highly virulent Type 1 RH strain (doubling time of ∼12 h (PDS) versus ∼6–8 h (RH)) (19Boothroyd J.C. Black M. Bonnefoy S. Hehl A. Knoll L.J. Manger I.D. Ortega-Barria E. Tomavo S. Philos. Trans. R. Soc. Lond-Biol. Sci. 1997; 352: 1347-1354Crossref PubMed Scopus (92) Google Scholar). For each time point, a microarray was used to compare cDNA prepared from mock-infected versus parasite-infected cells. Following hybridization, the data were filtered based on the criteria described above: spots were three times greater than background in at least one channel, of high quality, and displayed a 2-fold change or greater (relative to uninfected) in at least one time point during the experiment. The filtered data were organized into temporal gene expression clusters. This approach was previously used to classify cell-cycle-regulated genes in Saccharomyces cerevisiae and identify serum responsive genes in HFFs (21Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13268) Google Scholar, 22Iyer V.R. Eisen M.B. Ross D.T. Schuler G. Moore T. Lee J.C. f. Trent J.M. Staudt L.M. Hudson J. Boguske M.S. Lashkari D. Shalon D. Botstein D. Brown P.O. Science. 1999; 283: 83-87Crossref PubMed Scopus (1732) Google Scholar, 23Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher D. Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3915) Google Scholar). Clustering of the time course data indicated that two distinct gene expression patterns were present: genes that are modulated rapidly after infection (1–2 hpi) and those that are modulated later (>6 hpi) (Fig.2). Due to the large number of genes modulated during the time course, only data related to genes that have some functional information are shown and discussed. The full group, including ESTs and genes not discussed here, can be downloaded from J.C.B.'s website at Stanford University. During the first 2 h of infection, a total of 63 unique, known genes exhibited a 2-fold or greater increase in their abundance, whereas 15 unique, known genes exhibited a 2-fold or greater decrease. A high proportion of the induced genes (27 genes, 43%), are genes previously known to be associated with the immune response. These immunomodulatory molecules include chemokines (GRO1,GRO2, LIF, and MCP1), cytokines (IL-1β, IL-6), cell matrix and adhesion proteins (ICAM1 and matrix metalloproteinase 3), apoptotic (superoxide dismutase 2) and transcriptional regulatory factors (REL-B, NF-κB p105, I-κBα). Several additional cytokines previously shown to be important in T. gondiiinfection, such as β-interferon,IL-10, and IL-12 (24Schmitz J.L. Carlin J.M. Borden E.C. Byrne G.I. Infect. Immun. 1989; 57: 3254-3256Crossref PubMed Google Scholar, 25Hunter C.A. Subauste C.S. Van Cleave V.H. Remington J.S. Infect. Immun. 1994; 62: 2818-2824Crossref PubMed Google Scholar), were not among the genes spotted on the microarrays and therefore were not included in this analysis. Although TNF-α and IFN-γ, which is not normally expressed in HFFs, were not spotted on the microarrays used for this time course, subsequent microarray experiments, using microarrays that included TNF-α andIFN-γ, showed that they were not induced even by 24 hpi (not shown). Although the early induced genes are predominantly associated with the immune response, these represent a relatively small number of immune response genes spotted on the microarrays. For example, only 4 out of 24 genes encoding chemokines and 2 out of 41 genes encoding interleukins or their receptors were up-regulated byT. gondii 2 hpi. Although microarrays are a powerful tool to study global transcriptional responses to infection, it is important to confirm microarray data using independent methods such as Northern blot analysis, ribonuclease protection assay, or quantitative reverse transcription-polymerase chain reaction. Northern blots of representative genes from among those induced, repressed, or unchanged by infection were performed to verify the microaray data. RNA prepared from uninfected and infected cells (2 and 24 hpi) were used to make cDNA for hybridization to the microarrays and for analysis by Northern blotting using probes for an up-regulated gene (GRO1), a down-regulated gene (DKK1), and an unmodulated gene (β-actin). The Northern blot data were consistent with the microarrays providing an independent validation of the microarray data (Fig. 3). Although β-actin remained unchanged by both microarray and Northern blot analysis, it was observed that GRO1 was up-regulated andDKK1 was down-regulated with both techniques. Importantly, the Northern blot data corroborated the microarray GRO1induction trend, i.e. 8- and 20-fold induction (2 hpi) and 38- and 95-fold induction (24 hpi) (microarray and Northern, respectively). To determine whether the change in GRO1 mRNA levels reflects a change in the GRO1 protein produced, we used an ELISA-based assay to determine the amount of this chemokine in the medium of uninfected versus T. gondii-infected HFF cultures. The results indicated that 4 hpi of T. gondiistimulated an increase in the GRO1 concentration from ∼600 to ∼9000 pg/ml (data not shown). These results are in good agreement with the 8- to 20-fold induction of GRO1 mRNA indicated by the microarray and Northern blot data. To examine the late pattern of gene expression in greater detail, two independent 24-h infections were analyzed with microarrays. The data were filtered to identify genes whose spots were of high quality and in both experiments were up- or down-modulated at least 2-fold. Using these criteria, 567 unique genes were identified of which 376 corresponded to previously characterized genes with some functional information while 191 were ESTs or genes of unknown function. Categorizing the known genes by function indicated that numerous cellular processes are modulated during T. gondii infection. These included genes involved in carbohydrate and lipid metabolism (13.7%); transcriptional regulation (13.2%); protein synthesis, targeting, and degradation (12.3%); cell signaling (11.4%); inflammation (8.2%); cell adhesion and cytoskeleton (8.2%); nucleotide and amino acid metabolism (4.7%); cell cycle (4.1%); and apoptosis (3%). It is important to note that this type of functional annotation may be misleading, because some genes may be involved in more than one process but have been arbitrarily grouped into a single class. For example, although ICAM1 is an adhesion molecule, its expression is often associated with the inflammatory response (26van de Stolpe A. van der Saag P.T. J. Mol. Med. 1996; 74: 13-33Crossref PubMed Scopus (663) Google Scholar). Because the list of modulated known genes and unknown ESTs is too large to be presented in this report, the list can be downloaded from J.C.B.'s website at Stanford University. Examination of those down-regulated genes revealed no clear trends for any group of genes involved in a particular function or pathway. Examination of the up-regulated genes, however, showed some clear functional groupings and is discussed below. Similar to other intracellular parasites, T. gondii must scavenge from its host nutrients such as glucose and cholesterol (27Sinai A.P. Joiner K.A. Annu. Rev. Microbiol. 1997; 51: 415-462Crossref PubMed Scopus (200) Google Scholar). Interestingly, our results show that many enzymes associated with these pathways are up-regulated during infection. Glycolysis is a 10-step metabolic pathway that converts glucose to 2 molar equivalents of pyruvate with a net yield of 2 molar equivalents of ATP. Genes encoding each of the host glycolytic enzymes were present on these microarrays. Of these genes,hexokinase 2, phosphofructokinase 1, triose phosphate isomerase 1, phosphoglycerate kinase,phosphoglycerate mutase 1, and enolase 2 were significantly up-regulated ≥2-fold (Fig.4). However, glucose phosphate isomerase, aldolase A, and pyruvate kinase 1were not significantly changed. Glyceraldehyde-3-phosphate dehydrogenase was up-regulated (3.7-fold) in one experiment, and in the second, it was up-regulated ∼1.8-fold, which is below the minimal threshold of significance. Depending on the aerobic status of the host, pyruvate is further metabolized to either lactate or acetyl-CoA. Microarray data indicated that lactate dehydrogenase-A (muscle isoform) was up-regulated 24 hpi. Besides lactate dehydrogenase-A, two other lactate dehydrogenase isoforms have been previously characterized (lactate dehydrogenase-B (heart isoform) and lactate dehydrogenase-C (sperm isoform)) (28Ansari A.A. Baig M.A. Malling H.V. Biochem. Biophys. Res. Commun. 1981; 102: 93-99Crossref PubMed Scopus (3) Google Scholar). Of these, lactate dehydrogenase-B was not spotted on the microarrays, and the spot for lactate dehydrogenase-C was of insufficient intensity to be included in our analysis, suggesting that it is not expressed in HFFs. Acetyl-CoA, which is generated from pyruvate by the pyruvate dehydrogenase complex, can be utilized by the TCA cycle that is coupled to oxidative phosphorylation. We found that expression of pyruvate dehydrogenase complex genes was not significantly changed duringT. gondii infection. Moreover, pyruvate dehydrogenase kinase isoenzyme 1, which is a negative regulator of pyruvate dehydrogenase, was up-regulated ∼2.5-fold.Fructose-bisphosphatase, which catalyzes the rate-limiting reaction in gluconeogenesis, was not significantly altered 24 hpi. Genes encoding components of the nine TCA cycle holoenzymes, exceptsuccinyl-CoA synthase, were spotted on the microarrays, and none were significantly induced or repressed during infection. Similarly, no significant changes were observed in the expression of genes encoding enzymes involved in either amino acid catabolism, which yield either pyruvate or acetyl-CoA, or in the p" @default.
• W2048288056 created "2016-06-24" @default.
• W2048288056 creator A5012652764 @default.
• W2048288056 creator A5053925806 @default.
• W2048288056 creator A5075698928 @default.
• W2048288056 date "2001-06-01" @default.
• W2048288056 modified "2023-09-27" @default.
• W2048288056 title "Microarray Analysis Reveals Previously Unknown Changes in Toxoplasma gondii-infected Human Cells" @default.
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