Coxiella burnetii Nine Mile II proteins modulate gene expression of monocytic host cells during infection

<p>Abstract</p> <p>Background</p> <p><it>Coxiella burnetii </it>is an intracellular bacterial pathogen that causes acute and chronic disease in humans. Bacterial replication occurs within enlarged parasitophorous vacuoles (PV) of eukaryotic cells, the biogenesis and maintenance of which is dependent on <it>C. burnetii </it>protein synthesis. These observations suggest that <it>C. burnetii </it>actively subverts host cell processes, however little is known about the cellular biology mechanisms manipulated by the pathogen during infection. Here, we examined host cell gene expression changes specifically induced by <it>C. burnetii </it>proteins during infection.</p> <p>Results</p> <p>We have identified 36 host cell genes that are specifically regulated when <it>de novo </it><it>C. burnetii </it>protein synthesis occurs during infection using comparative microarray analysis. Two parallel sets of infected and uninfected THP-1 cells were grown for 48 h followed by the addition of chloramphenicol (CAM) to 10 μg/ml in one set. Total RNA was harvested at 72 hpi from all conditions, and microarrays performed using Phalanx Human OneArray™ slides. A total of 784 (mock treated) and 901 (CAM treated) THP-1 genes were up or down regulated ≥2 fold in the <it>C. burnetii </it>infected vs. uninfected cell sets, respectively. Comparisons between the complementary data sets (using >0 fold), eliminated the common gene expression changes. A stringent comparison (≥2 fold) between the separate microarrays revealed 36 host cell genes modulated by <it>C. burnetii </it>protein synthesis. Ontological analysis of these genes identified the innate immune response, cell death and proliferation, vesicle trafficking and development, lipid homeostasis, and cytoskeletal organization as predominant cellular functions modulated by <it>C. burnetii </it>protein synthesis.</p> <p>Conclusions</p> <p>Collectively, these data indicate that <it>C. burnetii </it>proteins actively regulate the expression of specific host cell genes and pathways. This is in addition to host cell genes that respond to the presence of the pathogen whether or not it is actively synthesizing proteins. These findings indicate that <it>C. burnetii </it>modulates the host cell gene expression to avoid the immune response, preserve the host cell from death, and direct the development and maintenance of a replicative PV by controlling vesicle formation and trafficking within the host cell during infection.</p

Structural and Functional Characterization of a Composite Conjugative Transposon in Streptococcus Pneumoniae

Extensive homology between the tet determinant and the flanking regions among most of the streptococcal conjugative transposons suggested that this novel class of mobile elements could have arisen by the 1nsertion of heterologous elements into a basic tet unit, as represented by the smallest known conjugative transposon, Tn916. However, the homology between Tn916 and the 65 kb Tn5253 was limited only to the tet reg1on and did not extend to any other region. Moreover, an 18 kb segment carry1ng the tet determinant that is totally internal to Tn5253 was found to be an independent conjugative transposon, Tn5251. We sought to determine the role of the sequences beyond Tn5251 w1thin Tn5253 in transposition. To this aim, the segment of DNA containing Tn5251 was deleted from within Tn5253 and the deletion mutant strain was used in filter-matings to determine whether the altered element retained 1ts transposit1on properties. The sequences beyond Tn5251 within Tn5253, designated Tn5252, transferred by filter-mat1ng to Streptococcus pneumoniae and Enterococcus faecalis recipient cells at a frequency comparable to that of the parental element. Differences in the structural and functional properties between Tn5251 and Tn5252 seemed to suggest that these belonged to two distinct classes of mobile elements. Moreover, Tn5253, originally detected in a clinical 1solate of ~- pneumoniae, is indeed a compos1te element comprised of two independent conjugative transposons, Tn5252 and Tn5251. These results can be extrapolated to the conclusion that the prototype for this type of element is perhaps a Tn5252-like transposon and not the tet carrying Tn916-like element.Microbiolog

Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs

BACKGROUND: The construction of cDNA libraries is a useful tool to understand gene expression in organisms under different conditions, but random sequencing of unbiased cDNA collections is laborious and can give rise to redundant EST collections. We aimed to isolate cDNAs of messages induced by switching Aspergillus nidulans from growth on glucose to growth on selected polysaccharides. Approximately 4,700 contigs from 12,320 ESTs were already available from a cDNA library representing transcripts isolated from glucose-grown A. nidulans during asexual development. Our goals were to expand the cDNA collection without repeated sequencing of previously identified ESTs and to find as many transcripts as possible that are specifically induced in complex polysaccharide metabolism. RESULTS: We have devised a Negative Subtraction Hybridization (NSH) method and tested it in A. nidulans. NSH entails screening a plasmid library made from cDNAs prepared from cells grown under a selected physiological condition with labeled cDNA probes prepared from another physiological condition. Plasmids with inserts that failed to hybridize to cDNA probes through two rounds of screening (i.e. negatives) indicate that they are transcripts present at low concentration in the labeled probe pool. Thus, these transcripts will be predominantly condition-specific, along with some rare transcripts. In a screen for transcripts induced by switching the carbon source from glucose to 12 selected polysaccharides, 3,532 negatives were isolated from approximately 100,000 surveyed colonies using this method. Negative clones were end-sequenced and assembled into 2,039 contigs, of which 1,722 were not present in the previously characterized glucose-grown cDNA library. Single-channel microarray hybridization experiments confirmed that the majority of the negatives represented genes that were differentially induced by a switch from growth in glucose to one or more of the polysaccharides. CONCLUSIONS: The Negative Subtraction Hybridization method described here has several practical benefits. This method can be used to screen any existing cDNA library, including full-length and pooled libraries, and does not rely on PCR or sequence information. In addition, NSH is a cost-effective method for the isolation of novel, full-length cDNAs for differentially expressed transcripts or enrichment of rare transcripts

Comparison of gene expression signatures of diamide, H(2)O(2 )and menadione exposed Aspergillus nidulans cultures – linking genome-wide transcriptional changes to cellular physiology

BACKGROUND: In addition to their cytotoxic nature, reactive oxygen species (ROS) are also signal molecules in diverse cellular processes in eukaryotic organisms. Linking genome-wide transcriptional changes to cellular physiology in oxidative stress-exposed Aspergillus nidulans cultures provides the opportunity to estimate the sizes of peroxide (O(2)(2-)), superoxide (O(2)(•-)) and glutathione/glutathione disulphide (GSH/GSSG) redox imbalance responses. RESULTS: Genome-wide transcriptional changes triggered by diamide, H(2)O(2 )and menadione in A. nidulans vegetative tissues were recorded using DNA microarrays containing 3533 unique PCR-amplified probes. Evaluation of LOESS-normalized data indicated that 2499 gene probes were affected by at least one stress-inducing agent. The stress induced by diamide and H(2)O(2 )were pulse-like, with recovery after 1 h exposure time while no recovery was observed with menadione. The distribution of stress-responsive gene probes among major physiological functional categories was approximately the same for each agent. The gene group sizes solely responsive to changes in intracellular O(2)(2-), O(2)(•- )concentrations or to GSH/GSSG redox imbalance were estimated at 7.7, 32.6 and 13.0 %, respectively. Gene groups responsive to diamide, H(2)O(2 )and menadione treatments and gene groups influenced by GSH/GSSG, O(2)(2- )and O(2)(•- )were only partly overlapping with distinct enrichment profiles within functional categories. Changes in the GSH/GSSG redox state influenced expression of genes coding for PBS2 like MAPK kinase homologue, PSK2 kinase homologue, AtfA transcription factor, and many elements of ubiquitin tagging, cell division cycle regulators, translation machinery proteins, defense and stress proteins, transport proteins as well as many enzymes of the primary and secondary metabolisms. Meanwhile, a separate set of genes encoding transport proteins, CpcA and JlbA amino acid starvation-responsive transcription factors, and some elements of sexual development and sporulation was ROS responsive. CONCLUSION: The existence of separate O(2)(2-), O(2)(•- )and GSH/GSSG responsive gene groups in a eukaryotic genome has been demonstrated. Oxidant-triggered, genome-wide transcriptional changes should be analyzed considering changes in oxidative stress-responsive physiological conditions and not correlating them directly to the chemistry and concentrations of the oxidative stress-inducing agent

Time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover

Background: Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study.Results: A. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14 days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2 days and then maintained a steady state of 4% of the total biomass for the next 12 days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14 days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance.Conclusions: Our results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1 day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulans is capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.Peer reviewedBiochemistry and Molecular BiologyMicrobiology and Molecular Genetic

Evidence of the role of tick subolesin in gene expression

Background: Subolesin is an evolutionary conserved protein that was discovered recently in Ixodes scapularis as a tick protective antigen and has a role in tick blood digestion, reproduction and development. In other organisms, subolesin orthologs may be involved in the control of developmental processes. Because of the profound effect of subolesin knockdown in ticks and other organisms, we hypothesized that subolesin plays a role in gene expression, and therefore affects multiple cellular processes. The objective of this study was to provide evidence for the role of subolesin in gene expression.Results: Two subolesin-interacting proteins were identified and characterized by yeast two-hybrid screen, co-affinity purification and RNA interference (RNAi). The effect of subolesin knockdown on the tick gene expression pattern was characterized by microarray analysis and demonstrated that subolesin RNAi affects the expression of genes involved in multiple cellular pathways. The analysis of subolesin and interacting protein sequences identified regulatory motifs and predicted the presence of conserved protein kinase C (PKC) phosphorylation sites.Conclusion: Collectively, these results provide evidence that subolesin plays a role in gene expression in ticks.Peer reviewedVeterinary PathobiologyBiochemistry and Molecular Biolog

Constitutive Modelling of Endothelium Denudation for Finite Element Simulation of Angioplasty

This study aims at characterizing and modelling the effect of mechanical factors on endothelial denudation during angioplasty, such as normal force between balloon and artery, stretching of arterial walls, and relative displacement between contacting surfaces. Friction damage was applied to porcine aorta samples with different contact forces, relative displacements, and biplanar stretching conditions. After the tests, endothelium denudation was quantified by isolating and counting the remaining endothelial cells. Using multiple-regression analysis, a constitutive model is proposed for integration in finite element software. This model will help optimize balloon and stent deployment conditions to minimize the amount of damage to the endothelium, and eventually to reduce the occurrence of restenosis.Peer reviewed: YesNRC publication: Ye

Gene expression profiling of human promyelocytic cells in response to infection with Anaplasma phagocytophilum

Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) causes human, equine and canine granulocytic anaplasmosis and tick-borne fever of ruminants. The rickettsia parasitizes granulocytes and bone marrow progenitor cells, and can be propagated in human promyelocytic and tick cell lines. In this study, microarrays of synthetic polynucleotides of 21 329 human genes were used to identify genes that are differentially expressed in HL-60 human promyelocytic cells in response to infection with A. phagocytophilum. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) of selected genes confirmed the results of the microarray analysis. Six genes in the A. phagocytophilum-infected cells were found to be upregulated greater than 30-fold, while expression of downregulated genes most often did not change more than sixfold. Genes that were found to be differentially regulated in infected cells were those essential for cellular mechanisms including growth and differentiation, cell transport, signalling and communication and protective response against infection, some of which are most likely necessary for infection and multiplication of A. phagocytophilum in host cells. The differentially regulated genes described herein provide new information on the gene expression profiles in A. phagocytophilum-infected HL-60 cells, thus expanding in a global manner the existing information on the response of mammalian cells to A. phagocytophilum infection.This research was supported by the Project No. 1669 of the Oklahoma Agricultural Experiment Station, the Endowed Chair for Food Animal Research (K.M. Kocan, College of Veterinary Medicine, Oklahoma State University) and NIH Centers for Biomedical Research Excellence through a subcontract to J. de la Fuente from the Oklahoma Medical Research Foundation, and the Oklahoma Center for the Advancement of Science and Technology, Applied Research Grants, AR00(1)-001 and ARO21-37. OSU Microarray Core and Bioinformatics Resource Facilities are supported by grants from NSF (EOS-0132534) and NIH (1P20RR16478-02 and 5P20RR15564-03). C. Almazán is supported by grants-in-aid from the CONACYT and Promep (University of Tamaulipas), Mexico and a grant from Pfizer Animal Health, Kalamazoo, Michigan, USA. V. Naranjo is founded by Consejería de Sanidad, Junta de Comunidades de Castilla – La Mancha, Spain.Peer reviewe