Analysis of Coxiella Burnetii Mediated Modulation of Host Cells During Infection

Coxiella burnetii is an obligate intracellular bacteria and the etiologic agent of Q fever. Although discovered over six decades ago, our understanding of the molecular mechanisms involved in disease development remains elementary. Few host cell processes actively modulated by C. burnetii have been identified. This study analyses host-cell pathways and processes that are specifically affected by C. burnetii proteins. It also defines C. burnetii induced temporal modulation of NF-kB activation throughout the infectious cycle. Additionally, it determines C. burnetii's growth cycle in an established tick cell line. First, the global expression of host cell mRNA was characterized following infection with C. burnetii Nine Mile Phase II and transient inhibition of bacterial protein synthesis with chloramphenicol. Using comparative microarray analysis, 36 host cell genes were identified to be distinctively modulated by C. burnetii proteins. Subsequent gene ontology analysis revealed expression changes in host cell functions such as innate immune response, cell death and proliferation, vesicle trafficking and development, lipid homeostasis, and cytoskeletal organization. A subset of pro-inflammatory cytokine genes was also identified whose expression is classically mediated through the NF-kB signaling pathway. This led to the demonstration that C. burnetii infection temporally modulates the activation of the NF-kB signaling pathway. Additionally, I have shown that C. burnetii readily infects Ixodes scapularis-derived cultured IDE8 cells, followed by a prolonged lag phase, then a doubling time similar to that in eukaryotic cells. Together these studies show that C. burnetii replicates and produces infectious progeny in arthropod cells, and temporally modulates mammalian host cell NF-kB signaling pathway as well as host cell gene expression in a bacterial protein synthesis specific manner.Department of Biochemistry and Molecular Biolog

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

Growth of Coxiella burnetii in the Ixodes scapularis–Derived IDE8 Tick Cell Line

Q fever, a zoonotic disease, is caused by a gram-negative intracellular bacterium, Coxiella burnetii. Although normally transmitted during exposure to infectious aerosols, C. burnetii is also found in arthropod vectors. In the environment, ticks are thought to play a crucial role in bacterial maintenance and transmission by infecting various mammalian species. However, the nature of the pathogen–tick relationship is not well defined. To determine C. burnetii's interactions with a cultured tick cell line, we introduced purified C. burnetii NMII into Ixodes scapularis–derived IDE8 cells and assayed for bacterial presence, replication, gene expression, and subsequent infectivity for mammalian cells. Tick cells were harvested at 24 h, 72 h, 7 days, and 11 days postinfection (PI). C. burnetii uptake and subsequent replication was demonstrated by indirect immunofluorescence assay, electron microscopy, and real-time polymerase chain reaction (PCR). When a genome equivalent multiplicity of infection of 30 was used, 30%–40% of exposed cells were seen to have small, rounded, vacuoles at 72 h PI, whereas at 7 and 11 days PI, 60%–70% of cells contained enlarged vacuoles harboring large numbers of bacteria. Quantitative PCR analysis of total genomic DNA confirmed that C. burnetii genome numbers increased significantly from 24 h to 11 days PI. Expression of C. burnetii type four secretion system homologs at 7 days PI was demonstrated by reverse transcriptase PCR. Finally, indirect immunofluorescence assay demonstrated that C. burnetii propagated within IDE8 cells were infectious for mammalian cells. These studies demonstrate the utility of cultured tick cell lines as a model to investigate C. burnetii's molecular interactions with its arthropod vectors

Hypertension from targeted ablation of chromogranin A can be rescued by the human ortholog

The secretory prohormone chromogranin A (CHGA) is overexpressed in essential hypertension, a complex trait with genetic predisposition, while its catecholamine release–inhibitory fragment catestatin is diminished, and low catestatin predicts augmented adrenergic pressor responses. These findings from studies on humans suggest a mechanism whereby diminished catestatin might increase the risk for hypertension. We generated Chga(–/–) and humanized mice through transgenic insertion of a human CHGA haplotype in order to probe CHGA and catestatin in vivo. Chga(–/–) mice displayed extreme phenotypic changes, including: (a) decreased chromaffin granule size and number; (b) elevated BP; (c) loss of diurnal BP variation; (d) increased left ventricular mass and cavity dimensions; (e) decreased adrenal catecholamine, neuropeptide Y (Npy), and ATP contents; (f) increased catecholamine/ATP ratio in the chromaffin granule; and (g) increased plasma catecholamine and Npy levels. Rescue of elevated BP to normalcy was achieved by either exogenous catestatin replacement or humanization of Chga(–/–) mice. Loss of the physiological “brake” catestatin in Chga(–/–) mice coupled with dysregulation of transmitter storage and release may act in concert to alter autonomic control of the circulation in vivo, eventuating in hypertension