A functional map of NFkappaB signaling identifies novel modulators and multiple system controls.

BACKGROUND: The network of signaling pathways that leads to activation of the NFkappaB transcription factors is a branched structure with different inputs and cross-coupling with other signaling pathways. How these signals are integrated to produce specific, yet diverse responses is not clearly understood. To identify the components and structural features of the NFkappaB network, a series of cell-based, genomic screens was performed using a library of approximately 14,500 full-length genes. RESULTS: A total of 154 positive and 88 negative modulators of NFkappaB signaling were identified. Using a series of dominant-negative constructs and functional assays, these modulators were mapped to the known NFkappaB signaling cascade. Most of the positive modulators acted upstream of the IkappaB kinase complex, supporting previous observations that the IkappaB kinases represent the primary point of convergence in the network. A number of negative modulators were localized downstream of the IkappaB kinase beta (IKBKB) subunit, suggesting that they form an additional layer of negative control within the system. The expression of the modulators at the RNA level was distributed disproportionately across tissues, providing flexibility in network structure, and the number of positive and negative modulators present in a given tissue was highly correlated, suggesting that positive and negative regulation is balanced at the tissue level. CONCLUSION: The relative locations of the modulators are consistent with an hourglass structure for the NFkappaB network that is characteristic of robust systems. The tissue distribution of the modulators and downstream location of the negative modulators serve as layers of control within the system that allow differential responses to different stimuli.

Application of genomic biomarkers to predict increased lung tumor incidence in 2-year rodent cancer bioassays.

Rodent cancer bioassays are part of a legacy of safety testing that has not changed significantly over the past 30 years. The bioassays are expensive, time consuming, and use hundreds of animals. Fewer than 1500 chemicals have been tested in a rodent cancer bioassay compared to the thousands of environmental and industrial chemicals that remain untested for carcinogenic activity. In this study, we used existing data generated by the National Toxicology Program (NTP) to identify gene expression biomarkers that can predict results from a rodent cancer bioassay. A set of 13 diverse chemicals was selected from those tested by the NTP. Seven chemicals were positive for increased lung tumor incidence in female B6C3F1 mice and six were negative. Female mice were exposed subchronically to each of the 13 chemicals, and microarray analysis was performed on the lung. Statistical classification analysis using the gene expression profiles identified a set of eight probe sets corresponding to six genes whose expression correctly predicted the increase in lung tumor incidence with 93.9% accuracy. The sensitivity and specificity were 95.2 and 91.8%, respectively. Among the six genes in the predictive signature, most were enzymes involved in endogenous and xenobiotic metabolism, and one gene was a growth factor receptor involved in lung development. The results demonstrate that increases in chemically induced lung tumor incidence in female mice can be predicted using gene biomarkers from a subchronic exposure and may form the basis of a more efficient and economical approach for evaluating the carcinogenic activity of chemicals.

Co-regulation of Salmonella enterica genes required for virulence and resistance to antimicrobial peptides by SlyA and PhoP/PhoQ.

Analysis of the transcriptome of slyA mutant Salmonella enterica serovar Typhimurium revealed that many SlyA-dependent genes, including pagC, pagD, ugtL, mig-14, virK, phoN, pgtE, pipB2, sopD2, pagJ and pagK, are also controlled by the PhoP/PhoQ regulatory system. Many SlyA- and PhoP/PhoQ-co-regulated genes have functions associated with the bacterial envelope, and some have been directly implicated in virulence and resistance to antimicrobial peptides. Purified His-tagged SlyA binds to the pagC and mig-14 promoters in regions homologous to a previously proposed 'SlyA-box'. The pagC promoter lacks a consensus PhoP binding site and does not bind PhoP in vitro, suggesting that the effect of PhoP on pagC transcription is indirect. Stimulation of pagC expression by PhoP requires SlyA. Levels of SlyA protein and mRNA are not significantly changed under low-magnesium PhoP-inducing conditions in which pagC expression is profoundly elevated, however, indicating that the PhoP/PhoQ system does not activate pagC expression by altering SlyA protein concentration. Models are proposed in which PhoP may control SlyA activity via a soluble ligand or SlyA may function as an anti-repressor to allow PhoP activation. The absence of almost all SlyA-activated genes from the Escherichia coli K12 genome suggests that the functional linkage between the SlyA and PhoP/PhoQ regulatory systems arose as Salmonella evolved its distinctive pathogenic lifestyle.

The ferritin-like Dps protein is required for Salmonella enterica serovar Typhimurium oxidative stress resistance and virulence.

Resistance to phagocyte-derived reactive oxygen species is essential for Salmonella enterica serovar Typhimurium pathogenesis. Salmonella can enhance its resistance to oxidants through the induction of specific genetic pathways controlled by SoxRS, OxyR, sigma(S), sigma(E), SlyA, and RecA. These regulons can be found in a wide variety of pathogenic and environmental bacteria, suggesting that evolutionarily conserved mechanisms defend against oxidative stress both endogenously generated by aerobic respiration and exogenously produced by host phagocytic cells. Dps, a ferritin-like protein found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress by sequestering iron and limiting Fenton-catalyzed oxyradical formation. In Escherichia coli and some other bacterial species, Dps has been shown to accumulate during stationary phase in a sigma(S)-dependent fashion, bind nonspecifically to DNA, and form a crystalline structure that compacts and protects chromatin from oxidative damage. In the present study, we provide evidence that Dps protects Salmonella from iron-dependent killing by hydrogen peroxide, promotes Salmonella survival in murine macrophages, and enhances Salmonella virulence. Reduced numbers of dps mutant bacteria in the livers and spleens of infected mice are consistent with a role of Dps in protecting Salmonella from oxidative stress encountered during infection.