Immunospecific responses to bacterial elongation factor Tu during Burkholderia infection and immunization

Burkholderia pseudomallei is the etiological agent of melioidosis, a disease endemic in parts of Southeast Asia and Northern Australia. Currently there is no licensed vaccine against infection with this biological threat agent. In this study, we employed an immunoproteomic approach and identified bacterial Elongation factor-Tu (EF-Tu) as a potential vaccine antigen. EF-Tu is membrane-associated, secreted in outer membrane vesicles (OMVs), and immunogenic during Burkholderia infection in the murine model of melioidosis. Active immunization with EF-Tu induced antigen-specific antibody and cell-mediated immune responses in mice. Mucosal immunization with EF-Tu also reduced lung bacterial loads in mice challenged with aerosolized B. thailandensis. Our data support the utility of EF-Tu as a novel vaccine immunogen against bacterial infection.     

Analysis of hepatic glycogen‐associated proteins

Glycogen particles are associated with a population of proteins that mediate its biological functions, including: management of glucose flux into and out of the glycogen particle, maintenance of glycogen structure and regulation of particle size, number, and cellular location. A survey of the glycogen‐associated proteome would be predicted to identify the relative representation of known members of this population, and associations with unexpected proteins that have the potential to mediate other functions of the glycogen particle. We therefore purified glycogen particles from both mouse and rat liver, using different techniques, and analyzed the resulting tryptic peptides by MS. We also specifically eluted glycogen‐binding proteins from the pellet using malto‐oligosaccharides. Comparison of the rat and mouse populations, and analysis of specifically eluted proteins allow some conclusions to be made about the hepatic glycogen sub‐proteome. With the exception of glycogen branching enzyme all glycogen metabolic proteins were detected. Novel associations were identified, including ferritin and starch‐binding domain protein 1, a protein that contains both a transmembrane endoplasmic reticulum signal peptide and a carbohydrate‐binding module. This study therefore provides insight into the organization of the glycogen proteome, identifies other associated proteins and provides a starting point to explore the dynamic nature and cellular distribution of this metabolically important protein population.     

Long-Term IGF-I Exposure Decreases Autophagy and Cell Viability

A reduction in IGF-I signaling has been found to increase lifespan in multiple organisms despite the fact that IGF-I is a trophic factor for many cell types and has been found to have protective effects against multiple forms of damage in acute settings. The increase in longevity seen in response to reduced IGF-I signaling suggests that there may be differences between the acute and chronic impact of IGF-I signaling. We have examined the possibility that long-term stimulation with IGF-I may have a negative impact at the cellular level using quiescent human fibroblasts. We find that fibroblast cells exposed to IGF-I for 14 days have reduced long-term viability as judged by colony forming assays, which is accompanied by an accumulation of senescent cells. In addition we observe an accumulation of cells with depolarized mitochondria and a reduction in autophagy in the long-term IGF-I treated cultures. An examination of mice with reduced IGF-I levels reveals evidence of enhanced autophagy and fibroblast cells derived from these mice have a larger mitochondrial mass relative to controls indicating that changes in mitochondrial turnover occurs in animals with reduced IGF-I. The results indicate that chronic IGF-I stimulation leads to mitochondrial dysfunction and reduced cell viability.     

CD28 Down-Regulation on Circulating CD4 T-Cells Is Associated with Poor Prognoses of Patients with Idiopathic Pulmonary Fibrosis

Background

Although the etiology of idiopathic pulmonary fibrosis (IPF) remains perplexing, adaptive immune activation is evident among many afflicted patients. Repeated cycles of antigen-induced proliferation cause T-cells to lose surface expression of CD28, and we hypothesized this process might also occur in IPF.

Methodology/Principal Findings

Peripheral blood CD4 T-cells from 89 IPF patients were analyzed by flow cytometry and cytokine multiplex assays, and correlated with clinical events. In comparison to autologous CD4⁺CD28⁺cells, the unusual CD4⁺CD28^null lymphocytes seen in many IPF patients had discordant expressions of activation markers, more frequently produced cytotoxic mediators perforin (2.4±0.8% vs. 60.0±7.4%, p<0.0001) and granzyme B (4.5±2.8% vs.74.9±6.5%, p<0.0001), produced greater amounts of many pro-inflammatory cytokines, and less frequently expressed the regulatory T-cell marker FoxP3 (12.9±1.1% vs. 3.3±0.6% p<0.0001). Infiltration of CD4⁺CD28^null T-cells in IPF lungs was confirmed by confocal microscopy. Interval changes of CD28 expression among subjects who had replicate studies were correlated with conterminous changes of their forced vital capacities (r_s = 0.49, p = 0.012). Most importantly, one-year freedom from major adverse clinical events (either death or lung transplantation) was 56±6% among 78 IPF patients with CD4⁺CD28⁺/CD4_total≥82%, compared to 9±9% among those with more extensive CD28 down-regulation (CD4⁺CD28⁺/CD4_total<82%) (p = 0.0004). The odds ratio for major adverse events among those with the most extensive CD28 down-regulation was 13.0, with 95% confidence intervals 1.6-111.1.

Conclusions/Significance

Marked down-regulation of CD28 on circulating CD4 T-cells, a result of repeated antigen-driven proliferations, is associated with poor outcomes in IPF patients. The CD4⁺CD28^null cells of these patients have potentially enhanced pathogenic characteristics, including increased productions of cytotoxic mediators and pro-inflammatory cytokines. These findings show proliferative T-cell responses to antigen(s) resulting in CD28 down-regulation are associated with progression and manifestations of IPF, and suggest assays of circulating CD4 T-cells may identify patients at greatest risk for clinical deterioration.     

Quantitative analysis of fitness and genetic interactions in yeast on a genome scale

Global quantitative analysis of genetic interactions is a powerful approach for deciphering the roles of genes and mapping functional relationships among pathways. Using colony size as a proxy for fitness, we developed a method for measuring fitness-based genetic interactions from high-density arrays of yeast double mutants generated by synthetic genetic array (SGA) analysis. We identified several experimental sources of systematic variation and developed normalization strategies to obtain accurate single- and double-mutant fitness measurements, which rival the accuracy of other high-resolution studies. We applied the SGA score to examine the relationship between physical and genetic interaction networks, and we found that positive genetic interactions connect across functionally distinct protein complexes revealing a network of genetic suppression among loss-of-function alleles.     

Cyclin-Dependent Kinase-Like Function Is Shared by the Beta- and Gamma- Subset of the Conserved Herpesvirus Protein Kinases

The UL97 protein of human cytomegalovirus (HCMV, or HHV-5 (human herpesvirus 5)), is a kinase that phosphorylates the cellular retinoblastoma (Rb) tumor suppressor and lamin A/C proteins that are also substrates of cellular cyclin-dependent kinases (Cdks). A functional complementation assay has further shown that UL97 has authentic Cdk-like activity. The other seven human herpesviruses each encode a kinase with sequence and positional homology to UL97. These UL97-homologous proteins have been termed the conserved herpesvirus protein kinases (CHPKs) to distinguish them from other human herpesvirus-encoded kinases. To determine if the Cdk-like activities of UL97 were shared by all of the CHPKs, we individually expressed epitope-tagged alleles of each protein in human Saos-2 cells to test for Rb phosphorylation, human U-2 OS cells to monitor nuclear lamina disruption and lamin A phosphorylation, or S. cerevisiae cdc28-13 mutant cells to directly assay for Cdk function. We found that the ability to phosphorylate Rb and lamin A, and to disrupt the nuclear lamina, was shared by all CHPKs from the beta- and gamma-herpesvirus families, but not by their alpha-herpesvirus homologs. Similarly, all but one of the beta and gamma CHPKs displayed bona fide Cdk activity in S. cerevisiae, while the alpha proteins did not. Thus, we have identified novel virally-encoded Cdk-like kinases, a nomenclature we abbreviate as v-Cdks. Interestingly, we found that other, non-Cdk-related activities reported for UL97 (dispersion of promyelocytic leukemia protein nuclear bodies (PML-NBs) and disruption of cytoplasmic or nuclear aggresomes) showed weak conservation among the CHPKs that, in general, did not segregate to specific viral families. Therefore, the genomic and evolutionary conservation of these kinases has not been fully maintained at the functional level. Our data indicate that these related kinases, some of which are targets of approved or developmental antiviral drugs, are likely to serve both overlapping and non-overlapping functions during viral infections.     

A scalable approach for discovering conserved active subnetworks across species

Overlaying differential changes in gene expression on protein interaction networks has proven to be a useful approach to interpreting the cell's dynamic response to a changing environment. Despite successes in finding active subnetworks in the context of a single species, the idea of overlaying lists of differentially expressed genes on networks has not yet been extended to support the analysis of multiple species' interaction networks. To address this problem, we designed a scalable, cross-species network search algorithm, neXus (Network-cross(X)-species-Search), that discovers conserved, active subnetworks based on parallel differential expression studies in multiple species. Our approach leverages functional linkage networks, which provide more comprehensive coverage of functional relationships than physical interaction networks by combining heterogeneous types of genomic data. We applied our cross-species approach to identify conserved modules that are differentially active in stem cells relative to differentiated cells based on parallel gene expression studies and functional linkage networks from mouse and human. We find hundreds of conserved active subnetworks enriched for stem cell-associated functions such as cell cycle, DNA repair, and chromatin modification processes. Using a variation of this approach, we also find a number of species-specific networks, which likely reflect mechanisms of stem cell function that have diverged between mouse and human. We assess the statistical significance of the subnetworks by comparing them with subnetworks discovered on random permutations of the differential expression data. We also describe several case examples that illustrate the utility of comparative analysis of active subnetworks.     

Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes

Background

Bacterial viruses (phages) play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage.

Methodology/Principal Findings

To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles), and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL) or of a whole genome shotgun library (WGSL), or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling.

Conclusions/Significance

These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.     

Expression of Long-chain Fatty Acyl-CoA Synthetase 4 in Breast and Prostate Cancers Is Associated with Sex Steroid Hormone Receptor Negativity

Previous studies have shown that key enzymes involved in lipid metabolic pathways are differentially expressed in normal compared with tumor tissues. However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established. Fatty acid synthase is overexpressed in a variety of cancers, including breast and prostate. The purpose of the present study was to examine the expression patterns of additional lipid metabolic enzymes in human breast and prostate cancers. This was accomplished by analysis of published expression databases, with confirmation by immunoblot assays. Our results indicate that the fatty acid-activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), is differentially expressed in human breast cancer as a function of estrogen receptor alpha (ER) status. In 10 separate studies, ACSL4 messenger RNA (mRNA) was overexpressed in ER-negative breast tumors. Of 50 breast cancer cell lines examined, 17 (89%) of 19 ER-positive lines were negative for ACSL4 mRNA expression and 20 (65%) of 31 ER-negative lines expressed ACSL4 mRNA. The inverse relationship between ER expression and ACSL4 expression was also observed for androgen receptor status in both breast and prostate cancers. Furthermore, loss of steroid hormone sensitivity, such as that observed in Raf1-transfected MCF-7 cells and LNCaP-AI cells, was associated with induction of ACSL4 expression. Ablation of ACSL4 expression inMDA-MB-231 breast cancer cells had no effect on cell proliferation; however, sensitivity to the cytotoxic effects of triacsin C was increased three-fold in the cells lacking ACSL4.