The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans

The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.     

High abundance synovial fluid proteome: distinct profiles in health and osteoarthritis

The development of increasingly high-throughput and sensitive mass spectroscopy-based proteomic techniques provides new opportunities to examine the physiology and pathophysiology of many biologic fluids and tissues. The purpose of this study was to determine protein expression profiles of high-abundance synovial fluid (SF) proteins in health and in the prevalent joint disease osteoarthritis (OA). A cross-sectional study of 62 patients with early OA (n = 21), patients with late OA (n = 21), and control individuals (n = 20) was conducted. SF proteins were separated by using one-dimensional PAGE, and the in-gel digested proteins were analyzed by electrospray ionization tandem mass spectrometry. A total of 362 spots were examined and 135 high-abundance SF proteins were identified as being expressed across all three study cohorts. A total of 135 SF proteins were identified. Eighteen proteins were found to be significantly differentially expressed between control individuals and OA patients. Two subsets of OA that are not dependent on disease duration were identified using unsupervised analysis of the data. Several novel SF proteins were also identified. Our analyses demonstrate no disease duration-dependent differences in abundant protein composition of SF in OA, and we clearly identified two previously unappreciated yet distinct subsets of protein profiles in this disease cohort. Additionally, our findings reveal novel abundant protein species in healthy SF whose functional contribution to SF physiology was not previously recognized. Finally, our studies identify candidate biomarkers for OA with potential for use as highly sensitive and specific tests for diagnostic purposes or for evaluating therapeutic response.     

Reductive effect of H(2) uptake and poly-beta-hydroxybutyrate formation on nitrogenase-mediated H(2) accumulation of Rhodobacter sphaeroides according to light intensity

Nitrogenase-mediated H(2) accumulation of Rhodobacter sphaeroides under photoheterotrophic conditions is reduced directly by the hydrogenase activity catalyzing H(2) uptake and indirectly by energy-demanding metabolic processes such as poly-beta-hydroxybutyrate (PHB) formation. H(2) accumulation of R. sphaeroides was examined during cell growth under illumination of 15, 7, and 3 W/m(2). Mutations in either hupSL (H(2)-uptake hydrogenase) or phbC (PHB synthase) had no effect on nitrogenase activity. The nitrogenase activity of R. sphaeroides grown at 15 W/m(2), however, was 70% higher than that of cells grown at 3 W/m(2), while the H(2)-uptake hydrogenase activity was approximately 3-fold higher in the same comparison. Accordingly, H(2) uptake by hydrogenase, monitored by measuring the difference in H(2) accumulation between a hupSL-deletion mutant and the corresponding parental strain, appeared to reach a maximum level as illumination was increased to 15 W/m(2). On the other hand, the surplus energy due to lack of PHB formation led to a fixed increase in H(2) accumulation independent of light intensity, reflecting the fact that the cellular PHB content was not changed significantly depending on light intensity. Therefore, H(2) uptake by hydrogenase should be suppressed to achieve higher H(2) accumulation of R. sphaeroides, especially at 15 W/m(2).     

Anthocyanin synthesis in a white flowering mutant of Petunia hybrida

In flower buds of the white flowering mutant W19 of Petunia hybrida four biologically active dihydroflavonol intermediates-dihydroquercetin-7-glucoside, dihydroquercetin-4-glucoside, dihydroquercetin, and dihydrokaempferol-7-glucoside-are accumulated. When dihydroquercetin was supplied to in vitro cultured corollas of the white flowering mutant W18, a mixture of cyanidin and delphinidin glycosides was produced, cyanidin-3-glucoside being the major pigment. The quantity of dihydroquercetin accumulated in W19 is very small, but this compound appears to be a more direct precursor of anthocyanins than the glucosides of dihydrokaempferol and dihydroquercetin. The conditions for pigment synthesis in W18 were optimalized. The quantitative uptake of dihydroquercetin was also studied. It was demonstrated that ca. 1/3 of the quantity present in the culture solution entered the corolla. From the absorbed dihydroquercetin only 14% was converted into anthocyanins. Complementation experiments to determine the biosynthetic sequence of the anthocyanin genes An1, An2, and An3 indicated that the genes An1 and An2 are indistinguishable by this technique.Abbreviation DHQ (+) dihydroquercetin     

Biological effects of G1 phase arrest compound,sesquicillin, in human breast cancer cell lines

Sesquicillin, isolated from fungal fermentation broth, strongly induced G1 phase arrest in human breast cancer cells. During G1 phase arrest, the expression level of cyclin D1, cyclin A, and cyclin E was decreased, and the expression of CDK (cyclin-dependent-kinase) inhibitor, protein p21(Waf1/Cip1), was increased in a time-dependent manner in a breast cancer cell MCF-7. Interestingly, the G1 phase arrest induced by sesquicillin also occurred independently of the tumor suppressor protein, p53. Sesquicillin inhibits the proliferation of MCF-7 via G1 phase arrest in association with the induction of CDK inhibitor protein, p21(Waf1/Cip1), and the reduction of G1 phase related-cyclin proteins.     

Correlative proteomics identify the key roles of stress tolerance strategies in Acinetobacter baumannii in response to polymyxin and human macrophages

The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections.     

Expression analysis of asthma candidate genes during human and murine lung development

Background

Little is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.

Objective

To investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.

Methods

Asthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.

Results

In total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.

Conclusions

Our data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.     

Interactome analysis of yeast glutathione peroxidase 3

Oxidative stress damages all cellular constituents, and therefore, cell has to possess various defense mechanisms to cope. Saccharomyces cerevisiae, widely used as a model organism for studying cellular responses to oxidative stress, contains three glutathione peroxidase (Gpx) proteins. Among them, Gpx3 plays a major defense role against oxidative stress in S. cerevisiae. In this study, in order to identify the new interaction proteins of Gpx3, we carried out two-dimensional gel electrophoresis after immunoprecipitation (IP-2DE), and MALDI-TOF mass spectrometry. The results showed that several proteins including protein disulfide isomerase, glutaredoxin 2, and SSY protein 3 specifically interact with Gpx3. These findings led us to suggest the possibility that Gpx3, known as a redox sensor and ROS scavenger, has another functional role by interacting with several proteins with various cellular functions.