Pseudomonas syringae Catalases Are Collectively Required for Plant Pathogenesis

The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 must detoxify plant-produced hydrogen peroxide (H(2)O(2)) in order to survive in its host plant. Candidate enzymes for this detoxification include the monofunctional catalases KatB and KatE and the bifunctional catalase-peroxidase KatG of DC3000. This study shows that KatG is the major housekeeping catalase of DC3000 and provides protection against menadione-generated endogenous H(2)O(2). In contrast, KatB rapidly and substantially accumulates in response to exogenous H(2)O(2). Furthermore, KatB and KatG have nonredundant roles in detoxifying exogenous H(2)O(2) and are required for full virulence of DC3000 in Arabidopsis thaliana. Therefore, the nonredundant ability of KatB and KatG to detoxify plant-produced H(2)O(2) is essential for the bacteria to survive in plants. Indeed, a DC3000 catalase triple mutant is severely compromised in its ability to grow in planta, and its growth can be partially rescued by the expression of katB, katE, or katG. Interestingly, our data demonstrate that although KatB and KatG are the major catalases involved in the virulence of DC3000, KatE can also provide some protection in planta. Thus, our results indicate that these catalases are virulence factors for DC3000 and are collectively required for pathogenesis.     

Mixotrophic and photoautotrophic cultivation of 14 microalgae isolates from Saskatchewan, Canada: potential applications for wastewater remediation for biofuel production

Northern regions are generally viewed as unsuitable for microalgal biofuel production due to unfavorable climate and solar insolation levels. However, these conditions can potentially be mitigated by coupling microalgal cultivation to industrial processes such as wastewater treatment. In this study, we have examined the biomass and lipid productivity characteristics of 14 microalgae isolates (Chlorophyta) from the Canadian province of Saskatchewan. Under both photoautotrophic and mixotrophic cultivation, a distinct linear trend was observed between biomass and lipid productivities in the 14 SK isolates. The most productive strain under cultivation in TAP media was Scenedesmus sp.-AMDD which displayed rates of biomass and fatty acid productivities of 80 and 30.7?mg?L^?1?day^?1, respectively. The most productive strain in B3NV media was Chlamydomonas debaryana-AMLs1b which displayed rates of biomass and fatty acid productivities of 51.7 and 5.9?mg?L^?1?day^?1, respectively. In 11 of the isolates tested, secondary municipal wastewater (MCWW) supported rates of biomass productivity between 21 and 33?mg?L^?1?day^?1 with Scenedesmus sp.-AMDD being the most productive. Three strains, Chlamydomonas debaryana-AMB1, Chlorella sorokiniana-RBD8 and Micractinium sp.-RB1b, showed large increases in biomass productivity when cultivated mixotrophically in MCWW supplemented with glycerol. High relative oleic acid content was detected in 10 of the 14 isolates when grown mixotrophically in media supplemented with acetate. There was no detectable effect on the fatty acid profiles in cells cultivated mixotrophically in glycerol-supplemented MCWW. These data indicate that biomass and lipid productivities are boosted by mixotrophic cultivation. Exploiting this response in municipal wastewater is a promising strategy for the production of environmentally sustainable biofuels.     

Differential regulation of CD4+ T helper cell responses by curcumin in experimental autoimmune encephalomyelitis

Nutraceuticals and phytochemicals are important regulators of human health and diseases. Curcumin is a polyphenolic phytochemical isolated from the rhizome of the plant Curcuma longa (turmeric) that has been traditionally used for the treatment of inflammation and wound healing for centuries. Systematic analyses have shown that curcumin exerts its beneficial effects through antioxidant, antiproliferative and anti-inflammatory properties. We and others have shown earlier that curcumin ameliorates experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. In this study, we show that C57BL/6 mice induced to develop EAE express elevated levels of interferon (IFN) γ and interleukin (IL)-17 in the central nervous system (CNS) and lymphoid organs that decreased significantly following in vivo treatment with curcumin. The EAE mice also showed elevated expression of IL-12 and IL-23 that decreased after treatment with curcumin. Ex vivo and in vitro treatment with curcumin resulted in a dose-dependent decrease in the secretion of IFNγ, IL-17, IL-12 and IL-23 in culture. The inhibition of EAE by curcumin was also associated with an up-regulation of IL-10, peroxisome proliferator activated receptor γ and CD4⁺CD25⁺Foxp3⁺ Treg cells in the CNS and lymphoid organs. These findings highlight that curcumin differentially regulates CD4⁺ T helper cell responses in EAE.     

SPO71 Mediates Prospore Membrane Size and Maturation in Saccharomyces cerevisiae

The mechanisms that control the size and shape of membranes are not well understood, despite the importance of these structures in determining organelle and cell morphology. The prospore membrane, a double lipid bilayer that is synthesized de novo during sporulation in S. cerevisiae, grows to surround the four meiotic products. This membrane determines the shape of the newly formed spores and serves as the template for spore wall deposition. Ultimately, the inner leaflet of the prospore membrane will become the new plasma membrane of the cell upon germination. Here we show that Spo71, a pleckstrin homology domain protein whose expression is induced during sporulation, is critical for the appropriate growth of the prospore membrane. Without SPO71, prospore membranes surround the nuclei but are abnormally small, and spore wall deposition is disrupted. Sporulating spo71Δ cells have prospore membranes that properly localize components to their growing leading edges yet cannot properly localize septin structures. We also found that SPO71 genetically interacts with SPO1, a gene with homology to the phospholipase B gene that has been previously implicated in determining the shape of the prospore membrane. Together, these results show that SPO71 plays a critical role in prospore membrane development.     

Clonal immune responses of Mycobacterium-specific γδ T cells in tuberculous and non-tuberculous tissues during M. tuberculosis infection

Background

We previously demonstrated that unvaccinated macaques infected with large-dose M.tuberculosis(Mtb) exhibited delays for pulmonary trafficking of Ag-specific αβ and γδ T effector cells, and developed severe lung tuberculosis(TB) and “secondary” Mtb infection in remote organs such as liver and kidney. Despite delays in lungs, local immunity in remote organs may accumulate since progressive immune activation after pulmonary Mtb infection may allow IFNγ-producing γδ T cells to adequately develop and traffic to lately-infected remote organs. As initial efforts to test this hypothesis, we comparatively examined TCR repertoire/clonality, tissue trafficking and effector function of Vγ2Vδ2 T cells in lung with severe TB and in liver/kidney without apparent TB.

Methodology/Principal Findings

We utilized conventional infection-immunity approaches in macaque TB model, and employed our decades-long expertise for TCR repertoire analyses. TCR repertoires in Vγ2Vδ2 T-cell subpopulation were broad during primary Mtb infection as most TCR clones found in lymphoid system, lung, kidney and liver were distinct. Polyclonally-expanded Vγ2Vδ2 T-cell clones from lymphoid tissues appeared to distribute and localize in lung TB granuloms at the endpoint after Mtb infection by aerosol. Interestingly, some TCR clones appeared to be more predominant than others in lymphocytes from liver or kidney without apparent TB lesions. TCR CDR3 spetratyping revealed such clonal dominance, and the clonal dominance of expanded Vγ2Vδ2 T cells in kidney/liver tissues was associated with undetectable or low-level TB burdens. Furthermore, Vγ2Vδ2 T cells from tissue compartments could mount effector function for producing anti-mycobacterium cytokine.

Conclusion

We were the first to demonstrate clonal immune responses of mycobacterium-specific Vγ2Vδ2 T cells in the lymphoid system, heavily-infected lungs and lately subtly-infected kidneys or livers during primary Mtb infection. While clonally-expanded Vγ2Vδ2 T cells accumulated in lately-infected kidneys/livers without apparent TB lesions, TB burdens or lesions appeared to impact TCR repertoires and tissue trafficking patterns of activated Vγ2Vδ2 T cells.     

Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.     

Radiative forcing of natural forest disturbances

Forest disturbances are major sources of carbon dioxide to the atmosphere, and therefore impact global climate. Biogeophysical attributes, such as surface albedo (reflectivity), further control the climate‐regulating properties of forests. Using both tower‐based and remotely sensed data sets, we show that natural disturbances from wildfire, beetle outbreaks, and hurricane wind throw can significantly alter surface albedo, and the associated radiative forcing either offsets or enhances the CO₂ forcing caused by reducing ecosystem carbon sequestration over multiple years. In the examined cases, the radiative forcing from albedo change is on the same order of magnitude as the CO₂ forcing. The net radiative forcing resulting from these two factors leads to a local heating effect in a hurricane‐damaged mangrove forest in the subtropics, and a cooling effect following wildfire and mountain pine beetle attack in boreal forests with winter snow. Although natural forest disturbances currently represent less than half of gross forest cover loss, that area will probably increase in the future under climate change, making it imperative to represent these processes accurately in global climate models.     

Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content

Top-down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.