Engineering xylose metabolism in thraustochytrid T18.

BACKGROUND: Thraustochytrids are heterotrophic, oleaginous, marine protists with a significant potential for biofuel production. High-value co-products can off-set production costs; however, the cost of raw materials, and in particular carbon, is a major challenge to developing an economical viable production process. The use of hemicellulosic carbon derived from agricultural waste, which is rich in xylose and glucose, has been proposed as a sustainable and low-cost approach. Thraustochytrid strain T18 is a commercialized environmental isolate that readily consumes glucose, attaining impressive biomass, and oil production levels. However, neither thraustochytrid growth capabilities in the presence of xylose nor a xylose metabolic pathway has been described. The aims of this study were to identify and characterize the xylose metabolism pathway of T18 and, through genetic engineering, develop a strain capable of growth on hemicellulosic sugars. RESULTS: Characterization of T18 performance in glucose/xylose media revealed diauxic growth and copious extracellular xylitol production. Furthermore, T18 did not grow in media containing xylose as the only carbon source. We identified, cloned, and functionally characterized a xylose isomerase. Transcriptomics indicated that this xylose isomerase gene is upregulated when xylose is consumed by the cells. Over-expression of the native xylose isomerase in T18, creating strain XI 16, increased xylose consumption from 5.2 to 7.6 g/L and reduced extracellular xylitol from almost 100% to 68%. Xylose utilization efficiency of this strain was further enhanced by over-expressing a heterologous xylulose kinase to reduce extracellular xylitol to 20%. Moreover, the ability to grow in media containing xylose as a sole sugar was dependent on the copy number of both xylose isomerase and xylulose kinase present. In fed-batch fermentations, the best xylose metabolizing isolate, XI-XK 7, used 137 g of xylose versus 39 g by wild type and produced more biomass and fatty acid. CONCLUSIONS: The presence of a typically prokaryotic xylose isomerase and xylitol production through a typically eukaryotic xylose reductase pathway in T18 is the first report of an organism naturally encoding enzymes from two native xylose metabolic pathways. Our newly engineered strains pave the way for the growth of T18 on waste hemicellulosic feedstocks for biofuel production.

Natural Killer T-cell Immunotherapy in Combination with Chemotherapy-Induced Immunogenic Cell Death Targets Metastatic Breast Cancer.

Natural killer T (NKT) cells are glycolipid-reactive lymphocytes that promote cancer control. In previous studies, NKT-cell activation improved survival and antitumor immunity in a postsurgical mouse model of metastatic breast cancer. Herein, we investigated whether NKT-cell activation could be combined with chemotherapeutic agents to augment therapeutic outcomes. Gemcitabine and cyclophosphamide analogues enhanced the potential immunogenicity of 4T1 mammary carcinoma cells by increasing the expression of antigen-presenting molecules (MHC-I, MHC-II, and CD1d) and promoting exposure or release of immunogenic cell death markers (calreticulin, HMGB1, and ATP). In 4T1 primary tumor and postsurgical metastasis models, BALB/c mice were treated with cyclophosphamide or gemcitabine. NKT cells were then activated by transfer of dendritic cells loaded with the glycolipid antigen α-galactosylceramide (α-GalCer). Chemotherapeutic treatments did not impact NKT-cell activation but enhanced recruitment into primary tumors. Cyclophosphamide, gemcitabine, or α-GalCer-loaded dendritic cell monotherapies decreased tumor growth in the primary tumor model and reduced metastatic burden and prolonged survival in the metastasis model. Combining chemotherapeutics with NKT-cell activation therapy significantly enhanced survival, with surviving mice exhibiting attenuated tumor growth following a second tumor challenge. The frequency of myeloid-derived suppressor cells was reduced by gemcitabine, cyclophosphamide, or α-GalCer-loaded dendritic cell treatments; cyclophosphamide also reduced the frequency of regulatory T cells. Individual treatments increased immune cell activation, cytokine polarization, and cytotoxic responses, although these readouts were not enhanced further by combining therapies. These findings demonstrate that NKT-cell activation therapy can be combined with gemcitabine or cyclophosphamide to target tumor burden and enhance protection against tumor recurrence. Cancer Immunol Res; 5(12); 1086-97. ©2017 AACR.

The bacterial pathogen-ubiquitin interface: lessons learned from Shigella.

Shigella species are the aetiological agents of shigellosis, a severe diarrhoeal disease that is a significant cause of morbidity and mortality worldwide. Shigellosis causes massive colonic destruction, high fever and bloody diarrhoea. Shigella pathogenesis is tightly linked to the ability of the bacterium to invade and replicate intracellularly within the colonic epithelium. Shigella uses a type 3 secretion system to deliver its effector proteins into the cytosol of infected cells. Among the repertoire of Shigella effectors, many are known to target components of the actin cytoskeleton to promote bacterial entry. An emerging alternate theme for effector function is the targeting of the host ubiquitin system. Ubiquitination is a post-translational modification restricted to eukaryotes and is involved in many essential host processes. By virtue of sheer number of ubiquitin-modulating effector proteins, it is clear that Shigella has invested heavily into subversion of the ubiquitin system. Understanding these host-pathogen interactions will inform us about the strategies used by successful pathogens and may also provide avenues for novel antimicrobial strategies.

Myopodin isoforms alter the chemokinetic response of PC3 cells in response to different migration stimuli via differential effects on Rho-ROCK signaling pathways.

The gene encoding myopodin, an actin binding protein, is commonly deleted in invasive, but not in indolent, prostate cancers. There are conflicting reports on the effects of myopodin expression on prostate cancer cell migration and invasion. The recent recognition that myopodin is expressed as four different isoforms further complicates our understanding of how this potentially important invasive prostate cancer biomarker affects tumor cell migration and invasion. We now show that myopodin affects the chemokinetic, rather than the chemotactic, properties of PC3 prostate cancer cells. Furthermore, all myopodin isoforms can either increase or decrease PC3 cell migration in response to different chemokinetic stimuli. These migration properties were reflected by differences in cell morphology and the relative dependence on Rho-ROCK signaling pathways induced by the environmental stimuli. Truncation analysis determined that a unique 9-residue C-terminal sequence in the shortest isoform and the conserved, PDZ domain-containing N-terminal region of the long isoforms both contribute to the ability of myopodin to alter the response of PC3 cells to chemokinetic stimuli. Matrigel invasion assays also indicated that myopodin primarily affects the migration, rather than the invasion, properties of PC3 cells. The correlation between loss of myopodin expression and invasive prostate cancer therefore reflects complex myopodin interactions with pathways that regulate the cellular migration response to diverse signals that may be present in a tumor microenvironment.