Generation of highly activated, antigen-specific tumor-infiltrating CD8+ T cells induced by a novel T cell-targeted immunotherapy.

The induction of tumor-targeted, cytotoxic T lymphocytes has been recognized as a key component to successful immunotherapy. DPX-based treatment was previously shown to effectively recruit activated CD8+ T cells to the tumor. Herein, we analyze the unique phenotype of the CD8+ T cells recruited into the tumor in response to DPX-based therapy, and how combination with checkpoint inhibitors impacts T cell response. C3-tumor-bearing mice were treated with cyclophosphamide (CPA) for seven continuous days every other week, followed by DPX treatment along with anti-CTLA-4 and/or anti-PD-1. Efficacy, immunogenicity, and CD8+ T cells tumor infiltration were assessed. The expression of various markers, including checkpoint markers, peptide specificity, and proliferation and activation markers, was determined by flow cytometry. tSNE analysis of the flow data revealed a resident phenotype of CD8+ T cells (PD-1+TIM-3+CTLA-4+) within untreated tumors, whereas DPX/CPA treatment induced recruitment of a novel population of CD8+ T cells (PD-1+TIM-3+CTLA-4-) within tumors. Combination of anti-CTLA-4 (ipilimumab) with DPX/CPA versus DPX/CPA alone significantly increased survival and inhibition of tumor growth, without changing overall systemic immunogenicity. Addition of checkpoint inhibitors did not significantly change the phenotype of the newly recruited cells induced by DPX/CPA. Yet, anti-CTLA-4 treatment in combination with DPX/CPA enhanced a non-antigen specific response within the tumor. Finally, the tumor-recruited CD8+ T cells induced by DPX/CPA were highly activated, antigen-specific, and proliferative, while resident phenotype CD8+ T cells, seemingly initially exhausted, were reactivated with combination treatment. This study supports the potential of combining DPX/CPA with ipilimumab to further enhance survival clinically.

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.

Comprehensive analysis of flagellin glycosylation in Campylobacter jejuni NCTC 11168 reveals incorporation of legionaminic acid and its importance for host colonization.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis. It relies on several virulence factors for host colonization, including glycosylated flagella. C. jejuni NCTC 11168 modifies its flagellins with pseudaminic acid derivatives. It is also presumed to modify these proteins with legionaminic acid, although no glycopeptide evidence was available at the onset of this study. The enzyme encoded by cj1319 can be used to make legionaminic acid in vitro, but the pathway for legionaminic acid synthesis partially inferred by knockout mutagenesis in Campylobacter coli VC167 excludes Cj1319. To address this contradiction, we examined the presence of legionaminic acid in flagellin glycopeptides of wild-type (WT) C. jejuni NCTC 11168 and of a cj1319 knockout mutant. We used high-energy collision-induced dissociation to obtain amino acid sequences while also visualizing signature sugar oxonium ions. Data analysis was performed with PEAKS software, and spectra were manually inspected for glycopeptide determination and verification. We showed that legionaminic acid is present on the flagellins of C. jejuni NCTC 11168 and that flagellin glycosylation is highly heterogeneous, with up to six different sugars singly present at a given site. We found that the cj1319 mutant produces more legionaminic acid than WT, thus excluding the requirement for Cj1319 for legionaminic acid synthesis. We also showed that this mutant has enhanced chicken colonization compared with WT, which may in part be attributed to the high content of legionaminic acid on its flagella.

Evaluating the pathogenic potential of environmental Escherichia coli by using the Caenorhabditis elegans infection model.

The detection and abundance of Escherichia coli in water is used to monitor and mandate the quality of drinking and recreational water. Distinguishing commensal waterborne E. coli isolates from those that cause diarrhea or extraintestinal disease in humans is important for quantifying human health risk. A DNA microarray was used to evaluate the distribution of virulence genes in 148 E. coli environmental isolates from a watershed in eastern Ontario, Canada, and in eight clinical isolates. Their pathogenic potential was evaluated with Caenorhabditis elegans, and the concordance between the bioassay result and the pathotype deduced by genotyping was explored. Isolates identified as potentially pathogenic on the basis of their complement of virulence genes were significantly more likely to be pathogenic to C. elegans than those determined to be potentially nonpathogenic. A number of isolates that were identified as nonpathogenic on the basis of genotyping were pathogenic in the infection assay, suggesting that genotyping did not capture all potentially pathogenic types. The detection of the adhesin-encoding genes sfaD, focA, and focG, which encode adhesins; of iroN2, which encodes a siderophore receptor; of pic, which encodes an autotransporter protein; and of b1432, which encodes a putative transposase, was significantly associated with pathogenicity in the infection assay. Overall, E. coli isolates predicted to be pathogenic on the basis of genotyping were indeed so in the C. elegans infection assay. Furthermore, the detection of C. elegans-infective environmental isolates predicted to be nonpathogenic on the basis of genotyping suggests that there are hitherto-unrecognized virulence factors or combinations thereof that are important in the establishment of infection.

Protein glycosylation in Helicobacter pylori: beyond the flagellins?

Glycosylation of flagellins by pseudaminic acid is required for virulence in Helicobacter pylori. We demonstrate that, in H. pylori, glycosylation extends to proteins other than flagellins and to sugars other than pseudaminic acid. Several candidate glycoproteins distinct from the flagellins were detected via ProQ-emerald staining and DIG- or biotin- hydrazide labeling of the soluble and outer membrane fractions of wild-type H. pylori, suggesting that protein glycosylation is not limited to the flagellins. DIG-hydrazide labeling of proteins from pseudaminic acid biosynthesis pathway mutants showed that the glycosylation of some glycoproteins is not dependent on the pseudaminic acid glycosylation pathway, indicating the existence of a novel glycosylation pathway. Fractions enriched in glycoprotein candidates by ion exchange chromatography were used to extract the sugars by acid hydrolysis. High performance anion exchange chromatography with pulsed amperometric detection revealed characteristic monosaccharide peaks in these extracts. The monosaccharides were then identified by LC-ESI-MS/MS. The spectra are consistent with sugars such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Ac7Ac) previously described on flagellins, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Am7Ac), bacillosamine derivatives and a potential legionaminic acid derivative (Leg5AmNMe7Ac) which were not previously identified in H. pylori. These data open the way to the study of the mechanism and role of protein glycosylation on protein function and virulence in H. pylori.

Cj1121c, a novel UDP-4-keto-6-deoxy-GlcNAc C-4 aminotransferase essential for protein glycosylation and virulence in Campylobacter jejuni.

Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins carry diacetamidobacillosamine (DAB), a sugar that is not found in humans. Hence, the enzymes responsible for DAB synthesis represent potential therapeutic targets. We describe the biochemical characterization of Cj1121c, a putative aminotransferase encoded by the general protein glycosylation locus, to assess its role in DAB biosynthesis. By using overexpressed and affinity-purified enzyme, we demonstrate that Cj1121c has pyridoxal phosphate- and glutamate-dependent UDP-4-keto-6-deoxy-GlcNAc C-4 transaminase activity and produces UDP-4-amino-4,6-dideoxy-GlcNAc. This is consistent with a role in DAB biosynthesis and distinguishes Cj1121c from Cj1294, a homologous UDP-2-acetamido-2,6-dideoxy-beta-l-arabino-4-hexulose C-4 aminotransferase that we characterized previously. We show that Cj1121c can also use this 4-keto-arabino sugar indirectly as a substrate, that Cj1121c and Cj1294 are active simultaneously in C. jejuni, and that the activity of Cj1121c is preponderant under standard growth conditions. Kinetic data indicate that Cj1121c has a slightly higher catalytic efficiency than Cj1294 with regard to the 4-keto-arabino substrate. By site-directed mutagenesis, we show that residues Glu-158 and Leu-131 are not essential for catalysis or for substrate specificity contrary to expectations. We further demonstrate that a cj1121c knock-out mutant is impaired for flagella-mediated motility, for invasion of intestinal epithelial cells, and for persistence in the chicken intestine, clearly demonstrating that Cj1121c is essential for host colonization and virulence. Finally, we show that cj1121c is necessary for protein glycosylation by lectin Western blotting. Collectively, these results validate Cj1121c as a promising drug target and provide the means to assay for inhibitors.