Stealth proteins: in silico identification of a novel protein family rendering bacterial pathogens invisible to host immune defense

There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC) is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.     

Discovery of novel indolinone-based,potent, selective and brain penetrant inhibitors of LRRK2

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common genetic cause of Parkinson’s disease (PD). The most frequent kinase-enhancing mutation is the G2019S residing in the kinase activation domain. This opens up a promising therapeutic avenue for drug discovery targeting the kinase activity of LRRK2 in PD. Several LRRK2 inhibitors have been reported to date. Here, we report a selective, brain penetrant LRRK2 inhibitor and demonstrate by a competition pulldown assay in vivo target engagement in mice.     

Moose summer and winter diets along a large scale gradient of forage availability in southern Norway

Studies on dietary functional responses in large herbivores are traditionally conducted by following individual animals. The method is very time-consuming, and hence, typically provides only a narrow array of forage species compositions. Here we use a range level approach to look at moose (Alces alces) selectivity for and utilization of forage species in relation to availability in both summer and winter. We compare 12 Norwegian ranges representing a large scale gradient in plant communities. The most important forage species in the diet were birches (Betula spp., comprising 43% of all trees browsed in summer and 27% in winter), rowan (Sorbus aucuparia, 25% of trees browsed in summer, 37% in winter), and bilberry (Vaccinum myrtillus, 42% of herbaceous epidermal fragments in summer feces). Selectivity for birches was positively related to its availability and negatively related to availability of rowan, Salix spp., and aspen (Populus tremula) together (all more selected for than birches). Multiple regression models including availability of several forage species were thus superior to single-species models in explaining the diet content of main forage plants. Selectivity for birches was also stronger in summer than in winter, while the opposite pattern was found for rowan. The finding is relevant for our evaluation of the quality of summer and winter ranges, and hence, their relative influence on population productivity. Our study underlines the need to incorporate species composition of available forage when quantifying dietary functional responses in selective herbivores such as moose. Furthermore, care should be taken when extrapolating data on moose diet across ranges or seasons.     

Mutagen specificity in the induction of karyotic versus cytoplasmic respiratory deficient mutants in yeast by nitrous acid and alkylating nitrosamides

Summary In the haploid eukaryotic organism Saccharomyces cerevisiae the induction of cytoplasmic and genic (karyotic) RD mutants was studied, using nitrous acid, nitrosomethylurethane (NMU) and nitrosoimidazolidone (NIL).The cytoplasmic or genic origin of the induced RD mutants was determined by prescreening in complementation tests with ^– and wild type tester strains. Among the mutants of all three agents we could thus score the incidence of three RD mutant types: genic, suppressive and cytoplasmic (both primary and secondary). The final identification of the cytoplasmic type was only possible through tetrad analysis, performed in the cases of HNO₂ and NMU.A distinct difference in cytoplasmic versus genic mutagen specificity was observed between HNO₂ and NMU. HNO₂ was unable to induce cytoplasmic RD mutants but it proved to be highly efficient in the induction of genic RD mutants. In contrast, NMU induced more cytoplasmic effects was it possible to detect mutagenic specificities which, solely on the basis of karyotic action, were not detectable.     

Diabetic liver injury from streptozotocin is regulated through the caspase-8 homolog cFLIP involving activation of JNK2 and intrahepatic immunocompetent cells

The endemic occurrence of obesity and the associated risk factors that constitute the metabolic syndrome have been predicted to lead to a dramatic increase in chronic liver disease. Non-alcoholic steatohepatitis (NASH) has become the most frequent liver disease in countries with a high prevalence of obesity. In addition, hepatic steatosis and insulin resistance have been implicated in disease progression of other liver diseases, including chronic viral hepatitis and hepatocellular carcinoma. The molecular mechanisms underlying the link between insulin signaling and hepatocellular injury are only partly understood. We have explored the role of the antiapoptotic caspase-8 homolog cellular FLICE-inhibitory protein (cFLIP) on liver cell survival in a diabetic model with hypoinsulinemic diabetes in order to delineate the role of insulin signaling on hepatocellular survival. cFLIP regulates cellular injury from apoptosis signaling pathways, and loss of cFLIP was previously shown to promote injury from activated TNF and CD95/Apo-1 receptors. In mice lacking cFLIP in hepatocytes (flip^−/−), loss of insulin following streptozotocin treatment resulted in caspase- and c-Jun N-terminal kinase (JNK)-dependent liver injury after 21 days. Substitution of insulin, inhibition of JNK using the SP600125 compound in vivo or genetic deletion of the mitogen-activated protein kinase (MAPK)9 (JNK2) in all tissues abolished the injurious effect. Strikingly, the difference in injury between wild-type and cFLIP-deficient mice occurred only in vivo and was accompanied by liver-infiltrating inflammatory cells with a trend toward increased amounts of NK1.1-positive cells and secretion of proinflammatory cytokines. Transfer of bone marrow from rag-1-deficient mice that are depleted from B and T lymphocytes prevented liver injury in flip^−/− mice. These findings support a direct role of insulin on cellular survival by alternating the activation of injurious MAPK, caspases and the recruitment of inflammatory cells to the liver. Thus, increasing resistance to insulin signaling pathways in hepatocytes appears to be an important factor in the initiation and progression of chronic liver disease.     

Dominant versus recessive behavior of a cold- and a heat-sensitive mammalian cell cycle variant in heterokaryons

A heat-sensitive (hs, arrested at 39.5 degrees C, termed 21-Ta) and a cold-sensitive (cs, arrested at 33 degrees C, termed 21-Fb) clonal cell cycle variant were isolated from the same clone of the P-815 murine mastocytoma line. At the respective nonpermissive temperatures, both the hs and the cs variant were reversibly arrested in G1 phase, and numbers of cells forming colonies upon reincubation at the permissive temperature remained nearly constant for at least 6 days. Cells arrested in G1 by incubation at the respective nonpermissive temperatures were fused to cells of another P-815 clone (31-S) that had been arrested by serum deprivation. Upon reincubation in medium containing 10% serum for 48 h at 39.5 degrees C, 21-Ta x 31-S heterokaryons, similar to 31-S x 31-S homokaryons, entered the S phase, whereas at 33 degrees C, 21-Fb x 31-S heterokaryons, similar to 21-Fb x 21-Fb homokaryons, remained arrested in G1, indicating a recessive expression of the hs and a dominant expression of the cs phenotype.     

Adaptive laboratory evolution of microbial co‐cultures for improved metabolite secretion

Adaptive laboratory evolution has proven highly effective for obtaining microorganisms with enhanced capabilities. Yet, this method is inherently restricted to the traits that are positively linked to cell fitness, such as nutrient utilization. Here, we introduce coevolution of obligatory mutualistic communities for improving secretion of fitness‐costly metabolites through natural selection. In this strategy, metabolic cross‐feeding connects secretion of the target metabolite, despite its cost to the secretor, to the survival and proliferation of the entire community. We thus co‐evolved wild‐type lactic acid bacteria and engineered auxotrophic Saccharomyces cerevisiae in a synthetic growth medium leading to bacterial isolates with enhanced secretion of two B‐group vitamins, viz., riboflavin and folate. The increased production was specific to the targeted vitamin, and evident also in milk, a more complex nutrient environment that naturally contains vitamins. Genomic, proteomic and metabolomic analyses of the evolved lactic acid bacteria, in combination with flux balance analysis, showed altered metabolic regulation towards increased supply of the vitamin precursors. Together, our findings demonstrate how microbial metabolism adapts to mutualistic lifestyle through enhanced metabolite exchange.     

A Tracer Bolus Method for Investigating Glutamine Kinetics in Humans

Glutamine transport between tissues is important for the outcome of critically ill patients. Investigation of glutamine kinetics is, therefore, necessary to understand glutamine metabolism in these patients in order to improve future intervention studies. Endogenous glutamine production can be measured by continuous infusion of a glutamine tracer, which necessitates a minimum measurement time period. In order to reduce this problem, we used and validated a tracer bolus injection method. Furthermore, this method was used to measure the glutamine production in healthy volunteers in the post-absorptive state, with extra alanine and with glutamine supplementation and parenteral nutrition. Healthy volunteers received a bolus injection of [1-¹³C] glutamine, and blood was collected from the radial artery to measure tracer enrichment over 90 minutes. Endogenous rate of appearance (endoR_a) of glutamine was calculated from the enrichment decay curve and corrected for the extra glutamine supplementation. The glutamine endoR_a of healthy volunteers was 6.1±0.9 µmol/kg/min in the post-absorptive state, 6.9±1.0 µmol/kg/min with extra alanyl-glutamine (p = 0.29 versus control), 6.1±0.4 µmol/kg/min with extra alanine only (p = 0.32 versus control), and 7.5±0.9 µmol/kg/min with extra alanyl-glutamine and parenteral nutrition (p = 0.049 versus control). In conclusion, a tracer bolus injection method to measure glutamine endoR_a showed good reproducibility and small variation at baseline as well as during parenteral nutrition. Additionally, we showed that parenteral nutrition including alanyl-glutamine increased glutamine endoR_a in healthy volunteers, which was not attributable to the alanine part of the dipeptide.