Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors

AAA+ proteases are frequently regulated by adaptors that modulate spatial and temporal control of protein turnover. Caulobacter crescentus is an alpha-proteobacterium which requires protein degradation by the AAA+ ClpXP protease for cell-cycle progression, and contains an adaptor (SspBalpha) that binds ssrA-tagged proteins and targets them to ClpXP. Here we determine the tag-binding specificity and crystal structure of SspBalpha. Despite poor sequence homology, the overall SspBalpha fold resembles orthologs from other bacteria. However, several structural features are specific to the SspBalpha subfamily, including the dimerization interface, binding surfaces optimized for ssrA-tag delivery, and residues in the tag-binding groove that act as selectivity gatekeepers for substrate recognition. Mutagenesis of these residues broadens specificity, creating a promiscuous adaptor that recognizes an expanded substrate repertoire. These results highlight general features of adaptor-mediated substrate recognition and shed light on design principles that underlie adaptor function.     

Development of a multiplex PCR for identification of vineyard mealybugs

A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus ficus (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26±2°C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6-8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs.     

Cooperation between 4-1BB and ICOS in the immune response to influenza virus revealed by studies of CD28/ICOS-deficient mice

CD28, ICOS, and 4-1BB each play distinct roles in the CD8 T cell response to influenza virus. CD28-/- mice are severely impaired in primary CD8 T cell expansion and fail to mount a secondary response to influenza. Influenza-specific CD8 T cells expand normally in ICOS-/- mice, with only a small and transient defect late in the primary response and an unimpaired secondary response. Conversely, 4-1BB/4-1BBL interaction is dispensable for the primary CD8 T cell response to influenza, but maintains CD8 T cell survival and controls the size of the secondary response. Previous results showed that a single dose of agonistic anti-4-1BB Ab at priming allowed partial restoration of primary CD8 T cell expansion and full recovery of the secondary CD8 T cell responses to influenza in CD28-/- mice. In this study we show that anti-4-1BB fails to correct the CD8 T cell defect in CD28-/-ICOS-/- mice, suggesting that ICOS partially compensates for CD28 in this model. In support of this hypothesis, we found that anti-4-1BB enhances ICOS expression on both T cell subsets and that anti-4-1BB and anti-ICOS can synergistically activate CD4 and CD8 T cells. Furthermore, ICOS and 4-1BB can cooperate to directly stimulate isolated CD28-/- CD8 T cells. These results reveal a novel interaction between the ICOS and 4-1BB costimulatory pathways as well as unexpected redundancy between CD28 and ICOS in primary CD8 T cell expansion. These findings have implications for costimulation of human T cell responses in diseases such as AIDS or rheumatoid arthritis, in which CD28- T cells accumulate.     

Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease

During chronic injury a population of bipotent hepatic progenitor cells (HPCs) become activated to regenerate both cholangiocytes and hepatocytes. Here we show in human diseased liver and mouse models of the ductular reaction that Notch and Wnt signaling direct specification of HPCs via their interactions with activated myofibroblasts or macrophages. In particular, we found that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signaling in HPCs and thus their biliary specification to cholangiocytes. Alternatively, during hepatocyte regeneration, macrophage engulfment of hepatocyte debris induced Wnt3a expression. This resulted in canonical Wnt signaling in nearby HPCs, thus maintaining expression of Numb (a cell fate determinant) within these cells and the promotion of their specification to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted.     

An IP3R3- and NPY-Expressing Microvillous Cell Mediates Tissue Homeostasis and Regeneration in the Mouse Olfactory Epithelium

Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3^+/−, and IP3R3^−/− mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3^−/− mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3^−/− mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3^−/− mice. The reductions in both NPY release and number of progenitor cells in IP3R3^−/− mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.     

Azole-resistant Aspergillus fumigatus is highly prevalent in the environment of Vietnam,with marked variability by land use type

Azole-resistant environmental Aspergillus fumigatus presents a threat to public health but the extent of this threat in Southeast Asia is poorly described. We conducted environmental surveillance in the Mekong Delta region of Vietnam, collecting air and ground samples across key land-use types, and determined antifungal susceptibilities of Aspergillus section Fumigati (ASF) isolates and azole concentrations in soils. Of 119 ASF isolates, 55% were resistant (or non-wild type) to itraconazole, 65% to posaconazole and 50% to voriconazole. Azole resistance was more frequent in A. fumigatus sensu stricto isolates (95%) than other ASF species (32%). Resistant isolates and agricultural azole residues were overrepresented in samples from cultivated land. cyp51A gene sequence analysis showed 38/56 resistant A. fumigatus sensu stricto isolates carried known resistance mutations, with TR₃₄/L98H most frequent (34/38).