Differential plague susceptibility in species and populations of prairie dogs

Laboratory trials conducted over the past decade at U.S. Geological Survey National Wildlife Health Center indicate that wild populations of prairie dogs (Cynomys spp.) display different degrees of susceptibility to experimental challenge with fully virulent Yersinia pestis, the causative agent of plague. We evaluated patterns in prairie dog susceptibility to plague to determine whether the historical occurrence of plague at location of capture was related to survival times of prairie dogs challenged with Y. pestis. We found that black‐tailed prairie dogs (Cynomys ludovicianus) from South Dakota (captured prior to the detection of plague in the state), Gunnison's prairie dogs (Cynomys gunnisoni) from Colorado, and Utah prairie dogs (Cynomys parvidens) from Utah were most susceptible to plague. Though the susceptibility of black‐tailed prairie dogs in South Dakota compared with western locations supports our hypothesis regarding historical exposure, both Colorado and Utah prairie dogs have a long history of exposure to plague. It is possible that for these populations, genetic isolation/bottle necks have made them more susceptible to plague outbreaks.     

Burrow Dusting or Oral Vaccination Prevents Plague-Associated Prairie Dog Colony Collapse

Plague impacts prairie dogs (Cynomys spp.), the endangered black-footed ferret (Mustela nigripes) and other sensitive wildlife species. We compared efficacy of prophylactic treatments (burrow dusting with deltamethrin or oral vaccination with recombinant “sylvatic plague vaccine” [RCN-F1/V307]) to placebo treatment in black-tailed prairie dog (C. ludovicianus) colonies. Between 2013 and 2015, we measured prairie dog apparent survival, burrow activity and flea abundance on triplicate plots (“blocks”) receiving dust, vaccine or placebo treatment. Epizootic plague affected all three blocks but emerged asynchronously. Dust plots had fewer fleas per burrow (P < 0.0001), and prairie dogs captured on dust plots had fewer fleas (P < 0.0001) than those on vaccine or placebo plots. Burrow activity and prairie dog density declined sharply in placebo plots when epizootic plague emerged. Patterns in corresponding dust and vaccine plots were less consistent and appeared strongly influenced by timing of treatment applications relative to plague emergence. Deltamethrin or oral vaccination enhanced apparent survival within two blocks. Applying insecticide or vaccine prior to epizootic emergence blunted effects of plague on prairie dog survival and abundance, thereby preventing colony collapse. Successful plague mitigation will likely entail strategic combined uses of burrow dusting and oral vaccination within large colonies or colony complexes.     

Biotite and microcline as potassium sources in ectomycorrhizal and non-mycorrhizal Pinus sylvestris seedlings

 The aim of this study was to investigate the role of plants colonised by two ectomycorrhizal fungi, Paxillus involutus and Suillus variegatus, in mobilising potassium (K) from biotite and microcline, two minerals common in acid to medium-acid bedrock. This was carried out in a 33-week pot study with seedlings of Pinus sylvestris growing in symbiosis with the fungi, where no K was added or where K was added in the form of biotite or microcline. The mineral additions were similar to those found in natural soils. All seedlings, including non-mycorrhizal, were able to access the K in biotite, leading to stimulated growth and K uptake relative to controls. Microcline addition induced growth depression in all seedlings except those colonised by P. involutus, which were stimulated. The soil solution from S. variegatus-colonised seedlings grown with biotite had higher concentrations of citric and oxalic acid. Citric acid concentration was positively correlated to the fungal biomass (ergosterol) in the soil, as well as to the foliar K in S. variegatus-colonised seedlings. Seedlings growing without K addition had low K concentrations in the shoot. Magnesium (Mg) concentrations were enhanced in seedlings with severe K shortage, indicating that Mg can substitute for K, while calcium concentrations did not vary significantly. Accepted: 18 January 1999     

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+2 dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+2 (translationally inactive) and high Mg+2 (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.     

Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics.     

Mutual control of membrane fission and fusion proteins

Membrane fusion and fission are antagonistic reactions controlled by different proteins. Dynamins promote membrane fission by GTP-driven changes of conformation and polymerization state, while SNAREs fuse membranes by forming complexes between t- and v-SNAREs from apposed vesicles. Here, we describe a role of the dynamin-like GTPase Vps1p in fusion of yeast vacuoles. Vps1p forms polymers that couple several t-SNAREs together. At the onset of fusion, the SNARE-activating ATPase Sec18p/NSF and the t-SNARE depolymerize Vps1p and release it from the membrane. This activity is independent of the SNARE coactivator Sec17p/alpha-SNAP and of the v-SNARE. Vps1p release liberates the t-SNAREs for initiating fusion and at the same time disrupts fission activity. We propose that reciprocal control between fusion and fission components exists, which may prevent futile cycles of fission and fusion.     

Apatite as a P source in mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings

The objectives of the study are firstly to test the ability of ectomycorrhizal pine seedlings to use apatite as a P source in comparison with non-mycorrhizal pine seedlings and secondly, to determine if there is a relation between exudation of organic acids and the ability to use apatite as a P source. Non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 4 different isolates of ectomycorrhizal fungi were grown for 220 days in sand/peat filled pots with apatite (Ca₅(F,OH)(PO₄)₃) as the sole P source. In an additional experiment, non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 2 different isolates of ectomycorrhizal fungi were grown without any P source for 250 days. All other nutrients were supplied in a balanced nutrient solution.Ectomycorrhizal seedlings grew less than non-mycorrhizal seedlings but ectomycorrhizal seedlings produced a large external mycelium not included in the biomass estimates. All seedlings in the present study had low shoot:root ratios compared to seedlings growing under optimal conditions. All seedlings grown with apatite as P source had higher foliar P concentrations (0.71–2.11 mg/g) than seedlings growing without any P source (0.57–0.75 mg/g) indicating a significant ability to use apatite as a P source. Seedlings colonized by Suillus variegatus and Paxillus involutus had higher concentrations and total contents of P in shoots compared with non-mycorrhizal seedlings, indicating significant improvement of P uptake by these fungi in comparison with non-mycorrhizal seedlings or seedlings colonized Piloderma croceum.No clear relationship between exudation of organic acids and uptake of P was found. Seedlings colonized by S. variegatus reduced the pH of the soil more than seedlings colonized by P. involutus or non-mycorrhizal seedlings. It is suggested that S. variegatus colonization improves the P uptake by reducing the pH of the soil while P. involutus improves P uptake by having a greater ability to absorb dissolved phosphate than non-mycorrhizal roots or roots colonized by the other fungi used in the study.     

Population differences in host immune factors may influence survival of Gunnison's prairie dogs (Cynomys gunnisoni) during plague outbreaks

Over the past 40 yr, epizootics of plague (Yersinia pestis) in northern Arizona have reduced populations of the Gunnison's prairie dog (Cynomys gunnisoni), with the exception of a large population found in the Aubrey Valley (AV). To examine potential mechanisms accounting for their survival, we collected prairie dog serum samples in 2005-2006 from AV and a neighboring population near Seligman (SE), Arizona. We quantified gene expression at 58 diverse immune proteins using a multiplexed enzyme-linked immunosorbent assay panel. We found a subset of proteins important in coagulation and inflammation (tissue factor [TF], calbindin [Cal], and thrombopoietin [TPO]) and T-cell responses (CD40L and CD40) that were present in AV at levels two to eight times greater than SE. These results suggest that AV and SE animals might differ in their ability to mount an immune response.     

Interaction of thrombin with sucrose octasulfate

The serine protease thrombin plays multiple roles in many important physiological processes, especially coagulation, where it functions as both a pro- and anticoagulant. The polyanionic glycosaminoglycan heparin modulates thrombin's activity through binding at exosite II. Sucrose octasulfate (SOS) is often used as a surrogate for heparin, but it is not known whether it is an effective heparin mimic in its interaction with thrombin. We have characterized the interaction of SOS with thrombin in solution and determined a crystal structure of their complex. SOS binds thrombin with a K(d) of ~1.4 μM, comparable to that of the much larger polymeric heparin measured under the same conditions. Nonionic (hydrogen bonding) interactions make a larger contribution to thrombin binding of SOS than to heparin. SOS binding to exosite II inhibits thrombin's catalytic activity with high potency but with low efficacy. Analytical ultracentrifugation shows that bovine and human thrombins are monomers in solution in the presence of SOS, in contrast to their complexes with heparin, which are dimers. In the X-ray crystal structure, two molecules of SOS are bound nonequivalently to exosite II portions of a thrombin dimer, in contrast to the 1:2 stoichiometry of the heparin-thrombin complex, which has a different monomer association mode in the dimer. SOS and heparin binding to exosite II of thrombin differ on both chemical and structural levels and, perhaps most significantly, in thrombin inhibition. These differences may offer paths to the design of more potent exosite II binding, allosteric small molecules as modulators of thrombin function.     

Mouse and human granzyme B have distinct tetrapeptide specificities and abilities to recruit the bid pathway

Granzyme B is an important mediator of cytotoxic lymphocyte granule-induced death of target cells, accomplishing this through cleavage of Bid and cleavage and activation of caspases as well as direct cleavage of downstream substrates. Significant controversy exists regarding the primary pathways used by granzyme B to induce cell death, perhaps arising from the use of different protease/substrate combinations in different studies. The primary sequence of human, rat, and mouse granzymes B is well conserved, and the substrate specificity and crystal structure of the human and rat proteases are extremely similar. Although little is known about the substrate specificity of mouse granzyme B, recent studies suggest that it may differ significantly from the human protease. In these studies we show that the specificities of human and mouse granzymes B differ significantly. Human and mouse granzyme B cleave species-specific procaspase-3 more efficiently than the unmatched substrates. The distinct specificities of human and mouse granzyme B highlight a previously unappreciated requirement for Asp(192) in the acquisition of catalytic activity upon cleavage of procaspase-3 at Asp(175). Although human granzyme B efficiently cleaves human or mouse Bid, these substrates are highly resistant to cleavage by the mouse protease, strongly indicating that the Bid pathway is not a major primary mediator of the effects of mouse granzyme B. These studies provide important insights into the substrate specificity and function of the granzyme B pathway in different species and highlight that caution is essential when designing and interpreting experiments with different forms of granzyme B.