Role of Tellurite Resistance Operon in Filamentous Growth of Yersinia pestis in Macrophages

Background

Yersinia pestis initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular Y. pestis can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of Y. pestis tellurite resistance proteins TerD and TerE from the terZABCDE operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in Escherichia coli exposed to tellurite. Therefore, in this study we examine the potential role of Y. pestis tellurite resistance operon in filamentous cellular morphology during macrophage infections.

Principal Findings

In vitro treatment of Y. pestis culture with sodium tellurite (Na₂TeO₃) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes terZAB abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a terZABCDE deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the terZABCDE deletion mutant with terCDE, but not terZAB, partially restored tellurite resistance. When the terZABCDE deletion mutant was complemented with terZAB or terCDE, Y. pestis exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an in vitro condition. Further in E. coli, expression of Y. pestis terZAB, but not terCDE, conferred a filamentous phenotype.

Conclusions

These findings support the role of Y. pestis terZAB mediation of the filamentous response phenotype; whereas, terCDE confers tellurite resistance. Although the beneficial role of filamentous morphological responses by Y. pestis during macrophage infections is yet to be fully defined, it may be a bacterial adaptive strategy to macrophage associated stresses.     

Dynamin-related protein 1 is required for normal mitochondrial bioenergetic and synaptic function in CA1 hippocampal neurons

Disrupting particular mitochondrial fission and fusion proteins leads to the death of specific neuronal populations; however, the normal functions of mitochondrial fission in neurons are poorly understood, especially in vivo, which limits the understanding of mitochondrial changes in disease. Altered activity of the central mitochondrial fission protein dynamin-related protein 1 (Drp1) may contribute to the pathophysiology of several neurologic diseases. To study Drp1 in a neuronal population affected by Alzheimer''s disease (AD), stroke, and seizure disorders, we postnatally deleted Drp1 from CA1 and other forebrain neurons in mice (CamKII-Cre, Drp1^lox/lox (Drp1cKO)). Although most CA1 neurons survived for more than 1 year, their synaptic transmission was impaired, and Drp1cKO mice had impaired memory. In Drp1cKO cell bodies, we observed marked mitochondrial swelling but no change in the number of mitochondria in individual synaptic terminals. Using ATP FRET sensors, we found that cultured neurons lacking Drp1 (Drp1KO) could not maintain normal levels of mitochondrial-derived ATP when energy consumption was increased by neural activity. These deficits occurred specifically at the nerve terminal, but not the cell body, and were sufficient to impair synaptic vesicle cycling. Although Drp1KO increased the distance between axonal mitochondria, mitochondrial-derived ATP still decreased similarly in Drp1KO boutons with and without mitochondria. This indicates that mitochondrial-derived ATP is rapidly dispersed in Drp1KO axons, and that the deficits in axonal bioenergetics and function are not caused by regional energy gradients. Instead, loss of Drp1 compromises the intrinsic bioenergetic function of axonal mitochondria, thus revealing a mechanism by which disrupting mitochondrial dynamics can cause dysfunction of axons.Mitochondrial dynamics – the balance between mitochondrial fission and fusion – regulates mitochondrial quality control by segregating poorly functioning mitochondria for degradation while mixing the contents of healthy mitochondria.^{1, 2} In neurons, fission uniquely facilitates movement of mitochondria down narrow distal axons.^{3, 4} Disruptions of this movement, and of other neuron-specific functions, may explain why systemic mutations in mitochondrial fusion and fission proteins specifically cause death of neurons. However, the roles and requirements of these proteins also differ between neuronal types.¹ For example, mutations in the fusion protein optic atrophy 1 cause degeneration of retinal ganglion neurons,⁵ and mutations in the fusion protein mitofusin-2 or the fission protein ganglioside-induced differentiation-associated protein 1 cause peripheral neuropathy (Charcot-Marie-Tooth types 2A and 4A^{6, 7}).There are several potential reasons why specific neurons have unique requirements for fission–fusion proteins. First, the functions of these proteins may be more critical in vulnerable neuronal populations. Recently, we showed that most midbrain DA neurons are uniquely vulnerable to loss of the central mitochondrial fission protein dynamin-related protein 1 (Drp1),⁴ a GTPase recruited to fission sites on the outer mitochondrial membrane.¹ Loss of Drp1 depletes axonal mitochondria, which is followed by axonal degeneration and neuronal death. However, a subpopulation of midbrain DA neurons survive, despite losing their axonal mitochondria, suggesting that they have lower needs for energy or other mitochondrial functions in their axons.⁴ Do unique requirements for mitochondrial dynamics underlie differential neuronal vulnerability? Do resistant neurons compensate with other fission or fusion mechanisms? Do the functions of fission differ between neurons? Notably, Drp1 may also have mitochondria-independent functions in synaptic vesicle release.⁸ Addressing these issues could help elucidate the physiological functions of mitochondrial dynamics in the nervous system and reveal how shifts in the fission–fusion balance contribute to selective neuronal death in neurodegenerative diseases, including Huntington''s disease, Parkinson''s disease and Alzheimer''s disease (AD),^{1, 4} and in other neurologic disorders, including stroke and epilepsy.^{9, 10, 11}To understand mitochondrial dynamics, it would be useful to know why mitochondrial fission is needed in the nervous system in the first place, and how loss of fission affects mitochondrial functions in specific cell types. Notably, Drp1 knockout did not change respiration or ATP levels in resuspended mouse embryonic fibroblasts (MEFs),^{12, 13} indicating that mitochondrial fission is not required for respiration in these cells. However, neuronal respiration may be more sensitive to Drp1 loss. Indeed, Drp1 loss markedly decreased the number of mitochondria in axons and the cell body in midbrain DA neurons in vivo,⁴ and reduced staining of complex I and IV activity in cerebellar neurons in vivo.¹⁴ However, it is unclear whether these changes translate into decreased ATP levels in neurons and, if so, whether this decrease compromises neuronal function. Furthermore, Drp1 loss caused cell death in cerebellar and most midbrain DA neurons,^{4, 14} which challenges our ability to dissociate the specific effects of Drp1 loss on mitochondrial function from other non-specific changes that accompany cell death.To learn how disrupting mitochondrial fission contributes to selective neurodegeneration, we studied the function of Drp1 in CA1 hippocampal neurons and its role in mitochondrial bioenergetics. Surprisingly, despite losing Drp1, most CA1 neurons survived for more than 1 year in vivo, although their function was compromised, leading to deficits in synaptic transmission and memory. To begin to understand how loss of Drp1 causes neuronal dysfunction, we examined the role of Drp1 in mitochondrial bioenergetics. We found that Drp1 is required to maintain normal mitochondrial-derived ATP levels specifically in axons (but not the cell body), and that the loss of this function is unrelated to the distribution of mitochondria within axons.     

Survey and prioritisation of potential biological control agents for prickly acacia (Acacia nilotica subsp. indica) in southern India

Prickly acacia, Acacia nilotica subsp. indica (Benth.) Brenan (Mimosaceae), a multi-purpose tree native to the Indian subcontinent, is a weed of national significance, widespread throughout the grazing areas of western Queensland and has the potential to spread throughout northern Australia. Biological control of prickly acacia has been in progress since the early 1980s, but with limited success to date. Based on genetic and climate matching studies, native surveys for potential biological control agents were conducted in 64 sites in Tamil Nadu state and eight sites in Karnataka state from November 2008 to December 2011. Surveys yielded 33 species of phytophagous insects (16 species of leaf-feeders, eight species of stem feeders, four species with leaf-feeding adults and root-feeding larvae, two stem-borers and bark-feeders and three flower-feeders) and two rust fungi. The number of species recorded at survey sites increased with the number of times the sites were surveyed. Using a scoring system based on field host range, geographic range, seasonal incidence and damage levels, we prioritised a scale insect (Anomalococcus indicus Ramakrishna Ayyar), two leaf-webbing caterpillars (Phycita sp. A and Phycita sp. B), a leaf weevil (Dereodus denticollis Boheman), a leaf beetle (Pachnephorus sp.), a gall-inducing rust (Ravenelia acacia-arabica Mundk. & Thirumalachari) and a leaf rust (Ravenelia evansii Syd. & P.) for detailed host specificity tests. The two rusts were sent to CABI-UK for preliminary host-specificity testing. Three insects (A. indicus, D. denticollis and Phycita sp. A) were imported into a quarantine facility in Brisbane, Australia where host-specificity tests are in progress.     

Effect of S-allylcysteine,a sulphur containing amino acid on iron metabolism in streptozotocin induced diabetic rats

It is suggested that iron may play a role in the pathogenesis of diabetes. Iron is not only chaperoned through its essential functional pathways, but it also causes damage to biological systems by catalyzing the production of reactive oxygen species. So, the parenchymal tissues of several organs are subject to cell injury and functional insufficiency due to excess deposition of iron. The present study investigated the effects of S-allylcysteine (SAC), a sulphur containing amino acid derived from garlic on the changes in iron metabolism induced by oxidative stress in tissues, as well as on serum biochemical parameters of streptozotocin (STZ)-induced diabetic rats. SAC was administered orally for 45 days to control and experimental diabetic rats. The effects of SAC on glucose, insulin, serum iron, ferritin, transferrin, serum bilirubin, heart heme oxygenase activity (HO) and δ-aminolevulinicacid dehydratase activity (δ-ALA-D) in liver and kidneys were studied. The levels of glucose, iron, ferritin, bilirubin and HO in liver were increased significantly (p < 0.05) whereas the levels of insulin, transferrin and δ-ALA-D in tissues were decreased in diabetic rats. Administration of SAC to diabetic rats showed a decrease in blood glucose, iron, ferritin, bilirubin and HO. In addition, the levels of insulin, transferrin and δ-ALA-D activity in tissues were increased in SAC treated diabetic rats. These findings suggest that S-allylcysteine could have a protective effect against alterations in oxidative stress induced iron metabolism in the diabetic state which was evidenced by the capacity of this natural antioxidant to modulate parameters of iron metabolism.     

Mucin (Muc) expression during pancreatic cancer progression in spontaneous mouse model: potential implications for diagnosis and therapy

Pancreatic cancer (PC) is a lethal malignancy primarily driven by activated Kras mutations and characterized by the deregulation of several genes including mucins. Previous studies on mucins have identified their significant role in both benign and malignant human diseases including PC progression and metastasis. However, the initiation of MUC expression during PC remains unknown because of lack of early stage tumor tissues from PC patients. In the present study, we have evaluated stage specific expression patterns of mucins during mouse PC progression in (KrasG12D;Pdx1-Cre (KC)) murine PC model from pancreatic intraepithelial neoplasia (PanIN) to pancreatic ductal adenocarcinoma (PDAC) by immunohistochemistry and quantitative real-time PCR. In agreement with previous studies on human PC, we observed a progressive increase in the expression of mucins particularly Muc1, Muc4 and Muc5AC in the pancreas of KC (as early as PanIN I) mice with advancement of PanIN lesions and PDAC both at mRNA and protein levels. Additionally, mucin expression correlated with the increased expression of inflammatory cytokines IFN-γ (p?相似文献   

Dominant Negative Mutations Affect Oligomerization of Human Pyruvate Kinase M2 Isozyme and Promote Cellular Growth and Polyploidy

This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM₂) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM₂ in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM₂ by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM₂ showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM₂ by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.     

Neutral sphingomyelinase-3 is a DNA damage and nongenotoxic stress-regulated gene that is deregulated in human malignancies

In this study, we report the characterization of a novel genotoxic and nongenotoxic stress-regulated gene that we had previously named as SKNY. Our results indicate that SKNY encodes the recently identified neutral sphingomyelinase-3 (nSMase3; hereafter SKNY is referred to as nSMase3). Examination of nSMase3 subcellular distribution reveals nSMase3 to localize to the endoplasmic reticulum (ER), and deletion of a COOH-terminal region containing its putative transmembrane domain and ER targeting signal partly alters its compartmentalization to the ER. Treatment with genotoxic Adriamycin and nongenotoxic tumor necrosis factor-alpha up-regulates endogenous nSMase3 expression, albeit with different kinetics. Tumor necrosis factor-alpha up-regulates nSMase3 expression within 2 h that lasts beyond 24 h and declines to control levels by 36 h. Adriamycin up-regulation of nSMase3 is transient, occurs within 30 min, and declines to control levels by 120 min. Prolonged treatment with Adriamycin by 24 h and beyond, however, causes a down-regulation in nSMase3 expression. Activation of wild-type p53 also down-regulates nSMase3 expression, suggesting that DNA damage-mediated nSMase3 down-regulation seems to occur partly through the tumor suppressor p53. Overexpression of exogenous nSMase3 sensitizes cells to Adriamycin-induced cell killing, a finding consistent with the proposed proapoptotic role of nSMase enzymes and nSMase-generated ceramide. We further investigated nSMase3 expression in various human malignancies and found its expression to be deregulated in several types of primary tumors when compared with their matching normal tissues. Collectively, our results have identified nSMase3 to be an important molecule that is linked to tumorigenesis and cellular stress response.     

Synthesis and in vitro cholesterol dissolution by 23- and 24-phosphonobile acids

A new class of 23- and 24-phosphonobile acids have been synthesized from bile acid and their in vitro cholesterol-dissolving efficiency have been estimated. 24-Phosphonobile salts (PBSs) are slightly more efficient in solubilizing cholesterol than 23-PBSs and natural bile salts. The cholesterol solubilizing power is influenced by the structure of PBSs, and is considerably reduced with an increase in the bulk pH.     

dbGAPs: A comprehensive database of genes and genetic markers associated with psoriasis and its subtypes

Psoriasis is a systemic hyperproliferative inflammatory skin disorder, although rarely fatal but significantly reduces quality of life. Understanding the full genetic component of the disease association may provide insight into biological pathways as well as targets and biomarkers for diagnosis, prognosis and therapy. Studies related to psoriasis associated genes and genetic markers are scattered and not easily amendable to data-mining. To alleviate difficulties, we have developed dbGAPs an integrated knowledgebase representing a gateway to psoriasis associated genomic data. The database contains annotation for 202 manually curated genes associated with psoriasis and its subtypes with cross-references. Functional enrichment of these genes, in context of Gene Ontology and pathways, provide insight into their important role in psoriasis etiology and pathogenesis. The dbGAPs interface is enriched with an interactive search engine for data retrieval along with unique customized tools for Single Nucleotide Polymorphism (SNP)/indel detection and SNP/indel annotations. dbGAPs is accessible at http://www.bmicnip.in/dbgaps/.     

Expression levels of genes involved in metal homeostasis,physiological adaptation,and growth characteristics of rice (Oryza sativa L.) genotypes under Fe and/or Al toxicity

Acid sulphate soil contains high amounts of iron (Fe) and aluminum (Al), and their contamination has been reported as major problems, especially in rainfed and irrigated lowland paddy fields. Rice is sensitive to Fe and Al grown in acid soil (pH < 5.5), leading to growth inhibition and grain yield loss. The objective of this study was to evaluate Fe and/or Al uptake, translocation, physiological adaptation, metal toxicity, and growth inhibition in rice genotypes grown in acid soil. Fe and Al in the root tissues of all rice genotypes were enriched depending on the exogenous application of either Fe or Al in the soil solution, leading to root growth inhibition, especially in the KDML105 genotype. Expression level of OsYSL1 in KDML105 was increased in relation to metal uptake into root tissues, whereas OsVIT2 was downregulated, leading to Fe (50.3 mg g⁻¹ DW or 13.1 folds over the control) and Al (4.8 mg g⁻¹ DW or 2.2 folds over the control) translocation to leaf tissues. Consequently, leaf greenness (SPAD), net photosynthetic rate (P_n), stomatal conductance (g_s), and transpiration rate (E) in the leaf tissues of genotype KDML105 under Fe + Al toxicity significantly declined by 28.4%, 35.3%, 55.6%, and 51.6% over the control, respectively. In Azucena (AZU; Fe/Al tolerant), there was a rapid uptake of Fe and Al by OsYSL1 expression in the root tissues, but a limited secretion into vacuole organelles by OsVIT2, leading to a maintenance of low level of toxicity driven by an enhanced accumulation of glutathione together with downregulation of OsGR expression level. In addition, Fe and Al restrictions in the root tissues of genotype RD35 were evident; therefore, crop stress index (CSI) of Fe + Al–treated plants was the maximum, leading to an inhibition of g_s (53.6% over the control) and E (49.0% over the control). Consequently, free proline, total phenolic compounds, and ascorbic acid in the leaf tissues of rice under Fe + Al toxicity significantly increased by 3.2, 1.2, and 1.5 folds over the control, respectively, indicating their functions in non-enzymatic antioxidant defense. Moreover, physiological parameters including leaf temperature (T_leaf) increment, high level of CSI (>0.6), SPAD reduction, photon yield of PSII (Φ_PSII) diminution, P_n, g_s, and E inhibition in rice genotype IR64 (Fe/Al-sensitive) under Fe + Al treatment were clearly demonstrated as good indicators of metal-induced toxicity. Our results on Fe- and/or Al-tolerant screening to find out the candidate genotypes will contribute to present screening and breeding efforts, which in turn help increase rice production in the Fe/Al-contaminated acid soil under lowland conditions.