Characterization of temperature inducible promoters from a novel rolling circle replicating plasmid of Enterococcus faecium DJ1

Limited studies have been performed on the characterization of small size plasmids of Enterococcus faecium with the intention of evaluating the strength of their promoters in Escherichia coli. The complete nucleotide sequence (3.825 Kb) and structural organization of E. faecium DJ1 cryptic plasmid pNJAKD is presented. Seven promoter sequences from the pNJAKD plasmid of E. faecium have been identified. The regions coding for the putative promoters were either amplified using PCR based techniques or chemically synthesized as oligonucleotides of different sizes. These were subsequently cloned in the pEGFP vector at the Pvu II site. The efficiency of putative promoter fragments were measured using the intensity of eGFP fluorescence in E. coli JM101, DH5α and BL21(DE3), among which AKD3 exhibited moderate to strongest promoter activity at temperatures of 30, 37, and 42°C.     

Retrotranslocation of prion proteins from the endoplasmic reticulum by preventing GPI signal transamidation

Neurodegeneration in diseases caused by altered metabolism of mammalian prion protein (PrP) can be averted by reducing PrP expression. To identify novel pathways for PrP down-regulation, we analyzed cells that had adapted to the negative selection pressure of stable overexpression of a disease-causing PrP mutant. A mutant cell line was isolated that selectively and quantitatively routes wild-type and various mutant PrPs for ER retrotranslocation and proteasomal degradation. Biochemical analyses of the mutant cells revealed that a defect in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an unprocessed GPI-anchoring signal sequence that directs both ER retention and efficient retrotranslocation of PrP. An unprocessed GPI signal was sufficient to impart ER retention, but not retrotranslocation, to a heterologous protein, revealing an unexpected role for the mature domain in the metabolism of misprocessed GPI-anchored proteins. Our results provide new insights into the quality control pathways for unprocessed GPI-anchored proteins and identify transamidation of the GPI signal sequence as a step in PrP biosynthesis that is absolutely required for its surface expression. As each GPI signal sequence is unique, these results also identify signal recognition by the GPI-transamidase as a potential step for selective small molecule perturbation of PrP expression.     

The Ubiquitin Proteasome System Plays a Role in Venezuelan Equine Encephalitis Virus Infection

Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.     

Bacillus anthracis-derived nitric oxide induces protein S-nitrosylation contributing to macrophage death

Bacillus anthracis, a causative agent of anthrax, is able to germinate and survive within macrophages. A recent study suggested that B. anthracis-derived nitric oxide (bNO) is a key aspect of bacterial defense that protects bacterial DNA from oxidative burst in the macrophages. However, the virulent effect of bNO in host cells has not been investigated. Here, we report that bNO contributes macrophage killing by S-nitrosylation of bioenergetic-relating proteins within mitochondria. Toxigenic Sterne induces expression of the bnos gene and produces bNO during early stage of infection. Nitroso-proteomic analysis coupled with a biotin-switch technique demonstrated that toxigenic infection induces protein S-nitrosylation in B. anthracis-susceptible RAW264.7. For each target enzyme tested (complex I, complex III and complex IV), infection by B. anthracis Sterne caused enzyme inhibition. Nω-nitro-l-arginine methyl ester, a NO synthase inhibitor, reduced S-nitrosylation and partially restored cell viability evaluated by intracellular ATP levels in macrophages. Our data suggest that bNO leads to energy depletion driven by impaired mitochondrial bioenergetic machinery that ultimately contributes to macrophage death. This novel mechanism of anthrax pathogenesis may offer specific approach to the development of therapeutics.