Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species

Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O₂⁻). In the present study, mutants of F. tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion F. tularensis mutant of sodC (ΔsodC) and a F. tularensis ΔsodC mutant with attenuated Fe-superoxide dismutase (sodB) gene expression (sodB ΔsodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-γ)-activated macrophages and a mouse model of respiratory tularemia. The F. tularensis ΔsodC and sodB ΔsodC mutants showed attenuated intramacrophage survival in IFN-γ-activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the sodC gene in the ΔsodC mutant or inhibiting the IFN-γ-dependent production of O₂⁻ or nitric oxide (NO) enhanced intramacrophage survival of the sod mutants. The ΔsodC and sodB ΔsodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type F. tularensis LVS. As observed for macrophages, the virulence of the ΔsodC mutant was restored in ifn-γ^−/−, inos^−/−, and phox^−/− mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of F. tularensis in mice.Francisella tularensis is considered a potential biological threat due to its extreme infectivity, ease of artificial dissemination via aerosols, and substantial capacity to cause illness and death. A hallmark of all F. tularensis subspecies is their ability to survive and replicate within macrophages (18) and other cell types (6, 11, 25, 28). While recent work has furthered our understanding of F. tularensis virulence mechanisms, little is known with respect to its ability to resist the microbicidal production of reactive oxygen species (ROS) or reactive nitrogen species (RNS).Superoxide dismutases (SODs) are metalloproteins that are classified according to their coordinating active site metals. SODs catalyze the dismutation of the highly reactive superoxide (O₂⁻) anion to hydrogen peroxide (H₂O₂) and O₂ (26). The dismutation of O₂⁻ prevents accumulation of microbicidal ROS and RNS in infected macrophages. Three major categories of SODs have been identified in bacteria and include Mn-, Fe-, and CuZn-containing SODs (SodA, SodB, and SodC, respectively) and are required for aerobic survival (27). The F. tularensis genome encodes SodB (FTL_1791) and SodC (FTL_0380). In several intracellular bacterial pathogens, SodC is an important virulence factor, and its localization to the periplasmic space protects bacteria from host-derived O₂⁻ and NO radicals (8, 9, 21, 32). Moreover, many virulent bacteria possess two copies of the sodC gene (4). The evolutionary maintenance of an extra sodC gene copy suggests that it serves some essential function in survival (4). As an intracellular pathogen, F. tularensis is exposed to ROS and RNS generated by inflammatory cells during the macrophage activation process, which suggests that SODs may play an important role in its intracellular survival and pathogenesis. We have demonstrated that decreases in SodB activity render F. tularensis sensitive to ROS and attenuate virulence in mice (2). However, the contribution of F. tularensis SodC in virulence and intramacrophage survival has not been defined. In this study we have constructed a F. tularensis sodC mutant (ΔsodC) and a F. tularensis sodBC double mutant (sodB ΔsodC) and determined that SodC in conjunction with SodB primarily protects the pathogen from host-derived ROS and is required for intramacrophage survival and virulence of F. tularensis in mice.     

Subdiffraction-limit study of Kaede diffusion and spatial distribution in live Escherichia coli

Photoactivation localization microscopy (PALM) is used to study the spatial distribution and diffusion of single copies of the protein Kaede in the cytoplasm of live Escherichia coli under moderate growth conditions (67 min doubling time). The spatial distribution of Kaede is uniform within the cytoplasm. The cytoplasmic radius of 380 ± 30 nm varies little from cell to cell. Single-particle tracking using 4 ms exposure times reveals negatively curved plots of mean-square displacement versus time. A detailed comparison with Monte Carlo simulations in a spherocylindrical volume shows that the curvature can be quantitatively understood in terms of free diffusion within a confining volume. The mean diffusion coefficient across cells is <D_Kaede> = 7.3 ± 1.1 μm²·s⁻¹, consistent with a homotetrameric form of Kaede. The distribution of squared displacements along the long axis for individual Kaede molecules is consistent with homogeneous diffusion. However, for longer cells, a spatial map of one-step estimates of the diffusion coefficient along x suggests that diffusion is ∼20–40% faster within nucleoids than in the ribosome-rich region lying between nucleoid lobes at the cell mid-plane. Fluorescence recovery after photobleaching yielded <D_FRAP> = 8.3 ± 1.6 μm²·s⁻¹, in agreement with the single-particle tracking results.     

Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics.

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.     

Diffusible Fraction of Serum Cholesterol and Atherogenesis

CHOLESTEROL is found in the blood as a structural component of lipoproteins concerned with the transport of other lipids¹. The high resolution nuclear magnetic resonance spectra of high density serum lipoproteins are similar to that observed when lipids are dissolved in organic solvents, or dispersed in water by bile salts or detergents, or in sonicated form. The lipid component in lipoproteins is therefore probably in an extremely fluid condition². If human serum is mixed with paraffin oil, some of the cholesterol diffuses into the oil without affecting the ultraviolet absorption spectrum of serum proteins. This procedure avoids any protein denaturing action used for cholesterol extraction^3–5. It therefore seems that serum cholesterol has two fractions, one strongly bound by lipoprotein structures and the other loosely bound and diffusible in an oil phase. In this article I designate the loosely bound fraction “diffusible”.     

Thermostable pullulanase from a mesophilic Bacillus cereus isolate and its mutant UV7.4

Summary A mesophilicBacillus cereus strain was isolated from soil. Since it produced fairly good amounts of extracellular pullulanase, mutations with ultraviolet light and nitrosoguanidine were done to improve the productivity. One of the second generation mutants, UV7.4, produced larger amounts of pullulanase at 37°C than most (mesophilic or thermophilic) organisms reported. The pullulanase was highly active and stable for up to one hour at 70°C. If developed further, UV7.4 can be used commercially for the saccharification of starch.     

Regulation of Vascular Tone,Angiogenesis and Cellular Bioenergetics by the 3-Mercaptopyruvate Sulfurtransferase/H2S Pathway: Functional Impairment by Hyperglycemia and Restoration by dl-α-Lipoic Acid

Hydrogen sulfide (H₂S), as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important biological source of H₂S. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have an adverse effect on cellular homeostasis. In the current study, given the importance of H₂S as a vasorelaxant, angiogenesis stimulator and cellular bioenergetic mediator, we first determined whether the 3-MST/H₂S system plays a physiological regulatory role in endothelial cells. Next, we tested whether a dysfunction of this pathway develops during the development of hyperglycemia and μmol/L to diabetes-associated vascular complications. Intraperitoneal (IP) 3-MP (1 mg/kg) raised plasma H₂S levels in rats. 3-MP (10 1 mmol/L) promoted angiogenesis in vitro in bEnd3 microvascular endothelial cells and in vivo in a Matrigel assay in mice (0.3–1 mg/kg). In vitro studies with bEnd3 cell homogenates demonstrated that the 3-MP-induced increases in H₂S production depended on enzymatic activity, although at higher concentrations (1–3 mmol/L) there was also evidence for an additional nonenzymatic H₂S production by 3-MP. In vivo, 3-MP facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased mitochondrial bioenergetic function. In vitro hyperglycemia or in vivo streptozotocin diabetes impaired angiogenesis, attenuated mitochondrial function and delayed wound healing; all of these responses were associated with an impairment of the proangiogenic and bioenergetic effects of 3-MP. The antioxidants dl-α-lipoic acid (LA) in vivo, or dihydrolipoic acid (DHLA) in vitro restored the ability of 3-MP to stimulate angiogenesis, cellular bioenergetics and wound healing in hyperglycemia and diabetes. We conclude that diabetes leads to an impairment of the 3-MST/H₂S pathway, and speculate that this may contribute to the pathogenesis of hyperglycemic endothelial cell dysfunction. We also suggest that therapy with H₂S donors, or treatment with the combination of 3-MP and lipoic acid may be beneficial in improving angiogenesis and bioenergetics in hyperglycemia.     

Trisomy 8 in leukemia: A GCRI experience

Trisomy of chromosome 8 is frequently reported in myeloid lineage disorders and also detected in lymphoid neoplasms as well as solid tumors suggesting its role in neoplastic progression in general. It is likely to be a disease-modulating secondary event with underlying cryptic aberrations as it has been frequently reported in addition to known abnormalities contributing to clinical heterogeneity and modifying prognosis. Here, we share our findings of trisomy 8 in leukemia patients referred for diagnostic and prognostic cytogenetic assessment. Total 60 cases of trisomy 8, as a sole anomaly or in addition to other chromosomal aberrations, were reported (January 2005-September 2008). Unstimulated bone marrow or blood samples were cultured, followed by GTG banding and karyotyping as per the ISCN 2005. Patients with +8 were chronic myeloid leukemia (CML) (36), acute myeloid leukemia (AML) (17), and acute lymphoblastic leukemia (ALL) (7). In 7 patients, trisomy 8 was the sole anomaly, whereas in 6 patients +8 was in addition to normal clone, in 47 patients, the +8 was in addition to t(9;22), t(15;17), and others, including 3 with tetrasomy 8. Only one patient showed constitutional +8. The present study will form the basis of further cumulative studies to correlate potential differential effects of various karyotypic anomalies on disease progression and survival following a therapeutic regime. To unravel the role of extra 8 chromosome, constitutional chromosomal analysis and uniparental disomy will be considered.     

T-bet deficiency facilitates airway colonization by Mycoplasma pulmonis in a murine model of asthma

Epidemiological and clinical evidence suggest a correlation between asthma and infection with atypical bacterial respiratory pathogens. However, the cellular and molecular underpinnings of this correlation remain unclear. Using the T-bet-deficient (T-bet(-/-)) murine model of asthma and the natural murine pathogen Mycoplasma pulmonis, we provide a mechanistic explanation for this correlation. In this study, we demonstrate the capacity of asthmatic airways to facilitate colonization by M. pulmonis and the capacity of M. pulmonis to exacerbate symptoms associated with acute and chronic asthma. This mutual synergism results from an inability of T-bet(-/-) mice to mount an effective immune defense against respiratory infection through release of IFN-gamma and the ability of M. pulmonis to trigger the production of Th2-type cytokines (e.g., IL-4 and IL-5), and Abs (e.g., IgG1, IgE, and IgA), eosinophilia, airway remodeling, and hyperresponsiveness; all pathophysiological hallmarks of asthma. The capacity of respiratory pathogens such as Mycoplasma spp. to dramatically augment the pathological changes associated with asthma likely explains their association with acute asthmatic episodes in juvenile patients and with adult chronic asthmatics, >50% of whom are found to be PCR positive for M. pneumoniae. In conclusion, our study demonstrates that in mice genetically predisposed to asthma, M. pulmonis infection elicits an inflammatory milieu in the lungs that skews the immune response toward the Th2-type, thus exacerbating the pathophysiological changes associated with asthma. For its part, airways exhibiting an asthmatic phenotype provide a fertile environment that promotes colonization by Mycoplasma spp. and one which is ill-equipped to kill and clear respiratory pathogens.     

Superoxide dismutase B gene (sodB)-deficient mutants of Francisella tularensis demonstrate hypersensitivity to oxidative stress and attenuated virulence

A Francisella tularensis live vaccine strain mutant (sodB(Ft)) with reduced Fe-superoxide dismutase gene expression was generated and found to exhibit decreased sodB activity and increased sensitivity to redox cycling compounds compared to wild-type bacteria. The sodB(Ft) mutant also was significantly attenuated for virulence in mice. Thus, this study has identified sodB as an important F. tularensis virulence factor.