The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions. Viruses belonging to several different families utilize or modulate the system for their advantage. Here we showed that the proteasome inhibitors MG132 and epoxomicin blocked a postentry step in vaccinia virus (VACV) replication. When proteasome inhibitors were added after virus attachment, early gene expression was prolonged and the expression of intermediate and late genes was almost undetectable. By varying the time of the removal and addition of MG132, the adverse effect of the proteasome inhibitors was narrowly focused on events occurring 2 to 4 h after infection, the time of the onset of viral DNA synthesis. Further analyses confirmed that genome replication was inhibited by both MG132 and epoxomicin, which would account for the effect on intermediate and late gene expression. The virus-induced replication of a transfected plasmid was also inhibited, indicating that the block was not at the step of viral DNA uncoating. UBEI-41, an inhibitor of the ubiquitin-activating enzyme E1, also prevented late gene expression, supporting the role of the ubiquitin-proteasome system in VACV replication. Neither the overexpression of ubiquitin nor the addition of an autophagy inhibitor was able to counter the inhibitory effects of MG132. Further studies of the role of the ubiquitin-proteasome system for VACV replication may provide new insights into virus-host interactions and suggest potential antipoxviral drugs.The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions (22). Proteins to be degraded are modified by the addition of multiple copies of the 76-amino-acid ubiquitin through the sequential activities of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3) (4, 12). The degradation is mediated by the 26S proteasome, a large multiprotein complex containing trypsin-, chymotrypsin-, and postglutamyl peptidyl hydrolytic-like protease activities. In addition, ubiquitylation has nondegradative roles in DNA repair, transcriptional regulation, signal transduction, endocytosis, and intracellular trafficking (48). Viruses belonging to several families utilize or modulate the ubiquitin-proteasome system (2, 13). The inhibition of proteasomal degradation prevents the entry of influenza virus (23) and mouse hepatitis virus (54); the early postentry steps of minute virus of mice (44) and herpes simplex virus (7); and the genome replication or expression of human coxsackie 3B virus (27), adenovirus (5), cytomegalovirus (20), infectious bursal disease virus (26), and vesicular stomatitis virus (40). In some cases the effects may be secondary to the activation of a cellular stress response and signaling pathway (24, 40, 52). Proteasomal inhibitors have an indirect effect on retroviruses and rhabdoviruses by depleting free ubiquitin needed to modify proteins for budding (16).Vaccinia virus (VACV), the repre...
We sought to visualize the site of Bacillus anthracis spore germination in vivo. For that purpose, we constructed a reporter plasmid with the lux operon under control of the spore small acid-soluble protein B (sspB) promoter. In B. subtilis, sspB-driven synthesis of luciferase during sporulation results in incorporation of the enzyme in spores. We observed that B. anthracis Sterne transformed with our sspBp::lux plasmid was only luminescent during germination. In contrast, Sterne transformed with a similarly constructed plasmid with lux expression under control of the protective antigen promoter displayed luminescence only during vegetative growth. We then infected A/J mice intranasally with spores that harbored the germination reporter. Mice were monitored for up to 14 days with the Xenogen In Vivo Imaging System. While luminescence only became evident in live animals at 18 h, dissection after sacrificing infected mice at earlier time points revealed luminescence in lung tissue at 30 min after intranasal infection. Microscopic histochemical and immunofluorescence studies on luminescent lung sections and imprints revealed that macrophages were the first cells in contact with the B. anthracis spores. By 6 h after infection, polymorphonuclear leukocytes with intracellular spores were evident in the alveolar spaces. After 24 h, few free spores were observed in the alveolar spaces; most of the spores detected by immunofluorescence were in the cytoplasm of interstitial macrophages. In contrast, mediastinal lymph nodes remained nonluminescent throughout the infection. We conclude that in this animal system, the primary site of B. anthracis spore germination is the lungs.
Bacillus collagen-like protein of anthracis (BclA) is the immunodominant glycoprotein on the exosporium of Bacillus anthracis spores. Here, we sought to assess the impact of BclA on spore germination in vitro and in vivo, surface charge, and interaction with host matrix proteins. For that purpose, we constructed a markerless bclA null mutant in B. anthracis Sterne strain 34F2. The growth and sporulation rates of the ⌬bclA and parent strains were nearly indistinguishable, but germination of mutant spores occurred more rapidly than that of wild-type spores in vitro and was more complete by 60 min. Additionally, the mean time to death of A/J mice inoculated subcutaneously or intranasally with mutant spores was lower than that for the wild-type spores even though the 50% lethal doses of the two strains were similar. We speculated that these in vitro and in vivo differences between mutant and wild-type spores might reflect the ease of access of germinants to their receptors in the absence of BclA. We also compared the hydrophobic and adhesive properties of ⌬bclA and wild-type spores. The ⌬bclA spores were markedly less water repellent than wild-type spores, and, probably as a consequence, the extracellular matrix proteins laminin and fibronectin bound significantly better to mutant than to wild-type spores. These studies suggest that BclA acts as a shield to not only reduce the ease with which spores germinate but also change the surface properties of the spore, which, in turn, may impede the interaction of the spore with host matrix substances.Bacillus anthracis is a gram-positive, spore-forming bacillus that can cause anthrax (15). The spore is the form of the organism found in its natural habitat, the soil, and is also the infectious form for herbivores, the typical vertebrate host for the bacterium, and humans (15). The B. anthracis spore is covered by a loose balloon-like membranous structure called the exosporium (8). BclA (for bacillus collagen-like protein of anthracis) was first described by Sylvestre et al. (23), who constructed an insertional bclA mutant and compared it to its wild-type parent. These investigators and, subsequently, others found that BclA is a glycoprotein and a major component of the hair-like projections that cover the exosporium (16,22,23,25). BclA is also an immunodominant marker on the outside of the spore (22). The finding that BclA does not play a significant role in the virulence of a Sterne-like strain for mice was first reported by Sylvestre et al. (23). Sterne strains contain pXO1 but not pX02 and are attenuated in humans and many other animals except certain mouse strains (26). In support of the findings of Sylvestre and colleagues, Bozue and coworkers recently constructed a bclA mutant of the fully virulent B. anthracis Ames strain and showed that the absence of BclA had no impact on the lethality of that strain for guinea pigs or mice (5). Whether BclA, the substance on the spore with which the host cells probably first interact, plays a more subtle role in B. anthracis patho...
Based on previous studies showing that host chemokines exert antimicrobial activities against bacteria, we sought to determine whether the interferon-inducible Glu-Leu-Arg-negative CXC chemokines CXCL9, CXCL10, and CXCL11 exhibit antimicrobial activities against Bacillus anthracis. In vitro analysis demonstrated that all three CXC chemokines exerted direct antimicrobial effects against B. anthracis spores and bacilli including marked reductions in spore and bacillus viability as determined using a fluorometric assay of bacterial viability and CFU determinations. Electron microscopy studies revealed that CXCL10-treated spores failed to undergo germination as judged by an absence of cytological changes in spore structure that occur during the process of germination. Immunogold labeling of CXCL10-treated spores demonstrated that the chemokine was located internal to the exosporium in association primarily with the spore coat and its interface with the cortex. To begin examining the potential biological relevance of chemokine-mediated antimicrobial activity, we used a murine model of inhalational anthrax. Upon spore challenge, the lungs of C57BL/6 mice (resistant to inhalational B. anthracis infection) had significantly higher levels of CXCL9, CXCL10, and CXCL11 than did the lungs of A/J mice (highly susceptible to infection). Increased CXC chemokine levels were associated with significantly reduced levels of spore germination within the lungs as determined by in vivo imaging. Taken together, our data demonstrate a novel antimicrobial role for host chemokines against B. anthracis that provides unique insight into host defense against inhalational anthrax; these data also support the notion for an innovative approach in treating B. anthracis infection as well as infections caused by other spore-forming organisms.Bacillus anthracis is a gram-positive, spore-forming bacterium that causes the disease anthrax. The infectious B. anthracis spore is a dormant, metabolically inactive form of the organism made up of distinct, concentric layers that collectively provide a highly structured casing capable of protecting the spore core from high temperature, UV irradiation, lytic digestion, and numerous reactive agents (31, 59). Spore germination is initiated through receptor-mediated interactions between soluble germinant molecules (typically nutrients such as single amino acids, sugars, or purine nucleosides) and germinant receptors located at the inner membrane of the dormant spore (20,36). Although the molecular mechanism(s) linking germinant binding to the loss of dormancy is undefined, germinant receptor engagement initiates a cascade of processes, including dipicolinic acid (DPA) release, that promote core rehydration and result in the controlled degradation of the protective spore structures; as germination concludes, metabolic activity resumes, and vegetative outgrowth is initiated (58). Fully virulent B. anthracis bacilli generate several virulence factors including an antiphagocytic, poly-D-glutamic acid capsule encode...
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