Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is apparently formed by a single glycoprotein, while the basal layer contains many different structural proteins and several enzymes. One of the enzymes is Alr, an alanine racemase capable of converting the spore germinant l-alanine to the germination inhibitor d-alanine. Unlike other characterized exosporium proteins, Alr is nonuniformly distributed in the exosporium and might have a second spore location. In this study, we demonstrated that expression of the alr gene, which encodes Alr, is restricted to sporulating cells and that the bulk of alr transcription and Alr synthesis occurs during the late stages of sporulation. We also mapped two alr promoters that are differentially active during sporulation and might be involved in the atypical localization of Alr. Finally, we constructed a Deltaalr mutant of B. anthracis that lacks Alr and examined the properties of the spores produced by this strain. Mature Deltaalr spores germinate more efficiently in the presence of l-alanine, presumably because of their inability to convert exogenous l-alanine to d-alanine, but they respond normally to other germinants. Surprisingly, the production of mature spores by the Deltaalr mutant is defective because approximately one-half of the nascent spores germinate and lose their resistance properties before they are released from the mother cell. This phenotype suggests that an important function of Alr is to produce D-alanine during the late stages of sporulation to suppress premature germination of the developing spore.
ABSTRACT Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-β- l -rhamnose α-1,3- l -rhamnosyl transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD -encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit β-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N -acyltransferase activity.
ABSTRACT The rise in quinolone resistance is threatening the clinical use of this important class of broad-spectrum antibacterials. Quinolones kill bacteria by increasing the level of DNA strand breaks generated by the type II topoisomerases gyrase and topoisomerase IV. Most commonly, resistance is caused by mutations in the serine and acidic amino acid residues that anchor a water-metal ion bridge that facilitates quinolone-enzyme interactions. Although other mutations in gyrase and topoisomerase IV have been reported in quinolone-resistant strains, little is known regarding their contributions to cellular quinolone resistance. To address this issue, we characterized the effects of the V96A mutation in the A subunit of Bacillus anthracis topoisomerase IV on quinolone activity. The results indicate that this mutation causes an ∼3-fold decrease in quinolone potency and reduces the stability of covalent topoisomerase IV-cleaved DNA complexes. However, based on metal ion usage, the V96A mutation does not disrupt the function of the water-metal ion bridge. A similar level of resistance to quinazolinediones (which do not use the bridge) was seen. V96A is the first topoisomerase IV mutation distal to the water-metal ion bridge demonstrated to decrease quinolone activity. It also represents the first A subunit mutation reported to cause resistance to quinazolinediones. This cross-resistance suggests that the V96A change has a global effect on the structure of the drug-binding pocket of topoisomerase IV.
The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer. Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization. Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization. The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion. A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed.
Gyrase appears to be the primary cellular target for quinolone antibacterials in multiple pathogenic bacteria, including Bacillus anthracis, the causative agent of anthrax. Given the significance of this type II topoisomerase as a drug target, it is critical to understand how quinolones interact with gyrase and how specific mutations lead to resistance. However, these important issues have yet to be addressed for a canonical gyrase. Therefore, we utilized a mechanistic approach to characterize interactions of quinolones with wild-type B. anthracis gyrase and enzymes containing the most common quinolone resistance mutations. Results indicate that clinically relevant quinolones interact with the enzyme through a water–metal ion bridge in which a noncatalytic divalent metal ion is chelated by the C3/C4 keto acid of the drug. In contrast to other bacterial type II topoisomerases that have been examined, the bridge is anchored to gyrase primarily through a single residue (Ser85). Substitution of groups at the quinolone C7 and C8 positions generated drugs that were less dependent on the water–metal ion bridge and overcame resistance. Thus, by analyzing the interactions of drugs with type II topoisomerases from individual bacteria, it may be possible to identify specific quinolone derivatives that can overcome target-mediated resistance in important pathogenic species.
Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively nontoxic prodrugs into membrane-permeant cytotoxic compounds with high bystander activity. In the present study, we examined tumor regressions resulting from treatment with E. coli PNP and fludarabine phosphate (F-araAMP), a clinically approved compound used in the treatment of hematologic malignancies. We tested bystander killing with an adenoviral construct expressing E. coli PNP and then more formally examined thresholds for the bystander effect, using both MuLv and lentiviral vectoring. Because of the importance of understanding the mechanism of bystander action and the limits to this anticancer strategy, we also evaluated in vivo variables related to the expression of E. coli PNP (level of E. coli PNP activity in tumors, ectopic expression in liver, percentage of tumor cells transduced in situ, and accumulation of active metabolites in tumors). Our results indicate that F-araAMP confers excellent in vivo dose-dependent inhibition of bystander tumor cells, including strong responses in subcutaneous human glioma xenografts when 95 to 97.5% of the tumor mass is composed of bystander cells. These findings define levels of E. coli PNP expression necessary for antitumor activity with F-araAMP and demonstrate new potential for a clinically approved compound in solid tumor therapy.
The pathogenesis of the human immunodeficiency virus (HIV)-associated cognitive/motor complex, or acquired immunodeficiency syndrome (AIDS) dementia complex, is unknown, but it afflicts over 50% of all patients infected with HIV-1. Because neurons are not directly infected with HIV-1, the causes of neuronal dysfunction are undoubtedly indirect. We investigated the role of the astrocyte in the development of AIDS dementia complex, focusing on cytokine and HIV-1 gp120 stimulation of Na+/H+ exchange (NHE) activity of primary rat astrocytes. Our results show that the cytokines tumor necrosis factor-alpha, interferon (IFN)-gamma, and interleukin (IL)-1 beta (all found to be elevated in the central nervous system of AIDS patients), can stimulate Na+/H+ exchange, but that transforming growth factor-beta, IL-2, and IL-6 do not. IFN-gamma and gp120-induced activation of Na+/H+ exchange appears to be mediated through activation of tyrosine kinase (TK), because TK inhibitors block the action of IFN-gamma and gp120. Additionally, gp120 induces tyrosine phosphorylation of two proteins (approximately 90 and 130 kDa), which is also inhibited by TK inhibitors. The predominant NHE isoform present in rat astrocytes is NHE-1; however, other isoforms are also present. We conclude that Na+/H+ exchange of rat astrocytes can be differentially stimulated by cytokines and HIV-1 gp120. We hypothesize that the resultant increase in intracellular pH with its concomitant changes in astrocyte membrane permeability properties produces an imbalance in the K+ and glutamate microenvironment of the neurons, leading to a rise in intraneuronal Ca2+ and eventual neuronal dysfunction and/or demise.
The development of conditionally replicative adenoviruses (CRAds) has presented a new platform for cancer gene therapy. The relative contribution of CRAd-related toxicity, and the cytotoxic immune response, especially the antigen specific cytotoxic T lymphocyte (CTL) response, in tumor cell killing is unclear. The anti-CRAd CTL may destroy tumor cells by specific killing; on the other hand, the spread of viral progeny may be limited by the clearance of infected cells. A better understanding of the effector mechanism of the CTL response would have implications in design of adenovirus-derived vectors, in particular, the potent CRAds. Previous studies indicated that FasL and TNF-α but not perforin, play a central role in immune-mediated adenoviral vector clearance, whereas CD28 is thought to be a critical molecule for the generation of effector cells. However, it has been difficult to clearly distinguish effector cell generation from its function due to the inability to specifically recognize Ad-specific CTLs. To investigate the effector mechanisms directly, we employed two novel approaches: tetramer staining and an antigen specific in vivo CTL assay. The Db-E1Bp MHC-I tetramer, that recognizes the immunodominant epitope of Ad E1B (192 VNIRNCCYI) is the first Ad-mouse tetramer. B6 mice, CD28KO mice and FasL deficient gld mice which are all on the H-2b background, as well as D2 mice (H-2d), were immunized with 4×108 i.u. of wild type Ad5. Eight days later, target cells from syngeneic mice were labeled with high concentration of CFSE (2μM; CFSEHi) and pulsed with E1Bp, or labeled with low concentration of CFSE (0.2μM; CFSELo), but unpulsed. Immunized mice were then adoptively transferred with identical numbers of CFSEHi and CFSELo cells. After 6 hours, lymphocytes were isolated from the spleen and liver and stained with fluorescent anti-CD8 and anti-CD44 antibodies and the Db-E1Bp tetramer. Flow cytometry results showed that in B6 mice, approximately 33% of the CD8+ T cells in liver and 11% in spleen are Db-E1Bp positive. The E1Bp-specific killing in the spleen of B6 mice was 93% whereas no Db-E1Bp+ CD8+ T cells or E1Bp-specific killing were observed in D2 mice. E1Bp-specific lysis was significantly correlated with the frequency of Db-E1Bp+CD8+ T cells in B6 mice (p<0.01). As expected, CD28 deficient mice exhibited an 80% reduction in generation of Db-E1Bp+CD8+ efffector T cells and E1Bp specific killing efficiency was reduced by 65% compared to B6 mice. Surprisingly, the E1Bp-specific killing efficiency in gld mice was only minimally reduced by 25% compared to B6 mice. There was also a 25% decrease in generation of Db-E1Bp+CD8+ T cells in gld mice. However, the specific cytotoxicity of individual CTL in both CD28KO and gld mice was compromised in that the percentage lysis per cell was lower than in B6. Our results indicate 1) CD28 plays an important role in generation of the specific CTL response to Ad; 2) FasL-dependent pathway plays a minor role in CTL response and also affects generation of CTLs; and 3) tetramer analysis of Ad-specific CTL and specific in vivo CTL assay can be used to distinguish generation and effector functions of CTLs.
Journal Article Structure and Assembly of the Bacillus anthracis Exosporium Get access Cynthia M Rodenburg, Cynthia M Rodenburg Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA Search for other works by this author on: Oxford Academic Google Scholar Sylvia A McPherson, Sylvia A McPherson Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA Search for other works by this author on: Oxford Academic Google Scholar Charles L Turnbough, Jr., Charles L Turnbough, Jr. Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA Search for other works by this author on: Oxford Academic Google Scholar Terje Dokland Terje Dokland Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 21, Issue S3, 1 August 2015, Pages 897–898, https://doi.org/10.1017/S1431927615005280 Published: 23 September 2015
