clpC operon regulates cell architecture and sporulation in Bacillus anthracis

The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.     

Engineering Anthrax Toxin Variants That Exclusively Form Octamers and Their Application to Targeting Tumors

Anthrax toxin protective antigen (PA) delivers its effector proteins into the host cell cytosol through formation of an oligomeric pore, which can assume heptameric or octameric states. By screening a highly directed library of PA mutants, we identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application.     

Capillary morphogenesis protein-2 is required for mouse parturition by maintaining uterine collagen homeostasis

Capillary morphogenesis protein-2 (CMG2) functions as an anthrax toxin receptor that plays an essential role in anthrax pathogenesis. Although mutations in CMG2 have been identified to cause two human autosomal recessive disorders, Juvenile Hyaline Fibromatosis and Infantile Systemic Hyalinosis, both characterized by excess hyaline material deposition in connective tissues, the physiologic function of CMG2 remains elusive. To study the roles of CMG2 in normal physiology, here we performed detailed histological analyses of the CMG2-null mice we generated previously. While no morphological or histological defects were observed in CMG2(-/-) male mice, CMG2(-/-) female mice were unable to produce any offspring due to a defect in parturition. We found that deletion of CMG2 resulted in a diffuse deposition of collagen within the myometrium of CMG2(-/-) females, causing remarkable morphological changes to their uteri. This collagen accumulation also led to loss of smooth muscle cells in the myometrium of CMG2(-/-) mice, apparently disabling uterine contractile function during parturition. As a consequence, even though pregnant CMG2(-/-) mice were able to carry the gestation to full term, they were unable to deliver pups. However, the fully-developed fetuses could be successfully delivered by Cesarean section and survived to adulthood when fostered. Our results demonstrate that CMG2 is not required for normal mouse embryonic development but is indispensable for murine parturition. In parallel to its role in anthrax toxin binding and internalization, herein we provide evidence that CMG2 may function as a collagen receptor which is essential for maintaining collagen homeostasis in the uterus.     

Anthrax Toxin Uptake by Primary Immune Cells as Determined with a Lethal Factor-β-Lactamase Fusion Protein

Background

To initiate infection, Bacillus anthracis needs to overcome the host innate immune system. Anthrax toxin, a major virulence factor of B. anthracis, impairs both the innate and adaptive immune systems and is important in the establishment of anthrax infections.

Methodology/Principal Findings

To measure the ability of anthrax toxin to target immune cells, studies were performed using a fusion of the anthrax toxin lethal factor (LF) N-terminal domain (LFn, aa 1–254) with β-lactamase (LFnBLA). This protein reports on the ability of the anthrax toxin protective antigen (PA) to mediate LF delivery into cells. Primary immune cells prepared from mouse spleens were used in conjunction with flow cytometry to assess cleavage and resulting FRET disruption of a fluorescent β-lactamase substrate, CCF2/AM. In spleen cell suspensions, the macrophages, dendritic cells, and B cells showed about 75% FRET disruption of CCF2/AM due to cleavage by the PA–delivered LFnBLA. LFnBLA delivery into CD4+ and CD8+ T cells was lower, with 40% FRET disruption. When the analyses were done on purified samples of individual cell types, similar results were obtained, with T cells again having lower LFnBLA delivery than macrophages, dendritic cells, and B cells. Relative expression levels of the toxin receptors CMG2 and TEM8 on these cells were determined by real-time PCR. Expression of CMG2 was about 1.5-fold higher in CD8+ cells than in CD4+ and B cells, and 2.5-fold higher than in macrophages.

Conclusions/Significance

Anthrax toxin entry and activity differs among immune cells. Macrophages, dendritic cells, and B cells displayed higher LFnBLA activity than CD4+ and CD8+ T cells in both spleen cell suspension and the purified samples of individual cell types. Expression of anthrax toxin receptor CMG2 is higher in CD4+ and CD8+ T cells, which is not correlated to the intracellular LFnBLA activity.     

Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a β-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.     

Role of N-terminal amino acids in the potency of anthrax lethal factor

Anthrax lethal factor (LF) is a Zn(+2)-dependent metalloprotease that cleaves several MAPK kinases and is responsible for the lethality of anthrax lethal toxin (LT). We observed that a recombinant LF (LF-HMA) which differs from wild type LF (LF-A) by the addition of two residues (His-Met) to the native Ala (A) terminus as a result of cloning manipulations has 3-fold lower potency toward cultured cells and experimental animals. We hypothesized that the "N-end rule", which relates the half-life of proteins in cells to the identity of their N-terminal residue, might be operative in the case of LF, so that the N-terminal residue of LF would determine the cytosolic stability and thereby the potency of LF. Mutational studies that replaced the native N-terminal residue of LF with known N-end rule stabilizing or destabilizing residues confirmed that the N-terminal residue plays a significant role in determining the potency of LT for cultured cells and experimental animals. The fact that a commercially-available LF preparation (LF-HMA) that is widely used in basic research studies and for evaluation of vaccines and therapeutics is 3-fold less potent than native LF (LF-A) should be considered when comparing published studies and in the design of future experiments.     

Killing of macrophages by anthrax lethal toxin: involvement of the N-end rule pathway

Macrophages from certain inbred mouse strains are rapidly killed (< 90 min) by anthrax lethal toxin (LT). LT cleaves cytoplasmic MEK proteins at 20 min and induces caspase-1 activation in sensitive macrophages at 50-60 min, but the mechanism of LT-induced death is unknown. Proteasome inhibitors block LT-mediated caspase-1 activation and can protect against cell death, indicating that the degradation of at least one cellular protein is required for LT-mediated cell death. Proteins can be degraded by the proteasome via the N-end rule, in which a protein's stability is determined by its N-terminal residue. Using amino acid derivatives that act as inhibitors of this pathway, we show that the N-end rule is required for LT-mediated caspase-1 activation and cell death. We also found that bestatin methyl ester, an aminopeptidase inhibitor protects against LT in vitro and in vivo and that the different inhibitors of the protein degradation pathway act synergistically in protecting against LT. We identify c-IAP1, a mammalian member of the inhibitor of apoptosis protein (IAP) family, as a novel N-end rule substrate degraded in macrophages treated with LT. We also show that LT-induced c-IAP1 degradation is independent of the IAP-antagonizing proteins Smac/DIABLO and Omi/HtrA2, but dependent on caspases.     

Anthrax lethal toxin-induced inflammasome formation and caspase-1 activation are late events dependent on ion fluxes and the proteasome

Anthrax lethal toxin (LT) is cytotoxic to macrophages from certain inbred mouse strains. The gene controlling macrophage susceptibility to LT is Nalp1b . Nalp1b forms part of the inflammasome, a multiprotein complex involved in caspase-1 activation and release of interleukin (IL)-1β and IL-18. We confirm the role of caspase-1 in LT-mediated death by showing that caspase inhibitors differentially protected cells against LT, with the degree of protection corresponding to each compound's ability to inhibit caspase-1. Caspase-1 activation and cytokine processing and release were late events inhibited by elevated levels of KCl and sucrose, by potassium channel blockers, and by proteasome inhibitors, suggesting that inflammasome formation requires a protein-degradation event and occurs downstream of LT-mediated potassium efflux. In addition, IL-18 and IL-1β release was dependent on cell death, indicating that caspase-1-mediated cytotoxicity is independent of these cytokines. Finally, inducing NALP3-inflammasome formation in LT-resistant macrophages did not sensitize cells to LT, suggesting that general caspase-1 activation cannot account for sensitivity to LT and that a Nalp1b-mediated event is specifically required for death. Our data indicate that inflammasome formation is a contributing, but not initiating, event in LT-mediated cytotoxicity and that earlier LT-mediated events leading to ion fluxes are required for death.