Bacillus anthracis spores influence ATP synthase activity in murine macrophages

Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis. To identify the mitochondrial proteins that are expressed differently in murine macrophages infected with spores of B. anthracis Sterne, proteomic and MALDI-TOF/MS analyses of uninfected and infected macrophages were conducted. As a result, 13 mitochondrial proteins with different expression patterns were discovered in the infected murine macrophages, and some were identified as ATP5b, NIAP-5, ras-related GTP binding protein B isoform CRAa, along with several unnamed proteins. Among these proteins, ATP5b is related to energy production and cytoskeletal rearrangement, whereas NIAP-5 causes apoptosis of host cells due to binding with caspase-9. Therefore, this paper focused on ATP5b, which was found to be downregulated following infection. The downregulated ATP5b also reduced ATP production in the murine macrophages infected with B. anthracis spores. Consequently, this study represents the first mitochondrial proteome analysis of infected macrophages.     

Human neutrophils kill Bacillus anthracis

Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.     

Immunosuppressive action of the lethal toxin of Bacillus anthracis in mice

In experiments on inbred mice infected with B. anthracis capsular strain 71/12 of Tsenkovsky's second vaccine B. anthracis lethal toxin introduced in mixture with spores has been shown to aggravate anthrax infection in CBA mice susceptible to anthrax, while producing a faint effect on the infectious process in BALB mice with hereditary resistance to anthrax. B. anthracis purified edema toxin has been found to produce a weaker aggravating effect with respect to anthrax infection than the lethal toxin. As revealed in these experiments, the capacity of the lethal toxin to suppress the activity of peritoneal macrophages in vitro is the more pronounced, the more resistant to anthrax are the mice used as the donors of these macrophages. The mechanism of hereditary immunity which may ensure resistance to infection in the presence of immunosuppression is discussed.     

Activation of the classical complement pathway by Bacillus anthracis is the primary mechanism for spore phagocytosis and involves the spore surface protein BclA

Interactions between spores of Bacillus anthracis and macrophages are critical for the development of anthrax infections, as spores are thought to use macrophages as vehicles to disseminate in the host. In this study, we report a novel mechanism for phagocytosis of B. anthracis spores. Murine macrophage-like cell line RAW264.7, bone marrow-derived macrophages, and primary peritoneal macrophages from mice were used. The results indicated that activation of the classical complement pathway (CCP) was a primary mechanism for spore phagocytosis. Phagocytosis was significantly reduced in the absence of C1q or C3. C3 fragments were found deposited on the spore surface, and the deposition was dependent on C1q and Ca(2+). C1q recruitment to the spore surface was mediated by the spore surface protein BclA, as recombinant BclA bound directly and specifically to C1q and inhibited C1q binding to spores in a dose-dependent manner. C1q binding to spores lacking BclA (ΔbclA) was also significantly reduced compared with wild-type spores. In addition, deposition of both C3 and C4 as well as phagocytosis of spores were significantly reduced when BclA was absent, but were not reduced in the absence of IgG, suggesting that BclA, but not IgG, is important in these processes. Taken together, these results support a model in which spores actively engage CCP primarily through BclA interaction with C1q, leading to CCP activation and opsonophagocytosis of spores in an IgG-independent manner. These findings are likely to have significant implications on B. anthracis pathogenesis and microbial manipulation of complement.     

Early interactions between fully virulent Bacillus anthracis and macrophages that influence the balance between spore clearance and development of a lethal infection

The role of macrophages in the pathogenesis of anthrax is unresolved. Macrophages are believed to support the initiation of infection by Bacillus anthracis spores, yet are also sporicidal. Furthermore, it is believed that the anthrax toxins suppress normal macrophage function. However, the significance of toxin effects on macrophages has not been addressed in an in vivo infection model. We used mutant derivatives of murine macrophage RAW264.7 cells that are toxin receptor-negative (R3D) to test the role of toxin-targeting of macrophages during a challenge with spores of the Ames strain of B. anthracis in both in vivo and in vitro models. We found that the R3D cells were able to control challenge with Ames when mice were inoculated with the cells prior to spore challenge. These findings were confirmed in vitro by high dose spore infection of macrophages. Interestingly, whereas the R3D cells provided a significantly greater survival advantage against spores than did the wild type RAW264.7 cells or R3D-complemented cells, the protection afforded the mutant and wild type cells was equivalent against a bacillus challenge. The findings appear to be the first specific test of the role of toxin targeting of macrophages during infection with B. anthracis spores.     

Species-specific peptide ligands for the detection of Bacillus anthracis spores

Currently available detectors for spores of Bacillus anthracis, the causative agent of anthrax, are inadequate for frontline use and general monitoring. There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of B. anthracis spores. Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores. By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to B. anthracis spores. We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to B. anthracis spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from, B. anthracis. These two peptides were used in tandem in simple assays to rapidly and unambiguously identify B. anthracis spores. We envision that these peptides can be used as sensors in economical and portable B. anthracis spore detectors that are essentially free of false-positive signals due to other environmental Bacillus spores.     

Bacillus anthracis interacts with plasmin(ogen) to evade C3b-dependent innate immunity

The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B. anthracis with the innate immune system through specific interaction of the spore surface with host proteins such as the complement system has heretofore attracted little attention. In order to assess the mechanisms by which B. anthracis evades the defense system, we employed a proteomic analysis to identify human serum proteins interacting with B. anthracis spores, and found that plasminogen (PLG) is a major surface-bound protein. PLG efficiently bound to spores in a lysine- and exosporium-dependent manner. We identified α-enolase and elongation factor tu as PLG receptors. PLG-bound spores were capable of exhibiting anti-opsonic properties by cleaving C3b molecules in vitro and in rabbit bronchoalveolar lavage fluid, resulting in a decrease in macrophage phagocytosis. Our findings represent a step forward in understanding the mechanisms involved in the evasion of innate immunity by B. anthracis through recruitment of PLG resulting in the enhancement of anti-complement and anti-opsonization properties of the pathogen.     

Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies

We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of na?ve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.     

Effect of Bacillus anthracis lethal toxin on human peripheral blood mononuclear cells

Lethal toxin (LeTx) plays a central role in anthrax pathogenesis, however a cytotoxicity of LeTx has been difficult to demonstrate in vitro. No cytolytic effect has been reported for human cells, in contrast to murine cell lines, indicating that cell lysis can not be considered as a marker of LeTx activity. We have recently shown that murine macrophage-like RAW 264.7 cells treated with LeTx or infected with anthrax spores underwent changes typical of apoptotic death. Here we demonstrate that cells from human peripheral blood display a proapoptotic behavior similar to murine cells. TUNEL assay detected a nucleosomal degradation typical of apoptosis in peripheral blood mononuclear cells (PBMC) treated with LeTx. Membrane staining with apoptotic dyes was detected in macrophages derived from monocytes in presence of LeTx. The toxin inhibited production of proinflammatory cytokines in PBMC stimulated with a preparation of Bacillus anthracis cell wall. Infection of PBMC with anthrax spores led to the appearance of a large population of cells stained positively for apoptosis, with a reduced capacity to eliminate spores and vegetative bacteria. The aminopeptidase inhibitor, bestatin, capable of protecting cells from LeTx, restored a bactericidal activity of infected cells. These findings may be explained by LeTx expression within phagocytes and support an important role of LeTx as an early intracellular virulence factor contributing to bacterial dissemination and disease progression.     

Bacillus anthracis internalization by human fibroblasts and epithelial cells

The current model for Bacillus anthracis dissemination in vivo focuses on macrophages as carriers. However, recent evidence suggested that other host cells may also play a role in the process. Here, we tested the possibility of B. anthracis being internalized by a human fibroblast cell line, HT1080 and an epithelial cell line, Caco-2. A combination of gentamicin protection assays, scanning and transmission electron microscopy (EM) and fluorescence microscopy was used. The results demonstrated for the first time that both spores and vegetative cells of B. anthracis Sterne strain 7702 were able to adhere to and be internalized by cultured HT1080 and Caco-2 cells. Spore adherence to and internalization by HT1080 cells were not affected by a germination inhibitor. This suggested that certain features on dormant spores were sufficient for these processes. Vegetative cell adherence to and internalization by both cell lines were growth phase-dependent. EM images suggested that vegetative cells may have the ability to escape phagocytic vacuoles. Finally, we showed that internalization of both spores and vegetative cells required active functions of the host cell cytoskeleton. These results raised the possibility that B. anthracis may disseminate in vivo by directly infecting non-phagocytic cells.     

Polymeric glycoconjugates protect and activate macrophages to promote killing of <Emphasis Type="Italic">Bacillus cereus</Emphasis> spores during phagocytosis

Diseases caused by Bacillus spores might be attenuated if macrophages were able to kill the spores on exposure. Glycoconjugate-bearing polymers, which have been shown to bind to Bacillus spores, were tested for modulation of phagocytosis of B. cereus spores. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores during phagocytosis. In the presence of glycoconjugates, however, the macrophages efficiently killed the organisms. The glycoconjugates were shown to have a protective influence, sparing macrophages from spore-induced cell death. Very low concentrations of the glycoconjugates prevented macrophage cell death, as shown by lactate dehydrogenase (LDH) release and trypan blue assays. Increased levels of inducible nitric oxide (NO) production by the macrophages in the presence of glycoconjugates suggested that the glycoconjugates provide an activation signal to the macrophages. These results suggest that glycoconjugates promote the killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level. Polymeric glycoconjugates may suggest novel approaches to improve existing vaccines as well as prevent and treat infections incurred through either B. cereus or B. anthracis spores.     

Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis

The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat.     

Modulation of cytokine production and enhancement of cell viability by TLR7 and TLR9 ligands during anthrax infection of macrophages

Inhalation of Bacillus anthracis, a bioterrorism agent, results in a high mortality rate despite appropriate antibiotic therapy. Macrophages appear to be a key factor in B. anthracis pathogenesis. The burst of pro-inflammatory cytokines from macrophages could be a major cause of death in anthrax. However, preactivation of Toll-like receptors (TLRs) could modify the host response. TLR ligands stimulate the release of activating cytokines but may also down-modulate the subsequent deleterious cytokine response to pathogens. We developed a cell culture model to measure macrophage responses to B. anthracis spores and bacilli. We found that germination from spores to bacilli produced a substantial stimulus for the secretion of the cytokines IL-6, TNF-alpha, IL-10, and IL-12 p40. Our studies showed that pretreatment of mouse macrophages with the TLR9 ligand ISS-1018, or the TLR7 ligands R-848 and IT-37, results in a substantial decrease in the subsequent secretion of IL-6 and TNF-alpha in response to B. anthracis infection of macrophages. Furthermore, the TLR7 and TLR9 ligands significantly decreased anthrax-induced cytotoxicity in the macrophages. These findings suggest that TLR ligands may contribute to the enhancement of innate immunity in B. anthracis infection by suppressing potentially deleterious pro-inflammatory cytokine responses and by improving macrophage viability.     

Monoclonal antibodies for Bacillus anthracis spore detection and functional analyses of spore germination and outgrowth

All members of the Bacillus genus produce endospores as part of their life cycle; however, it is not possible to determine the identity of spores by casual or morphological examination. The 2001 anthrax attacks demonstrated a need for fast, dependable methods for detecting Bacillus anthracis spores in vitro and in vivo. We have developed a variety of isotypes and specificities of mAbs that were able to distinguish B. anthracis spores from other Bacillus spores. The majority of Abs were directed toward BclA, a major component of the exosporium, although other components were also distinguished. These Abs did not react with vegetative forms. Some Abs distinguished B. anthracis spores from spores of distantly related species in a highly specific manner, whereas others discriminated among strains that are the closest relatives of B. anthracis. These Abs provide a rapid and reliable means of identifying B. anthracis spores, for probing the structure and function of the exosporium, and in the analysis of the life cycle of B. anthracis.     

Morphogenesis of the Bacillus anthracis spore

Bacillus spp. and Clostridium spp. form a specialized cell type, called a spore, during a multistep differentiation process that is initiated in response to starvation. Spores are protected by a morphologically complex protein coat. The Bacillus anthracis coat is of particular interest because the spore is the infective particle of anthrax. We determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence. One of these, cotE, has a striking function in B. anthracis: it guides the assembly of the exosporium, an outer structure encasing B. anthracis but not B. subtilis spores. However, CotE has only a modest role in coat protein assembly, in contrast to the B. subtilis orthologue. cotE mutant spores are fully virulent in animal models, indicating that the exosporium is dispensable for infection, at least in the context of a cotE mutation. This has implications for both the pathophysiology of the disease and next-generation therapeutics. CotH, which directs the assembly of an important subset of coat proteins in B. subtilis, also directs coat protein deposition in B. anthracis. Additionally, however, in B. anthracis, CotH effects germination; in its absence, more spores germinate than in the wild type. We also found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore. This function is very similar to that of the B. subtilis orthologue, which directs the assembly of the coat to the forespore. These results show that while B. anthracis and B. subtilis rely on a core of conserved morphogenetic proteins to guide coat formation, these proteins may also be important for species-specific differences in coat morphology. We further hypothesize that variations in conserved morphogenetic coat proteins may play roles in taxonomic variation among species.     

Identification of an in vivo inhibitor of Bacillus anthracis spore germination

Germination of Bacillus anthracis spores into the vegetative form is an essential step in anthrax pathogenicity. This process can be triggered in vitro by the common germinants inosine and alanine. Kinetic analysis of B. anthracis spore germination revealed synergy and a sequential mechanism between inosine and alanine binding to their cognate receptors. Because inosine is a critical germinant in vitro, we screened inosine analogs for the ability to block in vitro germination of B. anthracis spores. Seven analogs efficiently blocked this process in vitro. This led to the identification of 6-thioguanosine, which also efficiently blocked spore germination in macrophages and prevented killing of these cells mediated by B. anthracis spores. 6-Thioguanosine shows potential as an anti-anthrax therapeutic agent.     

Potential dissemination of Bacillus anthracis utilizing human lung epithelial cells

Dissemination of Bacillus anthracis spores from the lung is a critical early event in the establishment of inhalational anthrax. We recently reported that B. anthracis could adhere to and be internalized by cultured intestinal epithelial and fibroblast cells. Here, using gentamicin protection assays and/or electron microscopy, we found that Sterne strain 7702 spores were able to adhere to and subsequently be internalized by polarized A549 cells and primary human small airway epithelial cells. We showed for the first time that internalized spores were able to survive and that spores could translocate across an A549 cell barrier from the apical side to the basolateral side without disrupting the barrier integrity, suggesting a transcellular route. In addition, dormant spores of fully virulent Ames and UT500 strains were able to adhere to A549 cells at a frequency similar to that of 7702, whereas the capsule in germinated Ames and UT500 spores prevented adherence. Fluorescence microscopy also revealed that dormant Ames spores were internalized at a frequency similar to that of 7702. These findings highlight the possibility of a novel route of dissemination in which B. anthracis utilizes epithelial cells of the lung. The implications of these results to B. anthracis pathogenesis are discussed.     

Identification of Bacillus anthracis proteins associated with germination and early outgrowth by proteomic profiling of anthrax spores

The use of anthrax spores as a bioweapon has spurred efforts aimed at identifying key proteins expressed in Bacillus anthracis. Because spore germination and outgrowth occur prior to and are required for disease manifestations, blocking germination and early outgrowth with novel vaccines or inhibitors targeting critical B. anthracis germination and outgrowth-associated factors is a promising strategy in mitigating bioterror. By screening 587 paired protein spots that were isolated from dormant and germinating anthrax spores, respectively, we identified 10 spore proteins with statistically significant germination-associated increases and decreases. It is likely that proteins whose levels change during germination may play key roles in the germination and outgrowth processes, and they should be listed as priority targets for development of prophylactic and therapeutic agents against anthrax. The 31 new proteins identified in this study also complement an emerging proteomic database of B. anthracis.