Investigating the Influence of ANTXR2 Gene Mutations on Protective Antigen Binding for Heightened Anthrax Resistance.

Bacillus anthracis is the bacterium responsible for causing the zoonotic disease called anthrax. The disease presents itself in different forms like gastrointestinal, inhalation, and cutaneous. Bacterial spores are tremendously adaptable, can persist for extended periods and occasionally endanger human health. The Anthrax Toxin Receptor-2 (ANTXR2) gene acts as membrane receptor and facilitates the entry of the anthrax toxin into host cells. Additionally, mutations in the ANTXR2 gene have been linked to various autoimmune diseases, including Hyaline Fibromatosis Syndrome (HFS), Ankylosing Spondylitis (AS), Juvenile Hyaline Fibromatosis (JHF), and Infantile Systemic Hyalinosis (ISH). This study delves into the genetic landscape of ANTXR2, aiming to comprehend its associations with diverse disorders, elucidate the impacts of its mutations, and pinpoint minimal non-pathogenic mutations capable of reducing the binding affinity of the ANTXR2 gene with the protective antigen. Recognizing the pivotal role of single-nucleotide polymorphisms (SNPs) in shaping genetic diversity, we conducted computational analyses to discern highly deleterious and tolerated non-synonymous SNPs (nsSNPs) in the ANTXR2 gene. The Mutpred2 server determined that the Arg465Trp alteration in the ANTXR2 gene leads to altered DNA binding (p = 0.22) with a probability of a deleterious mutation of 0.808; notably, among the identified deleterious SNPs, rs368288611 (Arg465Trp) stands out due to its significant impact on altering the DNA-binding ability of ANTXR2. We propose these SNPs as potential candidates for hypertension linked to the ANTXR2 gene, which is implicated in blood pressure regulation. Noteworthy among the tolerated substitutions is rs200536829 (Ala33Ser), recognized as less pathogenic; this highlights its potential as a valuable biomarker, potentially reducing side effects on the host while also reducing binding with the protective antigen protein. Investigating these SNPs holds the potential to correlate with several autoimmune disorders and mitigate the impact of anthrax disease in humans.

Molecular characterization of an outbreak-involved Bacillus anthracis strain confirms the spillover of anthrax from West Africa.

BACKGROUND: Anthrax, a zoonotic disease caused by the spore-forming bacterium Bacillus anthracis, remains a major global public health concern, especially in countries with limited resources. Sierra Leone, a West African country historically plagued by anthrax, has almost been out of report on this disease in recent decades. In this study, we described a large-scale anthrax outbreak affecting both animals and humans and attempted to characterize the pathogen using molecular techniques. METHODS: The causative agent of the animal outbreak in Port Loko District, Sierra Leone, between March and May 2022 was identified using the nanopore sequencing technique. A nationwide active surveillance was implemented from May 2022 to June 2023 to monitor the occurrence of anthrax-specific symptoms in humans. Suspected cases were subsequently verified using quantitative polymerase chain reaction. Full-genome sequencing was accomplished by combining long-read and short-read sequencing methods. Subsequent phylogenetic analysis was performed based on the full-chromosome single nucleotide polymorphisms. RESULTS: The outbreak in Port Loko District, Sierra Leone, led to the death of 233 animals between March 26th and May 16th, 2022. We ruled out the initial suspicion of Anaplasma species and successfully identified B. anthracis as the causative agent of the outbreak. As a result of the government's prompt response, out of the 49 suspected human cases identified during the one-year active surveillance, only 6 human cases tested positive, all within the first month after the official declaration of the outbreak. The phylogenetic analysis indicated that the BaSL2022 isolate responsible for the outbreak was positioned in the A.Br.153 clade within the TransEuroAsian group of B. anthracis. CONCLUSIONS: We successfully identified a large-scale anthrax outbreak in Sierra Leone. The causative isolate of B. anthracis, BaSL2022, phylogenetically bridged other lineages in A.Br.153 clade and neighboring genetic groups, A.Br.144 and A.Br.148, eventually confirming the spillover of anthrax from West Africa. Given the wide dissemination of B. anthracis spores, it is highly advisable to effectively monitor the potential reoccurrence of anthrax outbreaks and to launch campaigns to improve public awareness regarding anthrax in Sierra Leone.

Host Cell Transcriptional Tuning with CRISPR/dCas9 to Mitigate the Effects of Toxin Exposure.

Anthrax infection is caused byBacillus anthracis, a bacterium that once established within the host releases lethal toxin (LeTx). Anthrax LeTx is internalized by the capillary morphogenesis protein 2/anthrax toxin receptor 2 (CMG2/ANTXR2) cell surface receptor on mammalian cells. Once inside the cell, LeTx cleaves mitogen-activated protein kinases (MAPKs), ultimately leading to cell death. Previous reports have shown that decreased expression of ANTXR2 reduces cell susceptibility to LeTx. By ablating the ANTXR2 gene in cells in vitro, we observed complete resistance to LeTx-induced cell death. Here, we directed CRISPR/dCas9-based tools to the ANTXR2 promoter to modulate ANTXR2 expression without altering the underlying gene sequence in human cell lines that express the receptor at high levels. We hypothesized that downregulating the expression of the ANTXR2 gene at the genomic level would mitigate the impact of toxin exposure. In one epigenetic editing approach, we employed the fusion of DNMT3A DNA methyltransferase and dCas9 (dCas9-DNMT3A) to methylate CpGs within the CpG island of the ANTXR2 promoter and found this repressed ANTXR2 gene expression resulting in significant resistance to LeTx-induced cell death. Furthermore, by multiplexing gRNAs to direct dCas9-DNMT3A to multiple sites in the ANTXR2 promoter, we applied a broader distribution of CpG methylation along the gene promoter resulting in enhanced repression and resistance to LeTx. In parallel, we directed the dCas9-KRAB-MeCP2 transcriptional repressor to the ANTXR2 promoter to quickly and robustly repress ANTXR2 expression. With this approach, in as little as two weeks, we created resistance to LeTx at a similar level to ANTXR2 gene-ablated cells. Overall, we present a transcriptional tuning approach to inhibit the effects of LeTx and provide a framework to repress toxin-binding cell surface receptors.

Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis.

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD50 and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

Anthrax lethal factor cleaves regulatory subunits of phosphoinositide-3 kinase to contribute to toxin lethality.

Anthrax lethal toxin (LT), produced by Bacillus anthracis, comprises a receptor-binding moiety, protective antigen and the lethal factor (LF) protease1,2. Although LF is known to cleave mitogen-activated protein kinase kinases (MEKs/MKKs) and some variants of the NLRP1 inflammasome sensor, targeting of these pathways does not explain the lethality of anthrax toxin1,2. Here we report that the regulatory subunits of phosphoinositide-3 kinase (PI3K)-p85α (PIK3R1) and p85ß (PIK3R2)3,4-are substrates of LF. Cleavage of these proteins in a proline-rich region between their N-terminal Src homology and Bcr homology domains disrupts homodimer formation and impacts PI3K signalling. Mice carrying a mutated p85α that cannot be cleaved by LF show a greater resistance to anthrax toxin challenge. The LF(W271A) mutant cleaves p85α with lower efficiency and is non-toxic to mice but can regain lethality when combined with PI3K pathway inhibitors. We provide evidence that LF targets two signalling pathways that are essential for growth and metabolism and that the disabling of both pathways is likely necessary for lethal anthrax infection.

Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018.

In Italy anthrax is an endemic disease, with a few outbreaks occurring almost every year. We surveyed 234 B. anthracis strains from animals (n = 196), humans (n = 3) and the environment (n = 35) isolated during Italian outbreaks in the years 1972-2018. Despite the considerable genetic homogeneity of B. anthracis, the strains were effectively differentiated using canonical single nucleotide polymorphisms (CanSNPs) assay and multiple-locus variable-number tandem repeat analysis (MLVA). The phylogenetic identity was determined through the characterization of 14 CanSNPs. In addition, a subsequent 31-loci MLVA assay was also used to further discriminate B. anthracis genotypes into subgroups. The analysis of 14 CanSNPs allowed for the identification of four main lineages: A.Br.011/009, A.Br.008/011 (respectively belonging to A.Br.008/009 sublineage, also known Trans-Eurasian or TEA group), A.Br.005/006 and B.Br.CNEVA. A.Br.011/009, the most common subgroup of lineage A, is the major genotype of B. anthracis in Italy. The MLVA analysis revealed the presence of 55 different genotypes in Italy. Most of the genotypes are genetically very similar, supporting the hypothesis that all strains evolved from a local common ancestral strain, except for two genotypes representing the branch A.Br.005/006 and B.Br.CNEVA. The genotyping analysis applied in this study remains a very valuable tool for studying the diversity, evolution, and molecular epidemiology of B. anthracis.

Investigation and source-tracing of an anthrax outbreak in Gansu Province, China.

Anthrax is an endemic disease in China. Cases are reported every year, especially in the northwestern areas. In August 2016, an outbreak of 21 cutaneous anthrax cases was reported in Min County, Gansu Province, China. In this study, the general characteristics of the anthrax outbreak are described. Two molecular typing methods, canonical single-nucleotide polymorphism (canSNP) and multiple-locus variable-number tandem repeat analysis with 15 markers (MLVA15), were used to investigate the possible source of transmission and to identify the genetic relationship among the strains/samples isolated in this outbreak as well as previous isolates. In this outbreak, all patients were infected through contact with diseased livestock or contaminated animal products. Livestock had been introduced into the local area shortly before the outbreak from Gannan Prefecture (in Gansu Province), Sichuan and Qinghai Provinces. In the molecular typing analysis, there were two canSNP subgroups found in Gansu, A.Br.001/002 and A.Br.Ames, and five MLVA15 genotypes were observed. The strains collected from the anthrax outbreak in Min County in 2016 belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-28 and MLVA15-30 genotype. Strains previously isolated from Sichuan, Inner Mongolia and Maqu County (in Gannan Prefecture, Gansu Province) were clustered with these outbreak-related strains/samples according to the MLVA15-30 genotype. The MLVA15-28 genotype was found in strains isolated from Gansu and Xinjiang in previous studies. Combining the epidemiological investigation and molecular typing results, we conclude that the patients in this outbreak were infected by a local pathogen present in the adjoining area of Gansu, Sichuan and Qinghai Provinces.

Gene expression profiling of primary human type I alveolar epithelial cells exposed to Bacillus anthracis spores reveals induction of neutrophil and monocyte chemokines.

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores must escape through the alveolar epithelial cell (AEC) barrier and migrate to regional lymph nodes, germinate and enter the circulatory system to cause disease. Several mechanisms to explain alveolar escape have been postulated, and all these tacitly involve the AEC barrier. In this study, we incorporate our primary human type I AEC model, microarray and gene enrichment analysis, qRT-PCR, multiplex ELISA, and neutrophil and monocyte chemotaxis assays to study the response of AEC to B. anthracis, (Sterne) spores at 4 and 24 h post-exposure. Spore exposure altered gene expression in AEC after 4 and 24 h and differentially expressed genes (±1.3 fold, p ≤ 0.05) included CCL4/MIP-1ß (4 h), CXCL8/IL-8 (4 and 24 h) and CXCL5/ENA-78 (24 h). Gene enrichment analysis revealed that pathways involving cytokine or chemokine activity, receptor binding, and innate immune responses to infection were prominent. Microarray results were confirmed by qRT-PCR and multiplex ELISA assays. Chemotaxis assays demonstrated that spores induced the release of biologically active neutrophil and monocyte chemokines, and that CXCL8/IL-8 was the major neutrophil chemokine. The small or sub-chemotactic doses of CXCL5/ENA-78, CXCL2/GROß and CCL20/MIP-3α may contribute to chemotaxis by priming effects. These data provide the first whole transcriptomic description of the human type I AEC initial response to B. anthracis spore exposure. Taken together, our findings contribute to an increased understanding of the role of AEC in the pathogenesis of inhalational anthrax.

[The comparative evaluation of effectiveness of laboratory techniques of diagnostic of anthrax and detection its agent in objects of environment].

The analysis of samples received during ictus of anthrax in the Stavropolskii kraii in 2013 permitted to study comparative effectiveness of regulated methods of laboratory diagnostic. The effectiveness of bacteriological, biological and molecular methods and necessity of their complex application for receiving optimal results are confirmed. The rapidity, effectiveness and specificity of polymerase chain reaction is emphasized. This method in case of absence of isolation of anthrax microbe can be the only method of confirming diagnose in people in aggregate with typical clinical picture and corresponding epidemic situation.

The Poly-γ-d-Glutamic Acid Capsule Surrogate of the Bacillus anthracis Capsule Is a Novel Toll-Like Receptor 2 Agonist.

Bacillus anthracis is a pathogenic Gram-positive bacterium that causes a highly lethal infectious disease, anthrax. The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of B. anthracis, along with exotoxins. PGA enables B. anthracis to escape phagocytosis and immune surveillance. Our previous study showed that PGA activates the human macrophage cell line THP-1 and human dendritic cells, resulting in the production of the proinflammatory cytokine interleukin-1ß (IL-1ß) (M. H. Cho et al., Infect Immun 78:387-392, 2010, http://dx.doi.org/10.1128/IAI.00956-09). Here, we investigated PGA-induced cytokine responses and related signaling pathways in mouse bone marrow-derived macrophages (BMDMs) using Bacillus licheniformis PGA as a surrogate for B. anthracis PGA. Upon exposure to PGA, BMDMs produced proinflammatory mediators, including tumor necrosis factor alpha (TNF-α), IL-6, IL-12p40, and monocyte chemoattractant protein 1 (MCP-1), in a concentration-dependent manner. PGA stimulated Toll-like receptor 2 (TLR2) but not TLR4 in Chinese hamster ovary cells expressing either TLR2 or TLR4. The ability of PGA to induce TNF-α and IL-6 was retained in TLR4(-/-) but not TLR2(-/-) BMDMs. Blocking experiments with specific neutralizing antibodies for TLR1, TLR6, and CD14 showed that TLR6 and CD14 also were necessary for PGA-induced inflammatory responses. Furthermore, PGA enhanced activation of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB), which are responsible for expression of proinflammatory cytokines. Additionally, PGA-induced TNF-α production was abrogated not only in MyD88(-/-) BMDMs but also in BMDMs pretreated with inhibitors of MAP kinases and NF-κB. These results suggest that immune responses induced by PGA occur via TLR2, TLR6, CD14, and MyD88 through activation of MAP kinase and NF-κB pathways.

Two Putative Polysaccharide Deacetylases Are Required for Osmotic Stability and Cell Shape Maintenance in Bacillus anthracis.

Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) ß-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.

[Characteristics of isogenic variants of Bacillus anthracis with various content of virulence plasmids].

AIM: Production and characteristics by main cultural-morphologic and antigenic properties of isogenic variants Bacillus anthracis, that differ by the presence of virulence plasmids. MATERIALS AND METHODS: B. anthracis 81/1, 575/122 virulent and B. anthracis STI, 55, Sterne vaccine strains were used in the study. Isogenic variants, that differ by the presence of virulence plasmids, were obtained by temperature elimination of plasmids, as well as during cultivation of anthrax strains in medium with kanamycin. The strains were characterized by cultural-morphologic, biochemical properties. The presence of virulence plasmids was determined by polymerase chain reaction method. Antigenic properties were studied in immune diffusion reaction with growing cultures with sera against protective antigen and S-layer proteins, electrophoresis, immune blotting. RESULTS: Isogenic variants were produced from virulent strains B. anthracis 81/1, 575/122 and vaccine strains STI, 55, Sterne: mono-plasmid toxin-producing (81/1 R01, 575/122 R01) and capsule-containing (81/1 R02, 575/122 R02), and plasmid-less (81/1 R00, 575/122 R00, STI R00, 55 R00, Sterne R00), that differ by the presence of virulence plasmids. Strains had typical cultural-morphologic properties, differed by biochemical and antigenic properties. Cultural filtrates of toxin-producing strains had protein of anthrax toxin; plasmid-less strains--had proteins, that had molecular masses corresponding to molecular masses of S-layer EA1 and Sap proteins. CONCLUSION: These strains may be used to study variability and proteomic analysis of anthrax causative agent, as well as for isolation of antigens with the aim of evaluating their immune diagnostic significance.

The central nervous system as target of Bacillus anthracis toxin independent virulence in rabbits and guinea pigs.

Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC) inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development.

Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the International Space Station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.

Targeted silencing of anthrax toxin receptors protects against anthrax toxins.

Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax.

Anthrax lethal toxin inhibits translation of hypoxia-inducible factor 1α and causes decreased tolerance to hypoxic stress.

Hypoxia is considered to be a contributor to the pathology associated with administration of anthrax lethal toxin (LT). However, we report here that serum lactate levels in LT-treated mice are reduced, a finding inconsistent with the anaerobic metabolism expected to occur during hypoxia. Reduced lactate levels are also observed in the culture supernatants of LT-treated cells. LT inhibits the accumulation of hypoxia-inducible factor (HIF)-1α, a subunit of HIF-1, the master regulator directing cellular responses to hypoxia. The toxin has no effect on the transcription or protein turnover of HIF-1α, but instead it acts to inhibit HIF-1α translation. LT treatment diminishes phosphorylation of eIF4B, eIF4E, and rpS6, critical components of the intracellular machinery required for HIF-1α translation. Moreover, blockade of MKK1/2-ERK1/2, but not p38 or JNK signaling, lowers HIF-1α protein levels in both normoxic and hypoxic conditions, consistent with a role for MKK1 and MKK2 as the major targets of LT responsible for the inhibition of HIF-1α translation. The physiological importance of the LT-induced translation blockade is demonstrated by the finding that LT treatment decreases the survival of hepatocyte cell lines grown in hypoxic conditions, an effect that is overcome by preinduction of HIF-1α. Taken together, these data support a role for LT in dysregulating HIF-1α and thereby disrupting homeostatic responses to hypoxia, an environmental characteristic of certain tissues at baseline and/or during disseminated infection with Bacillus anthracis.

Key tissue targets responsible for anthrax-toxin-induced lethality.

Bacillus anthracis, the causative agent of anthrax disease, is lethal owing to the actions of two exotoxins: anthrax lethal toxin (LT) and oedema toxin (ET). The key tissue targets responsible for the lethal effects of these toxins are unknown. Here we generated cell-type-specific anthrax toxin receptor capillary morphogenesis protein-2 (CMG2)-null mice and cell-type-specific CMG2-expressing mice and challenged them with the toxins. Our results show that lethality induced by LT and ET occurs through damage to distinct cell types; whereas targeting cardiomyocytes and vascular smooth muscle cells is required for LT-induced mortality, ET-induced lethality occurs mainly through its action in hepatocytes. Notably, and in contradiction to what has been previously postulated, targeting of endothelial cells by either toxin does not seem to contribute significantly to lethality. Our findings demonstrate that B. anthracis has evolved to use LT and ET to induce host lethality by coordinately damaging two distinct vital systems.

Genomic copy number variants: evidence for association with antibody response to anthrax vaccine adsorbed.

BACKGROUND: Anthrax and its etiologic agent remain a biological threat. Anthrax vaccine is highly effective, but vaccine-induced IgG antibody responses vary widely following required doses of vaccinations. Such variation can be related to genetic factors, especially genomic copy number variants (CNVs) that are known to be enriched among genes with immunologic function. We have tested this hypothesis in two study populations from a clinical trial of anthrax vaccination. METHODS: We performed CNV-based genome-wide association analyses separately on 794 European Americans and 200 African-Americans. Antibodies to protective antigen were measured at week 8 (early response) and week 30 (peak response) using an enzyme-linked immunosorbent assay. We used DNA microarray data (Affymetrix 6.0) and two CNV detection algorithms, hidden markov model (PennCNV) and circular binary segmentation (GeneSpring) to determine CNVs in all individuals. Multivariable regression analyses were used to identify CNV-specific associations after adjusting for relevant non-genetic covariates. RESULTS: Within the 22 autosomal chromosomes, 2,943 non-overlapping CNV regions were detected by both algorithms. Genomic insertions containing HLA-DRB5, DRB1 and DQA1/DRA genes in the major histocompatibility complex (MHC) region (chromosome 6p21.3) were moderately associated with elevated early antibody response (ß = 0.14, p = 1.78×10(-3)) among European Americans, and the strongest association was observed between peak antibody response and a segmental insertion on chromosome 1, containing NBPF4, NBPF5, STXMP3, CLCC1, and GPSM2 genes (ß = 1.66, p = 6.06×10(-5)). For African-Americans, segmental deletions spanning PRR20, PCDH17 and PCH68 genes on chromosome 13 were associated with elevated early antibody production (ß = 0.18, p = 4.47×10(-5)). Population-specific findings aside, one genomic insertion on chromosome 17 (containing NSF, ARL17 and LRRC37A genes) was associated with elevated peak antibody response in both populations. CONCLUSION: Multiple CNV regions, including the one consisting of MHC genes that is consistent with earlier research, can be important to humoral immune responses to anthrax vaccine adsorbed.