ERK and c-Myc signaling in host-derived tumor endothelial cells is essential for solid tumor growth.

The limited efficacy of the current antitumor microenvironment strategies is due in part to the poor understanding of the roles and relative contributions of the various tumor stromal cells to tumor development. Here, we describe a versatile in vivo anthrax toxin protein delivery system allowing for the unambiguous genetic evaluation of individual tumor stromal elements in cancer. Our reengineered tumor-selective anthrax toxin exhibits potent antiproliferative activity by disrupting ERK signaling in sensitive cells. Since this activity requires the surface expression of the capillary morphogenesis protein-2 (CMG2) toxin receptor, genetic manipulation of CMG2 expression using our cell-type-specific CMG2 transgenic mice allows us to specifically define the role of individual tumor stromal cell types in tumor development. Here, we established mice with CMG2 only expressed in tumor endothelial cells (ECs) and determined the specific contribution of tumor stromal ECs to the toxin's antitumor activity. Our results demonstrate that disruption of ERK signaling only within tumor ECs is sufficient to halt tumor growth. We discovered that c-Myc is a downstream effector of ERK signaling and that the MEK-ERK-c-Myc central metabolic axis in tumor ECs is essential for tumor progression. As such, disruption of ERK-c-Myc signaling in host-derived tumor ECs by our tumor-selective anthrax toxins explains their high efficacy in solid tumor therapy.

Bacillus anthracis induces NLRP3 inflammasome activation and caspase-8-mediated apoptosis of macrophages to promote lethal anthrax.

Lethal toxin (LeTx)-mediated killing of myeloid cells is essential for Bacillus anthracis, the causative agent of anthrax, to establish systemic infection and induce lethal anthrax. The "LeTx-sensitive" NLRP1b inflammasome of BALB/c and 129S macrophages swiftly responds to LeTx intoxication with pyroptosis and secretion of interleukin (IL)-1ß. However, human NLRP1 is nonresponsive to LeTx, prompting us to investigate B. anthracis host-pathogen interactions in C57BL/6J (B6) macrophages and mice that also lack a LeTx-sensitive Nlrp1b allele. Unexpectedly, we found that LeTx intoxication and live B. anthracis infection of B6 macrophages elicited robust secretion of IL-1ß, which critically relied on the NLRP3 inflammasome. TNF signaling through both TNF receptor 1 (TNF-R1) and TNF-R2 were required for B. anthracis-induced NLRP3 inflammasome activation, which was further controlled by RIPK1 kinase activity and LeTx-mediated proteolytic inactivation of MAP kinase signaling. In addition to activating the NLRP3 inflammasome, LeTx-induced MAPKK inactivation and TNF production sensitized B. anthracis-infected macrophages to robust RIPK1- and caspase-8-dependent apoptosis. In agreement, purified LeTx triggered RIPK1 kinase activity- and caspase-8-dependent apoptosis only in macrophages primed with TNF or following engagement of TRIF-dependent Toll-like receptors. Consistently, genetic and pharmacological inhibition of RIPK1 inhibited NLRP3 inflammasome activation and apoptosis of LeTx-intoxicated and B. anthracis-infected macrophages. Caspase-8/RIPK3-deficient mice were significantly protected from B. anthracis-induced lethality, demonstrating the in vivo pathophysiological relevance of this cytotoxic mechanism. Collectively, these results establish TNF- and RIPK1 kinase activity-dependent NLRP3 inflammasome activation and macrophage apoptosis as key host-pathogen mechanisms in lethal anthrax.

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Central Nervous System Antimicrobial Exposure and Proposed Dosing for Anthrax Meningitis.

BACKGROUND: The high mortality of systemic anthrax is likely a consequence of the severe central nervous system inflammation that occurs in anthrax meningitis. Effective treatment of such infections requires, at a minimum, adequate cerebrospinal fluid (CSF) antimicrobial concentrations. METHODS: We reviewed English medical literature and regulatory documents to extract information on serum and CSF exposures for antimicrobials with in vitro activity against Bacillus anthracis. Using CSF pharmacokinetic exposures and in vitro B. anthracis susceptibility data, we used population pharmacokinetic modeling and Monte Carlo simulations to determine whether a specific antimicrobial dosage would likely achieve effective CSF antimicrobial activity in patients with normal to inflamed meninges (ie, an intact to markedly disrupted blood-brain barrier). RESULTS: The probability of microbiologic success at achievable antimicrobial dosages was high (≥95%) for ciprofloxacin, levofloxacin (500 mg every 12 hours), meropenem, imipenem/cilastatin, penicillin G, ampicillin, ampicillin/sulbactam, doxycycline, and minocycline; acceptable (90%-95%) for piperacillin/tazobactam and levofloxacin (750 mg every 24 hours); and low (<90%) for vancomycin, amikacin, clindamycin, and linezolid. CONCLUSIONS: Prompt empiric antimicrobial therapy of patients with suspected or confirmed anthrax meningitis may reduce the high morbidity and mortality. Our data support using several ß-lactam-, fluoroquinolone-, and tetracycline-class antimicrobials as first-line and alternative agents for treatment of patients with anthrax meningitis; all should achieve effective microbiologic exposures. Our data suggest antimicrobials that should not be relied on to treat suspected or documented anthrax meningitis. Furthermore, the protein synthesis inhibitors clindamycin and linezolid can decrease toxin production and may be useful components of combination therapy.

Omadacycline is active in vitro and in vivo against ciprofloxacin-resistant Bacillus anthracis.

Bacillus anthracis, the causative agent of anthrax, is among the most likely bacterial pathogens to be used in a biological attack. Inhalation anthrax is a serious, life-threatening form of infection, and the mortality from acute inhaled anthrax can approach 100% if not treated early and aggressively. Food and Drug Administration-approved antibiotics indicated for post-exposure prophylaxis (PEP) or treatment of anthrax are limited. This study assessed the in vitro activity and in vivo efficacy of omadacycline and comparators against clinical isolates of B. anthracis, including a ciprofloxacin-resistant isolate. Minimum inhibitory concentrations (MICs) of omadacycline, ciprofloxacin, and doxycycline were determined against animal and human clinical isolates of B. anthracis, including the ciprofloxacin-resistant Ames strain BACr4-2. Mice were challenged with aerosolized BACr4-2 spores, and survival was monitored for 28 days post-challenge. Treatment was initiated 24 h after aerosol challenge and administered for 14 days. Omadacycline demonstrated in vitro activity against 53 B. anthracis isolates with an MIC range of ≤0.008-0.25 µg/mL, and an MIC50/MIC90 of 0.015/0.03 µg/mL. Consistent with this, omadacycline demonstrated in vivo efficacy in a PEP mouse model of inhalation anthrax caused by the Ames BACr4-2 ciprofloxacin-resistant B. anthracis isolate. Omadacycline treatment significantly increased survival compared with the vehicle control group and the ciprofloxacin treatment group. As antibiotic resistance rates continue to rise worldwide, omadacycline may offer an alternative PEP or treatment option against inhalation anthrax, including anthrax caused by antibiotic-resistant B. anthracis.

Rapid in vitro activity of telavancin against Bacillus anthracis and in vivo protection against inhalation anthrax infection in the rabbit model.

Inhalation anthrax is the most severe form of Bacillus anthracis infection, often progressing to fatal conditions if left untreated. While recommended antibiotics can effectively treat anthrax when promptly administered, strains engineered for antibiotic resistance could render these drugs ineffective. Telavancin, a semisynthetic lipoglycopeptide antibiotic, was evaluated in this study as a novel therapeutic against anthrax disease. Specifically, the aims were to (i) assess in vitro potency of telavancin against 17 B. anthracis isolates by minimum inhibitory concentration (MIC) testing and (ii) evaluate protective efficacy in rabbits infected with a lethal dose of aerosolized anthrax spores and treated with human-equivalent intravenous telavancin doses (30 mg/kg every 12 hours) for 5 days post-antigen detection versus a humanized dose of levofloxacin and vehicle control. Blood samples were collected at various times post-infection to assess the level of bacteremia and antibody production, and tissues were collected to determine bacterial load. The animals' body temperatures were also recorded. Telavancin demonstrated potent bactericidal activity against all strains tested (MICs 0.06-0.125 µg/mL). Further, telavancin conveyed 100% survival in this model and cleared B. anthracis from the bloodstream and organ tissues more effectively than a humanized dose of levofloxacin. Collectively, the low MICs against all strains tested and rapid bactericidal in vivo activity demonstrate that telavancin has the potential to be an effective alternative for the treatment or prophylaxis of anthrax infection.

Doxycycline, levofloxacin, and moxifloxacin are superior to ciprofloxacin in treating anthrax meningitis in rabbits and NHP.

Efficient treatment of anthrax-related meningitis in patients poses a significant therapeutic challenge. Previously, we demonstrated in our anthrax meningitis rabbit model that ciprofloxacin treatment is ineffective with most of the treated animals succumbing to the infection. Herein we tested the efficacy of doxycycline in our rabbit model and found it highly effective. Since all of our findings are based on a rabbit model, we test the efficacy of ciprofloxacin or doxycycline in a specific central nervous system (CNS) model developed in non-human primates (NHPs). Similar to rabbits, ciprofloxacin treatment was ineffective, while doxycycline protected the infected rhesus macaques (n = 2) from the lethal CNS Bacillus anthracis infection. To test whether the low efficacy of Ciprofloxacin is an example of low efficacy of all fluoroquinolones or only this substance, we treated rabbits that were inoculated intracisterna magna (ICM) with levofloxacin or moxifloxacin. We found that in contrast to ciprofloxacin, levofloxacin and moxifloxacin were highly efficacious in treating lethal anthrax-related meningitis in rabbits and NHP (levofloxacin). We demonstrated (in naïve rabbits) that this difference probably results from variances in blood-brain-barrier penetration of the different fluoroquinolones. The combined treatment of doxycycline and any one of the tested fluoroquinolones was highly effective in the rabbit CNS infection model. The combined treatment of doxycycline and levofloxacin was effective in an inhalation rabbit model, as good as the doxycycline mono-therapy. These findings imply that while ciprofloxacin is highly effective as a post-exposure prophylactic drug, using this drug to treat symptomatic patients should be reconsidered.

Rational corticosteroids administration and antibiotic treatment is key to managing cutaneous anthrax.

BACKGROUND: Anthrax is a global health concern, with cutaneous anthrax accounting for over 95% of cases and generally promising outcomes. Nonetheless, the absence of timely intervention can result in mortality rates of 10-40%. This research aims to explore the clinical presentations and phenotypic characteristics of cutaneous anthrax patients and evaluate the efficacy of various therapeutic approaches. METHODS: A retrospective study was performed on 76 cutaneous anthrax patients identified at three hospitals from 2017 to 2022. Patients were categorized based on their hospital stay into two groups: those hospitalized for at least seven days and those for shorter durations. We assessed their clinical and phenotypic profiles, including symptoms, general health status, and laboratory findings, alongside treatment outcomes, focusing on corticosteroids therapy and antibiotic regimens. RESULTS: The study encompassed 76 diagnosed individuals, predominantly young adult males (78.9%). A significant gender disparity was noted. Hormonal treatment markedly improved edema regression in patients (P < 0.002), highlighting its therapeutic value. The impact of various antibiotic treatments on disease progression differed significantly based on corticosteroids treatment status, with specific combinations showing more effectiveness in non-corticosteroids-treated patients. CONCLUSIONS: The predominance of young male adults among cutaneous anthrax cases was observed, with corticosteroids treatment significantly reducing edema duration. In cases where corticosteroids therapy is not utilized, employing piperacillin-tazobactam alone or in combination with quinolones effectively shortens the illness duration, suggesting a tailored approach to treatment can enhance patient outcomes.

Pan-genome analysis reveals novel chromosomal markers for multiplex PCR-based specific detection of Bacillus anthracis.

BACKGROUND: Bacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis. METHODS: The complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes. RESULT: The distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361. CONCLUSION: The study identified thirty chromosome-encoded genes specific to B. anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis.

Lysins as a powerful alternative to combat Bacillus anthracis.

This review gathers all, to the best of our current knowledge, known lysins, mainly bacteriophage-derived, that have demonstrated activity against Bacillus anthracis strains. B. anthracis is a spore-forming, toxin-producing bacteria, naturally dwelling in soil. It is best known as a potential biowarfare threat, an etiological agent of anthrax, and a severe zoonotic disease. Anthrax can be treated with antibiotics (ciprofloxacin, penicillin, doxycycline); however, their administration may take up even to 60 days, and different factors can compromise their effectiveness. Bacterial viruses, bacteriophages (phages), are natural enemies of bacteria and use their lytic enzymes, endolysins (lysins), to specifically kill bacterial cells. Harnessing the potential of lysins to combat bacterial infections holds promise for diminishing antibiotic usage and, consequently, addressing the escalating antibiotic resistance in bacteria. In this context, we list the lysins with the activity against B. anthracis, providing a summary of their lytic properties in vitro and the outcomes observed in animal models. Bacillus cereus strain ATCC 4342/RSVF1, a surrogate for B. anthracis, was also included as a target bacteria. KEY POINTS: • More than a dozen different B. anthracis lysins have been identified and studied. • They fall into three blocks regarding their amino acid sequence similarity and most of them are amidases. • Lysins could be used in treating B. anthracis infections.

Strengthening anthrax outbreak response and preparedness: simulation and stakeholder education in Namisindwa district, Uganda.

BACKGROUND: Anthrax is a zoonotic disease caused by Bacillus anthracis that poses a significant threat to both human health and livestock. Effective preparedness and response to anthrax outbreak at the district level is essential to mitigate the devastating impact of the disease to humans and animals. The current diseaae surveillance in animals and humans uses two different infrastructure systems with online platform supported by established diagnostic facilities. The differences in surveillance systems affect timely outbreak response especially for zoonotic diseases like anthrax. We therefore aimed to assess the feasibility of implementing a simulation exercise for a potential anthrax outbreak in a local government setting and to raise the suspicion index of different district stakeholders for a potential anthrax outbreak in Namisindwa District, Uganda. METHODS: We conducted a field-based simulation exercise and a health education intervention using quantitative data collection methods. The study participants mainly members of the District Taskforce (DTF) were purposively selected given their role(s) in disease surveillance and response at the sub-national level. We combined 26 variables (all dichotomized) assessing knowledge on anthrax and knowledge on appropriate outbreak response measures into an additive composite index. We then dichotomized overall score based on the 80% blooms cutoff i.e. we considered those scoring at least 80% to have high knowledge, otherwise low. We then assessed the factors associated with knowledge using binary logistic regression with time as a proxy for the intervention effect. Odds ratios (ORs) and 95% Confidence intervals (95%CI) have been reported. RESULTS: The overall district readiness score was 35.0% (24/69) and was deficient in the following domains: coordination and resource mobilization (5/16), surveillance (5/11), laboratory capacity (3/10), case management (4/7), risk communications (4/12), and control measures (4/13). The overall community readiness score was 7 out of 32 (22.0%). We noted poor scores of readiness in all domains except for case management (2/2). The knowledge training did not have an effect on the overall readiness score, but improved specific domains such as control measures. Instead tertiary education was the only independent predictor of higher knowledge on anthrax and how to respond to it (OR = 1.57, 95% CI = 1.07-2.31). Training did not have a significant association with overall knowledge improvement but had an effect on several individual knowledge aspects. CONCLUSION: We found that the district's preparedness to respond to a potential anthrax outbreak was inadequate, especially in coordination and mobilisation, surveillance, case management, risk communication and control measures. The health education training intervention showed increased knowledge levels compared to the pre-test and post-test an indicator that the health education sessions could increase the index of suspicion. The low preparedness underscores the urgency to strengthen anthrax preparedness in the district and could have implications for other districts. We deduce that trainings of a similar nature conducted regularly and extensively would have better effects. This study's insights are valuable for improving anthrax readiness and safeguarding public and animal health in similar settings.

Modelling climate change impacts on the spatial distribution of anthrax in Zimbabwe.

BACKGROUND: In Zimbabwe, anthrax is endemic with outbreaks being reported almost annually in livestock, wildlife, and humans over the past 40 years. Accurate modelling of its spatial distribution is key in formulating effective control strategies. In this study, an Ensemble Species Distribution Model was used to model the current and future distribution of anthrax occurrence in Zimbabwe. METHODS: Bioclimatic variables derived from the Beijing Climate Centre Climate System Model were used to model the disease. Collinearity testing was conducted on the 19 bioclimatic variables and elevation to remove redundancy. Variables that had no collinearity were used for anthrax habitat suitability modelling. Two future climate change scenarios for different Representative Concentration Pathways (RCP), RCP4.5 and RCP8.5 were used. Model evaluation was done using true skill, Kappa statistics and receiver operating characteristics. RESULTS: The results showed that under current bioclimatic conditions, eastern and western districts of Zimbabwe were modelled as highly suitable, central districts moderately suitable and southern parts marginally suitable for anthrax occurrence. Future predictions demonstrated that the suitable (8%) and highly suitable (7%) areas for anthrax occurrence would increase under RCP4.5 scenario. In contrast, a respective decrease (11%) and marginal increase (0.6%) of suitable and highly suitable areas for anthrax occurrence were predicted under the RCP8.5 scenario. The percentage contribution of the predictors varied for the different scenarios; Bio6 and Bio18 for the current scenario, Bio2, Bio4 and Bio9 for the RCP4.5 and Bio3 and Bio15 for the RCP8.5 scenarios. CONCLUSIONS: The study revealed that areas currently suitable for anthrax should be targeted for surveillance and prevention. The predicted future anthrax distribution can be used to guide and prioritise surveillance and control activities and optimise allocation of limited resources. In the marginally to moderately suitable areas, effective disease surveillance systems and awareness need to be put in place for early detection of outbreaks. Targeted vaccinations and other control measures including collaborative 'One Health' strategies need to be implemented in the predicted highly suitable areas. In the southern part where a high decrease in suitability was predicted, continued monitoring would be necessary to detect incursions early.

Complement C5 inhibition protects against hemolytic anemia and acute kidney injury in anthrax peptidoglycan-induced sepsis in baboons.

Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of Bacillus anthracis, leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections.

The NLRP1 and CARD8 inflammasomes.

Inflammasomes are multiprotein complexes that activate inflammatory cytokines and induce pyroptosis in response to intracellular danger-associated signals. NLRP1 and CARD8 are related germline-encoded pattern recognition receptors that form inflammasomes, but their activation mechanisms and biological purposes have not yet been fully established. Both NLRP1 and CARD8 undergo post-translational autoproteolysis to generate two non-covalently associated polypeptide chains. NLRP1 and CARD8 activators induce the proteasome-mediated destruction of the N-terminal fragment, liberating the C-terminal fragment to form an inflammasome. Here, we review the danger-associated stimuli that have been reported to activate NLRP1 and/or CARD8, including anthrax lethal toxin, Toxoplasma gondii, Shigella flexneri and the small molecule DPP8/9 inhibitor Val-boroPro, focusing on recent mechanistic insights and highlighting unresolved questions. In addition, we discuss the recently identified disease-associated mutations in NLRP1 and CARD8, the potential role that DPP9's protein structure plays in inflammasome regulation, and the emerging link between NLRP1 and metabolism. Finally, we summarize all of this latest research and consider the possible biological purposes of these enigmatic inflammasomes.

Risk perceptions and preventive preparedness toward anthrax re-emergence within the lens of one health in Northcentral Nigeria.

Anthrax has re-emerged in domestic ruminants in Nigeria with public health concerns. This study assessed stakeholders' perceptions and preventive preparedness toward further resurgence and spread at the human-animal-environment interface. A questionnaire-based cross-sectional study was conducted in Nigeria. Descriptive and analytical statistical analyses were performed at 95% confidence levels. All the 384 recruited stakeholders responded. The majority (96.2%) of animal health practitioners (AHPs) and 56.7% of pastoralists were aware of the recent anthrax re-emergence in Nigeria (p < 0.001). Noteworthy, 88.5% of the AHPs and 32.2% of pastoralists mentioned that anthrax has an environmental component in its transmission to humans and animals. From the environmental perspective, 87.7% of AHPs and 24.0% of pastoralists significantly perceived that soil and aerosol contamination with anthrax spores are highly plausible explanation routes for its re-emergence. Extreme weather events (high rainfall, flooding, winds, and drought) (p = 0.001); grazing of livestock on pastures grown on contaminated soil (p < 0.001), transboundary movement and trade of animals (p = 0.001); introduction of new animals into the herds without quarantine (p = 0.001); and bioterrorism (p < 0.001) were more likely to influence the re-emergence and spread of anthrax. To tackle gaps in knowledge and risk perceptions, and address the socio-economic and anthropogenic drivers, cooperation and collaborations through the lens of the One Health approach are needed. The partnership will promote an integrated disease surveillance system from planning to implementation for the realization of elimination or reduction of the burden of anthrax and other zoonoses in Nigeria and contribute to achieving food safety, food security, and public and ecosystem health.

Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry.

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.

The N-end rule ubiquitin ligase UBR2 mediates NLRP1B inflammasome activation by anthrax lethal toxin.

Anthrax lethal toxin (LT) is known to induce NLRP1B inflammasome activation and pyroptotic cell death in macrophages from certain mouse strains in its metalloprotease activity-dependent manner, but the underlying mechanism is unknown. Here, we establish a simple but robust cell system bearing dual-fluorescence reporters for LT-induced ASC specks formation and pyroptotic lysis. A genome-wide siRNA screen and a CRISPR-Cas9 knockout screen were applied to this system for identifying genes involved in LT-induced inflammasome activation. UBR2, an E3 ubiquitin ligase of the N-end rule degradation pathway, was found to be required for LT-induced NLRP1B inflammasome activation. LT is known to cleave NLRP1B after Lys44. The cleaved NLRP1B, bearing an N-terminal leucine, was targeted by UBR2-mediated ubiquitination and degradation. UBR2 partnered with an E2 ubiquitin-conjugating enzyme UBE2O in this process. NLRP1B underwent constitutive autocleavage before the C-terminal CARD domain. UBR2-mediated degradation of LT-cleaved NLRP1B thus triggered release of the noncovalent-bound CARD domain for subsequent caspase-1 activation. Our study illustrates a unique mode of inflammasome activation in cytosolic defense against bacterial insults.

Systems biology approach to understand the interplay between Bacillus anthracis and human host genes that leads to CVDs.

Humans infected with invasive Bacillus anthracis (B. anthracis) have a very poor prognosis and are at high risk for developing cardiovascular diseases (CVDs) and shock. Several bacterial elements probably have significant pathogenic roles in this pathogenic process of anthrax. In our current work, we have analysed the molecular level interactions between B. anthracis and human genes to understand the interplay during anthrax that leads to the CVDs. Our results have shown dense interactions between the functional partners in both host and the B. anthracis Gene interaction network (GIN). The functional enrichment analysis indicated that the clusters in the host GIN had genes related to hypoxia and autophagy in response to the lethal toxin; and genes related to adherens junction and actin cytoskeleton in response to edema toxin play a significant role in multiple stages of the disease. The B. anthracis genes BA_0530, guaA, polA, rpoB, ribD, secDF, metS, dinG and human genes ACTB, EGFR, EP300, CTNNB1, ESR1 have shown more than 50 direct interactions with the functional partners and hence they can be considered as hub genes in the network and they are observed to have important roles in CVDs. The outcome of our study will help to understand the molecular pathogenesis of CVDs in anthrax. The hub genes reported in the study can be considered potential drug targets and they can be exploited for new drug discovery.

Electrochemical, optical and mass-based immunosensors: A comprehensive review of Bacillus anthracis detection methods.

A biosensor is an analytical device whose main components include transducer and bioreceptor segments. The combination of biological recognition with the ligand is followed by transformation into physical or chemical signals. Many publications describe biological sensors as user-friendly, easy, portable, and less time-consuming than conventional methods. Among major categories of methods for the detection of Bacillus anthracis, such as culture-based microbiological method, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), microarray-based techniques sensors with bioreceptors have been highlighted which particular emphasis is placed on herein. There are several types of biosensors based on various chemical or physical transducers (e.g., electrochemical, optical, piezoelectric, thermal or magnetic electrodes) and the type of biological materials used (e.g., enzymes, nucleic acids, antibodies, cells, phages or tissues). In recent decades, antibody-based sensors have increasingly gained popularity due to their reliability, sensitivity and rapidness. The fundamental principle of antibody-based sensors is mainly based on the molecular recognition between antigens and antibodies. Therefore, immunosensors that detect B. anthracis surface antigens can provide a rapid tool for detecting anthrax bacilli and spores, especially in situ. This review provides a comprehensive summary of immunosensor-based methods using electrochemical, optical, and mass-based transducers to detect B. anthracis.

Case-control study of human anthrax outbreak investigation in farta woreda, South Gondar, Northwest Ethiopia.

BACKGROUND: Anthrax is a zoonotic disease caused by the Bacillus anthracis bacteria, which is one of the top five important livestock diseases and the second top priority zoonotic disease, next to rabies, in Ethiopia, which remains a major problem for animals and public health in Ethiopia. This study was conducted to verify the existence of the outbreak, determine risk factors, and implement measures to control the anthrax outbreak in Farta woreda, South Gondar zone, Northwest Ethiopia in 2019. METHODS: A community-based case-control study was conducted from March 25 to April 1, 2019. A structured questionnaire was used to collect data and for review of documents and discussion with livestock and health office staff. The collected data were analyzed by SPSS and presented in tables and graphs. RESULTS: A total of 20 human anthrax cases with an attack rate of 2.5 per 1000 population were reported from the affected kebele. The age of the cases ranged from 1 month to 65 years (median age = 37.5 years). Of the total cases, 66.7% were male and 77.8% were 15 and older. The probability of developing anthrax among people who had unvaccinated animals was higher than in those who didn't have unvaccinated animals with an AOR = 8.113 (95% CI 1.685-39.056) and the probability of getting anthrax in relation to people's awareness of anthrax was AOR = 0.114 (95% CI 0.025-0.524). CONCLUSION: An anthrax outbreak occurred in Wawa Mengera Kebele of Farta woreda. The presence of unvaccinated animals in a household was found to be a risk factor for anthrax cases. Timely animal vaccination and strengthening health education on the vaccination of animals, mode of transmission, and disposal of dead animals are essential for preventing anthrax cases.