A Sporulation-Independent Way of Life for Bacillus thuringiensis in the Late Stages of an Infection.

The formation of endospores has been considered the unique survival and transmission mode of sporulating Firmicutes due to the exceptional resistance and persistence of this bacterial form. However, nonsporulated bacteria (Spo-) were reported at the early stages following the death of a host infected with Bacillus thuringiensis, an entomopathogenic sporulating bacterium. Here, we investigated the characteristics of the bacterial population in the late stages of an infection in the B. thuringiensis/Galleria mellonella infection model. Using fluorescent reporters and molecular markers coupled to flow cytometry, we demonstrated that the Spo- cells persist and constitute about half of the population 2 weeks post-infection (p.i.). Protein synthesis and growth recovery assays indicated that they are in a metabolically slowed-down state. These bacteria were extremely resistant to the insect cadaver environment, which did not support growth of in vitro-grown vegetative cells and spores. A transcriptomic analysis of this subpopulation at 7 days p.i. revealed a signature profile of this state, and the expression analysis of individual genes at the cell level showed that more bacteria mount an oxidative stress response as their survival time increases, in agreement with the increase of the free radical level in the host cadaver and in the number of reactive oxygen species (ROS)-producing bacteria. Altogether, these data show for the first time that nonsporulated bacteria are able to survive for a prolonged period of time in the context of an infection and indicate that they engage in a profound adaptation process that leads to their persistence in the host cadaver. IMPORTANCE Bacillus thuringiensis is an entomopathogenic bacterium widely used as a biopesticide. It belongs to the Bacillus cereus group, comprising the foodborne pathogen B. cereus sensu stricto and the anthrax agent Bacillus anthracis. Like other Firmicutes when they encounter harsh conditions, these Gram-positive bacteria can form dormant cells called spores. Due to its highly resistant nature, the spore was considered the unique mode of long-term survival, eclipsing any other form of persistence. Breaking this paradigm, we observed that B. thuringiensis was able to persist in its host cadaver in a nonsporulated form for at least 14 days. Our results show that these bacteria survived in the cadaver environment, which proved hostile for actively growing bacteria by engaging in a profound adaptation process. Studying this facet of the life cycle of a sporulating bacterium provides new fundamental knowledge and might lead to the development of strategies to combat sporulating pathogenic species.

Anthrax hotspot mapping in Kenya support establishing a sustainable two-phase elimination program targeting less than 6% of the country landmass.

Using data collected from previous (n = 86) and prospective (n = 132) anthrax outbreaks, we enhanced prior ecological niche models (ENM) and added kernel density estimation (KDE) approaches to identify anthrax hotspots in Kenya. Local indicators of spatial autocorrelation (LISA) identified clusters of administrative wards with a relatively high or low anthrax reporting rate to determine areas of greatest outbreak intensity. Subsequently, we modeled the impact of vaccinating livestock in the identified hotspots as a national control measure. Anthrax suitable areas included high agriculture zones concentrated in the western, southwestern and central highland regions, consisting of 1043 of 1450 administrative wards, covering 18.5% country landmass, and hosting 30% of the approximately 13 million cattle population in the country. Of these, 79 wards covering 5.5% landmass and hosting 9% of the cattle population fell in identified anthrax hotspots. The rest of the 407 administrative wards covering 81.5% of the country landmass, were classified as low anthrax risk areas and consisted of the expansive low agricultural arid and semi-arid regions of the country that hosted 70% of the cattle population, reared under the nomadic pastoralism. Modelling targeted annual vaccination of 90% cattle population in hotspot administrative wards reduced > 23,000 human exposures. These findings support an economically viable first phase of anthrax control program in low-income countries where the disease is endemic, that is focused on enhanced animal and human surveillance in burden hotspots, followed by rapid response to outbreaks anchored on public education, detection and treatment of infected humans, and ring vaccination of livestock. Subsequently, the global anthrax elimination program focused on sustained vaccination and surveillance in livestock in the remaining few hotspots for a prolonged period (> 10 years) may be implemented.

Evaluation of sample processing methods to improve the detection of Bacillus anthracis in difficult sample matrices.

Large area sampling approaches have been developed and implemented by the US Environmental Protection Agency (EPA) to increase sample sizes, and potentially representativeness, in outdoor urban environments (e.g., concrete, asphalt, grass/landscaping). These sampling approaches could be implemented in response to an outdoor biological contamination incident or bioterrorism attack to determine the extent of contamination and for clearance following remediation. However, sample collection over large areas often contains an extensive amount of co-collected debris and native background microorganisms that interfere with the detection of biological threat agents. Sample processing methods that utilize basic laboratory equipment amenable to field deployment were selected and applied to turbid aqueous samples (TAS) to reduce particulates and native environmental organisms prior to culture and rapid viability-polymerase chain reaction (RV-PCR) analytical methods. Bacillus anthracis Sterne (BaS) spores were spiked into TAS collected by soil grab, wet vacuum collection from an outdoor concrete surface, or storm water runoff from an urban parking lot. The implementation of a sample processing method improved the sensitivity of culture and RV-PCR analytical methods for BaS spore detection in soil and wet vacuum TAS samples compared to baseline (minimal to no field processing methods applied). For soil, when the processing method was applied, samples with 15 colony forming units (CFU)/ml (60 CFU/g) and 1.5 CFU/mL (6 CFU/g) BaS spore load were detected using culture and RV-PCR, respectively. Most notably, the processing methods greatly improved the sensitivity of the RV-PCR analytical method for the wet vacuum TAS from no detection at the 1500 CFU/mL BaS spore load level to as low as 1.5 CFU/mL BaS spore load.

A recombinant chimeric protein of protective antigen and lethal factor from Bacillus anthracis in polymeric nanocapsules showed a strong immune response in mice: a potential high efficacy vaccine against anthrax.

Anthrax is a serious infectious disease caused by Bacillus anthracis, rod-shaped gram-positive bacteria. The disease infects both humans and animals and causes severe illness.  Many vaccines have been developed for anthrax, but the vaccine with very high efficacy is yet to be developed. To overcome the problems of efficacy posed by the existing vaccines, a recombinant chimeric fusion protein containing domain 1 of lethal factor (LFD1) and domain 4 of Bacillus anthracis protective antigen (PA4) was used as antigen in copolymeric nanocapsules (NCs). Accordingly, the solvent evaporation double emulsion method was used to produce NCs containing recombinant chimeric fusion protein (LFD1-PA4). Zeta sizer and potential of nanoparticles, nanoparticle loading efficiency, release pattern of recombinant protein, and the possible effect of polylactic acid-polyethylene glycol (PLA-PEG) nanoparticle production method were investigated. Mice were used to test and evaluate the immune response. The mean titer of antibody produced against loaded LFD1-PA4 compared to free form showed a significant difference. The difference in antibody titer between the groups of once injected, twice injected, and free antigen was significant, and the highest antibody titer was found in the mice twice injected. In addition, a single-time loaded injection showed significantly higher antibodies than the free form injection indicating that loaded LFD1-PA4 into PLA-PEG nanoparticles elicits a stronger immune response. This study showed that LFD1-PA4 fusion protein from Bacillus anthracis served as an active antigen in mice. Also, the nanocarrier (PLA-PEG) containing the antigen can stimulate the immune system in the mice, owing to their controlled release property.

Role of serine/threonine protein phosphatase PrpN in the life cycle of Bacillus anthracis.

Reversible protein phosphorylation at serine/threonine residues is one of the most common protein modifications, widely observed in all kingdoms of life. The catalysts controlling this modification are specific serine/threonine kinases and phosphatases that modulate various cellular pathways ranging from growth to cellular death. Genome sequencing and various omics studies have led to the identification of numerous serine/threonine kinases and cognate phosphatases, yet the physiological relevance of many of these proteins remain enigmatic. In Bacillus anthracis, only one ser/thr phosphatase, PrpC, has been functionally characterized; it was reported to be non-essential for bacterial growth and survival. In the present study, we characterized another ser/thr phosphatase (PrpN) of B. anthracis by various structural and functional approaches. To examine its physiological relevance in B. anthracis, a null mutant strain of prpN was generated and shown to have defects in sporulation and reduced synthesis of toxins (PA and LF) and the toxin activator protein AtxA. We also identified CodY, a global transcriptional regulator, as a target of PrpN and ser/thr kinase PrkC. CodY phosphorylation strongly controlled its binding to the promoter region of atxA, as shown using phosphomimetic and phosphoablative mutants. In nutshell, the present study reports phosphorylation-mediated regulation of CodY activity in the context of anthrax toxin synthesis in B. anthracis by a previously uncharacterized ser/thr protein phosphatase-PrpN.

DnaJ and ClpX Are Required for HitRS and HssRS Two-Component System Signaling in Bacillus anthracis.

Bacillus anthracis is the causative agent of anthrax. This Gram-positive bacterium poses a substantial risk to human health due to high mortality rates and the potential for malicious use as a bioterror weapon. To survive within the vertebrate host, B. anthracis relies on two-component system (TCS) signaling to sense host-induced stresses and respond to alterations in the environment through changes in target gene expression. HitRS and HssRS are cross-regulating TCSs in B. anthracis that respond to cell envelope disruptions and high heme levels, respectively. In this study, an unbiased and targeted genetic selection was designed to identify gene products that are involved in HitRS and HssRS signaling. This selection led to the identification of inactivating mutations within dnaJ and clpX that disrupt HitRS- and HssRS-dependent gene expression. DnaJ and ClpX are the substrate-binding subunits of the DnaJK protein chaperone and ClpXP protease, respectively. DnaJ regulates the levels of HitR and HitS to facilitate signal transduction, while ClpX specifically regulates HitS levels. Together, these results reveal that the protein homeostasis regulators, DnaJ and ClpX, function to maintain B. anthracis signal transduction activities through TCS regulation.

Treatment of experimental anthrax with pegylated circularly permuted capsule depolymerase.

Anthrax is considered one of the most dangerous bioweapon agents, and concern about multidrug-resistant strains has led to the development of alternative therapeutic approaches that target the antiphagocytic capsule, an essential virulence determinant of Bacillus anthracis, the causative agent. Capsule depolymerase is a γ-glutamyltransferase that anchors the capsule to the cell wall of B. anthracis. Encapsulated strains of B. anthracis can be treated with recombinant capsule depolymerase to enzymatically remove the capsule and promote phagocytosis and killing by human neutrophils. Here, we show that pegylation improved the pharmacokinetic and therapeutic properties of a previously described variant of capsule depolymerase, CapD-CP, when delivered 24 hours after exposure every 8 hours for 2 days for the treatment of mice infected with B. anthracis. Mice infected with 382 LD50 of B. anthracis spores from a nontoxigenic encapsulated strain were completely protected (10 of 10) after treatment with the pegylated PEG-CapD-CPS334C, whereas 10% of control mice (1 of 10) survived with control treatment using bovine serum albumin (P < 0.0001, log-rank analysis). Treatment of mice infected with five LD50 of a fully virulent toxigenic, encapsulated B. anthracis strain with PEG-CapD-CPS334C protected 80% (8 of 10) of the animals, whereas 20% of controls (2 of 10) survived (P = 0.0125, log-rank analysis). This strategy renders B. anthracis susceptible to innate immune responses and does not rely on antibiotics. These findings suggest that enzyme-catalyzed removal of the capsule may be a potential therapeutic strategy for the treatment of multidrug- or vaccine-resistant anthrax and other bacterial infections.

Mechanistic elucidation of germination potential and growth of Sesbania sesban seedlings with Bacillus anthracis PM21 under heavy metals stress: An in vitro study.

Soils contaminated with heavy metals such as Chromium (Cr) and Cadmium (Cd) severely impede plant growth. Several rhizospheric microorganisms support plant growth under heavy metal stress. In this study, Cr and Cd stress was applied to in vitro germinating seedlings of a Legume plant species, Sesbania sesban, and investigated the plant growth potential in presence and absence of Bacillus anthracis PM21 bacterial strain under heavy metal stress. The seedlings were exposed to different concentrations of Cr (25-75 mg/L) and Cd (100-200 mg/L) in Petri plates. Growth curve analysis of B. anthracis PM21 revealed its potential to adapt Cr and Cd stress. The bacteria supported plant growth by exhibiting ACC-deaminase activity (1.57-1.75 µM of α-ketobutyrate/h/mg protein), producing Indole-3-acetic acid (99-119 µM/mL) and exopolysaccharides (2.74-2.98 mg/mL), under heavy metal stress condition. Analysis of variance revealed significant differences in growth parameters between the seedlings with and without bacterial inoculation in metal stress condition. The combined Cr+Cd stress (75 + 200 mg/L) significantly reduced root length (70%), shoot length (24%), dry weight (54%) and fresh weight (57%) as compared to control. Conversely, B. anthracis PM21 inoculation to seedlings significantly increased (p ≤ 0.05) seed germination percentage (5%), root length (31%), shoot length (23%) and photosynthetic pigments (Chlorophyll a: 20%; Chlorophyll b: 16% and total chlorophyll: 18%), as compared to control seedlings without B. anthracis PM21 inoculation. The B. anthracis PM21 inoculation also enhanced activities of antioxidant enzymes, including superoxide dismutase (52%), peroxidase (66%), and catalase (21%), and decreased proline content (56%), electrolyte leakage (50%), and malondialdehyde concentration (46%) in seedlings. The B. anthracis PM21 inoculated seedlings of S. sesban exhibited significantly high (p ≤ 0.05) tissue deposition of Cr (17%) and Cd (16%) as compared to their control counterparts. Findings of the study suggested that B. anthracis PM21 endured metal stress through homeostasis of antioxidant activities, and positively impacted S. sesban growth and biomass. Further experiments in controlled conditions are necessary for investigating phytoremediation potential of S. sesban in metal-contaminated soils in presence of B. anthracis PM21 bacterial strain.

Common garlic (Allium sativum L.) has potent Anti-Bacillus anthracis activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrointestinal anthrax, a disease caused by Bacillus anthracis, remains an important but relatively neglected endemic disease of animals and humans in remote areas of the Indian subcontinent and some parts of Africa. Its initial symptoms include diarrhea and stomachache. In the current study, several common plants indicated for diarrhea, dysentery, stomachache or as stomachic as per traditional knowledge in the Indian subcontinent, i.e., Aegle marmelos (L.) Correa (Bael), Allium cepa L. (Onion), Allium sativum L. (Garlic), Azadirachta indica A. Juss. (Neem), Berberis asiatica Roxb. ex DC. (Daruharidra), Coriandrum sativum L. (Coriander), Curcuma longa L. (Turmeric), Cynodon dactylon (L.) Pers. (Bermuda grass), Mangifera indica L. (Mango), Morus indica L. (Black mulberry), Ocimum tenuiflorum L. (Ocimum sanctum L., Holy Basil), Ocimum gratissimum L. (Ram Tulsi), Psidium guajava L. (Guava), Zingiber officinale Roscoe (Ginger), were evaluated for their anti-Bacillus anthracis property. The usage of Azadirachta indica A. Juss. and Curcuma longa L. by Santals (India), and Allium sp. by biblical people to alleviate anthrax-like symptoms is well documented, but the usage of other plants is traditionally only indicated for different gastrointestinal disturbances/conditions. AIM OF THE STUDY: Evaluate the above listed commonly available edible plants from the Indian subcontinent that are used in the traditional medicine to treat gastrointestinal diseases including those also indicated for anthrax-like symptoms for the presence of potent anti-B. anthracis activity in a form amenable to use by the general population in the endemic areas. MATERIALS AND METHODS: Aqueous extracts made from fourteen plants indicated above were screened for their anti-B. anthracis activity using agar-well diffusion assay (AWDA) and broth microdilution methods. The Aqueous Garlic Extract (AGE) that displayed most potent anti-B. anthracis activity was assessed for its thermostability, stability under pH extremes encountered in the gastrointestinal tract, and potential antagonistic interaction with bile salts as well as the FDA-approved antibiotics used for anthrax control. The bioactive fractions from the AGE were isolated by TLC coupled bioautography followed by their characterization using GC-MS. RESULTS: Garlic (Allium sativum L.) extract was identified as the most promising candidate with bactericidal activity against B. anthracis. It consistently inhibited the growth of B. anthracis in AWDA and decreased the viable colony-forming unit counts in liquid-broth cultures by 6-logs within 6-12 h. The AGE displayed acceptable thermostability (>80% anti-B. anthracis activity retained on incubation at 50 °C for 12 h) and stability in gastric pH range (2-8). It did not antagonize the activity of FDA-approved antibiotics used for anthrax control. GC-MS analysis of the TLC separated bioactive fractions of AGE indicated the presence of previously unreported constituents such as phthalic acid derivatives, acid esters, phenyl group-containing compounds, steroids etc. CONCLUSION: The Aqueous Garlic Extract (AGE) displayed potent anti-B. anthracis activity. It was better than that displayed by Azadirachta indica A. Juss. (Neem) and Mangifera indica L., while Curcuma longa L. (Turmeric) did not show any activity under the assay conditions used. Further work should be undertaken to explore the possible application of AGE in preventing anthrax incidences in endemic areas.

Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin.

BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

Directed evolution reveals the mechanism of HitRS signaling transduction in Bacillus anthracis.

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.

Dual-emission of silicon nanoparticles encapsulated lanthanide-based metal-organic frameworks for ratiometric fluorescence detection of bacterial spores.

Dipicolinic acid (DPA) is employed as a significant biomarker to detect Bacillus anthracis, which can do serious damages to the health of human beings. Hence, it is crucial to develop a fast and highly efficient strategy for DPA monitoring. In this work, based on silicon nanoparticles (Si NPs) and terbium metal-organic frameworks (Tb-MOFs), a hybrid structure (Si NPs/Tb-MOFs) as a novel dual-emitting fluorescence probe was fabricated for ratiometric detection of DPA, where blue light-emitting Si NPs (Ex: 280 nm; Em: 422 nm) are encapsulated into green light-emitting Tb-MOFs (Ex: 280 nm; Em: 547 nm). The optical properties and chemical composition of the as-obtained Si NPs/Tb-MOFs were characterized in detail. The Si NPs/Tb-MOFs probe not merely possesses the merits of a facile synthesis method but also is an excellent fluorescence probe. The response time towards DPA is less than 30 s, revealing that the process of detecting DPA can be completed in such a short time. The limit of detection for DPA is 5.3 nM, which is four orders of magnitude lower than an infectious dosage of anthrax spores for human beings (60 µM). This dual-emitting Si NPs/Tb-MOFs probe with interference-free and self-calibrating properties may be a potential candidate for further development in medical diagnosis. Graphical abstract.

Isolation of two salt-tolerant strains from activated sludge and its COD degradation characteristics from saline organic wastewater.

The efficient biological treatment of saline wastewater has been limited by the low activities of microorganisms under saline conditions. High salinity poses unbalance osmotic stress across the cell wall and even leads to cell plasmolysis. In this work, we aim to isolate salt-tolerant bacterial strains from activated sludge, and apply them for degrading chemical oxygen demand (COD) of saline organic wastewater. Two salt-tolerant strains were screened and isolated from activated sludge, which was domesticated with salty water for over 300 days. The two strains were identified as Bacillus cereus (strain A) and Bacillus anthracis (strain B) through 16S rRNA sequencing. The degradation characteristics of strain A were explored. The results showed the relative membrane permeability of strain A remained stable under high salt stress, which glycine and proline play an important role to maintain cell osmotic. The protein and soluble sugar amounts of strain were increased by higher salt concentrations. In simulating saline wastewater, the optimum culture temperature, pH, salinity, influent COD concentration and inoculation amount of strain A were 35 °C, 9, 4%, 8000 mg L-1, 6%, respectively. Optimal conditions could provide guidance for the treatment of practical saline wastewater. The linear regression model of each impact factor built based on the result PB experiment revealed that cross-linking time has the most significant influence on COD removal for salt-tolerant strains. It will provide theoretical basis for biological treatment of saline organic wastewater.

Permafrost dynamics and the risk of anthrax transmission: a modelling study.

A recent outbreak of anthrax disease, severely affecting reindeer herds in Siberia, has been reportedly associated to the presence of infected carcasses or spores released from the active layer over permafrost, which is thawing and thickening at increasing rates, thus underlying the re-emerging nature of this pathogen in the Arctic region because of warming temperatures. Anthrax is a global zoonotic and epizootic disease, with a high case-fatality ratio in infected animals. Its transmission is mediated by environmental contamination through highly resistant spores which can persist in the soil for several decades. Here we develop and analyze a new epidemiological model for anthrax transmission that is specifically tailored to the Arctic environmental conditions. The model describes transmission dynamics including also herding practices (e.g. seasonal grazing) and the role of the active layer over permafrost acting as a long-term storage of spores that could be viable for disease transmission during thawing periods. Model dynamics are investigated through linear stability analysis, Floquet theory for periodically forced systems, and a series of simulations with realistic forcings. Results show how the temporal variability of grazing and active layer thawing may influence the dynamics of anthrax disease and, specifically, favor sustained pathogen transmission. Particularly warm years, favoring deep active layers, are shown to be associated with an increase risk of anthrax outbreaks, and may also foster infections in the following years. Our results enable preliminary insights into measures (e.g. changes in herding practice) that may be adopted to decrease the risk of infection and lay the basis to possibly establish optimal procedures for preventing transmission; furthermore, they elicit the need of further investigations and observation campaigns focused on anthrax dynamics in the Arctic environment.

Environmental determinants influencing anthrax distribution in Queen Elizabeth Protected Area, Western Uganda.

Bacillus anthracis, the bacteria that causes anthrax, a disease that primarily affects herbivorous animals, is a soil borne endospore-forming microbe. Environmental distribution of viable spores determines risky landscapes for herbivore exposure and subsequent anthrax outbreaks. Spore survival and longevity depends on suitable conditions in its environment. Anthrax is endemic in Queen Elizabeth Protected Area in western Uganda. Periodic historical outbreaks with significant wildlife losses date to 1950s, but B. anthracis ecological niche in the ecosystem is poorly understood. This study used the Maximum Entropy modeling algorithm method to predict suitable niche and environmental conditions that may support anthrax distribution and spore survival. Model inputs comprised 471 presence-only anthrax occurrence data from park management records of 1956-2010, and 11 predictor variables derived from the World Climatic and Africa Soil Grids online resources, selected considering the ecology of anthrax. The findings revealed predicted suitable niche favoring survival and distribution of anthrax spores as a narrow-restricted corridor within the study area, defined by hot-dry climatic conditions with alkaline soils rich in potassium and calcium. A mean test AUC of 0.94 and predicted probability of 0.93 for anthrax presence were registered. The five most important predictor variables that accounted for 93.8% of model variability were annual precipitation (70.1%), exchangeable potassium (12.6%), annual mean temperature (4.3%), soil pH (3.7%) and calcium (3.1%). The predicted suitable soil properties likely originate from existing sedimentary calcareous gypsum rocks. This has implications for long-term presence of B. anthracis spores and might explain the long history of anthrax experienced in the area. However, occurrence of suitable niche as a restricted hot zone offers opportunities for targeted anthrax surveillance, response and establishment of monitoring strategies in QEPA.

Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state.

Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first.

Frontline Science: Anthrax lethal toxin-induced, NLRP1-mediated IL-1ß release is a neutrophil and PAD4-dependent event.

Anthrax lethal toxin (LT) is a protease that activates the NLRP1b inflammasome sensor in certain rodent strains. Unlike better-studied sensors, relatively little is known about the priming requirements for NLRP1b. In this study, we investigate the rapid and striking priming-independent LT-induced release of IL-1ß in mice within hours of toxin challenge. We find IL-1ß release to be a NLRP1b- and caspase-1-dependent, NLRP3 and caspase-11-independent event that requires both neutrophils and peptidyl arginine deiminiase-4 (PAD4) activity. The simultaneous LT-induced IL-18 response is neutrophil-independent. Bone marrow reconstitution experiments in mice show toxin-induced IL-1ß originates from hematopoietic cells. LT treatment of neutrophils in vitro did not induce IL-1ß, neutrophil extracellular traps (NETs), or pyroptosis. Although platelets interact closely with neutrophils and are also a potential source of IL-1ß, they were unable to bind or endocytose LT and did not secrete IL-1ß in response to the toxin. LT-treated mice had higher levels of cell-free DNA and HMGB1 in circulation than PBS-treated controls, and treatment of mice with recombinant DNase reduced the neutrophil- and NLRP1-dependent IL-1ß release. DNA sensor AIM2 deficiency, however, did not impact IL-1ß release. These data, in combination with the findings on PAD4, suggest a possible role for in vivo NETs or cell-free DNA in cytokine induction in response to LT challenge. Our findings suggest a complex interaction of events and/or mediators in LT-treated mice with the neutrophil as a central player in induction of a profound and rapid inflammatory response to toxin.

Bacillus anthracis Poly-γ-D-Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation.

Bacillus anthracis poly-γ-D-glutamic acid (PGA) capsule is an essential virulent factor that helps the bacterial pathogen to escape host immunity. Like other encapsulated bacterial species, the B. anthracis capsule may also inhibit complement-mediated clearance and ensure bacterial survival in the host. Previous reports suggest that B. anthracis spore proteins inhibit complement activation. However, the mechanism through which the B. anthracis capsule imparts a survival advantage to the active bacteria has not been demonstrated till date. Thus, to evaluate the role of the PGA capsule in evading host immunity, we have undertaken the present head-to-head comparative study of the phagocytosis and complement activation of non-encapsulated and encapsulated B. anthracis strains. The encapsulated virulent strain exhibited resistance toward complement-dependent and complement-independent bacterial phagocytosis by human macrophages. The non-encapsulated Sterne strain was highly susceptible to phagocytosis by THP-1 macrophages, after incubation with normal human serum (NHS), heat-inactivated serum, and serum-free media, thus indicating that the capsule inhibited both complement-dependent and complement-independent opsonic phagocytosis. An increased binding of C3b and its subsequent activation to C3c and C3dg, which functionally act as potent opsonins, were observed with the non-encapsulated Sterne strain compared with the encapsulated strain. Other known mediators of complement fixation, IgG, C-reactive protein (CRP), and serum amyloid P component (SAP), also bound more prominently with the non-encapsulated Sterne strain. Studies with complement pathway-specific, component-deficient serum demonstrated that the classical pathway was primarily involved in mediating C3b binding on the non-encapsulated bacteria. Both strains equally bound the complement regulatory proteins C4BP and factor H. Importantly, we demonstrated that the negative charge of the PGA capsule was responsible for the differential binding of the complement proteins between the non-encapsulated and encapsulated strains. At lower pH closer to the isoelectric point of PGA, the neutralization of the negative charge was associated with an increased binding of C3b and IgG with the encapsulated B. anthracis strain. Overall, our data have demonstrated that the B. anthracis capsule inhibits complement fixation and opsonization resulting in reduced phagocytosis by macrophages, thus allowing the bacterial pathogen to evade host immunity.

Bacillus anthracis Responds to Targocil-Induced Envelope Damage through EdsRS Activation of Cardiolipin Synthesis.

Bacillus anthracis is a spore-forming bacterium that causes devastating infections and has been used as a bioterror agent. This pathogen can survive hostile environments through the signaling activity of two-component systems, which couple environmental sensing with transcriptional activation to initiate a coordinated response to stress. In this work, we describe the identification of a two-component system, EdsRS, which mediates the B. anthracis response to the antimicrobial compound targocil. Targocil is a cell envelope-targeting compound that is toxic to B. anthracis at high concentrations. Exposure to targocil causes damage to the cellular barrier and activates EdsRS to induce expression of a previously uncharacterized cardiolipin synthase, which we have named ClsT. Both EdsRS and ClsT are required for protection against targocil-dependent damage. Induction of clsT by EdsRS during targocil treatment results in an increase in cardiolipin levels, which protects B. anthracis from envelope damage. Together, these results reveal that a two-component system signaling response to an envelope-targeting antimicrobial induces production of a phospholipid associated with stabilization of the membrane. Cardiolipin is then used to repair envelope damage and promote B. anthracis viability.IMPORTANCE Compromising the integrity of the bacterial cell barrier is a common action of antimicrobials. Targocil is an antimicrobial that is active against the bacterial envelope. We hypothesized that Bacillus anthracis, a potential weapon of bioterror, senses and responds to targocil to alleviate targocil-dependent cell damage. Here, we show that targocil treatment increases the permeability of the cellular envelope and is particularly toxic to B. anthracis spores during outgrowth. In vegetative cells, two-component system signaling through EdsRS is activated by targocil. This results in an increase in the production of cardiolipin via a cardiolipin synthase, ClsT, which restores the loss of barrier function, thereby reducing the effectiveness of targocil. By elucidating the B. anthracis response to targocil, we have uncovered an intrinsic mechanism that this pathogen employs to resist toxicity and have revealed therapeutic targets that are important for bacterial defense against structural damage.

Coalescence modeling of intrainfection Bacillus anthracis populations allows estimation of infection parameters in wild populations.

Bacillus anthracis, the etiological agent of anthrax, is a well-established model organism. For B. anthracis and most other infectious diseases, knowledge regarding transmission and infection parameters in natural systems, in large part, comprises data gathered from closely controlled laboratory experiments. Fatal, natural anthrax infections transmit the bacterium through new host-pathogen contacts at carcass sites, which can occur years after death of the previous host. For the period between contact and death, all of our knowledge is based upon experimental data from domestic livestock and laboratory animals. Here we use a noninvasive method to explore the dynamics of anthrax infections, by evaluating the terminal diversity of B. anthracis in anthrax carcasses. We present an application of population genetics theory, specifically, coalescence modeling, to intrainfection populations of B. anthracis to derive estimates for the duration of the acute phase of the infection and effective population size converted to the number of colony-forming units establishing infection in wild plains zebra (Equus quagga). Founding populations are small, a few colony-forming units, and infections are rapid, lasting roughly between 1 d and 3 d in the wild. Our results closely reflect experimental data, showing that small founding populations progress acutely, killing the host within days. We believe this method is amendable to other bacterial diseases from wild, domestic, and human systems.