Bacillus strains efficiently secrete heterologous recombinant proteins as well as multiple proteases, with the latter destroying the former. Strains deleted of multiple secreted proteases are, therefore, preferred as protein expression hosts. We report the genome sequence of one such easily available strain, Bacillus subtilis KO7-S.

AAA+ proteins function as molecular machines that utilize ATP to perform diverse cellular functions, including protein homeostasis, stress regulation, and cell cycle/developmental processes. In this study, we identified a novel AAA+ ATPase BAS PrkA in B. anthracis Sterne 34F2 which has 88 % protein homology to Bacillus subtilis PrkA. Conserved domain analysis confirms BAS PrkA has an N-terminal AAA+ ATPase domain with characteristic Walker A and Walker B motifs and a conserved secondary region of homology (SRH) domain, along with a C-terminal cAMP-dependent protein kinase domain. Based on Alpha Fold3 predicted structure, we classified BAS PrkA as part of Clade III of the AAA+ superfamily. Contrary to the reported enzymatic activity in B. subtilis PrkA, we observed that BAS PrkA has negligible protease and kinase activity under in-vitro conditions. Nonetheless, BAS PrkA plays a significant role in regulating sporulation. It is temporally expressed during Stages II to VI during sporulation. A null mutant of BAS PrkA exhibits severe sporulation defects, with reduced spore viability, and down regulation of genes related to spore-coat formation. These phenotypes were restored in a complementation strain expressing BAS PrkA ectopically. Additionally, the null mutant strain showed compromised growth under ionic-osmotic stress conditions. Analysis of the BAS PrkA interactome revealed enrichment of two proteins, ProA and EzrA, that are implicated in osmotic stress response and the sporulation process, respectively. These findings show that BAS PrkA plays a critical role in sporulation and osmotic stress response in B. anthracis .

Hemolysin BL (HBL) is a tripartite α‑pore‑forming toxin and a major Bacillus cereus virulence factor. Although LITAF and its homolog CDIP1 were previously identified as host receptors that promote HBL‑induced cytolysis, mice lacking both proteins remain only partially protected, indicating the existence of additional determinants of susceptibility. Using genome‑wide CRISPR knockout screens in Litaf-/-/Cdip1-/- mouse embryonic fibroblasts and human HT1080 cells, we identify caveolin‑1 (CAV1), a caveola‑associated membrane protein, as a third conserved host factor required for HBL cytolytic activity. Confocal imaging and co‑immunoprecipitation analyses show that CAV1 colocalizes with HBL and physically associates with the toxin at the plasma membrane. Notably, mice lacking all three factors (Litaf/Cdip1/Cav1 triple knockouts) are completely resistant to HBL toxin challenge, establishing CAV1 as an additional host factor mediating HBL toxin cytotoxicity. These findings define a tripartite host factor system required for HBL toxin activity and position CAV1 as an additional host determinant to B. cereus virulence.

Bacillus strains efficiently secrete heterologous recombinant proteins as well as multiple proteases, with the latter destroying the former. Strains deleted of multiple secreted proteases are therefore preferred as protein expression hosts. We report the genome sequence of one such easily available strain, Bacillus anthracis BH500.

Double-stranded (ds) RNAs are major structural components of the transcriptome, hallmarks of viral infection, and primary triggers of innate immune responses. The J2 monoclonal antibody is the gold-standard method to discover and map endogenous dsRNAs across subcellular locations and cell surfaces, detect exogenous RNAs in viral infection, and surveil mRNA prophylactics and therapeutics for inflammatory dsRNAs. To define its epitope, specificity, and mechanism, we determine a 2.85 Å co-crystal structure of J2 antigen-binding fragment (Fab) bound to dsRNA. J2 uses its heavy and light chains in tandem to track the dsRNA minor groove, recognizing a staggered 8-bp duplex. J2 is highly selective for dsRNAs, requires 14 bp for robust binding, and exhibits greatly diminished binding for GC-rich dsRNAs. J2 and the R-loop-specific S9.6 antibody share a common recognition strategy distinct from intracellular dsRNA-binding proteins. This study provides mechanistic insights into dsRNA recognition and establishes a framework for reliable application and data interpretation of the J2 antibody in RNA discovery. The J2 antibody is widely used for detecting double-stranded RNAs. Here, Bou-Nader et al. define its nucleic acid specificity and recognition mechanism by solving its co-crystal structure bound to dsRNA, establishing a framework for its reliable use in RNA detection.

Double-stranded (ds) RNAs are major structural components of the transcriptome, hallmarks of viral infection, and primary triggers of innate immune responses. The J2 monoclonal antibody is the gold-standard method to discover and map endogenous dsRNAs across subcellular locations and cell surfaces, detect exogenous RNAs in viral infection, and surveil mRNA prophylactics and therapeutics for inflammatory dsRNAs. To define its epitope, specificity, and mechanism, we determine a 2.85 Å co-crystal structure of J2 antigen-binding fragment (Fab) bound to dsRNA. J2 uses its heavy and light chains in tandem to track the dsRNA minor groove, recognizing a staggered 8-bp duplex. J2 is exquisitely selective for dsRNAs, requires 14 bp for robust binding, and exhibits greatly diminished binding for GC-rich dsRNAs. J2 and R-loop-specific S9.6 antibody share a common recognition strategy distinct from intracellular dsRNA-binding proteins. This study provides mechanistic insights into dsRNA recognition and establishes a framework for reliable application and data interpretation of the J2 antibody in RNA discovery.

Endothelial permeability induced by the potent adenylate cyclase edema toxin (ET) is central to bacterial dissemination and lethal vascular collapse during infections caused by Bacillus anthracis. Antibiotic and antitoxin treatments are ineffective against anthrax lethal toxemia once high doses of toxins have been released. This study uncovers a critical cAMP-dependent disruption of the F-actin network by ET in human brain microvascular endothelial cells (HBMECs), mediated by Rac1 and cofilin. Rac1 activation by ET leads to a loss of cell area and monolayer permeability. These effects are preceded by the rapid cAMP-independent activation of IGF1R and EGFR and their respective downstream effectors PI3K/AKT and MEK/ERK, which contribute to F-actin remodeling and permeability induced by ET. Consistent with these findings, Rac1, PI3K, and MEK inhibitors prevent ET-induced edema in a mouse footpad model, providing the in vivo pre-clinical validation of their therapeutic potential.

Anthrax lethal toxin (LT) and edema toxin (ET) are two of the major virulence factors of Bacillus anthracis, the causative pathogen of anthrax disease. While the roles of LT in anthrax pathogenesis have been extensively studied, the pathogenic mechanism of ET remains poorly understood. ET is a calmodulin-dependent adenylate cyclase that elevates intracellular cAMP by converting ATP to cAMP. Thus, it was postulated that the ET-induced in vivo toxicity is mediated by certain cAMP-dependent events. However, mechanisms linking cAMP elevation and ET-induced damage have not been established. Cholera toxin is another bacterial toxin that increases cAMP. This toxin is known to cause severe intestinal fluid secretion and dehydration by cAMP-mediated activation of protein kinase A (PKA), which in turn activates cystic fibrosis transmembrane conductance regulator (CFTR). The cAMP-activated PKA phosphorylation of CFTR on the surface of intestinal epithelial cells leads to an efflux of chloride ions accompanied by secretion of H2O into the intestinal lumen, causing rapid fluid loss, severe diarrhea and dehydration. Due to similar in vivo effects, it was generally believed that ET and cholera toxin would exhibit a similar pathogenic mechanism. Surprisingly, in this work, we found that cAMP-mediated PKA/CFTR activation is not essential for ET to exert its in vivo toxicity. Instead, our data suggest that ET-induced ATP depletion may play an important role in the toxin's pathogenesis.

Since the discovery by Fleming that a mold could inhibit the growth of bacteria, each new antibiotic developed to treat infections quickly lost its efficacy due to the emergence of resistant strains. As a result, the ongoing threat by antibiotic-resistant pathogens would benefit from new strategies to combat bacterial infections. An ideal drug is one which is efficacious, can limit selective pressure against the pathogen, and potentially augment the currently available antibiotics to restore their efficacy. Targeting virulence factors used by bacteria to establish infections has the potential to meet these goals. Anthrax edema factor (EF), an adenylate cyclase secreted by Bacillus anthracis, which causes anthrax, is an example of this type of virulence factor. Our previous work showed that the activity of EF can be blocked with small molecule covalent inhibitors targeting the catalytic site. The current report extends this work with the discovery of enzyme-activated-substrate inhibitors which display improved drug-like properties and stability.

Pathogenic Bacillus anthracis strains carry two plasmids - pX01, which encodes a tripartite protein exotoxin complex (PA, LF, and EF); and pX02, which encodes a poly-D-gamma-glutamic acid capsule. A multidomain transcription factor, AtxA, regulates the expression of these virulence genes. AtxA has two DNA-binding Helix-Turn-Helix (HTH) domains, two phosphoenolpyruvate: carbohydrate phosphotransferase system regulatory domains (PRD1 and PRD2), and a putative EIIB domain (a component of PTS sugar transport EII-complexes). Previous studies showed that glucose and CO2 increase AtxA-dependent toxin gene transcription, along with histidine phosphorylation of PRD1 and PRD2. Our transcriptional profiling of virulence factors, PA secretion, and fluorescent reporter strain analyses confirms a synergistic effect of glucose and CO2 on AtxA-dependent toxin production. Deletion of AtxA (ΔatxA) significantly reduced glucose uptake in bacteria, suggesting that AtxA may act within the glucose-PTS system. Mutation analysis of the EIIB domain of AtxA identified the cysteine at position 402 as essential for the transcriptional activity of AtxA. Deletion of glucose PTS permease PtsG (ΔptsG) significantly reduced the expression of PA, LF, and EF. Loss of PtsG also caused attenuation in a mouse model of infection. Intracellular imaging using FLIM confirms a physical interaction of PtsG and AtxA through EIIB domain of AtxA. Using phosphomimetic and phosphoablative mutants of AtxA, we confirmed that the physical interaction of PtsG and AtxA is essential for AtxA activity. Finally, the synergy between glucose and CO2 was targeted by deleting pyruvate carboxylase Pyc (Δpyc), which regulates anaplerosis. This deletion confirms that Pyc stimulates the level of phosphoenolpyruvate (PEP) and increases the phosphorelay in glucose-PTS to enhance AtxA activity. Therefore, we propose that a histidine-phosphorelay from PEP regulates AtxA via PTS enzymatic activity, impacting AtxA activity through physical interaction of AtxA and PtsG. Finally, we propose AtxA as an integral component of the glucose-PTS, where transcriptional activity of AtxA is regulated by environmental signals including glucose and CO2.

Lethal toxin (LT), the major virulence factor of Bacillus anthracis, proteolytically inactivates MEKs and disables downstream ERK, p38 and JNK pathway signalling leading to tissue damage and mortality. Therapies for LT-induced damage after host cell internalization of the toxin are lacking. Here we constructed MEK variants in which the LT proteolytic site was modified: MEK2(P10V/A11D), MEK3(I27D) and MEK6(I15D). These variants were resistant to proteolysis by LT. Expression in cells enabled sustained activation of ERK and p38 pathways and promoted cell survival upon LT treatment. Survival of LT- or B. anthracis-challenged MEK variant transgenic mice also increased compared with controls. We found that LT-mediated disruption of both ERK and p38 pathway is essential for anthrax pathogenesis. We show that engagement of upstream receptor tyrosine kinases reactivated the LT-disrupted ERK pathway, as did administering a cocktail of EGF, GM-CSF and FGF2 growth factors, which significantly increased survival of LT- or B. anthracis-challenged mice. These findings offer potential towards developing damage-limiting therapeutic strategies for anthrax.

Mammary tumors are the most frequent type of neoplasms in intact female dogs. New therapies that target neoplastic cells without affecting normal cells are highly sought. The Bacillus anthracis toxin has been reengineered to target tumor cells that express urokinase plasminogen activators and metalloproteinases. In previous studies carried out in our laboratory, the reengineered anthrax toxin had inhibitory effects on canine oral mucosal melanoma and canine osteosarcoma cells. In this study, five canine neoplastic epithelial cell lines (four adenocarcinomas and one adenoma) and one non-neoplastic canine mammary epithelial cell line were treated with different concentrations of reengineered anthrax toxin components. Cell viability was quantified using an MTT assay and half-maximal inhibitory concentration (IC50) values. Cell lines were considered sensitive when the IC50 was lower than 5000 ng/ml. One canine mammary adenocarcinoma cell line and one mammary adenoma cell line showed significantly decreased viability after treatment, whereas the non-neoplastic cell line was resistant. We conclude that the reengineered anthrax toxin may be considered a targeted therapy for canine mammary neoplasms while preserving normal canine mammary epithelial cells.

Pathogenic B. anthracis carry two plasmids: pX01, which encodes a tripartite protein exotoxin complex (PA, LF, and EF); and pX02, which encodes a poly-D-glutamic acid capsule. A multidomain transcription factor, AtxA, regulates the expression of these virulence genes. AtxA has two DNA-binding HTH domains, two PTS (Phosphoenolpyruvate Sugar Phosphotransferase System) regulatory domains (PRD1 and PRD2), and a putative EIIB domain (a component of PTS sugar transport EII-complexes). Earlier studies showed that glucose and carbon dioxide increase AtxA-dependent toxin gene transcription, as does histidine phosphorylation of PRD1 and PRD2 of AtxA. We propose that a histidine-phosphorelay originating from phosphoenolpyruvate (PEP) regulates AtxA via enzymatic activity in the PTS, and thus pathways regulating the intracellular concentration of PEP impact AtxA activity. The anaplerotic response in bacteria, activated under high carbon dioxide concentrations, is one mechanism for control of the concentrations of PEP and other TCA intermediates. Our analyses of transcriptional profiling of virulence factors, secretion of PA, and use of a fluorescent reporter strain suggest a synergistic effect of carbon dioxide and sugars on AtxA-dependent toxin production. Deletion of AtxA (ΔatxA) significantly reduced glucose uptake in bacteria, suggesting that AtxA may act within the glucose-PTS system. Mutation analysis of the EIIB domain of AtxA identified the cysteine at position 402 as essential for the transcriptional activity of AtxA. Deletion of glucose PTS permease ptsG (ΔptsG) significantly reduced the expression of PA, LF, and EF. Loss of ptsG also caused attenuation in a mouse model of infection. However, deletion of pyruvate carboxylase pyc (Δpyc), regulating anaplerosis, had minimal effect on toxin production. Biochemical and metabolic profiling of ΔatxA, ΔptsG, Δpyc, and ΔptsG/Δpyc strains showed a significant shift in the glycolytic and anaplerotic intermediates, implicating glucose as a primary stimulus for anthrax toxin production through AtxA. Subsequently, the environmental CO2 will stimulate the anaplerotic flux and alter the phosphorelay in glucose-PTS to enhance AtxA activity. Finally, we propose AtxA as an integral component of the glucose-PTS, where transcriptional activity of AtxA is regulated by environmental signals like glucose and CO2. This research was supported by the intramural research programs of the National Institute of Allergy and Infectious Diseases of National Institutes of Health.

Bacillus anthracis causes anthrax through a combination of bacterial infection and toxemia. As a major virulence factor of B. anthracis, anthrax lethal toxin (LT) is a zinc-dependent metalloproteinase, exerting its cytotoxicity through proteolytic cleavage of the mitogen-activated protein kinase kinases, thereby shutting down the MAPK pathways. Anthrax lethal toxin induces host lethality mostly by targeting the cardiovascular system. Although the enzymatic activity and the molecular targets of LT have long been known, the detailed mechanisms underlying cellular/tissue/organ toxicity are still poorly understood. In this work, we sought to investigate the mechanism of LT-induced cellular damage in the cardiovascular system. We demonstrate for the first time that anthrax lethal toxin has potent inhibitory effects on the central metabolism of cardiomyocytes and endothelial cells. This is likely due to the observed downregulating of c-Myc expression through the toxin-induced inhibition of the ERK pathway. Since c-Myc is a master transcription factor controlling the expression of many rate-limiting metabolic enzymes in glycolysis and the tricarboxylic acid cycle, LT's downregulation of c-Myc may lead to the observed bioenergetic collapse, particularly, in cardiomyocytes. Since cardiac cell contraction requires continuous production of large amounts of ATP, potent inhibition of the bioenergetics of cardiomyocytes would be incompatible with life. Thus, LT-induced lethality through targeting cardiomyocytes and endothelial cells appears to be a consequence of a bioenergetic collapse, likely due to the toxin's potent inhibitory activity on the MEK-ERK-c-Myc-metabolic/bioenergetic axis within these target cells of cardiovascular system.IMPORTANCEAnthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis, the causative pathogen of anthrax disease. Anthrax lethal toxin is a metalloproteinase that cleaves and inactivates MEKs, thereby shutting down MAPK pathways, leading to host mortality primarily through targeting of the cardiovascular system. However, the detailed mechanisms underlying the toxin's cellular and tissue toxicity are still poorly understood. Here, we found that anthrax lethal toxin has potent inhibitory activity on glycolysis and oxidative phosphorylation of cardiomyocytes and endothelial cells. These effects appear to be the consequence of downregulation of c-Myc, a master transcription factor that controls many rate-limiting enzymes of glycolysis and the tricarboxylic acid cycle. With the high demand on energy for cardiac contraction, the potent inhibition of cardiomyocyte metabolism by LT would be incompatible with life. This work provides critical insights into why the cardiovascular system is the major in vivo target of LT-induced lethality.

Supplementary Data from Matrix Metalloproteinase–Activated Anthrax Lethal Toxin Inhibits Endothelial Invasion and Neovasculature Formation during In vitro Morphogenesis

Between two banks of the River Main in Frankfurt, Germany, a new high-rise building is planned with a challenging structural concept. The geotechnical boundary conditions, such as the location of the building, nearby sensitive traffic and infrastructures and quay walls defines the complexity of the nature of the project. The quay walls are more than 100 years and should be kept unaffected during and after the construction phases. The high-rise building will have a height of about 75 m and will be founded on a Combined Pile-Raft Foundation (CPRF) in the tertiary, soft marl. For analysis of the stability and the serviceability of the heavily eccentric loaded foundation of the new high-rise building and the existing structures and for the investigation of the soil-structure interaction complex three-dimensional, non-linear numerical simulations have been carried out. The paper introduces into a very complex construction project in which the realistic consideration of the soil-structure interaction is very important for the serviceability of the foundation of the planned high-rise building as well as for the surrounding infrastructure.

Diagnosis of infectious agents is increasingly done by the detection of unique nucleic acid sequences, typically using methods such as PCR that specifically amplify these sequences. A largely neglected alternative approach is to use antibodies that recognize nucleic acids. The unique monoclonal antibody S9.6 recognizes DNA-RNA hybrids in a largely sequence-independent manner. S9.6 has been used in several cases for the analysis of nucleic acids. Extending our recent determination of the structure of S9.6 Fab bound to a DNA-RNA hybrid, we have developed reagents and methods for the sensitive detection of specific DNA and RNA sequences. To facilitate the use in diagnostics, we conjugated the S9.6 Fab to the highly active and well-characterized reporter enzyme human-secreted embryonic alkaline phosphatase (SEAP). Two approaches were utilized for conjugation. The first used sortase A (SrtA), which generates a covalent peptide bond between short amino acid sequences added to recombinantly produced S9.6 Fab and SEAP. The second approach was to genetically fuse the S9.6 Fab and SEAP so that the two are produced as a single molecule. Using these two antibody-SEAP proteins, we developed a simplified ELISA format for the identification of synthetic DNA-RNA hybrids, which can be optimized for detecting nucleic acids of pathogens, as well as for other applications. We successfully used this immunosorbent assay, HC-S, to identify DNA-RNA hybrids in solution with high specificity and sensitivity.

The detection of pathogens is critical for clinical diagnosis and public health surveillance. Detection is usually done with nucleic acid-based tests (NATs) and rapid antigen tests (e.g., lateral flow assays [LFAs]). Although NATs are more sensitive and specific, their use is often limited in resource-poor settings due to specialized requirements. To address this limitation, we developed a rapid DNA-RNA Hybrid Capture immunoassay (HC) that specifically detects RNA from pathogens. This assay utilizes a unique monoclonal antibody, S9.6, which binds DNA-RNA hybrids. Biotinylated single-stranded DNA probes are hybridized to target RNAs, followed by hybrid capture on streptavidin and detection with S9.6. The HC-ELISA assay can detect as few as 10 4 RNA molecules that are 2.2 kb in length. We also adapted this assay into a LFA format, where captured Bacillus anthracis rpoB RNA of 3.5 kb length was detectable from a bacterial load equivalent to 10 7 CFU per 100 mg of mouse tissue using either HC-ELISA or HC-LFA. Importantly, we also demonstrated the versatility of HC by detecting other pathogens, including SARS-CoV2 and Toxoplasma gondii , showing its potential for broad pathogen detection. Notably, HC does not require amplification of the target nucleic acid and utilizes economical formats like ELISA and LFA, making it suitable for use in sentinel labs for pathogen detection or as a molecular tool in basic research laboratories. Our study highlights the potential of HC as a sensitive and versatile method for RNA-based pathogen detection.

Protective antigen channel (PA63) is the central component of anthrax tripartite exotoxin, responsible for delivery of the enzymatic lethal and edema components of the toxin into the host cell cytosol. The channel, which is more than three times longer than the lipid bilayer membrane and has a 6-Å limiting diameter, is thought to provide sophisticated unfoldase and translocase machinery for foreign protein transport. The toxin can be reengineered, one component at a time, to be adapted for targeted cancer treatment. To reach high efficiency, it is important to understand the toxin translocation process at the molecular level.