c-di-AMP accumulation impairs toxin expression of Bacillus anthracis by down-regulating potassium importers.

The Gram-positive bacterium Bacillus anthracis is the causative agent of anthrax and a bioterrorism threat worldwide. As a crucial second messenger in many bacterial species, cyclic di-AMP (c-di-AMP) modulates various key processes for bacterial homeostasis and pathogenesis. Overaccumulation of c-di-AMP alters cellular growth and reduces anthrax toxin expression as well as virulence in Bacillus anthracis by unresolved underlying mechanisms. In this report, we discovered that c-di-AMP binds to a series of receptors involved in potassium uptake in B. anthracis. By analyzing Kdp and Ktr mutants for osmotic stress, gene expression, and anthrax toxin expression, we also showed that c-di-AMP inhibits Kdp operon expression through binding to the KdpD and ydaO riboswitch; up-regulating intracellular potassium promotes anthrax toxin expression in c-di-AMP accumulated B. anthracis. Decreased anthrax toxin expression at high c-di-AMP occurs through the inhibition of potassium uptake. Understanding the molecular basis of how potassium uptake affects anthrax toxin has the potential to provide new insight into the control of B. anthracis.IMPORTANCEThe bacterial second messenger cyclic di-AMP (c-di-AMP) is a conserved global regulator of potassium homeostasis. How c-di-AMP regulates bacterial virulence is unknown. With this study, we provide a link between potassium uptake and anthrax toxin expression in Bacillus anthracis. c-di-AMP accumulation might inhibit anthrax toxin expression by suppressing potassium uptake.

Flexible RNA aptamers as inhibitors of Bacillus anthracis ribosomal protein S8: Insights from molecular dynamics simulations.

Bacillus anthracis, the causative agent of anthrax, poses a substantial threat to public health and national security, and is recognized as a potential bioweapon due to its capacity to form resilient spores with enduring viability. Inhalation or ingestion of even minute quantities of aerosolized spores can lead to widespread illness and fatalities, underscoring the formidable lethality of the bacterium. With an untreated mortality rate of 100%, Bacillus anthracis is a disconcerting candidate for bioterrorism. In response to this critical scenario, we employed state-of-the-art computational tools to conceive and characterize flexible RNA aptamer therapeutics tailored for anthrax. The foundational structure of the flexible RNA aptamers was designed by removing the C2'-C3' in each nucleotide unit. Leveraging the crystal structure of Bacillus anthracis ribosomal protein S8 complexed with an RNA aptamer, we explored the structural, dynamic, and energetic aspects of the modified RNA aptamer - S8 protein complexes through extensive all-atom explicit-solvent molecular dynamics simulations (400 ns, 3 replicas each), followed by drawing comparisons to the control system. Our findings demonstrate the enhanced binding competencies of the flexible RNA aptamers to the S8 protein via better shape complementarity and improved H-bond network compared to the control RNA aptamer. This research offers valuable insights into the development of RNA aptamer therapeutics targeting Bacillus anthracis, paving the way for innovative strategies to mitigate the impact of this formidable pathogen.

Split fluorescent protein-mediated multimerization of cell wall binding domain for highly sensitive and selective bacterial detection.

Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial binding, and thus, have potential as biorecognition molecules for nondestructive bacterial detection. Here, a novel design for a self-complementing split fluorescent protein (FP) complex is proposed, where a multimeric FP chain fused with specific CBDs ((FP-CBD)n) is assembled inside the cell, to improve sensitivity by enhancing the signal generated upon Staphylococcus aureus or Bacillus anthracis binding. Flow cytometry shows enhanced fluorescence on the cell surface with increasing FP stoichiometry and surface plasmon resonance reveals nanomolar binding affinity to isolated peptidoglycan. The breadth of function of these complexes is demonstrated through the use of CBD modularity and the ability to attach enzymatic detection modalities. Horseradish peroxidase-coupled (FP-CBD)n complexes generate a catalytic amplification, with the degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 103 cells/droplet (approximately 0.1 ng S. aureus or B. anthracis) within 15 min on a polystyrene surface. These fusion proteins can be multiplexed for simultaneous detection. Multimeric split FP-CBD fusions enable use as a biorecognition molecule with enhanced signal for use in bacterial biosensing platforms.

Dachshund Homolog 1: Unveiling Its Potential Role in Megakaryopoiesis and Bacillus anthracis Lethal Toxin-Induced Thrombocytopenia.

Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34-megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis.

Visualization of immune pathways that enhance the neutralizing antibody response to vaccines after primary immunization.

We examined the relationship between the association of a vaccine antigen with immune cells in secondary lymphoid organs shortly after immunization and the resulting neutralizing antibody response induced by that antigen using three antigenic forms of anthrax protective antigen (PA) that induce qualitatively different antibody responses. The three PA forms used were wild-type PA, which binds to anthrax toxin receptors and elicits a robust antibody response that includes both neutralizing and non-neutralizing antibodies; a receptor-binding-deficient (RBD) mutant form of PA, which does not bind cellular receptors and elicits only barely detectable antibody responses; and an engineered chimeric form of PA, which binds cholera toxin receptors and elicits a robust total antibody response but a poor neutralizing antibody response. We found that both wild-type PA and the PA chimera associated with immune cells in secondary lymphoid organs after immunization, but the RBD mutant PA exhibited minimal association, revealing a relationship between antigen binding to toxin receptors on immune cells after immunization and subsequent antibody responses. A portion of wild-type PA that bound to immune cells was cell surface-associated and maintained its native conformation. Much lower amounts of conformationally intact PA chimera were associated with immune cells after immunization, correlating with the lower neutralizing antibody response elicited by the PA chimera. Thus, binding of an antigen to receptors on immune cells in secondary lymphoid organs after immunization and maintenance of conformational integrity of the cell-associated antigen help dictate the magnitude of the resulting neutralizing antibody response, but not necessarily the total antibody response.IMPORTANCEMany vaccines protect by the induction of antibodies that neutralize the action of the pathogen. Here, we followed the fate of three antigenic forms of a vaccine antigen in secondary lymphoid organs after immunization to investigate events leading to a robust neutralizing antibody response. We found that the magnitude of the neutralizing antibody response, but not the total antibody response, correlates with the levels of conformationally intact antigen associated with immune cells in secondary lymphoid organs after primary immunization. We believe that these results provide important insights into the genesis of neutralizing antibody responses induced by vaccine antigens and may have implications for vaccine design.

Bacillus anthracis, "la maladie du charbon", Toxins, and Institut Pasteur.

Institut Pasteur and Bacillus anthracis have enjoyed a relationship lasting almost 120 years, starting from its foundation and the pioneering work of Louis Pasteur in the nascent fields of microbiology and vaccination, and blooming after 1986 following the molecular biology/genetic revolution. This contribution will give a historical overview of these two research eras, taking advantage of the archives conserved at Institut Pasteur. The first era mainly focused on the production, characterisation, surveillance and improvement of veterinary anthrax vaccines; the concepts and technologies with which to reach a deep understanding of this research field were not yet available. The second period saw a new era of B. anthracis research at Institut Pasteur, with the anthrax laboratory developing a multi-disciplinary approach, ranging from structural analysis, biochemistry, genetic expression, and regulation to bacterial-host cell interactions, in vivo pathogenicity, and therapy development; this led to the comprehensive unravelling of many facets of this toxi-infection. B. anthracis may exemplify some general points on how science is performed in a given society at a given time and how a scientific research domain evolves. A striking illustration can be seen in the additive layers of regulations that were implemented from the beginning of the 21st century and their impact on B. anthracis research. B. anthracis and anthrax are complex systems that raise many valuable questions regarding basic research. One may hope that B. anthracis research will be re-initiated under favourable circumstances later at Institut Pasteur.