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.

Smartphone-assisted mobile fluorescence sensor for self-calibrated detection of anthrax biomarker, Cu2+, and cysteine in food analysis.

Dipicolinic acid (DPA), as a biomarker for Bacillus anthracis, is highly toxic at trace levels. Rapid and on-site quantitative detection of DPA is essential for maintaining food safety and public health. This work develops a dual-channel self-calibrated fluorescence sensor constructed by the YVO4:Eu and Tb-ß-diketone complex for rapid visual detection of DPA. This sensor exhibits high selectivity, fast response time, excellent detection sensitivity, and the detection limit is as low as 4.5 nM in the linear range of 0-16 µM. A smartphone APP and portable ultraviolet lamp can assemble a mobile fluorescence sensor for on-site analysis. Interestingly, adding Cu2+ ions can quench the fluorescence intensity of Tb3+. In contrast, the addition of cysteine can restore the fluorescence, allowing the accurate detection of Cu2+ ions and cysteine in environmental water and food samples. This work provides a portable sensor that facilitates real-time analysis of multiple targets in food and the environment.

Anthrax Toxin: Model System for Studying Protein Translocation.

Dedicated translocase channels are nanomachines that often, but not always, unfold and translocate proteins through narrow pores across the membrane. Generally, these molecular machines utilize external sources of free energy to drive these reactions, since folded proteins are thermodynamically stable, and once unfolded they contain immense diffusive configurational entropy. To catalyze unfolding and translocate the unfolded state at appreciable timescales, translocase channels often utilize analogous peptide-clamp active sites. Here we describe how anthrax toxin has been used as a biophysical model system to study protein translocation. The tripartite bacterial toxin is composed of an oligomeric translocase channel, protective antigen (PA), and two enzymes, edema factor (EF) and lethal factor (LF), which are translocated by PA into mammalian host cells. Unfolding and translocation are powered by the endosomal proton gradient and are catalyzed by three peptide-clamp sites in the PA channel: the α clamp, the ϕ clamp, and the charge clamp. These clamp sites interact nonspecifically with the chemically complex translocating chain, serve to minimize unfolded state configurational entropy, and work cooperatively to promote translocation. Two models of proton gradient driven translocation have been proposed: (i) an extended-chain Brownian ratchet mechanism and (ii) a proton-driven helix-compression mechanism. These models are not mutually exclusive; instead the extended-chain Brownian ratchet likely operates on ß-sheet sequences and the helix-compression mechanism likely operates on α-helical sequences. Finally, we compare and contrast anthrax toxin with other related and unrelated translocase channels.

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.

Localization of the CotY and ExsY proteins to the exosporium basal layer of Bacillus anthracis.

Spores are an infectious form of the zoonotic bacterial pathogen, Bacillus anthracis. The outermost spore layer is the exosporium, comprised of a basal layer and an external glycoprotein nap layer. The major structural proteins of the inner basal layer are CotY (at the mother cell central pole or bottlecap) and ExsY around the rest of the spore. The basis for the cap or noncap specificity of the CotY and ExsY proteins is currently unknown. We investigated the role of sequence differences between these proteins in localization during exosporium assembly. We found that sequence differences were less important than the timing of expression of the respective genes in the positioning of these inner basal layer structural proteins. Fusion constructs with the fluorescent protein fused at the N-terminus resulted in poor incorporation whereas fusions at the carboxy terminus of CotY or ExsY resulted in good incorporation. However, complementation studies revealed that fusion constructs, although accurate indicators of protein localization, were not fully functional. A model is presented that explains the localization patterns observed. Bacterial two-hybrid studies in Escherichia coli hosts were used to examine protein-protein interactions with full-length and truncated proteins. The N-terminus amino acid sequences of ExsY and CotY appear to be recognized by spore proteins located in the spore interspace, consistent with interactions seen with ExsY and CotY with the interspace proteins CotE and CotO, known to be involved with exosporium attachment.

Structure of the Anthrax Protective Antigen D425A Dominant Negative Mutant Reveals a Stalled Intermediate State of Pore Maturation.

The tripartite protein complex produced by anthrax bacteria (Bacillus anthracis) is a member of the AB family of ß-barrel pore-forming toxins. The protective antigen (PA) component forms an oligomeric prepore that assembles on the host cell surface and serves as a scaffold for binding of lethal and edema factors. Following endocytosis, the acidic environment of the late endosome triggers a pH-induced conformational rearrangement to promote maturation of the PA prepore to a functional, membrane spanning pore that facilitates delivery of lethal and edema factors to the cytosol of the infected host. Here, we show that the dominant-negative D425A mutant of PA stalls anthrax pore maturation in an intermediate state at acidic pH. Our 2.7 Å cryo-EM structure of the intermediate state reveals structural rearrangements that involve constriction of the oligomeric pore combined with an intramolecular dissociation of the pore-forming module. In addition to defining the early stages of anthrax pore maturation, the structure identifies asymmetric conformational changes in the oligomeric pore that are influenced by the precise configuration of adjacent protomers.

Halogen-Dance-Based Synthesis of Phosphonomethoxyethyl (PME) Substituted 2-Aminothiazoles as Potent Inhibitors of Bacterial Adenylate Cyclases.

A series of acyclic nucleoside phosphonates (ANPs) was designed as inhibitors of bacterial adenylate cyclases (ACs), where adenine was replaced with 2-amino-4-arylthiazoles. The target compounds were prepared using the halogen dance reaction. Final AC inhibitors were evaluated in cell-based assays (prodrugs) and cell-free assays (phosphono diphosphates). Novel ANPs were potent inhibitors of adenylate cyclase toxin (ACT) from Bordetella pertussis and edema factor (EF) from Bacillus anthracis, with substantial selectivity over mammalian enzymes AC1, AC2, and AC5. Six of the new ANPs were more potent or equipotent ACT inhibitors (IC50 =9-18 nM), and one of them was more potent EF inhibitor (IC50 =12 nM), compared to adefovir diphosphate (PMEApp) with IC50 =18 nM for ACT and IC50 =36 nM for EF. Thus, these compounds represent the most potent ACT/EF inhibitors based on ANPs reported to date. The potency of the phosphonodiamidates to inhibit ACT activity in J774A.1 macrophage cells was somewhat weaker, where the most potent derivative had IC50 =490 nM compared to IC50 =150 nM of the analogous adefovir phosphonodiamidate. The results suggest that more efficient type of phosphonate prodrugs would be desirable to increase concentrations of the ANP-based active species in the cells in order to proceed with the development of ANPs as potential antitoxin therapeutics.

Antibiotic resistance modifying ability of phytoextracts in anthrax biological agent Bacillus anthracis and emerging superbugs: a review of synergistic mechanisms.

BACKGROUND AND OBJECTIVES: The chemotherapeutic management of infections has become challenging due to the global emergence of antibiotic resistant pathogenic bacteria. The recent expansion of studies on plant-derived natural products has lead to the discovery of a plethora of phytochemicals with the potential to combat bacterial drug resistance via various mechanisms of action. This review paper summarizes the primary antibiotic resistance mechanisms of bacteria and also discusses the antibiotic-potentiating ability of phytoextracts and various classes of isolated phytochemicals in reversing antibiotic resistance in anthrax agent Bacillus anthracis and emerging superbug bacteria. METHODS: Growth inhibitory indices and fractional inhibitory concentration index were applied to evaluate the in vitro synergistic activity of phytoextract-antibiotic combinations in general. FINDINGS: A number of studies have indicated that plant-derived natural compounds are capable of significantly reducing the minimum inhibitory concentration of standard antibiotics by altering drug-resistance mechanisms of B. anthracis and other superbug infection causing bacteria. Phytochemical compounds allicin, oleanolic acid, epigallocatechin gallate and curcumin and Jatropha curcas extracts were exceptional synergistic potentiators of various standard antibiotics. CONCLUSION: Considering these facts, phytochemicals represents a valuable and novel source of bioactive compounds with potent antibiotic synergism to modulate bacterial drug-resistance.

Solution Structures of Bacillus anthracis Protective Antigen Proteins Using Small Angle Neutron Scattering and Protective Antigen 63 Ion Channel Formation Kinetics.

We are studying the structures of bacterial toxins that form ion channels and enable macromolecule transport across membranes. For example, the crystal structure of the Staphylococcus aureus α-hemolysin (α-HL) channel in its functional state was confirmed using neutron reflectometry (NR) with the protein reconstituted in membranes tethered to a solid support. This method, which provides sub-nanometer structural information, could also test putative structures of the Bacillus anthracis protective antigen 63 (PA63) channel, locate where B. anthracis lethal factor and edema factor toxins (LF and EF, respectively) bind to it, and determine how certain small molecules can inhibit the interaction of LF and EF with the channel. We report here the solution structures of channel-forming PA63 and its precursor PA83 (which does not form channels) obtained with small angle neutron scattering. At near neutral pH, PA83 is a monomer and PA63 a heptamer. The latter is compared to two cryo-electron microscopy structures. We also show that although the α-HL and PA63 channels have similar structural features, unlike α-HL, PA63 channel formation in lipid bilayer membranes ceases within minutes of protein addition, which currently precludes the use of NR for elucidating the interactions between PA63, LF, EF, and potential therapeutic agents.

Bottom-up fabrication of a proteasome-nanopore that unravels and processes single proteins.

The precise assembly and engineering of molecular machines capable of handling biomolecules play crucial roles in most single-molecule methods. In this work we use components from all three domains of life to fabricate an integrated multiprotein complex that controls the unfolding and threading of individual proteins across a nanopore. This 900 kDa multicomponent device was made in two steps. First, we designed a stable and low-noise ß-barrel nanopore sensor by linking the transmembrane region of bacterial protective antigen to a mammalian proteasome activator. An archaeal 20S proteasome was then built into the artificial nanopore to control the unfolding and linearized transport of proteins across the nanopore. This multicomponent molecular machine opens the door to two approaches in single-molecule protein analysis, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then addressed either as fragmented peptides or intact polypeptides.

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.

Titratable transmembrane residues and a hydrophobic plug are essential for manganese import via the Bacillus anthracis ABC transporter MntBC-A.

All extant life forms require trace transition metals (e.g., Fe2/3+, Cu1/2+, and Mn2+) to survive. However, as these are environmentally scarce, organisms have evolved sophisticated metal uptake machineries. In bacteria, high-affinity import of transition metals is predominantly mediated by ABC transporters. During bacterial infection, sequestration of metal by the host further limits the availability of these ions, and accordingly, bacterial ABC transporters (importers) of metals are key virulence determinants. However, the structure-function relationships of these metal transporters have not been fully elucidated. Here, we used metal-sensitivity assays, advanced structural modeling, and enzymatic assays to study the ABC transporter MntBC-A, a virulence determinant of the bacterial human pathogen Bacillus anthracis. We find that despite its broad metal-recognition profile, MntBC-A imports only manganese, whereas zinc can function as a high-affinity inhibitor of MntBC-A. Computational analysis shows that the transmembrane metal permeation pathway is lined with six titratable residues that can coordinate the positively charged metal, and mutagenesis studies show that they are essential for manganese transport. Modeling suggests that access to these titratable residues is blocked by a ladder of hydrophobic residues, and ATP-driven conformational changes open and close this hydrophobic seal to permit metal binding and release. The conservation of this arrangement of titratable and hydrophobic residues among ABC transporters of transition metals suggests a common mechanism. These findings advance our understanding of transmembrane metal recognition and permeation and may aid the design and development of novel antibacterial agents.

Spore-Associated Proteins Involved in c-di-GMP Synthesis and Degradation of Bacillus anthracis.

Bis-(3'-5')-cyclic-dimeric GMP (c-di-GMP) is an important bacterial regulatory signaling molecule affecting biofilm formation, toxin production, motility, and virulence. The genome of Bacillus anthracis, the causative agent of anthrax, is predicted to encode ten putative GGDEF/EAL/HD-GYP-domain containing proteins. Heterologous expression in Bacillus subtilis hosts indicated that there are five active GGDEF domain-containing proteins and four active EAL or HD-GYP domain-containing proteins. Using an mCherry gene fusion-Western blotting approach, the expression of the c-di-GMP-associated proteins was observed throughout the in vitro life cycle. Of the six c-di-GMP-associated proteins found to be present in sporulating cells, four (CdgA, CdgB, CdgD, and CdgG) contain active GGDEF domains. The six proteins expressed in sporulating cells are retained in spores in a CotE-independent manner and thus are not likely to be localized to the exosporium layer of the spores. Individual deletion mutations involving the nine GGDEF/EAL protein-encoding genes and one HD-GYP protein-encoding gene did not affect sporulation efficiency, the attachment of the exosporium glycoprotein BclA, or biofilm production. Notably, expression of anthrax toxin was not affected by deletion of any of the cdg determinants. Three determinants encoding proteins with active GGDEF domains were found to affect germination kinetics. This study reveals a spore association of cyclic-di-GMP regulatory proteins and a likely role for these proteins in the biology of the B. anthracis spore. IMPORTANCE The genus Bacillus is composed of Gram-positive, rod shaped, soil-dwelling bacteria. As a mechanism for survival in the harsh conditions in soil, the organisms undergo sporulation, and the resulting spores permit the organisms to survive harsh environmental conditions. Although most species are saprophytes, Bacillus cereus and Bacillus anthracis are human pathogens and Bacillus thuringiensis is an insect pathogen. The bacterial c-di-GMP regulatory system is an important control system affecting motility, biofilm formation, and toxin production. The role of c-di-GMP has been studied in the spore-forming bacilli Bacillus subtilis, Bacillus amyloliquefaciens, B. cereus, and B. thuringiensis. However, this regulatory system has not heretofore been examined in the high-consequence zoonotic pathogen of this genus, B. anthracis.

A ratiometric lanthanide-free fluorescent probe based on two-dimensional metal-organic frameworks and carbon dots for the determination of anthrax biomarker.

A lanthanide-free fluorescent probe has been constructed for the first time based on two-dimensional metal-organic frameworks (2D MOFs) and carbon dots (CDs) for ratiometric determination of dipicolinic acid (DPA), the biomarker of Bacillus anthracis. The fluorescence intensity at 659 nm increased due to the release of organic ligands TCPP resulting from the selective interaction between DPA and Zn2+ of 2D MOFs. CDs provided a reference signal at 445 nm which was almost unaffected, realizing self-calibration DPA sensing. F659/F445 versus the concentration of DPA shows good linear relationships in the range 0.01-0.2 µM and 0.2-10 µM under 390-nm excitation, with a detection limit of 7 nM. The ratiometric probe was prepared from 2D lanthanide-free MOFs so that the drawbacks of lanthanide-based probes were overcome. The proposed sensing system was successfully applied to the determination of DPA in spiked biological samples. These results suggest that a novel, simple, and selective strategy of determining DPA with 2D lanthanide-free MOFs is implemented. Graphical abstract Zn-TCPP nanosheets and a blue carbon dots (b-CDs) are synthesized to construct the ratiometric probe, which can exhibit fluorescence at 445and 659 nm with 390-nm excitation. Dipicolinic acid (DPA) can deprive the junction ions of Zn-TCPP nanosheets, triggering the collapse ofZn-TCPP nanosheets. The fluorescence at 659 nm is enhanced due to the release of TCPP, while the peak of b-CDs at 445 nm is almost not affected. Thus, the fluorescence intensity ratio (F659/F445) can serve as the response signal for sensitive DPA sensing.

Gold nanocluster-europium(III) ratiometric fluorescence assay for dipicolinic acid.

A ratiometric fluorescence assay was designed for determination of dipicolinic acid (DPA), a spore-specific compound which is used as a biomarker for Bacillus anthracis spores for food and medical product safety analysis. The dual-channel fluorescence probe integrates two fluorescent materials, Eu3+ ion and gold nanocluster (Au NC). The Au NC is used as a reference channel to measure background noise and the Eu3+ ion as the DPA-specific response signal channel. The probe was prepared through simply combing bovine serum albumin (BSA)-scaffolded Eu3+ ion and Au NCs. When excited at 530 nm, in the presence of DPA, the fluorescence signals of Eu3+ ion at 595, 617, and 695 nm increased significantly while the 650 nm signal of Au NC reference remained relatively constant. This fluorescence probe has good photo-stability and also displays good selectivity and high sensitivity for DPA with a low detection limit of 0.8 µM. The linear range of the ratiometric probe for DPA is 1-50 µM. For determination of DPA released during the germination of Bacillus subtilis spores, the detection results were in agreement with measurements by conventional calorimetry assay. The method may have potential for measuring the level of contamination and germination by spores. Graphical Abstract Dual-channel fluorescence biosensor was designed to detect dipicolinic acid, a spore-specific compound which is used as a biomarker for Bacillus anthracis spores for food and medical product safety analysis.

Assembly and Function of the Anthrax Toxin Protein Translocation Complex.

Anthrax toxin is a major virulence factor of Bacillus anthracis, a Gram-positive bacterium which can form highly stable spores that are the causative agents of the disease, anthrax. While chiefly a disease of livestock, spores can be "weaponized" as a bio-terrorist agent, and can be deadly if not recognized and treated early with antibiotics. The intracellular pathways affected by the enzymes are broadly understood and are not discussed here. This chapter focuses on what is known about the assembly of secreted toxins on the host cell surface and how the toxin is delivered into the cytosol. The central component is the "Protective Antigen", which self-oligomerizes and forms complexes with its pay-load, either Lethal Factor or Edema Factor. It binds a host receptor, CMG2, or a close relative, triggering receptor-mediated endocytosis, and forms a remarkably elegant yet powerful machine that delivers toxic enzymes into the cytosol, powered only by the pH gradient across the membrane. We now have atomic structures of most of the starting, intermediate and final assemblies in the infectious process. Together with a major body of biophysical, mutational and biochemical work, these studies reveal a remarkable story of both how toxin assembly is choreographed in time and space.

Effect of pH and denaturants on the fold and metal status of anthrax lethal factor.

Anthrax lethal factor (LF) is a critical component of the anthrax toxin, and functions intracellularly as a zinc-dependent endopeptidase targeting proteins involved in maintaining critical host signaling pathways. To reach the cytoplasm, LF requires to be unfolded and guided through the narrow protective antigen pore in a pH-dependent process. The current study sought to address the question as to whether LF is capable of retaining its metal ion when exposed to a low-pH environment (similar to that found in late endosomes) and an unfolding stress (induced by urea). Using a combination of tryptophan fluorescence spectroscopy and chelation studies, we show that a decrease in the pH value (from 7.0 to 5.0) leads to a pronounced shift in the onset of structural alterations in LF to lower urea concentrations. More importantly, the enzyme was found to retain its Zn2+ ion beyond the unfolding transitions monitored by Trp fluorescence, a finding indicative of tight metal binding to LF in a non-native state. In addition, an analysis of red-edge excitation shift (REES) spectra suggests the protein to maintain residual structure (a feature necessary for metal binding) even at very high denaturant concentrations. Furthermore, studies using the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) revealed LF's Zn2+ ion to become accessible to complexation at urea concentrations in between those required to cause structural changes and metal dissociation. This phenomenon likely originates from the conversion of a PAR-inaccessible (closed) to a PAR-accessible (open) state of LF at intermediate denaturant concentrations.

Novel Synthesis of Thiolated Gold Nanoclusters Induced by Lanthanides for Ultrasensitive and Luminescent Detection of the Potential Anthrax Spores' Biomarker.

In this study, we reported a facile, one-pot, and "green" synthesis of glutathione-protected gold nanoclusters (GSH@AuNCs) initiated by samarium (Sm3+) lanthanides for the first time. Sm3+ lanthanides more efficiently induced the formation of GSH@AuNCs with significantly enhanced luminescence than other lanthanides or heavy metal ions (Cd2+, Pb2+) did. Using this strategy, a detection for Sm3+ was made with a linearity range of (10.0-100.0 µM) and a limit of detection (LOD) of 0.5 µM. The Sm3+-based GSH@AuNCs were characterized by eco-friendliness, photostability, and low-cost synthesis with low biological toxicity and had great potential in the application for biosensing and bioimaging. They were successfully employed in the detection of dipicolinic acid (DPA), a well-reported biomarker for sensing potential infection by strongly hazardous anthrax spores. A good linear response was obtained for DPA detection ranging from 1.0 to 120.0 µM with a low LOD of 0.1 µM, which was much lower (600 times) than the infectious dosage of anthrax spores (6 × 10-5 M). The detection was due to the strong binding affinity and strong chelation capability of DPA to Sm3+ lanthanides, which caused the dissociation of the aggregates with an obvious decrease or even a turning-off effect of their luminescence.

Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting.

Sortases are a group of enzymes displayed on the cell-wall of Gram-positive bacteria. They are responsible for the attachment of virulence factors onto the peptidoglycan in a transpeptidation reaction through recognition of a pentapeptide substrate. Most housekeeping sortases recognize one specific pentapeptide motif; however, Streptococcus pyogenes sortase A (SpSrtA WT) recognizes LPETG, LPETA and LPKLG motifs. Here, we examined SpSrtA's flexible substrate specificity by investigating the role of the ß7/ß8 loop in determining substrate specificity. We exchanged the ß7/ß8 loop in SpSrtA with corresponding ß7/ß8 loops from Staphylococcus aureus (SaSrtA WT) and Bacillus anthracis (BaSrtA WT). While the BaSrtA-derived variant showed no enzymatic activity toward either LPETG or LPETA substrates, the activity of the SaSrtA-derived mutant toward the LPETA substrate was completely abolished. Instead, the mutant had an improved activity toward LPETG, the preferred substrate of SaSrtA WT.

Highly accurate classification of biological spores by culture medium for forensic attribution using multiple chemical signature types and machine learning.

Future proliferation of biological expertise and new technology may increasingly lower the difficulty to produce biological organisms for misuse. Rapid attribution of a biological attack is needed to quickly identify the person or lab responsible and prevent additional attacks by enabling the apprehension of suspects. Here, triplicate batches of Bacillus anthracis Sterne strain (BaSt) spores were grown in a total of seven amateur and professional media. Multiple orthogonal analytical signatures (peptides, metabolites, lipids by fatty acid methyl ester (FAME) analysis, bulk organic profile, and trace elements) were collected from the BaSt spores. The proteomics and metabolomics analyses identified promising attribution signature compounds that are unique to each of the seven production methods. In addition, while each of the signature types showed varying degrees of value individually for attributing BaSt spores to the culture medium used to prepare them, fusing results from all five signatures types to increase sourcing robustness and using a random forest sourcing algorithm yielded 100% hold-one-batch-out cross-validation classification accuracy and an average relative source probability for the correct source 5.5× higher than the most probable incorrect source. These preliminary results provide a proof-of-concept for the development of forensic examinations that can attribute biological agents to production methods for use in future investigations.