Anthracis Sterne Research Articles

Discovery of Prenyltransferase Inhibitors with In Vitro and In Vivo Antibacterial Activity.

Cis-prenyltransferases such as undecaprenyl diphosphate synthase (UPPS) and decaprenyl diphosphate synthase (DPPS) are essential enzymes in bacteria and are involved in cell wall biosynthesis. UPPS and DPPS are absent in the human genome, so they are of interest as targets for antibiotic development. Here, we screened a library of 750 compounds from National Cancer Institute Diversity Set V for the inhibition of Mycobacterium tuberculosis DPPS and found 17 hits, and then IC50s were determined using dose-response curves. Compounds were tested for growth inhibition against a panel of bacteria, for in vivo activity in a Staphylococcus aureus/Caenorhabditis elegans model, and for mammalian cell toxicity. The most active DPPS inhibitor was the dicarboxylic acid redoxal (compound 10), which also inhibited undecaprenyl diphosphate synthase (UPPS) as well as farnesyl diphosphate synthase. 10 was active against S. aureus, Clostridiodes difficile, Bacillus anthracis Sterne, and Bacillus subtilis, and there was a 3.4-fold increase in IC50 on addition of a rescue agent, undecaprenyl monophosphate. We found that 10 was also a weak protonophore uncoupler, leading to the idea that it targets both isoprenoid biosynthesis and the proton motive force. In an S. aureus/C. elegans in vivo model, 10 reduced the S. aureus burden 3 times more effectively than did ampicillin.

Vaccine Delivery with a Detoxified Bacterial Toxin.

It is still a challenge to develop needle-free mucosal vaccines. Despite progress in the development of the influenza vaccine, it must be reformulated annually because of antigenic changes in circulating influenza viral strains. Due to seasonal drift and shift of circulating strains, the influenza vaccine does not always match the circulating strains, and included adjuvants are not sufficient to induce a protective effect with long-lived memory cells. The adjuvants play a major role in the immune responses to a vaccine. Interestingly, the Bacillus anthracis detoxified anthrax edema toxin, which composes of protective antigen PA and N-fragment of edema factor (EFn), has shown improved effects for humoral and cellular immune responses. Here we describe the design of a universal influenza vaccine construct that consists of three tandem M2e repeats of the influenza antigen plus HA2 and detoxified toxin EFn, which is associated with the PA component, as well as the techniques used to corroborate protection. We present two major parts of description to demonstrate the vaccine strategy, using detoxified anthrax toxin for intranasal delivery of influenza antigen: (1) vaccine candidate design, production, and purification; (2) influenza virus microneutralization assay and cellular responses and lethal challenge with influenza viruses and B. anthracis Sterne spores. In the methods detailed here, we used different versions of the M2e-HA2 proteins.

Characterization of the UK anthrax vaccine and human immunogenicity

ABSTRACT The manufacture of the UK Anthrax vaccine (AVP) focuses on the production of Protective Antigen (PA) from the Bacillus anthracis Sterne strain. Although used for decades, several of AVP’s fundamental properties are poorly understood, including its exact composition, the extent to which proteins other than PA may contribute to protection, and whether the degree of protection varies between individuals. This study involved three innovative investigations. Firstly, the composition of AVP was analyzed using liquid chromatography tandem mass-spectrometry (LC-MS/MS), requiring the development of a novel desorption method for releasing B. anthracis proteins from the vaccine’s aluminum-containing adjuvant. Secondly, computational MHC-binding predictions using NetMHCIIpan were made for the eight most abundant proteins of AVP, for the commonest HLA alleles in multiple ethnic groups, and for multiple B. anthracis strains. Thirdly, antibody levels and toxin neutralizing antibody (TNA) levels were measured in sera from AVP human vaccinees for both PA and Lethal Factor (LF). It was demonstrated that AVP is composed of at least 138 B. anthracis proteins, including PA (65%), LF (8%) and Edema Factor (EF) (3%), using LC-MS/MS. NetMHCIIpan predicted that peptides from all eight abundant proteins are likely to be presented to T cells, a pre-requisite for protection; however, the number of such peptides varied considerably between different HLA alleles. These analyses highlight two important properties of the AVP vaccine that have not been established previously. Firstly, the effectiveness of AVP within humans may not depend on PA alone; there is compelling evidence to suggest that LF has a protective role, with computational predictions suggesting that additional proteins may be important for individuals with specific HLA allele combinations. Secondly, in spite of differences in the sequences of key antigenic proteins from different B. anthracis strains, these are unlikely to affect the cross-strain protection afforded by AVP.

Protective antibody response following oral vaccination with microencapsulated Bacillus Anthracis Sterne strain 34F2 spores

An oral vaccine against anthrax (Bacillus anthracis) is urgently needed to prevent annual anthrax outbreaks that are causing catastrophic losses in free-ranging livestock and wildlife worldwide. The Sterne vaccine, the current injectable livestock vaccine, is a suspension of live attenuated B. anthracis Sterne strain 34F2 spores (Sterne spores) in saponin. It is not effective when administered orally and individual subcutaneous injections are not a practical method of vaccination for wildlife. In this study, we report the development of a microencapsulated oral vaccine against anthrax. Evaluating Sterne spore stability at varying pH’s in vitro revealed that spore exposure to pH 2 results in spore death, confirming that protection from the gastric environment is of main concern when producing an oral vaccine. Therefore, Sterne spores were encapsulated in alginate and coated with a protein shell containing poly-L-lysine (PLL) and vitelline protein B (VpB), a non-immunogenic, proteolysis resistant protein isolated from Fasciola hepatica. Capsule exposure to pH 2 demonstrated enhanced acid gel character suggesting that alginate microcapsules provided the necessary protection for spores to survive the gastric environment. Post vaccination IgG levels in BALBc/J mouse serum samples indicated that encapsulated spores induced anti-anthrax specific responses in both the subcutaneous and the oral vaccination groups. Furthermore, the antibody responses from both vaccination routes were protective against anthrax lethal toxin in vitro, suggesting that further optimization of this vaccine formulation may result in a reliable oral vaccine that will conveniently and effectively prevent anthrax in wildlife populations.

Metabolic Profiling of Volatile Organic Compounds (VOCs) Emitted by the Pathogens Francisella tularensis and Bacillus anthracis in Liquid Culture

We conducted comprehensive (untargeted) metabolic profiling of volatile organic compounds (VOCs) emitted in culture by bacterial taxa Francisella tularensis (F. tularensis) subspecies novicida and Bacillus anthracis (B. anthracis) Sterne, surrogates for potential bacterial bioterrorism agents, as well as selective measurements of VOCs from their fully virulent counterparts, F. tularensis subspecies tularensis strain SCHU S4 and B. anthracis Ames. F. tularensis and B. anthracis were grown in liquid broth for time periods that covered logarithmic growth, stationary, and decline phases. VOCs emitted over the course of the growth phases were collected from the headspace above the cultures using solid phase microextraction (SPME) and were analyzed using gas chromatography-mass spectrometry (GC-MS). We developed criteria for distinguishing VOCs originating from bacteria versus background VOCs (originating from growth media only controls or sampling devices). Analyses of collected VOCs revealed methyl ketones, alcohols, esters, carboxylic acids, and nitrogen- and sulfur-containing compounds that were present in the bacterial cultures and absent (or present at only low abundance) in control samples indicating that these compounds originated from the bacteria. Distinct VOC profiles where observed for F. tularensis when compared with B. anthracis while the observed profiles of each of the two F. tularensis and B. anthracis strains exhibited some similarities. Furthermore, the relative abundance of VOCs was influenced by bacterial growth phase. These data illustrate the potential for VOC profiles to distinguish pathogens at the genus and species-level and to discriminate bacterial growth phases. The determination of VOC profiles lays the groundwork for non-invasive probes of bacterial metabolism and offers prospects for detection of microbe-specific VOC biomarkers from two potential biowarfare agents.

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.

Antibacterial, antibiofilm activity and cytotoxicity of crude extracts of Ptaeroxylon obliquum (Ptaeroxylaceae) used in South African ethnoveterinary medicine against Bacillus anthracis Sterne vaccine strain

Anthrax, a zoonotic disease and a potential biological weapon for terrorism is caused by Bacillus anthracis, a Gram‐positive, sporulating bacterium. Anthrax is endemic in South Africa and primarily affects livestock and wildlife species. Problems with the control of anthrax include growing resistance to present antibiotics and disinfectants or decontaminants coupled with low antibiotic access in rural and poor settings. Therefore, it is imperative to search for new antimicrobials, especially from natural sources that could offer effective and potent alternatives. Medicinal plants are putative sources of new antimicrobials. Ptaeroxylon obliquum (Ptaeroxylaceae) is used traditionally in South Africa as an anthrax remedy in livestock. Here, the antibacterial and antibiofilm activity as well as cytotoxicity of the bark and fresh leaf crude extracts of P. obliquum was determined.A two‐fold serial microdilution assay was used to determine the minimum inhibitory concentration (MIC) of the acetone and chloroform extracts of bark and the chloroform extract of fresh leaves of the plant against B. anthracis Sterne vaccine strain. The crystal violet assay was used to determine antibiofilm activity while cytotoxicity was determined using a tetrazolium‐based colorimetric (MTT) assay against Caco‐2 intestinal cells.The MIC of the extracts ranged from 0.005 – 0.039 mg/mL compared to the positive control gentamicin (MIC = 4.9 × 10−5) with acetone bark extract being the most active (MIC = 0.005 mg/mL). Activity of extracts against B. anthracis biofilms was high (>50 % antibiofilm activity) at 0 h (prevention of biofilms; ranged between 87–99 %), and 24 h (destruction of pre‐formed biofilms; ranged between 58–93 %). The chloroform fresh leaf extract had the best antibiofilm activity. The acetone bark extract had the best total antibacterial activity (yield (mg/g)/MIC (mg/mL)) of 21 500 g/mL. This means that the mass extracted from 1 g of the acetone extract can be diluted by 21 500 times and still inhibit bacterial growth. The 50% lethal concentration (LC50) of the extracts against Caco‐2 cells ranged between 0.03–0.19 mg/mL with the chloroform fresh leaf extract being relatively the safest (LC50 = 0.19 mg/mL). The acetone bark extract had the best selectivity index (LC50/MIC) of 6. This shows that the extract has higher toxicity against the bacteria than to mammalian cells.The results support the use of the plant against anthrax in South African ethnoveterinary medicine. The extracts may be potential alternatives to chemicals as disinfectants or decontaminants, especially in rural areas where chemicals are not accessible to the farmers. The compounds responsible for anti‐B. anthracis activity, and their mechanism(s) of action, is the focus of future studies.Support or Funding InformationThe University of Pretoria is thanked for providing research support

Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection.

In vivo functionalization of diatom biosilica frustules by genetic manipulation requires careful consideration of the overall structure and function of complex fusion proteins. Although we previously had transformed Thalassiosira pseudonana with constructs containing a single domain antibody (sdAb) raised against the Bacillus anthracis Sterne strain, which detected an epitope of the surface layer protein EA1 accessible in lysed spores, we initially were unsuccessful with constructs encoding a similar sdAb that detected an epitope of EA1 accessible in intact spores and vegetative cells. This discrepancy limited the usefulness of the system as an environmental biosensor for B. anthracis. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3T8 had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3T8 targeting peptide to the C-terminus of the fusion protein resulted in successful detection of EA1 with both sdAbs. Homology modeling of antigen binding by the two sdAbs supported the hypothesis that the rescue of antigen binding in the previously dysfunctional sdAb was due to removal of steric hindrances between the antigen binding loops and the diatom biosilica for that particular sdAb.

Development of a rapid immunochromatographic assay for detection of surface array protein (Sap), a potent biomarker of Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax poses serious threat to human health. Genetically, B. anthracis is very close to the other species of this group. Hence, it is important to develop a rapid screening test for B. anthracis. Surface array protein (Sap), a surface layer protein produced by B. anthracis is considered an important biomarker for its detection as it is excreted in the growth media. In the present study, we have developed an Immunochromatography test (ICT) strip for detection of Sap, for rapid screening of B. anthracis strains. The recombinant Sap was produced and used for production of mouse monoclonal antibodies. The antibodies were conjugated with colloidal gold for preparation of ICT. For determination of sensitivity and specificity, different Bacillus species were grown in culture broth, heat inactivated to kill the live bacteria and tested on ICT. The B. anthracis Sterne could be detected after 3 h of growth. Sample testing by ICT takes only 2 min in comparison to 4–5 h in sandwich plate ELISA. The results corroborated that the ICT can be used for rapid screening of B. anthracis isolates. The proposed method is rapid, safe and user friendly for detection of B. anthracis culture.

Galleria mellonella as an Infection Model for Bacillus anthracis Sterne.

Understanding bacterial virulence provides insight into the molecular basis behind infection and could identify new drug targets. However, assessing potential virulence determinants relies on testing in an animal model. The mouse is a well-validated model but it is constrained by the ethical and logistical challenges of using vertebrate animals. Recently the larva of the greater wax moth Galleria mellonella has been explored as a possible infection model for a number of pathogens. In this study, we developed G. mellonella as an infection model for Bacillus anthracis Sterne. We first validated two different infection assays, a survival assay and a competition assay, using mutants containing disruptions in known B. anthracis virulence genes. We next tested the utility of G. mellonella to assess the virulence of transposon mutants with unknown mutations that had increased susceptibility to hydrogen peroxide in in vitro assays. One of these transposon mutants also displayed significantly decreased virulence in G. mellonella. Further investigation revealed that this mutant had a disruption in the petrobactin biosynthesis operon (asbABCDEF), which has been previously implicated in both virulence and defense against oxidative stress. We conclude that G. mellonella can detect attenuated virulence of B. anthracis Sterne in a manner consistent with that of mammalian infection models. Therefore, G. mellonella could serve as a useful alternative to vertebrate testing, especially for early assessments of potential virulence genes when use of a mammalian model may not be ethical or practical.

Identification of Bacillus and Yersinia species and hoax agents by protein profiling using microfluidic capillary electrophoresis with peak detection algorithms

ABSTRACT Bacillus anthracis and Yersinia pestis are biological agents that pose an increasing concern to national security if deliberately disseminated. Hoax agents, including suspicious white powders and environmental bacterial species, can also cause disruption. In either scenario it is of high importance to rapidly and accurately identify any suspicious powder as hazardous or hoax. Protein profiling, using microfluidic capillary electrophoresis, provides a rapid (less than 40 minutes), reliable and field-based screening method. Two commonly encountered hoax agents, three Bacillus species (including B. anthracis Sterne strain), two Yersinia species and E. coli were profiled on the Experion™ System (Bio-Rad). Peak detection algorithms were developed for the identification of protein peaks in electropherograms. Boolean logic paths were then employed to predict the electrophoretic pattern of samples. Parameters assessed included variation within and between Experion™ Pro260 chips and the ability to discriminate between samples over time intervals, between operators and between field and laboratory analyses. Classification with optimal Boolean logic paths reported no misclassification with an accuracy of 100% for B. anthracis Sterne strain, B. thuringiensis (powder and culture-based), B. cereus and plain wheat flour. Overall there was 75% correct identification for the eight sample types tested.

An integrated experimental-computational approach for predicting virulence in New Zealand white rabbits and humans following inhalation exposure to Bacillus anthracis spores.

Inhalation of Bacillus anthracis spores can lead to an anthrax infection that can be fatal. Previously published mathematical models have extrapolated kinetic rates associated with bacterial growth in New Zealand White (NZW) rabbits to humans, but to date, actual measurements of the underlying processes associated with anthrax virulence between species have not been conducted. To address this knowledge gap, we have quantified species-specific rate constants associated with germination, proliferation, and immune cell inactivation of B. anthracis Sterne using an in vitro test platform that includes primary lung epithelial and immune cells. The generated data was then used to develop a physiologically based biokinetic model (PBBK) which quantitatively compares bacterial growth and mean time to death under lethal conditions in rabbits and humans. Simulations based upon our in vitro data and previously published in vivo data from rabbits indicate that disease progression is likely to be faster in humans than in NZW rabbits under comparable total deposited dose conditions. With the computational framework established, PBBK parameters can now be refined using experimental data for lethal B. anthracis strains (e.g. Ames) under identical conditions in future studies. The PBBK model can also be linked to existing aerosol dosimetry models that account for species-specific differences in aerosol deposition patterns to further improve the human health risk assessment of inhalation anthrax.

Discovery of Lipophilic Bisphosphonates That Target Bacterial Cell Wall and Quinone Biosynthesis.

We report that alkyl-substituted bisphosphonates have activity against Bacillus anthracis Sterne (0.40 μg/mL), Mycobacterium smegmatis (1.4 μg/mL), Bacillus subtilis (1.0 μg/mL), and Staphylococcus aureus (13 μg/mL). In many cases, there is no effect of serum binding, as well as low activity against a human embryonic kidney cell line. Targeting of isoprenoid biosynthesis is involved with 74 having IC50 values of ∼100 nM against heptaprenyl diphosphate synthase and 200 nM against farnesyl diphosphate synthase. B. subtilis growth inhibition was rescued by addition of farnesyl diphosphate, menaquinone-4 (MK-4), or undecaprenyl phosphate (UP), and the combination of MK-4 and UP resulted in a 25× increase in ED50, indicating targeting of both quinone and cell wall biosynthesis. Clostridioides difficile was inhibited by 74, and since this organism does not synthesize quinones, cell wall biosynthesis is the likely target. We also solved three X-ray structures of inhibitors bound to octaprenyl diphosphate and/or undecaprenyl diphosphate synthases.

Bacillus anthracis Sterne Strain 34f2 Vaccine Antibody Dose Response by Subcutaneous and Oral Administration

Anthrax (Bacillus anthracis) is a zoonotic disease endemic to environments worldwide. Spores, the dormant form of the bacteria, can survive for decades in nature’s harshest environments and maintain their viability to cause disease. Outbreaks are common in free-ranging livestock and wildlife, thus making anthrax an economically and ecologically important disease. The currently available vaccine to protect livestock is a suspension of B. anthracis Sterne Strain 34F2 spores in saponin (Sterne vaccine). However, it is only available as a subcutaneous injection which is an impractical method of prevention for wildlife. Oral vaccination is the ideal method for free-ranging wildlife, but the Sterne vaccine has never been thoroughly evaluated for oral administration. The current study evaluated the antibody titers induced in mice by subcutaneous or oral vaccination with three different doses of the Sterne vaccine. Results described here show a gradual increase in antibody titers at each time point following subcutaneous vaccination with all vaccine doses. In contrast, no antibody response was detected from any dose or any time point after oral vaccination. Taken together, these results suggest that the Sterne vaccine is only effective as a subcutaneous injection and that an alternate oral anthrax vaccine formulation must be developed to allow for efficient vaccination of free-ranging livestock and wildlife.

Laboratory Validation and Initial Field Testing of an Unobtrusive Bioaerosol Detector for Health Care Settings

ABSTRACT Microorganisms can be transmitted from infected to healthy people as an aerosol. Military bioaerosol detectors currently used by soldiers or first responders may potentially be utilized in health care settings as part of a strategy to prevent the spread of airborne infectious diseases. The goal of this study was to conduct initial laboratory and field validation of an inexpensive and unobtrusive TACBIO detector and compare its performance with that of an expensive bioaerosol detection instrument, the Ultraviolet Aerodynamic Particle Sizer (UV-APS). The laboratory validation test used three bacterial clusters (Bacillus thuringiensis [Bt], Bacillus anthracis Sterne [BaS], and Bacillus atrophaeus var. globigii [Bg]) generated at controlled rates by an ink jet aerosol generator (IJAG). The detection efficiency of the UV-APS was ≥ 99% for all particle generation rates and species. The TACBIO detector exhibited a slightly lower detection efficiency but was still able to detect > 88% of Bt and BaS and 62.7–81.7% of Bg. Field validation conducted with simultaneous UV-APS and TACBIO sampling in an occupied hospital clinic showed both instruments closely tracking each other in detecting fluorescent particles > 1.5 µm in diameter. During a 6 hour sampling period, fluorescent particle (> 1.5 µm) concentrations showed wide short term variation connected to nearby human activity while smaller nonfluorescent particles displayed more gradual changes. These results indicate the usefulness of an unobtrusive environmental aerosol sampler in health care settings, motivating future field characterization and validation studies.

Quality control of batches of bacteriological examination kits «Selective differential-diagnostic medium for recovery of Bacillus anthracis and closely related bacilli of Bacillus cereus group»

Possibilities of quality control of experimental series of a set of reagents for bacteriological studies «Selective differential diagnostic environment for the isolation of the causative agent of anthrax and closely related bacilli of the Bacillus cereus group» is justified. Findings of the investigation of growth properties of the selective medium showed typical colonial morphology of virulent strains of Bacillus anthracis (B. anthracis 81/1, B. anthracis 1(CО), B. anthracis 1193, B. anthracis И-363, B. anthracis 1283) and its vaccine strains B. anthracis CTI-1, B. anthracis Ihtiman, B. anthracis Sterne 34 F2, B. anthracis 55, B. anthracis 71/12 - II Tsenkovsky’s vaccine). The colour of the batches of selective medium and Hottinger’s agar around the Bacillus anthracis colonies was unchanged. Whereas, the colour of selective medium around colonies of closely related saprophytes of genus Bacillus discoloured to bright-yellow, allowing to differentiate colonies of anthracic micro germs and closely related microorganisms. Presence of inhibiting properties of the selective medium was confirmed using heterologous germ cultures (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Proteus vulgaris HX-19) which showed no growth in all series of the tested medium. Meanwhile, the typical colonial morphology of strains of these microorganisms was observed on Hottinger’s agar.

Petrobactin Protects against Oxidative Stress and Enhances Sporulation Efficiency in Bacillus anthracis Sterne.

Bacillus anthracis is a Gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is required for vegetative growth of B. anthracis under iron-depleted conditions and in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficient in vitro sporulation of B. anthracis requires petrobactin, that the petrobactin biosynthesis operon (asbA to -F) is induced prior to sporulation, and that the siderophore itself associates with spores. Petrobactin is also required for oxidative stress protection during late-stage growth and for wild-type levels of sporulation in sporulation medium. Sporulation in bovine blood was found to be petrobactin dependent. Collectively, the in vitro contributions of petrobactin to sporulation as well as growth imply that petrobactin may be required for B. anthracis transmission via the spore during natural infections, in addition to its key known functions during active anthrax infections.IMPORTANCEBacillus anthracis causes the disease anthrax, which is transmitted via its dormant, spore phase. However, conversion from bacillus to spore is a complex, energetically costly process that requires many nutrients, including iron. B. anthracis requires the siderophore petrobactin to scavenge iron from host environments. We show that, in the Sterne strain, petrobactin is required for efficient sporulation, even when ample iron is available. The petrobactin biosynthesis operon is expressed during sporulation, and petrobactin is biosynthesized during growth in high-iron sporulation medium, but instead of being exported, the petrobactin remains intracellular to protect against oxidative stress and improve sporulation. It is also required for full growth and sporulation in blood (bovine), an essential step for anthrax transmission between mammalian hosts.

Flexible Peptide Linkers Enhance the Antimicrobial Activity of Surface-Immobilized Bacteriolytic Enzymes.

Chemical linkers are frequently used in enzyme immobilization to improve enzyme flexibility and activity, whereas peptide linkers, although ubiquitous in protein engineering, are much less explored in enzyme immobilization. Here, we report peptide-linker-assisted noncovalent immobilization of the bacteriolytic enzyme lysostaphin (Lst) to generate anti- Staphylococcus aureus surfaces. Lst was immobilized through affinity tags onto a silica surface (glass slides) and nickel nitrilotriacetic acid (NiNTA) agarose beads via silica-binding peptides (SiBPs) or a hexahistidine tag (His-tag) fused at the C-terminus of Lst, respectively. By inserting specific peptide linkers upstream of the SiBP or His-tag, the immobilized enzymes killed >99.5% of S. aureus ATCC 6538 cells (108 CFU/mL) within 3 h in buffer and could be reused multiple times without significant loss of activity. In contrast, immobilized Lst without a peptide linker was less active/stable. Molecular modeling of Lst-linker-affinity tag constructs illustrated that the presence of the peptide linkers enhanced the molecular flexibility of the proximal Lst binding domain, which interacts with the bacterial substrate, and such increased flexibility correlated with increased antimicrobial activity. We further show that Lst immobilized onto NiNTA beads retained the ability to kill ∼99% of a 108 CFU/mL microbial challenge even in the presence of 1% of a commercial anionic surfactant, C12-14 alcohol EO 3:1 sodium sulfate, when the Lst construct contained a decapeptide linker containing glycine, serine, and alanine residues. This linker-assisted immobilization strategy could be extended to an unrelated lytic enzyme, the endolysin PlyPH, to target Bacillus anthracis Sterne cells either in buffer or in the presence of anionic surfactants. Our approach, therefore, provides a facile route to the use of antimicrobial enzymes on surfaces.

New technology for development of host-targeted therapeutic vaccine against anthrax

Abstract Anthrax is a serious disease caused by Bacillus anthracis, a bacterium that can form spores for long-term survival and secrete anthrax toxins including protective antigen (PA), lethal factor (LF), and edema factor (EF). However, current anthrax postexposure treatments are inadequate at later stages of infection. Previously, our research have shown RNA inhibition (RNAi) on host cells silences anthrax toxin receptors (ATRs): tumor endothelial marker 8 (TEM8), capillary morphogenesis protein 2 (CMG2). This approach was protective against the cytotoxicity of anthrax toxins, indicating that ATR-targeted RNAi can be used as a therapy against anthrax. However, inefficient cytosolic delivery and toxicity of siRNA delivery vehicles limit the use siRNA as therapeutics. In this study, we have developed a detoxified anthrax edema toxin, which consists of PA and nontoxic N-terminal fragment of EF (EFn) conjugated with a peptide nona-D-arginine residues (EFn-9dR) to enable siRNA binding. The detoxified toxin was able to deliver specific siRNA to induce cmg2 gene silencing in different cell lines and C57BL/6 mice, and provide significant protection against anthrax lethal toxin challenge in vitro and in mice. Survived mice from toxin challenge were fully protected against lethal challenge with B. anthracis Sterne spores. The immune protective mechanism is mainly due to the significantly high serum neutralizing antibody response against anthrax toxins in these mice. These results suggest that detoxified anthrax toxin provides a tool for delivery of host-targeted siRNA into anthrax pathogenesis-associated host cells, and this new system could potentially be developed as a lifesaving postexposure therapeutic vaccine against anthrax.

Transcriptional profiling of the clpX mutant in Bacillus anthracis reveals regulatory connection with the lrgAB operon.

ClpX functions as either an independent chaperone or a component of the ClpXP protease, a conserved intracellular protease that acts as a global regulator in the bacterial cell by degrading regulatory proteins, stress response proteins and rate-limiting enzymes. Previously, we found that loss of clpX in Bacillus anthracis Sterne leads to increased susceptibility to antimicrobial agents that target the cell envelope. The aim of this study was to identify genes within the regulatory network of clpX that contribute to antimicrobial resistance. Using microarray analysis, we found 119 genes that are highly differentially expressed in the ∆clpX mutant, with the majority involved in metabolic, transport or regulatory functions. Several of these differentially expressed genes, including glpF, sigM, mrsA, lrgA and lrgB, are associated with cell wall-active antibiotics in other bacterial species. We focused on lrgA and lrgB, which form the lrgAB operon and are downregulated in ∆clpX, because loss of lrgAB increases autolytic activity and penicillin susceptibility in Staphylococcus aureus. While we observed no changes in autolytic activity in either ∆clpX or ∆lrgAB B. anthracis Sterne, we find that both mutants have increased susceptibility to the antimicrobial peptide LL-37 and daptomycin. However, phenotypes between ∆clpX and ∆lrgAB are not identical as ∆clpX also displays increased susceptibility to penicillin and nisin but ∆lrgAB does not. Therefore, while decreased expression of lrgAB may be partially responsible for the increased antimicrobial susceptibility seen in the ∆clpX mutant, disruption of other pathways must also contribute to this phenotype.