Efficient Genome Editing by a Miniature CRISPR-AsCas12f1 Nuclease in Bacillus anthracis

A miniature CRISPR-Cas12f has been demonstrated to serve as an effective genome editing tool in gram negative bacteria as well as human cells. Here, we developed an alternative method to edit the genome of Bacillus anthracis based on the AsCas12f1 nuclease from Acidibacillus sulfuroxidans. When the htrA gene on the chromosome and the lef gene on the plasmid pXO1 were selected as targets, the CRISPR-AsCas12f1 system showed very high efficiency (100%). At the same time, a high efficiency was observed for large-fragment deletion. Our results also indicated that the length of the homologous arms of the donor DNA had a close relationship with the editing efficiency. Furthermore, a two-plasmid CRISPR-AsCas12f1 system was also constructed and combined with the endonuclease I-SceI for potential multi-gene modification. This represents a novel tool for mutant strain construction and gene function analyses in B. anthracis and other Bacillus cereus group bacteria.

Antimicrobial Activity and Degradation Ability Study on Nanoparticle-Enriched Formulations Specially Designed for the Neutralization of Real and Simulated Biological and Chemical Warfare Agents

The present work reveals a comprehensive decontamination study on real and simulated biological and chemical warfare agents (BCWA). The emphasis was on evaluating the antimicrobial activity against real biological warfare agents, such as Bacillus anthracis, and also the capacity of neutralizing real chemical warfare agents, such as mustard gas or soman, by employing three different types of organic solutions enriched with ZnO, TiO2, and zeolite nanoparticles, specially designed for decontamination applications. The capacity of decontaminating BCWA was evaluated through specific investigation tools, including surface monitoring with the swabs method, minimum inhibitory (MIC) and minimum bactericidal concentration (MBC) evaluations, time-kill tests for microorganisms, and GC-MS for monitoring chemical agents on different types of surfaces (glass, painted metal, rubber, and cotton butyl rubber). These tests revealed high decontamination factors for BCWA even after only 10 min, accomplishing the requirements imposed by NATO standards. At the completion of the decontamination process, the formulations reached 100% efficacy for Bacillus anthracis after 10–15 min, for soman after 20–30 min, and for mustard gas in an interval comprised between 5 and 24 h depending on the type of surface analyzed.

Bacillus anthracis induces NLRP3 inflammasome activation and caspase-8–mediated apoptosis of macrophages to promote lethal anthrax

Lethal toxin (LeTx)-mediated killing of myeloid cells is essential for Bacillus anthracis, the causative agent of anthrax, to establish systemic infection and induce lethal anthrax. The “LeTx-sensitive” NLRP1b inflammasome of BALB/c and 129S macrophages swiftly responds to LeTx intoxication with pyroptosis and secretion of interleukin (IL)-1β. However, human NLRP1 is nonresponsive to LeTx, prompting us to investigate B. anthracis host–pathogen interactions in C57BL/6J (B6) macrophages and mice that also lack a LeTx-sensitive Nlrp1b allele. Unexpectedly, we found that LeTx intoxication and live B. anthracis infection of B6 macrophages elicited robust secretion of IL-1β, which critically relied on the NLRP3 inflammasome. TNF signaling through both TNF receptor 1 (TNF-R1) and TNF-R2 were required for B. anthracis-induced NLRP3 inflammasome activation, which was further controlled by RIPK1 kinase activity and LeTx-mediated proteolytic inactivation of MAP kinase signaling. In addition to activating the NLRP3 inflammasome, LeTx-induced MAPKK inactivation and TNF production sensitized B. anthracis-infected macrophages to robust RIPK1- and caspase-8–dependent apoptosis. In agreement, purified LeTx triggered RIPK1 kinase activity- and caspase-8–dependent apoptosis only in macrophages primed with TNF or following engagement of TRIF-dependent Toll-like receptors. Consistently, genetic and pharmacological inhibition of RIPK1 inhibited NLRP3 inflammasome activation and apoptosis of LeTx-intoxicated and B. anthracis-infected macrophages. Caspase-8/RIPK3-deficient mice were significantly protected from B. anthracis-induced lethality, demonstrating the in vivo pathophysiological relevance of this cytotoxic mechanism. Collectively, these results establish TNF- and RIPK1 kinase activity–dependent NLRP3 inflammasome activation and macrophage apoptosis as key host–pathogen mechanisms in lethal anthrax.

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.

BrnQ-type Branched-chain Amino Acid Transporters Influence Bacillus anthracis Growth and Virulence

Bacillus anthracis, the anthrax agent, exhibits robust proliferation in diverse niches of mammalian hosts. Metabolic attributes of B. anthracis that permit rapid growth in multiple mammalian tissues have not been established. We posit that branched-chain amino acid (BCAA: Isoleucine, leucine and valine) metabolism is key to B. anthracis pathogenesis. Increasing evidence indicates relationships between B. anthracis virulence and expression of BCAA-related genes. Expression of some BCAA-related genes is altered during culture in bovine blood in vitro and the bacterium exhibits valine auxotrophy in a blood serum mimic medium. Transcriptome analyses have revealed that the virulence regulator AtxA, that positively affects expression of the anthrax toxin and capsule genes, negatively regulates genes predicted to be associated with BCAA biosynthesis and transport. Here, we show that B. anthracis growth in defined media is severely restricted in the absence of exogenous BCAAs, indicating that BCAA transport is required for optimal growth in vitro. We demonstrate functional redundancy among multiple BrnQ-type BCAA transporters. Three transporters are associated with isoleucine and valine transport, and deletion of one, BrnQ3, attenuates virulence in a murine model for anthrax. Interestingly, an ilvD-null mutant lacking dihydroxy-acid dehydratase, an enzyme essential for BCAAs synthesis, exhibits unperturbed growth when cultured in media containing BCAAs, but is highly attenuated in the murine model. Finally, our data show that BCAAs enhance AtxA activity in a dose-dependent manner, suggesting a model in which BCAAs serve as a signal for virulence gene expression.

Bacillus anthracis Phylogeography: New Clues From Kazakhstan, Central Asia

This article describes Bacillus anthracis strains isolated in Kazakhstan since the 1950s until year 2016 from sixty-one independent events associated with anthrax in humans and animals. One hundred and fifty-four strains were first genotyped by Multiple Locus VNTR (variable number of tandem repeats) Analysis (MLVA) using 31 VNTR loci. Thirty-five MLVA31 genotypes were resolved, 28 belong to the A1/TEA group, five to A3/Sterne-Ames group, one to A4/Vollum and one to the B clade. This is the first report of the presence of the B-clade in Kazakhstan. The MLVA31 results and epidemiological data were combined to select a subset of seventy-nine representative strains for draft whole genome sequencing (WGS). Strains from Kazakhstan significantly enrich the known phylogeny of the Ames group polytomy, including the description of a new branch closest to the Texas, United States A.Br.Ames sublineage stricto sensu. Three among the seven currently defined branches in the TEA polytomy are present in Kazakhstan, “Tsiankovskii”, “Heroin”, and “Sanitary Technical Institute (STI)”. In particular, strains from the STI lineage are largely predominant in Kazakhstan and introduce numerous deep branching STI sublineages, demonstrating a high geographic correspondence between “STI” and Kazakhstan, Central Asia. This observation is a strong indication that the TEA polytomy emerged after the last political unification of Asian steppes in the fourteenth century of the Common Era. The phylogenetic analysis of the Kazakhstan data and of currently available WGS data of worldwide origin strengthens our understanding of B. anthracis geographic expansions in the past seven centuries.

Phytoremediation of Cadmium Contaminated Soil Using Sesbania sesban L. in Association with Bacillus anthracis PM21: A Biochemical Analysis

Sustainable food production to feed nine to 10 billion people by 2050 is one of the greatest challenges we face in the 21st century. Due to anthropogenic activities, cadmium (Cd) contamination is ubiquitous with deleterious effects on plant and soil microbiota. In the current study, the phytoremediation potential of Sesbania sesban L. was investigated in Cd-spiked soil inoculated with Bacillus anthracis PM21. The Cd-spiked soil drastically reduced important plant attributes; however, inoculation of B. anthracis PM21 significantly (p ≤ 0.05) enhanced root length (17.21%), shoot length (15.35%), fresh weight (37.02%), dry weight (28.37%), chlorophyll a (52.79%), chlorophyll b (48.38%), and total chlorophyll contents (17.65%) at the Cd stress level of 200 mg/kg as compared to the respective control. In addition, bacterial inoculation improved superoxide dismutase (11.98%), peroxidase (12.16%), catalase (25.26%), and relative water content (16.66%) whereas it reduced proline content (16.37%), malondialdehyde content (12.67%), and electrolyte leakage (12.5%). Inoculated plants showed significantly (p ≤ 0.05) higher Cd concentration in the S. sesban root (118.6 mg/kg) and shoot (73.4 mg/kg) with a translocation (0.61) and bioconcentration factor (0.36), at 200 mg/kg Cd. Surface characterization of bacteria through Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) predicted the involvement of various functional groups and cell surface morphology in the adsorption of Cd ions. Amplification of the CzcD gene in strain PM21, improved antioxidant activities, and the membrane stability of inoculated S. sesban plants conferred Cd tolerance of strain PM21. In addition, the evaluated bacterial strain B. anthracis PM21 revealed significant plant growth-promoting potential in S. sesban; thus, it can be an effective candidate for phyto-remediation of Cd-polluted soil.

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.