Sharon Ehrlich

HtrA is a major virulence determinant of Bacillus anthracis.

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1+, pXO2+), or in the ΔVollum (pXO1‐, pXO2‐, nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation‐associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 108 spores, in the guinea pig model of anthrax), as compared with the parental wild‐type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans‐complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.

Identification and characterization of novel and potent transcription promoters of Francisella tularensis.

ABSTRACT Two alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potent Francisella tularensis promoters. Of the 26,000 F. tularensis strain LVS gfp library clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr). Out of a total of 2,000 chloramphenicol-resistant LVS clones isolated from the cat promoter library, we arbitrarily selected 40 for further analysis. Over 80% of these clones carry unique F. tularensis DNA sequences which appear to drive a wide range of protein expression, as determined by specific chloramphenicol acetyltransferase (CAT) Western dot blot and enzymatic assays. The DNA sequence information for the 33 unique and novel F. tularensis promoters reported here, along with the results of in silico and primer extension analyses, suggest that F. tularensis possesses classical Escherichia coli σ70-related promoter motifs. These motifs include the −10 (TATAAT) and −35 [TTGA(C/T)A] domains and an AT-rich region upstream from −35, reminiscent of but distinct from the E. coli upstream region that is termed the UP element. The most efficient promoter identified (Pbfr) appears to be about 10 times more potent than the F. tularensis groEL promoter and is probably among the strongest promoters in F. tularensis. The battery of promoters identified in this work will be useful, among other things, for genetic manipulation in the background of F. tularensis intended to gain better understanding of the mechanisms involved in pathogenesis and virulence, as well as for vaccine development studies.

Quantitative profiling of the in vivo enzymatic activity of ricin reveals disparate depurination of different pulmonary cell types.

The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template. We found that maximal depurination after intranasal exposure of mice to 2LD50 ricin was reached 48h, where nearly 40% of the ribosomes have been depurinated and that depurination can be halted by post-exposure administration of anti-ricin antibodies. We next demonstrated that the effect of ricin intoxication on different cell types populating the lungs differs greatly, and that outstandingly high levels of damage (80% depurination), were observed in particular for pulmonary epithelial cells. Finally, we found that the magnitude of depurination induced by the related plant-derived toxin abrin, was significantly lower in comparison to ricin, and can be attributed mostly to reduced depurination of pulmonary epithelial cells by abrin. This study provides for the first time vital information regarding the scope and timing of the catalytic performance of ricin and abrin in the lungs of intact animals.

Potent anti-edematous and protective effects of ciprofloxacin in pulmonary ricinosis

ABSTRACT The plant toxin ricin is considered a biological threat agent of concern and is most toxic when inhaled. Pulmonary exposure to a lethal dose of ricin can be redressed by treatment with antiricin antibodies; however, late antitoxin intervention is of limited efficacy. This limitation is associated with overt lung damage, clinically manifested as severe pulmonary inflammation, which develops over time. Increased evidence indicates that ciprofloxacin, a broad-spectrum antimicrobial agent, possesses immunomodulatory properties. Here we demonstrate that while antiricin antibody administration at late hours after intranasal exposure to ricin confers limited protection to mice, highly efficient protection can be achieved by adding ciprofloxacin to the antibody treatment. We further demonstrate that parameters associated with lung injury, in particular, pulmonary proinflammatory cytokine production, neutrophil migration, and edema, are sharply reduced in ricin-intoxicated mice that were treated with ciprofloxacin. The presented data highlight the potential clinical application of ciprofloxacin as a beneficial immunomodulatory agent in the course of ricin intoxication.

Acetylcholinesterase-Fc Fusion Protein (AChE-Fc): A Novel Potential Organophosphate Bioscavenger with Extended Plasma Half-Life

Acetylcholinesterase (AChE) is the physiological target of organophosphate nerve agent compounds. Currently, the development of a formulation for prophylactic administration of cholinesterases as bioscavengers in established risk situations of exposure to nerve agents is the incentive for many efforts. While cholinesterase bioscavengers were found to be highly effective in conferring protection against nerve agent exposure in animal models, their therapeutic use is complicated by short circulatory residence time. To create a bioscavenger with prolonged plasma half-life, compatible with biotechnological production and purification, a chimeric recombinant molecule of HuAChE coupled to the Fc region of human IgG1 was designed. The novel fusion protein, expressed in cultured cells under optimized conditions, maintains its full enzymatic activity, at levels similar to those of the recombinant AChE enzyme. Thus, this novel fusion product retained its binding affinity toward BW284c5 and propidium, and its bioscavenging reactivity toward the organophosphate-AChE inhibitors sarin and VX. Furthermore, when administered to mice, AChE-Fc exhibits exceptional circulatory residence longevity (MRT of 6000 min), superior to any other known cholinesterase-based recombinant bioscavengers. Owing to its optimized pharmacokinetic performance, high reactivity toward nerve agents, and ease of production, AChE-Fc emerges as a promising next-generation organophosphate bioscavenger.

Protective Immunity against Lethal F. tularensis holarctica LVS Provided by Vaccination with Selected Novel CD8+ T Cell Epitopes

Recently we described an unbiased bacterial whole-genome immunoinformatic analysis aimed at selection of potential CTL epitopes located in “hotspots” of predicted MHC-I binders. Applying this approach to the proteome of the facultative intra-cellular pathogen Francisella tularensis resulted in identification of 170 novel CTL epitopes, several of which were shown to elicit highly robust T cell responses. Here we demonstrate that by DNA immunization using a short DNA fragment expressing six of the most prominent identified CTL epitopes a potent and specific CD8+ T cell responses is being induced, to all encoded epitopes, a response not observed in control mice immunized with the DNA vector alone Moreover, this CTL-specific mediated immune response prevented disease development, allowed for a rapid clearance of the bacterial infection and provided complete protection against lethal challenge (10LD50) with F. tularensis holarctica Live Vaccine Strain (LVS) (a total to 30 of 30 immunized mice survived the challenge while all control DNA vector immunized mice succumbed). Furthermore, and in accordance with these results, CD8 deficient mice could not be protected from lethal challenge after immunization with the CTL-polyepitope. Vaccination with the DNA poly-epitope construct could even protect mice (8/10) against the more demanding pulmonary lethal challenge of LVS. Our approach provides a proof-of-principle for selecting and generating a multi-epitpoe CD8 T cell-stimulating vaccine against a model intracellular bacterium.

Next-Generation Bacillus anthracis Live Attenuated Spore Vaccine Based on the htrA - (High Temperature Requirement A) Sterne Strain

Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis. Live attenuated vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔhtrA strain secretes functional anthrax toxins but is 10–104-fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits). In spite of this attenuation, double or even single immunization with SterneΔhtrA spores elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization.

Proteomic Studies of Bacillus anthracis Reveal In Vitro CO2-Modulation and Expression During Infection of Extracellular Proteases

A comparative proteomic study of secretomes of virulent and avirulent Bacillus anthracis strains in various culturing conditions, including those encountered in the host (high CO2/bicarbonate), enabled identification of approximately 70 proteins representing collectively more than 99% of the secretome. In-vivo expression of 50 proteins was established by 2-dimension Western-analysis using anti B. anthracis immune sera. Many of the abundant proteins harbor features characteristic of virulence determinants and exhibit different patterns of expression. In minimal medium, virulent and avirulent B. anthracis strains manifest similar protein signatures and the metalloprotease NprA, (previously suggested to act in the context of a starvation-induced mechanism), represents 90% of the total secretome. Under high CO2/bicarbonate, NprA is repressed (possibly by a mechanism which preserves toxin integrity), while other proteins, including the bacterial toxins, are induced. One of the immunogens observed to be induced under high CO2-tension, was HtrA. We investigated the phenotype associated with disruption of HtrA by biochemical and proteomic approaches. The HtrA- bacteria are severely affected in their ability to respond to stress and fail to secrete the most abundant extracellular protease NprA. Most surprisingly, HtrA- cells do not possess the characteristic S-layer. This unique phenotype may have important implications for the role of HtrA in manifestation of B. anthracis virulence. Furthermore, the data show that distinct CO2/bicarbonate responsive chromosome-and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.

A novel swine model of ricin-induced acute respiratory distress syndrome

ABSTRACT Pulmonary exposure to the plant toxin ricin leads to respiratory insufficiency and death. To date, in-depth study of acute respiratory distress syndrome (ARDS) following pulmonary exposure to toxins is hampered by the lack of an appropriate animal model. To this end, we established the pig as a large animal model for the comprehensive study of the multifarious clinical manifestations of pulmonary ricinosis. Here, we report for the first time, the monitoring of barometric whole body plethysmography for pulmonary function tests in non-anesthetized ricin-treated pigs. Up to 30 h post-exposure, as a result of progressing hypoxemia and to prevent carbon dioxide retention, animals exhibited a compensatory response of elevation in minute volume, attributed mainly to a large elevation in respiratory rate with minimal response in tidal volume. This response was followed by decompensation, manifested by a decrease in minute volume and severe hypoxemia, refractory to oxygen treatment. Radiological evaluation revealed evidence of early diffuse bilateral pulmonary infiltrates while hemodynamic parameters remained unchanged, excluding cardiac failure as an explanation for respiratory insufficiency. Ricin-intoxicated pigs suffered from increased lung permeability accompanied by cytokine storming. Histological studies revealed lung tissue insults that accumulated over time and led to diffuse alveolar damage. Charting the decline in PaO2/FiO2 ratio in a mechanically ventilated pig confirmed that ricin-induced respiratory damage complies with the accepted diagnostic criteria for ARDS. The establishment of this animal model of pulmonary ricinosis should help in the pursuit of efficient medical countermeasures specifically tailored to deal with the respiratory deficiencies stemming from ricin-induced ARDS. Summary: Lung injury induced by intratracheal instillation of ricin to swine exhibits the full set of parameters defining ARDS.

A novel live attenuated anthrax spore vaccine based on an acapsular Bacillus anthracis Sterne strain with mutations in the htrA, lef and cya genes

We recently reported the development of a novel, next-generation, live attenuated anthrax spore vaccine based on disruption of the htrA (High Temperature Requirement A) gene in the Bacillus anthracis Sterne veterinary vaccine strain. This vaccine exhibited a highly significant decrease in virulence in murine, guinea pig and rabbit animal models yet preserved the protective value of the parental Sterne strain. Here, we report the evaluation of additional mutations in the lef and cya genes, encoding for the toxin components lethal factor (LF) and edema factor (EF), to further attenuate the SterneΔhtrA strain and improve its compatibility for human use. Accordingly, we constructed seven B. anthracis Sterne-derived strains exhibiting different combinations of mutations in the htrA, cya and lef genes. The various strains were indistinguishable in growth in vitro and in their ability to synthesise the protective antigen (PA, necessary for the elicitation of protection). In the sensitive murine model, we observed a gradual increase (ΔhtrA<ΔhtrAΔcya<ΔhtrAΔlef<ΔhtrAΔlefΔcya) in attenuation - up to 108-fold relative to the parental Sterne vaccine strain. Most importantly, all various SterneΔhtrA derivative strains did not differ in their ability to elicit protective immunity in guinea pigs. Immunisation of guinea pigs with a single dose (109 spores) or double doses (>107spores) of the most attenuated triple mutant strain SterneΔhtrAlefMUTΔcya induced a robust immune response, providing complete protection against a subsequent respiratory lethal challenge. Partial protection was observed in animals vaccinated with a double dose of as few as 105spores. Furthermore, protective immune status was maintained in all vaccinated guinea pigs and rabbits for at least 40 and 30weeks, respectively.