Anthrax Toxins Induce Shock in Rats by Depressed Cardiac Ventricular Function

Anthrax infections are frequently associated with severe and often irreversible hypotensive shock. The isolated toxic proteins of Bacillus anthracis produce a non-cytokine-mediated hypotension in rats by unknown mechanisms. These observations suggest the anthrax toxins have direct cardiovascular effects. Here, we characterize these effects. As a first step, we administered systemically anthrax lethal toxin (LeTx) and edema toxin (EdTx) to cohorts of three to twelve rats at different doses and determined the time of onset, degree of hypotension and mortality. We measured serum concentrations of the protective antigen (PA) toxin component at various time points after infusion. Peak serum levels of PA were in the µg/mL range with half-lives of 10–20 minutes. With doses that produced hypotension with delayed lethality, we then gave bolus intravenous infusions of toxins to groups of four to six instrumented rats and continuously monitored blood pressure by telemetry. Finally, the same doses used in the telemetry experiments were given to additional groups of four rats, and echocardiography was performed pretreatment and one, two, three and twenty-four hours post-treatment. LeTx and EdTx each produced hypotension. We observed a doubling of the velocity of propagation and 20% increases in left ventricular diastolic and systolic areas in LeTx-treated rats, but not in EdTx-treated rats. EdTx-but not LeTx-treated rats showed a significant increase in heart rate. These results indicate that LeTx reduced left ventricular systolic function and EdTx reduced preload. Uptake of toxins occurs readily into tissues with biological effects occurring within minutes to hours of serum toxin concentrations in the µg/mL range. LeTx and EdTx yield an irreversible shock with subsequent death. These findings should provide a basis for the rational design of drug interventions to reduce the dismal prognosis of systemic anthrax infections

PhD

dissertationSqualene:hopene cyclases (SHCs) are bacterial enzymes that convert squalene into hopanoids, a function analogous to the action of oxidosqualene cyclases (OSCs) in eukaryotic steroid and triterpenoid biosynthesis. Selective inhibitors for these two enzymes would uniquely suppress the committed step in triterpene biosynthesis, thus acting as cholesterol lowering drugs as well as antifungal or antimicrobial agents demonstrating minimal side effects. Ro48-8071, a benzophenone-containing hypocholesteremic drug, is a selective, potent, photoactivatable inhibitor of SHC. Identification of the binding site of Ro48-8071 for SHC from Alicyclobacillus acidocaldarius is described herein, Edman degradation of a peptide fragment of covalently modified SHC confirmed that Ala44 was specifically modified. Molecular modeling, using X-ray derived protein, coordinates and a single point constraint for the inhibitor, was then employed to define the enzyme inhibitor complex. Subsequent mutagenesis studies provide evidence that the nucleophilicity and positioning of Glu45 is crucial for its stabilization of the carbocation intermediates. Asp or Ala substitution resulted in significant lower cyclization activity, in addition to altered products ratios. Replacement of the conserved "DDTAV V" motif is SHC with the DCTAEA" motif from OSC changes the substrate specificity. This mutant cyclizes, 2,3-oxidosqualene but cannon process squalene. Mono- and pent acyclic 3-hydrosy triterpenes were isolated and characterized from the cyclization mixture for this mutant. (3S)-29-Methylidene-2,3-oxidosqualene (29-MOS) was a mechanism-based irreversible inhibitor of both OSC and SHC. A peptide fragment of SHC was identified to bind 29-MOS, and the specific residues were further defined. A dammarene derivative was isolated from the incubation mixture as a major cyclization product of 29-MOS. Sequence comparison led to the identification of a 501-residue protein from M. tuberculosis that was homologous to SHC and OSC. This gene was cloned and express in E. coli, but no cyclization activity for squalene and OS could be detected. However, this protein was found to be specifically labeled by [3H]Ro18-8071, and it was also shown to cyclize 2,3:22,23-dioxidosqualence (DOS) into a novel product. Catalytic function of this enzyme was further discussed

Anthrax Toxins Induce Shock in Rats by Depressed Cardiac Ventricular Function

Anthrax infections are frequently associated with severe and often irreversible hypotensive shock. The isolated toxic proteins of Bacillus anthracis produce a non-cytokine-mediated hypotension in rats by unknown mechanisms. These observations suggest the anthrax toxins have direct cardiovascular effects. Here, we characterize these effects. As a first step, we administered systemically anthrax lethal toxin (LeTx) and edema toxin (EdTx) to cohorts of three to twelve rats at different doses and determined the time of onset, degree of hypotension and mortality. We measured serum concentrations of the protective antigen (PA) toxin component at various time points after infusion. Peak serum levels of PA were in the µg/mL range with half-lives of 10–20 minutes. With doses that produced hypotension with delayed lethality, we then gave bolus intravenous infusions of toxins to groups of four to six instrumented rats and continuously monitored blood pressure by telemetry. Finally, the same doses used in the telemetry experiments were given to additional groups of four rats, and echocardiography was performed pretreatment and one, two, three and twenty-four hours post-treatment. LeTx and EdTx each produced hypotension. We observed a doubling of the velocity of propagation and 20% increases in left ventricular diastolic and systolic areas in LeTx-treated rats, but not in EdTx-treated rats. EdTx-but not LeTx-treated rats showed a significant increase in heart rate. These results indicate that LeTx reduced left ventricular systolic function and EdTx reduced preload. Uptake of toxins occurs readily into tissues with biological effects occurring within minutes to hours of serum toxin concentrations in the µg/mL range. LeTx and EdTx yield an irreversible shock with subsequent death. These findings should provide a basis for the rational design of drug interventions to reduce the dismal prognosis of systemic anthrax infections

Survival of Sprague Dawley rats (250 to 300 g) treated with single intravenous bolus infusion of LeTx (0.03 mg PA+0.015 mg LF) or EdTx (0.15 mg PA+0.075 mg EF) and monitored for two weeks.

<p>These are the same doses used for telemetry and ECHO experiments. N = 12 for each experiment. Kaplan-Meier graphs prepared.</p

Boxplots at different times post anthrax toxin bolus iv infusion of echocardiographic parameters:

<p>A. LeTx treated rats at zero, one and two hr post-infusion, velocity of propagation was 24±5, 34±7 and 46±17 cm/sec, respectively, with significant difference (P = 0.05 at one hr compared to controls and P = 0.007 at two hr compared to controls); B. LeTx treated rats at zero and one hr post-infusion, left ventricular diastolic area was 0.98±0.07 and 1.15±0.06, respectively, with significant difference (P = 0.01); C. LeTx treated rats at zero and one hr post-infusion, left ventricular systolic area was 0.79±0.08 and 0.98±0.10 cm, respectively, with significant difference (P = 0.02); D. EdTx treated rats at zero, one and two hr post-infusion, heart rate was 326±49, 371±28, and 393±10 beats per min, respectively, with significant difference (P = 0.05 at one hr and P = 0.001 at two hr compared to controls). The box represents the middle 50% of the data. The line through the box represents the median. The line (whiskers) extending from the box represent the upper and lower 25% of the data. The line of each plot connects the means of the sample.</p

Mortality of Anthrax Toxins^*

*<p>n = the number of animals used in each dose study</p

Serum levels of PA after single bolus intravenous infusions of different doses of LeTx to Sprague Dawley rats (250 to 300 g).

<p>Concentrations of toxin components measured by ELISA as described in the text. Peak concentrations and half-lives shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000466#pone-0000466-t002" target="_blank">Table 2</a>.</p

Anthrax toxins in serum samples^*

*<p>Three animals were used in each dose study; ND, not determined.</p