Effective antiprotease-antibiotic treatment of experimental anthrax

Abstract

Background: Inhalation anthrax is characterized by a systemic spread of the challenge agent, Bacillus anthracis. It causes severe damage, including multiple hemorrhagic lesions, to host tissues and organs. It is widely believed that anthrax lethal toxin secreted by proliferating bacteria is a major cause of death, however, the pathology of intoxication in experimental animals is drastically different from that found during the infectious process. In order to close a gap between our understanding of anthrax molecular pathology and the most prominent clinical features of the infectious process we undertook bioinformatic and experimental analyses of potential proteolytic virulence factors of B. anthracis distinct from lethal toxin.

Methods: Secreted proteins (other than lethal and edema toxins) produced by B. anthracis were tested for tissue-damaging activity and toxicity in mice. Chemical protease inhibitors and rabbit immune sera raised against B. anthracis proteases were used to treat mice challenged with B. anthracis (Sterne) spores.

Results: B. anthracis strain delta Ames (pXO1-, pXO2-) producing no lethal and edema toxins secrets a number of metalloprotease virulence factors upon cultivation under aerobic conditions, including those with hemorrhagic, caseinolytic and collagenolytic activities, belonging to M4 and M9 thermolysin and bacterial collagenase families, respectively. These factors are directly toxic to DBA/2 mice upon intratracheal administration at 0.5 mg/kg and higher doses. Chemical protease inhibitors (phosphoramidon and 1, 10-phenanthroline), as well as immune sera against M4 and M9 proteases of B. anthracis, were used to treat mice challenged with B. anthracis (Sterne) spores. These substances demonstrate a substantial protective efficacy in combination with ciprofloxacin therapy initiated as late as 48 h post spore challenge, compared to the antibiotic alone.

Conclusion: Secreted proteolytic enzymes are important pathogenic factors of B. anthrasis, which can be considered as effective therapeutic targets in the development of anthrax treatment and prophylactic approaches complementing anti-lethal toxin therapy.

Figure 1

SDS-PAGE of BACS fractions separated on size exclusion column (A) and Western blots: fractions (A) with specific antisera a-M4EL (B), BACS with a-M4AC (C, left lane), BACS with a-M4EP (C, right lane), BACS with a-M9Coll (D, right lane), and zymograms of caseinolytic and gelatinolytic activities of BACS (D, left and center lanes, correspondingly). Molecular masses (KDa) of the marker proteins are indicated by arrows. In A, s denoted BACS, and numbers above correspond to column fractions. In zymogram gels 3 μl of BACS were loaded.

Figure 2

Hemorrhagic activity of culture supernatants (A), its graphic representation (B) and inhibition with chemical inhibitors (C). 100 μl of secreted proteins (20 to 100 μg) were subcutaneously (sc) injected into the femoral artery region for observation of hemorrhagic changes. The fur over the femoral artery region was removed to allow observation of a 1.5 to 2.5 cm² area of skin. Control mice were administered equal amount of phosphate-buffered saline. Error bars indicate standard deviations.

Figure 3

Survival of mice upon intratracheal injection of BACS.

Figure 4

Protection of mice against B. anthracis (Sterne) infection by administration of ciprofloxacin and its combination with phosphoramidon for 10 days beginning 24 h and 48 h post spore challenge.

Figure 5

Protection of mice against B. anthracis (Sterne) infection by administration of ciprofloxacin or its combination with phenanthroline for 10 days beginning 24 h and 48 h post spore challenge.

Figure 6

Post exposure efficacy of hyperimmune rabbit sera in mice challenged with B. anthracis (Sterne). Treatment with sera and ciprofloxacin was initiated 24 h post exposure and continued for 10 days once daily.