Non-identity of the Phospholipase of Bacillus anthracis with the Anthrax Toxin

Abstract

Filtrates from suitable cultures of Bacillus anthracis contain a phospholipase which slowly hydrolyses the phospholipid in egg-yolk broth but has no action on free egg-lecithin. This enzyme does not appear to be the anthrax toxin. McGaughey & Chu (1948) showed that Bacillus anthracis produced an opalescence when growing in a medium containing egg yolk, but the effect was less than that produced by B. cereus and B. mycoides. B. anthracisgrowing in the blood of infected guinea-pigs produces an extracellular toxin which kills the host in secondary shock (Smith, Keppie, Stanley & Harris-Smith, 1955 ; Smith, Keppie & Stanley, 1955); a lethal amount of this toxin is only produced by the growth of a large number of organisms in the blood during the terminal phase of anthrax. Lecithinases, responsible for the egg-yolk reaction of certain bacteria, are involved in their toxigenicity (MacFarlane, 1955) and when injected can produce the symptoms of shock (Berg, Levinson & Wang, 1951). A possible relationship between the egg-yolk reaction and the toxin of B. anthracis has therefore been investigated. First, the egg-yolk reaction was examined to see whether the opalescence produced by B. anthracis was due to an extracellular product of the organism and whether this product was a lecithinase. Then, the toxic plasma of infected guinea-pigs was investigated for the presence of such an enzyme arid for a connexion between it and the toxic action. METHODS Organism.A spore suspension of Bacillus anthracis, strain N.P., as used in all previous work (Smith et al. 1955) was the source of organisms. Egg-yolk broth (E.Y.B.).Two egg yolks/l. 1 yo peptone solution containing 0 . 5 % NaCl and a t pH 7.4.Plasma containing the anthrm toxin. This was collected from guinea-pigs dying of anthrax as described by Smith et al. (1955).Anthrax antiserum. This was prepared in the horse by injecting the 'Sterne' strain of Bacillus anthracis (Belton & Strange, 1954).Testfor phospholipase activity. A sterile mixture of the solution to be tested ( 2 ml.) and E.Y.B. (2 ml., containing c. 50pg. ether-soluble P) was incubated at 37" and pH 7.4. After cooling the mixture, it was extracted with ether (3 ml.) and the P content of the ethereal extract determined by the method of Fiske & SubbaKow (1925). Activities are expressed as the percentage decrease in the ether-soluble P produced by the test materials in a stated time. Controls incubated for 72 hr. a t 37" did not show a decrease in ether-soluble P.

Bacillus anthracisPhospholipases C Facilitate Macrophage-Associated Growth and Contribute to Virulence in a Murine Model of Inhalation Anthrax

“…Transcript was detected for plcB, smcA, and plcA in vitro during B. anthracis infections of cultured macrophages (15). In addition, plasma from guinea pigs dying of anthrax displayed higher levels of PLC activity versus culture filtrates of B. anthracis, implying that they may play a role in the disease (45). Recently, it was shown by ectopic expression of each B. anthracis PLC gene in Escherichia coli and L. monocytogenes that the genes encode functional proteins with activities similar to their corresponding B. cereus orthologues (25,40).…”

SEPARATION OF THE TOXIN OF BACILLUS CEREUS INTO TWO COMPONENTS AND NONIDENTITY OF THE TOXIN WITH PHOSPHOLIPASE

“…Attempts to produce antisera in rabbits, guinea pigs, and rats were unsuccessful. Zwartouw and Smith (1956) found that filtrates of the NP strain of B. anthracis hydrolyzed the phospholipid in egg-yolk broth but not free egg lecithins or cephalins. Their preparations did not hemolyze guinea pig red blood cells and did not produce a skin reaction in guinea pigs.…”

LECITHINASE FROM BACILLUS ANTHRACIS

“…The possibility that lecithinase has a role in the disease of anthrax has not been extensively investigated. Zwartouw and Smith (1956) stated that B. anthracis culture filtrates hydrolyzed phospholipid in egg yolk broth but did not hydrolyze free egg lecithin and that the phospholipase was not anthrax toxin. Preliminary toxicity tests performed with lecithinase from an avirulent strain have indicated that B. anthracis lecithinase was nontoxic to mice and that it may not be a contributing factor in the damage of 1958]…”