Retrocyclins Kill Bacilli and Germinating Spores of Bacillus anthracis and Inactivate Anthrax Lethal Toxin

Abstract

-defensins are cyclic octadecapeptides encoded by the modified ␣-defensin genes of certain nonhuman primates. The recent demonstration that human ␣-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of -defensins on Bacillus anthracis (Sterne). We tested rhesus -defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of -defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of -defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that -defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.Under normal circumstances Bacillus anthracis causes human infections only in individuals exposed to infected farm animals or their spore-contaminated products. The virulence of B. anthracis primarily derives from the hardiness of its spores, an anti-phagocytic capsule that surrounds its vegetative cells (1), and two secreted binary toxins: lethal toxin (LeTx) 3 and edema toxin (EdTx). Both toxins contain protective antigen (PA, 83 kDa). LeTx also contains lethal factor (LF, 90 kDa), and EdTx contains edema factor (EF, 89 kDa). The genes for all three toxin components, PA, LF, and EF, reside on the pXO1 plasmid (2), and those responsible for capsule synthesis exist on the pXO2 plasmid (3). Both of these plasmids are required for in vivo virulence (3).EF is an adenylate cyclase (4) and LF is a zinc-dependent metalloprotease that selectively attacks certain MAPK kinases (5, 6). PA is required to allow both of the other toxin components to enter host cells (7). When PA binds a cellular receptor (8), it is cleaved into PA63 (63 kDa) and PA20 (20 kDa). The PA20 diffuses away, and the residual receptor-bound PA63 molecules self-associate into ring-shaped heptamers (9) that bind EF or LF with high affinity (10 -12). Oligomerization of PA63 leads to endocytosis, which transports the complexes to an acidic compartment (13-15). Here, the heptameric pre-pore changes into an integral-membrane pore (16, 17) that translocates EF or LF into the cytosol (18). Immu...

Through the Looking Glass, Mechanistic Insights from Enantiomeric Human Defensins

“…For these reasons, D-defensins were not expected to "specifically" interact with LF with high affinity. The fact that D-defensins were effective inhibitors of LF, as was the case with D-retrocyclins (38), suggests that defensin association with LF and many other proteins may be driven by forces that act in a nonspecific and non-directional fashion.…”

“…B. anthracis spores engulfed by human neutrophils germinated intracellularly, only to then be killed effectively by HNPs (37). Inhibition of LF and killing of vegetative cells of B. anthracis by retrocyclins, putative hominid homologues of rhesus monkey -defensins encoded by human pseudogenes, have also been reported (38).…”

“…Reduction of the three disulfides followed by S-alkylation in retrocyclin-1 also dramatically reduced the ability of the -defensin to inhibit LF (38). To better understand the effect of disulfide bonding on LF inhibition, we characterized the unstructured forms of HNP1, HD5, and HBD3, in which all six Cys residues were simultaneously replaced by either Ala (in HNP1) or ␣-aminobutyric acid (in HD5 and HBD3).…”

Treatment of Experimental Anthrax with Recombinant Capsule Depolymerase

“…However, we did not observe a decrease in bacterial viability during incubation of CapD-treated bacilli in mouse serum (data not shown). Some antimicrobial peptides or defensins are bactericidal for B. anthracis (29,36,41), and capsule does confer some resistance (33). Future studies will determine if CapD treatment sensitizes bacilli to antimicrobial peptides.…”

Functional Determinants of Human Enteric α-Defensin HD5

“…However, a recent study failed to find a correlation between ␣-defensin membrane activity and killing of the Gram-positive Staphylococcus aureus, instead attributing cell death to lipid II binding and sequestration (11), a mode of action reminiscent of the lantibiotic peptide nisin (33,34) and other defensins (35)(36)(37). Defensins also neutralize secreted bacterial toxins and virulence factors (1, 38 -42), including anthrax lethal toxin (43)(44)(45), which is a binary complex of two proteins secreted by B. anthracis: lethal factor (LF) 4 and protective antigen (46). LF is a Zn 2ϩ -dependent protease that induces cell death in macrophages through disruption of signaling pathways, and its inhibition has relevance for the development of therapies to treat anthrax exposure (47).…”