Testing nucleoside analogues as inhibitors of Bacillus anthracis spore germination in vitro and in macrophage cell culture.

Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then produce toxins and establish infection. Thus, germination is a crucial step for the initiation of pathogenesis. B. anthracis spore germination is activated by a wide variety of amino acids and purine nucleosides. Inosine and l-alanine are the two most potent nutrient germinants in vitro. Recent studies have shown that germination can be hindered by isomers or structural analogues of germinants. 6-Thioguanosine (6-TG), a guanosine analogue, is able to inhibit germination and prevent B. anthracis toxin-mediated necrosis in murine macrophages. In this study, we screened 46 different nucleoside analogues as activators or inhibitors of B. anthracis spore germination in vitro. These compounds were also tested for their ability to protect the macrophage cell line J774a.1 from B. anthracis cytotoxicity. Structure-activity relationship analysis of activators and inhibitors clarified the binding mechanisms of nucleosides to B. anthracis spores. In contrast, no structure-activity relationships were apparent for compounds that protected macrophages from B. anthracis-mediated killing. However, multiple inhibitors additively protected macrophages from B. anthracis.

Dissecting interactions between nucleosides and germination receptors in Bacillus cereus 569 spores.

Bacillus cereus 569 spores germinate either with inosine as a sole germinant or with a combination of nucleosides and L-alanine. Whereas the inosine-only germination pathway requires the presence of two different germination receptors (GerI and GerQ) to be activated, the nucleoside/alanine germination pathway only needs one of the two receptors. To differentiate how nucleoside recognition varies between the inosine-only germination pathway and the nucleoside/alanine germination pathway, we tested 61 purine analogues as agonists and antagonists of the two pathways in wild-type, DeltagerI and DeltagerQ spores. The structure-activity relationships of germination agonists and antagonists suggest that the inosine-only germination pathway is restricted to recognize a single germinant (inosine), but can be inhibited in predictable patterns by structurally distinct purine nucleosides. B. cereus spores encoding GerI as the only nucleoside receptor (DeltagerQ mutant) showed a germination inhibition profile similar to wild-type spores treated with inosine only. Thus, GerI seems to have a well-organized binding site that recognizes inosine and inhibitors through specific substrate-protein interactions. Structure-activity analysis also showed that the nucleoside/alanine germination pathway is more promiscuous toward purine nucleoside agonists, and is only inhibited by hydrophobic analogues. B. cereus spores encoding GerQ as the only nucleoside receptor (DeltagerI mutant) behaved like wild-type spores treated with inosine and L-alanine. Thus, the GerQ receptor seems to recognize substrates in a more flexible binding site through non-specific interactions. We propose that the GerI receptor is responsible for germinant detection in the inosine-only germination pathway. On the other hand, supplementing inosine with l-alanine allows bypassing of the GerI receptor to activate the more flexible GerQ receptor.

Potential use of inhibitors of bacteria spore germination in the prophylactic treatment of anthrax and Clostridium difficile-associated disease.

Spore germination is the first step in establishing Bacillus and Clostridium infections. Germination is triggered by the binding of small molecules by the resting spore. Subsequently, the activated spore secretes dipicolinic acid and calcium, the spore core is rehydrated and spore structures are degraded. Inhibition of any of the germination-related events will prevent development to the vegetative stage. Inhibition of spore germination has been studied intensively in the prevention of food spoilage. In this perspective, we propose that similar approaches could be used in the prophylactic control of Bacillus anthracis and Clostridium difficile infections. Inhibition of B. anthracis spore germination could protect military and first-line emergency personnel at high risk for anthrax exposure. Inhibition of C. difficile could prevent human C. difficile-associated disease during antibiotic treatment of immunocompromised patients.