Testing Nucleoside Analogues as Inhibitors of Bacillus anthracis Spore Germination In Vitro and in Macrophage Cell Culture

Abstract

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.

Solution structures of purine base analogues 6-chloroguanine, 8-azaguanine and allopurinol

“…Analogues of purines have been identified as lead compounds for therapy against a number of diseases (Adachi, Motomatsu, & Yara, 1979;Alvarez, Lee, & Abel-Santos;Cairo & Bishop, 2004;Cassera, Zhang, Hazleton, & Schramm, 2011;Cunico et al, 2006;Hosaka, Suzuki, & Kumada, 2011;Keough et al, 2006;Ogilvie et al, 1984;Yang et al, 2010). Purine base analogues can also be used to identify interactions of individual functional groups in the natural substrate in the protein complex.…”

Requirements for In Vitro Germination of Paenibacillus larvae Spores

“…The nature of the spore germination signals has been studied widely in Bacilli and Clostridia (21,27,28,(44)(45)(46)(47)(48)(49)(50), but the triggers for P. larvae spore germination have not been identified. In order to cultivate P. larvae, specialized media have been produced (1,31,(51)(52)(53).…”

“…The complexity of germination signals varies and commonly requires several types of germinants (22). We, and others, have also identified molecules that can inhibit spore germination (23)(24)(25)(26)(27)(28).…”

Comparison of in vitro methods for the production of Paenibacillus larvae endospores

“…While three procedures for in vitro sporulation of P. larvae have been commonly used ( (Dingman, 1983;Dingman and Stahly, 1983;Genersch et al, 2005) and recently described in (de Graaf et al, 2013)), these methods do not yield large enough numbers of spores of sufficient purity for studies that seek to identify chemical inhibitors and/or germinants of P. larvae spores (Akoachere et al, 2007;Alvarez et al, 2010;Howerton et al, 2011;Powell, 1950;Ramirez and AbelSantos, 2010). Consequently, a new method was developed ["UNLV"; (Alvarado et al, 2013)].…”