Identification of an in Vivo Inhibitor of Bacillus anthracis Spore Germination

Abstract

Germination of Bacillus anthracis spores into the vegetative form is an essential step in anthrax pathogenicity. This process can be triggered in vitro by the common germinants inosine and alanine. Kinetic analysis of B. anthracis spore germination revealed synergy and a sequential mechanism between inosine and alanine binding to their cognate receptors. Because inosine is a critical germinant in vitro, we screened inosine analogs for the ability to block in vitro germination of B. anthracis spores. Seven analogs efficiently blocked this process in vitro. This led to the identification of 6-thioguanosine, which also efficiently blocked spore germination in macrophages and prevented killing of these cells mediated by B. anthracis spores. 6-Thioguanosine shows potential as an anti-anthrax therapeutic agent. Germination of Bacillus anthracis spores into the vegetative form is an essential step in anthrax pathogenicity. This process can be triggered in vitro by the common germinants inosine and alanine. Kinetic analysis of B. anthracis spore germination revealed synergy and a sequential mechanism between inosine and alanine binding to their cognate receptors. Because inosine is a critical germinant in vitro, we screened inosine analogs for the ability to block in vitro germination of B. anthracis spores. Seven analogs efficiently blocked this process in vitro. This led to the identification of 6-thioguanosine, which also efficiently blocked spore germination in macrophages and prevented killing of these cells mediated by B. anthracis spores. 6-Thioguanosine shows potential as an anti-anthrax therapeutic agent. The 2001 anthrax bioterrorist attacks rekindled interest in the anthrax causative agent, Bacillus anthracis. The spores derived from B. anthracis survive exposure to extreme temperatures and harsh chemicals (1Nicholson W.L. Munakata N. Horneck G. Melosh H.J. Setlow P. Microbiol. Mol. Biol. Rev. 2000; 64: 548-572Crossref PubMed Scopus (1413) Google Scholar, 2Setlow P. J. Appl. Microbiol. 2006; 101: 514-525Crossref PubMed Scopus (1007) Google Scholar) and represent an infectious agent with significant potential for biowarfare and bioterrorism. The severity of anthrax pathogenicity is dependent on the uptake route. Following inhalation, the most toxic route, B. anthracis spores are phagocytosed by alveolar macrophages in the lungs of infected hosts (3Guidi-Rontani C. Weber-Levy M. Labruyere E. Mock M. Mol. Microbiol. 1999; 31: 9-17Crossref PubMed Scopus (274) Google Scholar, 4Guidi-Rontani C. Levy M. Ohayon H. Mock M. Mol. Microbiol. 2001; 42: 931-938Crossref PubMed Scopus (145) Google Scholar). During the migration of infected macrophages to regional lymph nodes, B. anthracis spores germinate into the vegetative form (4Guidi-Rontani C. Levy M. Ohayon H. Mock M. Mol. 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Cell Microbiol. 2003; 5: 15-23Crossref PubMed Scopus (93) Google Scholar). The transformation process from a dormant spore to a fully vegetative bacterium is a critical initial step in anthrax pathogenicity (3Guidi-Rontani C. Weber-Levy M. Labruyere E. Mock M. Mol. Microbiol. 1999; 31: 9-17Crossref PubMed Scopus (274) Google Scholar, 11Dixon T.C. Fadl A.A. Koehler T.M. Swanson J.A. Hanna P.C. Cell Microbiol. 2000; 2: 453-463Crossref PubMed Scopus (194) Google Scholar). Therefore, drugs that prevent germination of phagocytosed spores are attractive candidates for controlling the anthrax disease. The transformation into vegetative bacteria is a two-step process in vitro. Following activation of spores by heavy metals, heat, or hydrostatic pressure, bacterium-specific germinants trigger germination (12Splittstoesser D.F. Farkas D.F. J. Bacteriol. 1966; 92: 995-1001Crossref PubMed Google Scholar). Different bacterial species use an array of germinants, which include amino acids and nucleosides (13Hills G.M. Biochem. J. 1949; 45: 353-362Crossref PubMed Scopus (15) Google Scholar, 14Warren S.C. Gould G.W. Biochim. Biophys. Acta. 1968; 170: 341-350Crossref PubMed Scopus (20) Google Scholar, 15Yousten A.A. Can. J. Microbiol. 1975; 21: 1192-1197Crossref PubMed Scopus (8) Google Scholar). The exact mechanism by which these compounds induce germination is not clearly understood. It has been proposed that germinants trigger signal transduction pathways upon binding to specific receptors (16Wolgamott G.D. Durham N.N. Can. J. Microbiol. 1971; 17: 1043-1048Crossref PubMed Scopus (11) Google Scholar, 17Fisher N. Hanna P. J. Bacteriol. 2005; 187: 8055-8062Crossref PubMed Scopus (81) Google Scholar). Mutational analysis of the B. anthracis genome led to the identification of germination operons, which control the germination process (17Fisher N. Hanna P. J. Bacteriol. 2005; 187: 8055-8062Crossref PubMed Scopus (81) Google Scholar, 18Hornstra L.M. de Vries Y.P. Wells-Bennik M.H. de Vos W.M. Abee T. Appl. Environ. Microbiol. 2006; 72: 44-53Crossref PubMed Scopus (65) Google Scholar). Based on these mutational assays, seven germination (ger) operons have been identified in B. anthracis (19Read T.D. Peterson S.N. Tourasse N. Baillie L.W. Paulsen I.T. Fraser C.M. Nature. 2003; 423: 81-86Crossref PubMed Scopus (676) Google Scholar). Moreover, four distinct amino acid- and nucleoside-dependent germination pathways have been described for B. anthracis (17Fisher N. Hanna P. J. Bacteriol. 2005; 187: 8055-8062Crossref PubMed Scopus (81) Google Scholar). These pathways are purportedly initiated by binding of specific germinants to their corresponding receptors during the initial phase of germination (20Moir A. Corfe B.M. Behravan J. CMLS Cell. Mol. Life Sci. 2002; 59: 403-409Crossref PubMed Scopus (164) Google Scholar). Efficient germination of B. anthracis spores in vitro requires the presence of purine ribonucleosides and amino acid co-germinants. Although specific amino acids like alanine are able to germinate B. anthracis spores at unphysiologically high concentrations, the presence of a second co-germinant, like inosine, can promote germination at lower concentrations (21Ireland J.A. Hanna P.C. J. Bacteriol. 2002; 184: 1296-1303Crossref PubMed Scopus (99) Google Scholar). B. anthracis spores have a dense, highly structured morphology and scatter light more strongly than their vegetative form. Upon germination, there is a marked decrease in the optical density at 600 nm. Analysis of optical density has been applied extensively to monitor the germination process. The kinetics of spore germination have been fitted to an exponential equation that describes the changes in optical density as spores germinate (22Woese C.R. Vary J.C. Halvorson H.O. Proc. Natl. Acad. Sci. U. S. A. 1968; 59: 869-875Crossref PubMed Scopus (33) Google Scholar). Previous reports postulated that spore germination kinetics could be described using a rapid equilibrium approach (23Massa M.R. Cardillo M.R. Tarsi R. Burberi S. Nuovi. Ann. Ig. Microbiol. 1975; 26: 450-460PubMed Google Scholar). More recently, Ireland and Hannah (21Ireland J.A. Hanna P.C. J. Bacteriol. 2002; 184: 1296-1303Crossref PubMed Scopus (99) Google Scholar) demonstrated that B. anthracis germination with alanine follows complex kinetics. It has been observed that Bacillus subtilis, Bacillus cereus, and Bacillus megaterium spore germination can be blocked by alkyl alcohols (24Trujillo R. Laible N. Appl. Microbiol. 1970; 20: 620-623Crossref PubMed Google Scholar), ion channel blockers (25Mitchell C. Skomurski J.F. Vary J.C. FEMS Microbiol. Lett. 1986; 34: 211-214Crossref Google Scholar), protease inhibitors (26Boschwitz H. Milner Y. Keynan A. Halvorson H.O. Troll W. J. 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All of these potential Bacillus germination inhibitors have been tested in vitro but, to our knowledge, their ability to protect mammalian cells from B. anthracis-mediated killing has not been tested. In the present study, we examined the kinetics of the germination process triggered by the common in vitro germinants, inosine and alanine. We first determined kinetic parameters for germination using inosine and alanine as co-germinants. Michaelis-Menten analysis revealed that B. anthracis spore germination follows a sequential mechanism and that germinants show remarkable synergy. Because inosine is a strong germinant in vitro, we screened nucleoside analogs against inosine/alanine-mediated spore germination. These assays led to the identification of a series of germination inhibitors. One of these compounds, 6-thioguanosine (6-TG), 5The abbreviations used are: 6-TG, 6-thioguanosine; 6-MMPR, 6-methylmercapto-purine riboside; m.o.i., multiplicity of infection; PI, propidium iodide; v, germination rates.5The abbreviations used are: 6-TG, 6-thioguanosine; 6-MMPR, 6-methylmercapto-purine riboside; m.o.i., multiplicity of infection; PI, propidium iodide; v, germination rates. was also able to inhibit spore germination in macrophages and prevent anthrax-mediated cell death. 6-TG therefore shows potential as an anti-anthrax agent. Chemicals and Equipment—Unless noted, all chemicals were obtained from Sigma-Aldrich. Spore germination was monitored on a Biomate 5 spectrophotometer at 580 nm or a Labsystems iEMS 96-well plate reader (ThermoElectron Corporation, Waltham, MA) fitted with a 540-nm cut-off filter. Bacterial Strains and Spore Preparation—The B. anthracis Sterne 34F2 strain was a gift from Dr. Arturo Casadevall, Albert Einstein College of Medicine. The Sterne strain lacks the pXO2 plasmid for encapsulation but contains the pXO1 plasmid for the production of virulence factors. For spore preparation, the Sterne strain was grown on BD Biosciences nutrient agar for 5 days followed by harvesting in ice-cold water. After three washing steps, spores were separated from vegetative and partially sporulated forms by centrifugation through a 20–50% Histo-Denz gradient. The spore pellet was washed six times with water and stored at 4 °C. Analysis of Germination—Spore germination was monitored spectrophotometrically whereby the loss in light diffraction following the addition of inosine and alanine was reflected by decreased optical density at 580 nm (for Biomate 5 spectrophotometer) or above 540 nm (for Labsystems iEMS 96-well plate reader). Spores were heat-activated at 70 °C for 30 min before resuspension in germination buffer (50 mm Tris-HCl, pH 7.5, 10 mm NaCl) to an A580 of 1. The spore suspension was monitored for auto-germination at A580 for 1 h. Only spores that did not auto-germinate were used for subsequent assays in 96-well format. All germination experiments were carried out in 96-well plates in a total volume of 200 μl/well. Every experiment was done in triplicate with at least two different spore preparations. Spores were allowed to germinate upon exposure to varying concentrations of both germinants in the presence of 1 mm d-cycloserine, which inhibits alanine racemase activity in the spores. For inosine titrations, spores were exposed to varying concentrations of inosine (0.1, 0.175, 0.25, 0.5, 1, 2.5, and 5 mm) at specific constant alanine concentrations (0.075, 0.1, 0.15, and 0.25 mm). For alanine titrations, spores were exposed to varying concentrations of alanine (0.05, 0.1, 0.133, 0.2, 0.5, 1, and 5 mm) at specific constant inosine concentrations (1, 2.5, 5, and 10 μm). The concentration range for both co-germinants was selected to avoid data clusters in the double reciprocal plots. Spore germination was evaluated based on decrease in A540 at room temperature each minute for 35 min. The germination of each at each was as a of the by the obtained at the of germination. were against All show of than Germination were determined for the of germinants Germination were as the of the following the initial phase of All were in The data were as double reciprocal of germinant All were fitted using the analysis from the to and of spores were in 1 of germination analogs were to 10 mm Spore were for min at room temperature germination was inosine and alanine were to and mm Germination was monitored as that were to spore at A580 obtained at were against the of The data were fitted using the of to D. Clin. 20: PubMed Scopus Google Scholar). were from a equation J. 2001; PubMed Scopus Google Scholar). Germination and were in as inhibitors of germination. on inosine spores were exposed to varying concentrations of inosine (0.1, 0.175, 0.25, 0.5, 1, 2.5, and 5 mm) at specific constant 0.25, 0.5, and 1 mm) was at study on alanine spores were exposed to varying concentrations of alanine 0.1, and mm) at specific constant 0.25, 0.5, and 1 mm) was at All assays were carried out in All were fitted using the analysis from the to and Spore Germination by macrophages were washed three times in and at on 96-well plates in with and After the cells the was with with germination inhibitors. Spores were to the at an of The plates were at for 10 min at 4 °C and to a °C with to of In our did not germination from spores. following spore of cells were by a propidium A. Brojatsch J. 2005; PubMed Scopus Google Scholar, A. Lisanti M.P. Brojatsch J. Cell 2006; 5: PubMed Scopus Google Scholar). After in propidium was to each and the plates for 10 min. was in a plate reader at nm Life of Spore Germination by cells were on or Spores were to the macrophages at an of and cells were at °C in the presence or of 6-TG for h. 5 a acid was used to both cells and the vegetative form of B. propidium was used to were with an Spore germination is the first step in B. anthracis that germination, the of vegetative could block Efficient germination of B. anthracis spores can be by ribonucleosides and amino inosine and alanine (17Fisher N. Hanna P. J. Bacteriol. 2005; 187: 8055-8062Crossref PubMed Scopus (81) Google Scholar, J.A. Hanna P.C. J. Bacteriol. 2002; 184: 1296-1303Crossref PubMed Scopus (99) Google Scholar, T.D. Hanna P.C. J. Bacteriol. 2003; PubMed Scopus Google Scholar). We determined the kinetics of the germination process triggered by inosine and alanine as as by inosine with the that inosine analogs could as germination inhibitors. Spores derived from the B. anthracis Sterne strain were used for germination Germination of heat-activated spores was initiated by the addition of inosine and alanine and monitored at 540 nm. The optical density during the of germination. The spores were allowed to germinate upon exposure to varying concentrations of both germinants in the presence of concentrations of Upon the addition of inosine and alanine to we observed a rapid in germination, at concentrations of both We observed an phase during the germination and germination were from the changes in optical density during the phase of the The germination were used for Michaelis-Menten of the germination with germinant of germinant binding to specific studies were carried out using a of germinant concentrations, and the data were in double reciprocal and Analysis of and Scholar). parameters were determined from the of the with the the of the fitted with the allowed the of an reciprocal of at alanine concentrations a of that in the the at different for germination with alanine each at different that alanine binding the of spores for these and of inosine at alanine concentrations were to be and The that the germination with alanine concentrations that germinants to Moreover, the of the at a strongly the that both germinants are present on their to the synergy in both and The for inosine at the and alanine concentration tested were to 35 and This an in the of the spore for inosine a alanine concentration a remarkable between the reciprocal analysis of the of alanine on spore germination a of that the at different that the of spores for alanine with inosine concentrations the of germinant the germinant is at specific This is with a sequential which requires binding of both and binding of the first binding of the second kinetic analysis that the germination process requires inosine binding to a specific spore Therefore, we that inosine analogs with and prevent germination. For assays, we selected inosine and analogs B. 2004; PubMed Scopus Google Scholar, R. R. N. R. B. J. Med. 34: PubMed Scopus Google Scholar, S. Investig. 2005; PubMed Scopus Google Scholar). The inosine analogs were based on a in of a The 6-MMPR, has a to the 6-TG, and are analogs with and amino of the The inosine has at and constant for different nucleosides were obtained in the presence of concentrations of inosine and alanine. at the and at the not in a For B. anthracis spores were with nucleoside analogs for min. Germination was initiated by the addition of inosine and alanine. Germination were in the presence of nucleoside inhibitors as with obtained with spores. at the optical density of spores was than that of spores. this we identified germination inhibitors were using the of the D. Clin. 20: PubMed Scopus Google Scholar). The of the and the for inosine were used to the of the nucleoside using a equation J. 2001; PubMed Scopus Google Scholar). The inosine did not with germination, in the presence of alanine not In efficiently blocked spore germination with a of the presence of a at allowed binding to the germination and with the germination process. The 6-TG, and have at and and efficiently blocked spore germination in the presence of Therefore, at and are for and with at and on the other did not with spore germination. The obtained for the inosine analogs are in 1. The nucleoside analogs 6-TG and that blocked germination of B. anthracis are used in the of and and their are M. M. M. M. D. 1999; 59: Google Scholar, de D.J. C.J. J. 2003; PubMed Scopus Google Scholar). We used 6-TG and for kinetic analysis using double reciprocal plots. the of the nucleoside we the studies using alanine at inosine at and nucleoside analogs at concentrations for the to the that with This that the of the spores for inosine with the germination inosine, both the of germination and the of alanine for spores in the presence of 6-TG, like 6-MMPR, with inosine for Michaelis-Menten analysis of and 6-TG of germination that both nucleoside analogs inhibit germination by with inosine binding to cognate Moreover, both and 6-TG were also able to induce spore germination in the presence of alanine and in the of inosine both the germination and the total decrease in optical density were lower than observed using inosine as a These that and 6-TG are We that the germination potential of and 6-TG is to their ability to inhibit germination. inosine analogs with the germination inosine for binding and prevented spore germination. A and requires of B. anthracis spores and subsequent germination G. Young H.A. S. J. 2005; PubMed Scopus Google Scholar). In to in vitro assays, factors that promote germination of B. anthracis macrophages are factors have been postulated to a in germination amino acids and nucleosides Hanna P.C. 2003; PubMed Scopus Google Scholar, J.A. Hanna P.C. 2002; PubMed Scopus Google Scholar). We tested the nucleoside analogs that blocked germination in in vitro assays also prevented germination of spores phagocytosed by This the tested inosine analogs represent potential against B. anthracis Because the vegetative form of the B. anthracis spore efficiently we determined following spore using propidium We infected macrophages with B. anthracis spores at an to cell of to 1. a marked following anthrax and inosine 6-TG killing mediated by B. anthracis spores. macrophages with and propidium show than cells exposure to B. anthracis spores show of cells were exposure to B. anthracis spores. The vegetative form of B. anthracis is by cells exposure to B. anthracis spores show that the of cells is to the vegetative B. anthracis cells are of all nucleoside that efficiently blocked germination in vitro, 6-TG prevented germination and in 6-TG efficiently blocked killing by B. anthracis spores at concentrations between 1 and a of macrophages was in the presence of the 6-TG concentrations, to the ability of 6-TG to as a of B. anthracis germination was in the presence of the germination inhibitors 6-MMPR, or at the concentrations used and data not was examined by The is is to to the shows in cells and in were with and more than of macrophages to B. anthracis spores in and macrophages and and This is with the rapid in following B. anthracis also vegetative which were present in macrophages B. anthracis The addition of 6-TG prevented the killing of B. macrophages by a of vegetative bacteria were in macrophages and alanine are germinants of B. anthracis in vitro. Kinetic analysis of the germination process revealed that germination requires binding of inosine and alanine and that the germinants show significant synergy. inosine concentrations are in mammalian cells as this nucleoside is a of the purine and is to acid J. A. J. 1985; PubMed Scopus Google Scholar). high alanine concentrations in the range germination in macrophages at nucleoside the of inosine in germination of B. we screened inosine analogs for their ability to with this process. This led to the identification of inhibitors that blocked germination in vitro. Kinetic analysis revealed that the inosine analogs 6-TG and with inosine for spore It is not that these inhibitors are able to germinate B. anthracis in the presence of with a lower than of all inosine analogs that blocked germination in vitro, 6-TG prevented germination in Germination in macrophages is a complex and process. least four germination pathways have been described in B. anthracis (17Fisher N. Hanna P. J. Bacteriol. 2005; 187: 8055-8062Crossref PubMed Scopus (81) Google Scholar), germination triggered by a of amino acids It is that inosine is the germinant cells and that 6-TG germination by with inosine concentrations be the for the high of 6-TG at germination of B. anthracis spores in experiments to factors that B. anthracis germination in It to be 6-TG efficiently blocked germination in nucleoside analogs tested with It is that the to block germination in is to a of for the nucleoside analogs or to rapid of these all inhibitors tested were able to with inosine in vitro, is that the inosine receptors are not the of germination 6-TG not other nucleoside also with germinants present in macrophages other than These that receptors to spore germination in Seven different germination operons have been identified for B. anthracis. operons, and have been to germination (17Fisher N. Hanna P. J. Bacteriol. 2005; 187: 8055-8062Crossref PubMed Scopus (81) Google Scholar, J.A. Hanna P.C. J. Bacteriol. 2002; 184: 1296-1303Crossref PubMed Scopus (99) Google Scholar). using germination inhibitors as 6-TG to that germination of B. anthracis in we that the nucleoside 6-TG inhibits B. anthracis spore germination in macrophages and efficiently cell mediated by these spores. 6-TG analogs with and germination These could represent against anthrax The potential of 6-TG to prevent germination and in infected with B. anthracis spores to be tested. We and for critical of the and We and for