| Spores, the infectious agents of anthrax, are traditionally prepared in the laboratory over 4 to 5 days. We observed that addition of 1.5 mM Fe(NO3)3 to glucose-free chemically defined medium (CDM) induced 'rapid' sporulation -- complete within ∼30 to 36 hr. Although 'rapid' spores had a different set of spore coat proteins than did classically prepared spores, both spore preparations interacted similarly with anti-spore antibody, looked similar by electron microscopy, were equally resistance to harsh conditions, and infected human macrophages similarly. CDM+Fe can thus be used to prepare BA spores saving considerable time in spore research studies.;Anthrolysin O is a cholesterol dependent cytolysin of Bacillus anthracis (BA) that lyses human phagocytes and epithelial cells in vitro, and has been implicated in anthrax pathogenesis. We studied regulation of ALO expression using CDM, and observed that glucose increased ALO activity in BA culture supernatants. The increase in ALO activity was not due to increased transcription of alo, but rather due to decreased expression of ALO-cleaving metalloproteases by BA.;Soluble host defense peptides perform multiple innate immune functions including killing bacteria, neutralizing toxins and activating innate immune cells. We studied a new serum lipopeptide (discovered by collaborator James Thacker), called 1-peptidyl-2-arachidonoyl-3-stearoyl-sn-glycerol (pDAG) and its 18 amino acid peptide moiety, acetyl-ALYDKGYTSKEQKDCVGI (acALY-18). We determined the effects of synthetic pDAG and acALY-18 on human macrophages, platelets and neutrophils in vitro by measuring cytokine expression and secretion, by RT-PCR and ELISA. Neither pDAG nor acALY-18 were directly bactericidal; they were not toxic (≤1 mug/ml), and; they did not cause or inhibit platelet aggregation. However, acALY-18 selectively activated alpha-granule secretion from platelets, inducing 5-fold increased release of TGF-beta and PDGF-AB. pDAG induced increased IL-1beta, IL-6, IL-8, IL-18, IL-33 and NALP-3 mRNA expression by human macrophages. Neither pDAG nor acALY-18 significantly affected human neutrophil function, including chemotaxis and degranulation. In summary, we have identified an exciting new drug-like molecule that has many possible clinical uses. pDAG and acALY-18 act on the host, thus there is less concern of bacteria developing resistance to them. They may eventually be valuable additions to anti-infective therapy. |
