Bacillus anthracis requires siderophore biosynthesis for growth in macrophages and mouse virulence

Systemic anthrax infections can be characterized as proceeding in stages, beginning with an early intracellular establishment stage within phagocytes that is followed by extracelluar stages involving massive bacteraemia, sepsis and death. Because most bacteria require iron, and the host limits iron availability through homeostatic mechanisms, we hypothesized that B. anthracis requires a high-affinity mechanism of iron acquisition during its growth stages. Two putative types of siderophore synthesis operons, named B acillus a nthracis c atechol, bac (anthrabactin), and a nthrax s iderophore b iosynthesis, asb (anthrachelin), were identified. Directed gene deletions in both anthrabactin and anthrachelin pathways were generated in a B. anthracis (Sterne) 34F2 background resulting in mutations in asbA and bacCEBF . A decrease in siderophore production was observed during iron-depleted growth in both the δ asbA and δ bacCEBF strains, but only the δ asbA strain was attenuated for growth under these conditions. In addition, the δ asbA strain was severely attenuated both for growth in macrophages (Mφ) and for virulence in mice. In contrast, the δ bacCEBF strain did not differ phenotypically from the parental strain. These findings support a requirement for anthrachelin but not anthrabactin in iron assimilation during the intracellular stage of anthrax.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72033/1/j.1365-2958.2003.03861.x.pd

Role of the <italic>asb</italic> operon in <italic>Bacillus anthracis</italic> pathogenesis.

Systemic anthrax infections can be characterized as proceeding in stages, beginning with an early intracellular establishment stage within phagocytes that is followed by extracelluar stages involving massive bacteremia, sepsis and death. Since most bacteria require iron, and the host limits iron availability through homeostatic mechanisms, I hypothesize that B. anthracis requires a high-affinity mechanism of iron acquisition during its growth stages. Two putative types of siderophore synthesis operons, named Bacillus anthracis catechol, bac, and anthrax siderophore biosynthesis, asb, are encoded on the B. anthracis chromosome. Directed gene deletions in both bac and asb clusters were generated in a B. anthracis (Sterne) 34F2 background resulting in mutations in asbA and bacCEBF. A decrease in siderophore production was observed during iron-depleted growth in both the DeltaasbA and DeltabacCEBF strains, but only the DeltaasbA strain is attenuated during iron-starved growth. In addition, the DeltaasbA strain is attenuated both for growth in macrophages and for virulence in mice. In contrast, the DeltabacCEBF strain does not differ phenotypically from the parental strain for mouse virulence, iron-starved culture or macrophage growth. Further, asb expression is upregulated in response to iron deprivation while the asbA deletion, negatively, and anthrachelin, positively, influence the expression of an asb transcriptional fusion in addition to iron-depleted growth kinetics. Taken together, these findings suggest a requirement for asb expression for iron acquisition during the early growth stages of infection in macrophages. Anthrachelin synthesis is attributable to the B. anthracis siderophore synthetic enzymes encoded by the asb operon that is upregulated in response to iron deprivation, though direct evidence relating the asb encoded synthetic pathway to the production and nature of anthrachelin is unavailable. However, the work in this thesis provides evidence that the B. anthracis asb plays a role in virulence equivalent to the described anthrax virulence factors encoded on the pXO1 and pXO2 plasmids of B. anthracis. This work is the first to address the influence of B. anthracis iron acquisition during anthrax and may lead to a better understanding of the requirement for iron acquisition by anthrachelin in addition to the role iron plays as a regulatory signal during anthrax.Ph.D.Biological SciencesGeneticsHealth and Environmental SciencesImmunologyMicrobiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124639/2/3150170.pd

Petrobactin Protects against Oxidative Stress and Enhances Sporulation Efficiency in Bacillus anthracis Sterne

Bacillus anthracis causes the disease anthrax, which is transmitted via its dormant, spore phase. However, conversion from bacillus to spore is a complex, energetically costly process that requires many nutrients, including iron. B. anthracis requires the siderophore petrobactin to scavenge iron from host environments. We show that, in the Sterne strain, petrobactin is required for efficient sporulation, even when ample iron is available. The petrobactin biosynthesis operon is expressed during sporulation, and petrobactin is biosynthesized during growth in high-iron sporulation medium, but instead of being exported, the petrobactin remains intracellular to protect against oxidative stress and improve sporulation. It is also required for full growth and sporulation in blood (bovine), an essential step for anthrax transmission between mammalian hosts.Bacillus anthracis is a Gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is required for vegetative growth of B. anthracis under iron-depleted conditions and in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficient in vitro sporulation of B. anthracis requires petrobactin, that the petrobactin biosynthesis operon (asbA to -F) is induced prior to sporulation, and that the siderophore itself associates with spores. Petrobactin is also required for oxidative stress protection during late-stage growth and for wild-type levels of sporulation in sporulation medium. Sporulation in bovine blood was found to be petrobactin dependent. Collectively, the in vitro contributions of petrobactin to sporulation as well as growth imply that petrobactin may be required for B. anthracis transmission via the spore during natural infections, in addition to its key known functions during active anthrax infections

Genome Sequence of the Attenuated Carbosap Vaccine Strain of \u3ci\u3eBacillus anthracis\u3c/i\u3e

The Bacillus anthracis Carbosap genome, which includes the pXO1 and pXO2 plasmids, has been shown to encode the major B. anthracis virulence factors, yet this strain’s attenuation has not yet been explained. Here we report the draft genome sequence of this strain, and a comparison to fully virulent B. anthracis

Biosynthetic Analysis of the Petrobactin Siderophore Pathway from Bacillus anthracis

The asbABCDEF gene cluster from Bacillus anthracis is responsible for biosynthesis of petrobactin, a catecholate siderophore that functions in both iron acquisition and virulence in a murine model of anthrax. We initiated studies to determine the biosynthetic details of petrobactin assembly based on mutational analysis of the asb operon, identification of accumulated intermediates, and addition of exogenous siderophores to asb mutant strains. As a starting point, in-frame deletions of each of the genes in the asb locus (asbABCDEF) were constructed. The individual mutations resulted in complete abrogation of petrobactin biosynthesis when strains were grown on iron-depleted medium. However, in vitro analysis showed that each asb mutant grew to a very limited extent as vegetative cells in iron-depleted medium. In contrast, none of the B. anthracis asb mutant strains were able to outgrow from spores under the same culture conditions. Provision of exogenous petrobactin was able to rescue the growth defect in each asb mutant strain. Taken together, these data provide compelling evidence that AsbA performs the penultimate step in the biosynthesis of petrobactin, involving condensation of 3,4-dihydroxybenzoyl spermidine with citrate to form 3,4-dihydroxybenzoyl spermidinyl citrate. As a final step, the data reveal that AsbB catalyzes condensation of a second molecule of 3,4-dihydroxybenzoyl spermidine with 3,4-dihydroxybenzoyl spermidinyl citrate to form the mature siderophore. This work sets the stage for detailed biochemical studies with this unique acyl carrier protein-dependent, nonribosomal peptide synthetase-independent biosynthetic system

AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosynthesis

Bacterial pathogens need to scavenge iron from their host for growth and proliferation during infection. They have evolved several strategies to do this, one being the biosynthesis and excretion of small, high-affinity iron chelators known as siderophores. The biosynthesis of siderophores is an important area of study, not only for potential therapeutic intervention but also to illuminate new enzyme chemistries. Two general pathways for siderophore biosynthesis exist: the well-characterized nonribosomal peptide synthetase (NRPS)-dependent pathway and the NRPS-independent siderophore (NIS) pathway, which relies on a different family of sparsely investigated synthetases. Here we report structural and biochemical studies of AcsD from Pectobacterium (formerly Erwinia) chrysanthemi, an NIS synthetase involved in achromobactin biosynthesis. The structures of ATP and citrate complexes provide a mechanistic rationale for stereospecific formation of an enzyme-bound (3R)-citryladenylate, which reacts with L-serine to form a likely achromobactin precursor. AcsD is a unique acyladenylate-forming enzyme with a new fold and chemical catalysis strategy