Genetic analysis of petrobactin transport in Bacillus anthracis.

Iron acquisition mechanisms play an important role in the pathogenesis of many infectious microbes. In Bacillus anthracis, the siderophore petrobactin is required for both growth in iron-depleted conditions and for full virulence of the bacterium. Here we demonstrate the roles of two putative petrobactin binding proteins FatB and FpuA (encoded by GBAA5330 and GBAA4766 respectively) in B. anthracis iron acquisition and pathogenesis. Markerless deletion mutants were created using allelic exchange. The Delta fatB strain was capable of wild-type levels of growth in iron-depleted conditions, indicating that FatB does not play an essential role in petrobactin uptake. In contrast, Delta fpuA bacteria exhibited a significant decrease in growth under low-iron conditions when compared with wild-type bacteria. This mutant could not be rescued by the addition of exogenous purified petrobactin. Further examination of this strain demonstrated increased levels of petrobactin accumulation in the culture supernatants, suggesting no defect in siderophore synthesis or export but, instead, an inability of Delta fpuA to import this siderophore. Delta fpuA spores were also significantly attenuated in a murine model of inhalational anthrax. These results provide the first genetic evidence demonstrating the role of FpuA in petrobactin uptake.

The role of Bacillus anthracis germinant receptors in germination and virulence.

Nutrient-dependent germination of Bacillus anthracis spores is stimulated when receptors located in the inner membrane detect combinations of amino acid and purine nucleoside germinants. B. anthracis produces five distinct germinant receptors, GerH, GerK, GerL, GerS and GerX. Otherwise isogenic mutant strains expressing only one of these receptors were created and tested for germination and virulence. The GerH receptor was necessary and sufficient for wild-type levels of germination with inosine-containing germinants in the absence of other receptors. GerK and GerL were sufficient for germination in 50 mM L-alanine. When mutants were inoculated intratracheally, any receptor, except for GerX, was sufficient to allow for a fully virulent infection. In contrast, when inoculated subcutaneously only the GerH receptor was able to facilitate a fully virulent infection. These results suggest that route of infection determines germinant receptor requirements. A mutant lacking all five germinant receptors was also attenuated and exhibited a severe germination defect in vitro. Together, these data give us a greater understanding of the earliest moments of germination, and provide a more detailed picture of the signals required to stimulate this process.

Transcriptional profiling of Bacillus anthracis Sterne (34F2) during iron starvation.

Lack of available iron is one of many environmental challenges that a bacterium encounters during infection and adaptation to iron starvation is important for the pathogen to efficiently replicate within the host. Here we define the transcriptional response of B. anthracis Sterne (34F(2)) to iron depleted conditions. Genome-wide transcript analysis showed that B. anthracis undergoes considerable changes in gene expression during growth in iron-depleted media, including the regulation of known and candidate virulence factors. Two genes encoding putative internalin proteins were chosen for further study. Deletion of either gene (GBAA0552 or GBAA1340) resulted in attenuation in a murine model of infection. This attenuation was amplified in a double mutant strain. These data define the transcriptional changes induced during growth in low iron conditions and illustrate the potential of this dataset in the identification of putative virulence determinants for future study.

The germination-specific lytic enzymes SleB, CwlJ1, and CwlJ2 each contribute to Bacillus anthracis spore germination and virulence.

The bacterial spore cortex is critical for spore stability and dormancy and must be hydrolyzed by germination-specific lytic enzymes (GSLEs), which allows complete germination and vegetative cell outgrowth. We created in-frame deletions of three genes that encode GSLEs that have been shown to be active in Bacillus anthracis germination: sleB, cwlJ1, and cwlJ2. Phenotypic analysis of individual null mutations showed that the removal of any one of these genes was not sufficient to disrupt spore germination in nutrient-rich media. This finding indicates that these genes have partially redundant functions. Double and triple deletions of these genes resulted in more significant defects. Although a small subset of DeltasleB DeltacwlJ1 spores germinate with wild-type kinetics, for the overall population there is a 3-order-of-magnitude decrease in the colony-forming efficiency compared with wild-type spores. DeltasleB DeltacwlJ1 DeltacwlJ2 spores are unable to complete germination in nutrient-rich conditions in vitro. Both DeltasleB DeltacwlJ1 and DeltasleB DeltacwlJ1 DeltacwlJ2 spores are significantly attenuated, but are not completely devoid of virulence, in a mouse model of inhalation anthrax. Although unable to germinate in standard nutrient-rich media, spores lacking SleB, CwlJ1, and CwlJ2 are able to germinate in whole blood and serum in vitro, which may explain the persistent low levels of virulence observed in mouse infections. This work contributes to our understanding of GSLE activation and function during germination. This information may result in identification of useful therapeutic targets for the disease anthrax, as well as provide insights into ways to induce the breakdown of the protective cortex layer, facilitating easier decontamination of resistant spores.

Structural and functional analysis of AsbF: origin of the stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis.

Petrobactin, a virulence-associated siderophore produced by Bacillus anthracis, chelates ferric iron through the rare 3,4-isomer of dihydroxybenzoic acid (3,4-DHBA). Most catechol siderophores, including bacillibactin and enterobactin, use 2,3-DHBA as a biosynthetic subunit. Significantly, siderocalin, a factor involved in human innate immunity, sequesters ferric siderophores bearing the more typical 2,3-DHBA moiety, thereby impeding uptake of iron by the pathogenic bacterial cell. In contrast, the unusual 3,4-DHBA component of petrobactin renders the siderocalin system incapable of obstructing bacterial iron uptake. Although recent genetic and biochemical studies have revealed selected early steps in petrobactin biosynthesis, the origin of 3,4-DHBA as well as the function of the protein encoded by the final gene in the B. anthracis siderophore biosynthetic (asb) operon, asbF (BA1986), has remained unclear. In this study we demonstrate that 3,4-DHBA is produced through conversion of the common bacterial metabolite 3-dehydroshikimate (3-DHS) by AsbF-a 3-DHS dehydratase. Elucidation of the cocrystal structure of AsbF with 3,4-DHBA, in conjunction with a series of biochemical studies, supports a mechanism in which an enolate intermediate is formed through the action of this 3-DHS dehydratase metalloenzyme. Structural and functional parallels are evident between AsbF and other enzymes within the xylose isomerase TIM-barrel family. Overall, these data indicate that microbial species shown to possess homologs of AsbF may, like B. anthracis, also rely on production of the unique 3,4-DHBA metabolite to achieve full viability in the environment or virulence within the host.

Transcriptional profiling of Bacillus anthracis during infection of host macrophages.

The interaction between Bacillus anthracis and the mammalian phagocyte is one of the central stages in the progression of inhalational anthrax, and it is commonly believed that the host cell plays a key role in facilitating germination and dissemination of inhaled B. anthracis spores. Given this, a detailed definition of the survival strategies used by B. anthracis within the phagocyte is critical for our understanding of anthrax. In this study, we report the first genome-wide analysis of B. anthracis gene expression during infection of host phagocytes. We developed a technique for specific isolation of bacterial RNA from within infected murine macrophages, and we used custom B. anthracis microarrays to characterize the expression patterns occurring within intracellular bacteria throughout infection of the host phagocyte. We found that B. anthracis adapts very quickly to the intracellular environment, and our analyses identified metabolic pathways that appear to be important to the bacterium during intracellular growth, as well as individual genes that show significant induction in vivo. We used quantitative reverse transcription-PCR to verify that the expression trends that we observed by microarray analysis were valid, and we chose one gene (GBAA1941, encoding a putative transcriptional regulator) for further characterization. A deletion strain missing this gene showed no phenotype in vitro but was significantly attenuated in a mouse model of inhalational anthrax, suggesting that the microarray data described here provide not only the first comprehensive view of how B. anthracis survives within the host cell but also a number of promising leads for further research in anthrax.

Transcriptional profiling of the Bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation.

The life cycle of Bacillus anthracis includes both vegetative and endospore morphologies which alternate based on nutrient availability, and there is considerable evidence indicating that the ability of this organism to cause anthrax depends on its ability to progress through this life cycle in a regulated manner. Here we report the use of a custom B. anthracis GeneChip in defining the gene expression patterns that occur throughout the entire life cycle in vitro. Nearly 5,000 genes were expressed in five distinct waves of transcription as the bacteria progressed from germination through sporulation, and we identified a specific set of functions represented within each wave. We also used these data to define the temporal expression of the spore proteome, and in doing so we have demonstrated that much of the spore's protein content is not synthesized de novo during sporulation but rather is packaged from preexisting stocks. We explored several potential mechanisms by which the cell could control which proteins are packaged into the developing spore, and our analyses were most consistent with a model in which B. anthracis regulates the composition of the spore proteome based on protein stability. This study is by far the most comprehensive survey yet of the B. anthracis life cycle and serves as a useful resource in defining the growth-phase-dependent expression patterns of each gene. Additionally, the data and accompanying bioinformatics analyses suggest a model for sporulation that has broad implications for B. anthracis biology and offer new possibilities for microbial forensics and detection.