Bacillus anthracis begins its infectious cycle as a metabolically dormant cell type, the endospore. Upon entry into a host, endospores rapidly differentiate into vegetative bacilli through the process of germination, thus initiating anthrax. Elucidation of the signals that trigger germination and the receptors that recognize them is critical to understanding the pathogenesis of B. anthracis. Individual mutants deficient in each of the seven putative germinant receptor-encoding loci were constructed via temperature-dependent, plasmid insertion mutagenesis and used to correlate these receptors with known germinant molecules. These analyses showed that the GerK and GerL receptors are jointly required for the alanine germination pathway and also are individually required for recognition of either proline and methionine (GerK) or serine and valine (GerL) as cogerminants in combination with inosine. The germinant specificity of GerS was refined from a previous study in a nonisogenic background since it was required only for germination in response to aromatic amino acid cogerminants. The gerA and gerY loci were found to be dispensable for recognition of all known germinant molecules. In addition, we show that the promoter of each putative germinant receptor operon, except that of the gerA locus, is active during sporulation. A current model of B. anthracis endospore germination is presented.Bacillus anthracis, like many Bacillus and Clostridium species, is capable of differentiation between two distinct cellular morphologies: the vegetative cell and the endospore. The endospore is metabolically dormant and provides the cell with the ability to withstand environmental conditions disadvantageous for vegetative life (28). B. anthracis endospores are thought to be the sole infectious cell morphotype, and their inoculation into a suitable host organism initiates the development of anthrax disease. Germination of the endospore, which is the regulated resumption of metabolic activity within the bacterial cell, is the earliest pathogenic event after endospore entry into the body (5, 6, 10, 11).Germination has been well studied in a variety of endospore-forming species, and much is known regarding the specific signals that initiate the process (21). The signaling molecules vary widely among species, but in general small molecule nutrients, termed germinants, are recognized by receptors located within the inner endospore membrane (2,12,13,22,24,25,27). Activation of germinant receptors initiates a series of complex biophysical processes, which subsequently activate intracellular proteases and extracellular hydrolases that facilitate cellular differentiation to the vegetative form (28). The mechanism(s) of signal transduction from germinant receptors to downstream degradative enzymes remains poorly understood.In B. anthracis, five distinct germination pathways have been recognized (16, 31). The alanine germination pathway (Ala) requires only the presence of L-alanine in concentrations above 30 mM, which are thought to be conside...

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.Bacillus anthracis, the causative agent of anthrax, has come under increased scrutiny in recent years because of its potential role as a bioweapon (35). In the environment, B. anthracis exists primarily as a metabolically dormant endospore, and in this morphology the bacterium is both highly infectious and resistant to a wide range of harsh conditions (42). When the spores are inhaled, they reach the alveolar spaces of the lung, where they are efficiently taken up by resident phagocytes (5, 9, 48). It is commonly believed that the host cells then migrate across the alveolocapillary barrier, transporting the intracellular bacteria into the lymphatic system (26,40). During this time, the bacteria germinate, transforming from spores into vegetative bacilli, which begin to replicate within the phagocytes (15, 50). Eventually, the bacteria kill the phagocytes and escape into the extracellular environment, and the resulting sepsis ultimately leads to death of the host (23,24,43,52,57).Since the progression of anthrax is typically quite rapid once the systemic phase of the infection begins (16, 24), successful intervention depends on early diagnosis and treatment. Given this fact, it is particularly important from a therapeutic standpoint that the early events in anthrax are well understood. Most of these events occur within the context of the h...

Bacillus anthracis is a National Institute of Allergy and Infectious Diseases Category A priority pathogen and the causative agent of the deadly disease anthrax. We applied a transposon mutagenesis system to screen for novel chromosomally encoded B. anthracis virulence factors. This approach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and proteolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and markedly reduced in vivo survival of the fully virulent B. anthracis Ames upon intraperitoneal challenge in guinea pigs. The extracellular proteolytic activity dependent upon ClpX function was linked to degradation of cathelicidin antimicrobial peptides, a front-line effector of innate host defense. B. anthracis lacking ClpX were rapidly killed by cathelicidin and α-defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirming cathelicidin to be a critical element of innate defense against the pathogen. We conclude that ClpX is an important factor allowing B. anthracis to subvert host immune clearance mechanisms, and thus represents a novel therapeutic target for prevention or therapy of anthrax, a foremost biodefense concern.

In the environment, the gram-positive bacterium Bacillus anthracis persists as a metabolically dormant endospore. Upon inoculation into the host the endospores germinate and outgrow into vegetative bacilli able to cause disease. The dramatic morphogenic changes to the bacterium during germination and outgrowth are numerous and include major rearrangement of and modifications to the bacterial surface. Such modifications occur during a time in the B. anthracis infectious cycle when the bacterium must guard against a multitude of innate immune mediators. The dltABCD locus of B. anthracis encodes a cell wall D-alanine esterification system that is initiated by transcriptional activation during endospore outgrowth. The level of transcription from the dltABCD operon determined B. anthracis resistance to cationic antibacterial peptides during vegetative growth and cationic peptide, enzymatic, and cellular mediators of innate immunity during outgrowth. Mutation of dltABCD was also attenuating in a mouse model of infection. We propose that the dltABCD locus is important for protection of endosporeforming bacteria from environmental assault during outgrowth and that such protection may be critical during the establishment phase of anthrax.

Several models of anthrax pathogenesis suggest that early in the infectious process Bacillus anthracis endospores germinate and outgrow into vegetative bacilli within phagocytes before being released into the blood. Here, we define the respective contributions of three phospholipases C (PLCs) to the pathogenesis of B. anthracis. Genetic deletions of the PLCs were made in the Sterne 7702 background, resulting in the respective loss of their activities. The PLCs were redundant both in tissue culture and in murine models of anthrax. Deletion of all three PLC genes was required for attenuation of virulence in mice after intratracheal inoculation. This attenuation may be attributed to the inability of the PLC-null strain to grow in association with the macrophage. Complementation of these defects in both models of anthrax was achieved by expression of the PLC genes in trans. The functional redundancy between PLCs in the virulence of B. anthracis implies that their activities are important for anthrax pathogenesis.