bacillus anthracis ( anthrax ) spores were distributed through the postal system as biological weapons in the us in the autumn of 2001 , resulting in cases of cutaneous and inhalational anthrax . it has been reported that the mortality associated with inhalational anthrax was 45% ( 1 ) . there are three clinical forms of anthrax : cutaneous , inhalational and gastrointestinal ( 2 ) . ciprofloxacin has become a treatment of choice for all clinical forms of anthrax owing to potential resistance to penicillins and tetracyclines , and demonstrated efficacy in a primate model ( 1 ) . additionally , it is reported that the former soviet union engineered penicillin and tetracycline resistant anthrax spores ( 3 ) . the initial stage of infection occurs when pathogenic spores interact with macrophages at the site of host entry ( 4 ) . once inhaled , spores germinate while associated with alveolar macrophages traveling en route to regional lymph nodes . during systemic anthrax infection , late stage events include vegetative growth in the blood to very high titers and synthesis of the anthrax toxins , causing disease symptoms and ultimately death 5 . , 6 .. contemporary anthrax therapy focuses on three - component toxins : protective antigen ( pa ) , lethal factor ( lf ) , and edema factor ( ef ) , which are produced by vegetative cells during the late stage of anthrax infection ( 7 ) . targeting pa has been variably effective , and the ability of existing pa - targeting methods to protect humans against inhalational anthrax that might be released by biological weapons is an open question . targeting the early stages of anthrax infection ( macrophage interaction , germination , and early outgrowth ) may be more effective than targeting pa , lf , and/or ef in counteracting anthrax in that the upstream events that lead to their production could be prevented . first , the interaction between spores and macrophages is an upstream event in the life cycle of anthrax . the interruption of upstream events ( macrophage interaction , germination and early outgrowth ) precludes downstream events including the production of pa , lf , or ef . second , pa , lf , and ef are encoded by a plasmid in anthrax . therefore , by replacing this plasmid with others that encode virulent toxins ( e.g. , tetanus toxin , cobra toxin , etc . ) , bioterrorists could potentially introduce mutant anthrax spores that produce toxins other than pa / lf / ef . third , vegetative cells may develop antibiotic resistance over time or , bioterrorists might incorporate antibiotic - resistance genes into such cells . lastly , in contrast to toxins and antibiotic resistance that are prone to change , macrophage interaction and germination factors are highly conserved and less likely to be altered ( 8) . thus , blocking upstream events with vaccines and/or inhibitory drugs may prevent the use of anthrax spores as bioweapons . the objective of this article is to draw your attention that while the vast majority of anti - anthrax works center around pa / lf / ef , proteins expressed during the early anthrax infection are potential targets for the development of anti - anthrax vaccines or drugs . bacillus anthracis , the causative agent of anthrax , is an endospore - forming , gram - positive , aerobic , and rod - shaped bacterium ( 9 ) . the dormant spores of anthrax are formed during sporulation , a process triggered by starvation or harsh environment . they have high resistance to heat , radiation , desiccation , ph extremes and toxic chemicals ( 10 ) . as shown in figure 1 the mature spore is made up of different zones , an exosporium on the outside with multi - layered spore coats below , a cortex , and a central core . between the center core and the coat the spore also has an outer membrane covering the coat that may correspond to the regular bacterial cell wall peptidoglycan . anthrax can infect the host through intradermal inoculation , ingestion , or inhalation of the spore . in vitro and in vivo studies have demonstrated that the dormant spores monitor the environment until conditions are favorable for growth . the germination process consists of an irreversible series of degradative reactions that result in the breaking of spore dormancy . although still poorly understood , it appears to be essential for the development of the diseases caused by anthrax . once inhaled by animals or humans , anthrax spores interact with alveolar macrophages , which have been known to play a central role in innate and acquired immune responses . they act as guard cells to help clear invading bacteria from the lung alveoli 5 . , 6 .. although understanding of cellular and molecular interactions between anthrax spores and alveolar macrophages at the early stages of infection is incomplete , it is known that the majority of inhaled spores are rapidly and efficiently phagocytosed by alveolar macrophages via recruitment of f - actin ( 12 ) . this is followed by extracellular multiplication , together with the production of a capsule and toxins ( including pa , lf , and ef ) . thus the events occurring at the early stage of anthrax infection , including spore / macrophage interaction , germination , and early outgrowth , may be promising immunologic and pharmacologic targets against this organism . arrest of the anthrax life cycle at its onset would necessarily preclude all downstream events including the lethal manifestations of bacteremia and toxemia . currently , there are no anti - anthrax drugs that are marketed for commercial use . the us anthrax vaccine that was licensed in 1970 is made from the filtrate of a non - encapsulated attenuated strain ( 13 ) . this vaccine has unknown efficacy , and often contains impurities that can trigger local pain and edema as well as other undesirable side effects , such as headaches and malaise ( 14 ) . in addition , proteomics may also help to detect unexpected changes in protein expression that may be missed by conventional biochemical techniques ( 15 ) . specifically two - dimensional ( 2d ) electrophoresis gels can be used to separate thousands of proteins quantitatively over a wide dynamic range . matrix - assisted laser - desorption - ionization - time - of - flight mass spectrometry ( maldi - tof ms ; perseptive biosystems inc . , framingham , usa ) and quadrupole - time - of - flight ( q - tof ) ms / ms ( micromass , manchester , uk ) with high sensitivity have been commonly used to identify and sequence specific proteins by generating peptides or even amino acid mass  fingerprints  of proteins , which are then searched against a comprehensive computer database ( 16 ) . identification of spore proteins represents a crucial step towards the development of anti - anthrax vaccines and drugs . many anthrax proteins including membrane proteins , spore coat proteins , and exosporium glycoproteins have been identified via 2d electrophoresis or a variety of mass spectrometric methods , including the multidimensional chromatography tandem mass spectrometry and ion trap time - of - flight hybrid mass spectrometer ( qit - tof ms ; ref . , 22 . ) . to identify proteins involved in the early stage of anthrax infection , we constructed 2d protein profiles associated with anthrax germination and early outgrowth ( 11 ) . by screening 587 paired protein spots that were isolated from dormant and germinating anthrax spores , respectively , we identified 10 spore proteins with statistically significant germination associated increases and decreases . in this review , we will describe two proteases ( immune inhibitor a and gpr - like spore protease ) and alanine racemase that were differentially altered during the germination and early outgrowth of anthrax spores . based on information in the literature and findings in our laboratory , we propose that these three proteins ( immune inhibitor a , gpr - like spore protease , and alanine racemase ) may play vital roles during the early stages of anthrax infection . developing vaccines and/or inhibitory drugs targeting these three proteins may be an effective strategy to prevent or halt the development of the disease that this organism produces . over 500 proteins from the dormant sterne spore strain were reproducibly displayed across an isoelectric focusing ( ief ) range of 4 to 7 . a total of 50 protein spots ( figure 2a ) , corresponding to 36 different anthrax proteins , were successfully identified or sequenced by maldi - tof ms or q - tof 2 ms / ms ( 11 ) . genemark predicted six hypothetical proteins derived from the anthrax genome and these were coded as unknown proteins . following a protein sequence database search , one ( spot 1 ) of the six hypothetical proteins was shown to share 96% homology with immune inhibitor a ( gi|9858110 ) of bacillus thuringiensis 
 23 . , 24 .. the level of immune inhibitor a was increased nearly two folds during germination and early outgrowth of anthrax ( figure 2 ) . to identify this protein spot , the spot was cut from the silver - stained gels , in - gel digested with the trypsin enzyme , and then analyzed on a perseptive voyager - de maldi - tof ms . peptide fingerprint mass spectra generated from maldi - tof ms were interpreted and matched to swiss - prot database entries using mascot ( http://www.matrixscience.com ) . sixteen peaks within the peptide mass fingerprint of spot 1 matched those of a protein with accession number np_843763 in the swiss - prot database ( figure 3 ) . the protein named as a hypothetical protein predicted by genemark is in fact a predicted protein from the anthrax genome ( 25 ) . by using a q - tof 2 ms / ms mass spectrometer , we have also determined the internal sequence of this predicated protein at amino acids between 450 and 463 with m / z value at 1465.77 ( figure 4 ) . all 14 amino acid residues ( giglatyldqsvtk ) from spot 1 were thus determined . more interestingly , the entire amino acid sequence of spot 1 of anthrax shares a high identity with immune inhibitor a of bacillus thuringiensis ( figure 4 ) . both of these proteins contain a hexxh motif , which is defined as a zinc - binding domain of a metalloprotease ( 23 ) , indicating that spot 1 may be a zinc dependent metalloprotease . immune inhibitor a is a secreted virulence protease from bacillus thuringiensis and it specifically degrades two antibacterial proteins ( cecropins and attacins ) produced by an insect host ( 26 ) , suggesting that it may contribute to the overall virulence of bacillus thuringiensis . bacillus thuringiensis is highly resistant to the insect immune system due to its production of two factors , inhibitor a and inhibitor b , which selectively block the humoral defense system developed by insects against e. coli and bacillus cereus 
 ( 24 ) . immune inhibitor a is known to be a metalloprotease with similarity to the thermolysin of bacillus thermoproteolyticus , the elastase of pseudomonas aeruginosa , and the protease e-15 from serratia 
 ( 23 ) . it has been shown , using the transcriptional immune inhibitor a-lacz fusion construct , that expression of this protein occurs at the onset of sporulation ( 27 ) . immune inhibitor a has also been shown to be present as an active 73-kda polypeptide on the exosporium of bacillus cereus 
 ( 28 ) . these results demonstrate for the first time that immune inhibitor a is expressed in anthrax ( fig . 3 ) . anthrax harbors the pxo1 plasmid , which has been known to encode anthrax toxins including pa / lf / ef . the pxo1 plasmid sequence of anthrax is highly conserved among closely related bacterial species including bacillus thuringiensis 
 ( 29 ) , suggesting that anthrax may possess many toxins , which are highly similar to other bacillus species . the level of spot 2 was increased during spore germination and early outgrowth ( figure 2 ) . mass spectrometry matched this spot with protein accession number np_846769 in the swiss - prot database . since this protein shared 91% sequence identity with the spore protease of bacillus cereus and 67% identity with the small , acid soluble proteins ( sasp ) degradation spore germinate protease ( gpr ) precursor of bacillus subtilis 
 ( 30 ) , we assigned it gpr - like spore protease . in the center of the dormant spore is the spore core that contains most of the spore s enzymes , ribosomes and dna ( 30 ) . enzymes that are present in the dormant spore core are inactive , probably due to very low water content in the core . it has been shown that one of the mechanisms to protect the dormant spore against external damaging effects is saturation of the spore chromosome with low molecular weight proteins termed sasp ( 31 ) . in the early stage of spore amino acids are then released for protein synthesis , allowing dna transcription and also releasing energy for metabolism during the very early stages of germination and outgrowth . studies in our laboratory have demonstrated that the expression of gpr - like spore protease is significantly increased during germination and early outgrowth ( figure 2 ) . the three - dimensional structure and molecular mechanism of gpr have recently been extensively investigated ( 10 ) . gpr - like spore protease could be an ideal target for interventions to influence the early stage of anthrax life cycle . alanine racemase ( spot 3 , figure 2 ) was found to be strongly absorbed into the exosporium . alanine and inosine are the two major chemical triggers of spore germination ( 32 ) . alanine racemase converts l - ananine to d - alanine , a germination inhibitor and essential component of the peptidoglycan found within the cell wall and spore cortex 11 . , 32 .. its activity is generally higher in spores than in vegetative cells 11 . , 33 .. the expression level of alanine racemase was decreased during spore germination and early outgrowth 11 . , 32 . , supporting the hypothesis that inhibition of this enzyme may be one of the prerequisites for germination of anthrax spores . expression of alanine racemase in exosporium may allow inactivation of this enzyme by specific antibodies or inhibitory drugs without the requirement of penetrating a physical barrier . inhibition of alanine racemase may therefore minimize the survival potential of anthrax by triggering premature germination of spores in a suboptimal environment . it is thus possible to decontaminate an anthrax - spore - covered area by sprinkling inhibitors targeting this enzyme in conjunction with specific amino acids and nucleoside combinations that trigger the gera - like sensor operons ( 18 ) to induce death of anthrax through pre - mature spore germination . an approach using 2d gels in conjunction with mass spectrometry has been employed in an attempt to look for candidate vaccines and/or drugs that specifically target proteins expressed at the early stage of anthrax infection . the amount of protein in a 2d gel may be altered by numerous events including changes in extractability from dormant and germinated / early outgrowth spores . nonetheless , proteins characterized by 2d gels have a high probability of acting as targets , given the magnitude of changes that are occurring during germination . this makes it likely that they play a critical role in germination , outgrowth , maintenance , and stabilization of spores . further studies to determine the functions and distribution of anthrax proteins that have been identified are essential to the design of appropriate anti - anthrax vaccines and/or drugs . immune inhibitor a has also been shown to be present on the exosporium of bacillus cereus 
 ( 28 ) , but its location in bacillus anthracis is unknown . if immune inhibitor a is located within the spores , antibodies or anti - anthrax drugs may be unable to gain access to this protein . the same challenge is also present for gpr - like spore protease , which has a high sequence homology with gpr , the protease expressed within the spore core of bacillus cereus . since the spore core is protected by an inner membrane and other physical layers outside of the core , it will be necessary to design an intervention that can penetrate all of these barriers to reach the spore core and react with the gpr - like spore protease . there are other challenges in the development of vaccines / drugs targeting the early stages of anthrax infection . for example , once spores are inhaled , portions of immune inhibitor a and alanine racemase may remain intracellular before macrophages are lysed by anthrax toxins and/or immune inhibitor a. alternatively , discharge of immune inhibitor a and alanine racemase from dormant spores may occur within alveolar macrophages where they are beyond the reach of circulating antibodies or drugs . it has been reported that multiple signal pathways may be required for the breakdown of dormant anthrax spores ( 34 ) . many molecules including immune inhibitor a , gpr - like spore protease , and alanine racemase may coordinate to trigger the early - outgrowth of anthrax spores . additionally , immune inhibitor a may contribute to the cytotoxicity of anthrax spores by working together with pa / lf / ef . if that is the case , then multivalent vaccines or drugs that combine pa / lf / ef and immune inhibitor a may be a novel method of developing an effective anti - anthrax remedy . the development of vaccines and/or inhibitory drugs targeting specific proteins expressed during the early stage of anthrax infection may be an effective strategy to halt the attack and spread of the disease ( figure 5 ) . blocking upstream processes ( macrophage interaction , germination , and early outgrowth ) that occur early during an infection would prevent downstream events including the production of its three natural toxins , pa , lf and ef . the three anthrax proteins ( immune inhibitor a , gpr - like spore protease , and alanine racemase ) highlighted in this review have differential expressions during spore germination and outgrowth . designing anti - anthrax vaccines and/or drugs primarily targeting these three proteins rather than pa , lf and ef may provide a safer and more effective approach to counteract anthrax .
in this review , we advance a new concept in developing vaccines and/or drugs to target specific proteins expressed during the early stage of bacillus anthracis ( anthrax ) infection and address existing challenges to this concept . 
 three proteins ( immune inhibitor a , gpr - like spore protease , and alanine racemase ) initially identified by proteomics in our laboratory were found to have differential expressions during anthrax spore germination and early outgrowth . 
 other studies of different bacillus strains indicate that these three proteins are involved in either germination or cytotoxicity of spores , suggesting that they may serve as potential targets for the design of anti - anthrax vaccines and drugs .