78 Bacillus anthracis, a bug with attitude! Les Baillie* and Timothy D Read† The sequencing of the Bacillus anthracis genome and species in this group based on 16S rRNA sequences. virulence plasmids represents the greatest advance in anthrax However, amplified fragment length polymorphism research in the past 100 years. The data will provide the (AFLP) and multiple-locus VNTR (variable number tan- foundation of all future work on this organism and will be dem repeat) analysis (MLVA analysis) have provided clear invaluable to researchers in their battle to understand the basis evidence that B. anthracis can be distinguished reliably of the host–microbe interaction. from other members of the bacilli [5,6••]. In practical terms, the demonstration of virulence constitutes the principle Addresses point of difference between typical strains of B. anthracis *Pathobiology, Biomedical Sciences, DERA Porton Down, Salisbury and those of other anthrax-like organisms [4•,7]. SP4 0JQ, UK; e-mail: [email protected] †The Institute for Genomic Research, 9712 Medical Center Drive, Disease in man Rockville, MD 20850, USA; e-mail: [email protected] Man generally acquires the disease directly, from contact Current Opinion in Microbiology 2001, 4:78–81 with infected livestock (known as non-industrial anthrax) 1369-5274/01/$ — see front matter or indirectly in industrial occupations concerned with © 2001 Elsevier Science Ltd. All rights reserved. processing animal products (known as industrial anthrax) [4•]. Abbreviations EF oedema factor Three forms of the disease are recognised in humans: cuta- LF lethal factor neous, pulmonary (inhalation) and gastrointestinal PA protective antigen infection. The gastrointestinal and pulmonary forms are regarded as being most frequently fatal, owing to the fact Introduction that they can go unrecognised until it is too late to instigate In this review, we will describe Bacillus anthracis, its viru- effective treatment with the agents currently available lence factors and what is known about the basis of the [8••]. There is an obvious need to develop therapies that host–pathogen interaction. The work that is in progress to can be used to treat these individuals. determine the genetic sequence of the organism will also be described and the exploitation of this data discussed. Cutaneous anthrax This form accounts for the majority of human cases (>95%) Anthrax is a disease caused by the bacterium B. anthracis. [8••] and is usually caused by the handling of infected ani- Although primarily a disease of animals, it can also infect mals or their products. The organism gains access through man, sometimes with fatal consequences. The disease has a break in the skin, and forms a primary lesion within two been evident since biblical times: the fifth and sixth plagues to seven days. A ring of vesicles develops around the cen- of Egypt (Exodus, chapter 9) are considered to have been tral papule, which ulcerates and rapidly dries to form a anthrax. More recently, the organism was instrumental in the characteristic black lesion. Most such carbuncular cases founding of two modern sciences: bacteriology (Koch, 1877) recover without treatment, but in 20% of cases, the infec- and immunology (Pasteur, 1881) [1]. Since then, little atten- tion will progress into a generalised septicaemia with tion has been focused on understanding the biology of the poor prognosis [8••,9]. organism, save for the fact that it possesses properties that make it ideally suited as a biological weapon. It forms heat- Pulmonary (inhalation) anthrax resistant spores that are easy to produce using commercially Although commonly used, the term ‘pulmonary anthrax’ is available technology and can infect via the aerosol route. a misnomer. The lung is not the primary site of infection and a better description is ‘inhalation anthrax’. Following It has been reported that at the time of the Gulf War, Iraq inhalation, spores are phagocytosed by alveolar produced large quantities of anthrax spores and had macrophages and transported to hilar and tracheobronchial deployed SCUD/Al-Hussein missiles equipped with lymph nodes, where the spores germinate and multiplica- biological weapons warheads [2,3•]. tion of vegetative bacilli occurs [10•]. Fatal bacteraemia and toxaemia then ensue, with a mortality rate of >80% [8••,9]. The organism B. anthracis is the only obligate pathogen within the genus Gastrointestinal anthrax Bacillus, which comprises the Gram-positive aerobic or fac- Gastrointestinal anthrax is extremely rare and occurs ultatively anaerobic spore-forming, rod-shaped bacteria. It mainly in Africa, the Middle East and central and south- is frequently convenient to class B. anthracis informally ern Asia. It is rarely seen in man where the disease is within the ‘B. cereus group’, which, on the basis of pheno- infrequent or rare in livestock. Most cases of intestinal type, comprises B. cereus, B. anthracis, B. thuringiensis and anthrax result from eating insufficiently cooked meat from B. mycoides [4•]. It is not possible to discriminate between anthrax-infected animals [11]. Bacillus anthracis, a bug with attitude! Baillie and Read 79 Pathogenesis the chemotactic response of polymorphonuclear leucocytes Despite the early understanding of the cause of anthrax, and may inhibit subsequent phagocytosis [6••,13]. the pathogenic process in humans is ill-defined. Animal studies have shown that the interaction of the organism Protective antigen with the macrophage is the key event in the disease PA derives its name from the fact that it is the key protective process [12–14]. The spore appears to require the environ- immunogen in the current human vaccine [18]. The ability ment of the macrophage to stimulate germination. The of PA to transport proteins into the cytoplasm of eukaryotic resulting vegetative cells are able to survive the harsh envi- cells has been exploited to deliver antigens to the major ronment of the macrophage and escape the cell by means histocompatability complex (MHC) class 1 pathway [19•]. as yet undetermined. It is known that the organism expresses a toxin called lethal factor (LF), to which the Capsule macrophage is particularly sensitive. At low levels, LF Fully pathogenic strains produce an antiphagocytic capsule stimulates the production of the shock-inducing media- composed of poly-D-glutamic acid. The genes controlling tors, tumour necrosis factor α and interleukin-1β. At higher capsule synthesis — CapB, CapC and CapA — are located levels, the toxin causes the death of the macrophage and on pXO2. An additional gene, dep, located downstream the release of the cytokines into the blood stream. Thus, it from the Cap genes, may also play a role in virulence [20]. is likely that death is caused by a combination of massive bacteraemia and cytokine-induced shock [12,13]. Coordinate regulation of major virulence factors Capsule and toxin synthesis and the expression of a num- By understanding the biology of this interaction, it may be ber of genes encoding nontoxins of unknown function are possible to design therapeutic approaches that tip the bal- enhanced in the presence of serum, CO2 and bicarbonate ance in favour of the macrophage and stop the infection [4•,7,21,22]. This effect is mediated via two trans-acting before it starts. regulators: acpA, located on pXO2 (which activates expres- sion of capB), and atxA, located on pXO1 (which activates Virulence factors expression of the toxin genes and also appears to regulate It is likely that the organism expresses a range of virulence acpA transcription to some degree). This system enables factors. The two major factors, a tripartite toxin and an the organism to sense changes in the host environment and antiphagocytic capsule composed of poly-D-glutamic acid, upregulate the expression of virulence genes. are encoded by genes carried on two plasmids, pXO1 and pXO2. The loss of either plasmid results in a marked Minor virulence factors reduction in virulence [7]. In addition to the major factors already described, the organism expresses other plasmid- and chromosome- The tripartite anthrax toxin is considered to be the major encoded genes that contribute to the pathogenesis of the virulence factor. The three proteins of the exotoxin are organism [14,20,23–25,26•,27,28]. protective antigen (PA), LF and oedema factor (EF). The toxins follow the A–B model, in which the A moiety is the Plasmid-encoded virulence factors catalytic domain and the B moiety is the receptor-binding The first step toward genomic characterisation of B. anthracis domain. PA acts as the B moiety and binds to an as yet was the sequencing of the pXO1 virulence-associated unknown cell-surface receptor, whereupon it is cleaved by megaplasmid from an isolate of the Sterne strain by Rich the cell-surface protease furin to release an amino-terminal Okinaka’s group at the Los Alamos National Laboratory 20 kDa fragment. Following proteolytic activation, PA [29••]. The 181654 base pair (bp) sequence contained 143 forms a membrane-inserting heptamer that translocates identifiable open reading frames (ORFs), which comprised the toxic enzymes, LF and EF, into the cytosol [15]. only 61% of the plasmid [29••]. The pXO1 G+C content (32.5%) is close to that of the chromosome (34%) and the Lethal factor codon usage in pXO1 genes matches that of the putative set LF is the central effector of shock and death from anthrax. of B. anthracis generated in the preliminary stage of genome It contains a thermolysin-like active site and zinc-binding sequencing (TD Read, unpublished data). This suggests consensus motif HexxH, and has been shown to act as a that most genes acquired horizontally originated in bacteria Zn2+ metalloprotease on a variety of substrates, including closely related to B. anthracis. The pXO1 plasmid includes a peptide hormones and mitogen-activated protein kinase pathogenicity island of 44.8 kilobases (kb), bounded by kinase [16,17]. It is now believed that the intracellular IS1617 elements in inverted orientation, which contains the hydrolysis of important host protein substrates is responsible lef, cya, pag, atxA and pagR genes [7,22].