Anthrax toxin is an essential virulence factor for Bacillus anthracis, the causative pathogen underlying anthrax infection, whose proximal mechanisms of action have been recently reviewed [1,2]. This toxin is composed of three subunits, edema factor (EF), lethal factor (LF), and protective antigen (PA). EF has adenylate cyclase activity resulting in tissue edema, whereas LF is a zinc metalloproteinase that targets specific mitogen-activated protein kinase kinases (MKKs). These toxin enzymes require the host receptor-binding component of anthrax toxin, PA, for intracellular entry into target cells. Much research attention has focused on the biology of anthrax LF, which when combined with its receptor-binding partner, PA, is termed lethal toxin (LT). Administration of purified LT to mice recapitulates many of the major clinical features of anthrax infection [3]. Although anthrax LT can result in the caspase-1-dependent release of cytokines such as IL-1β and IL-18, this effect is species- and strain-specific [3,4,5]. Instead, the predominant effect of anthrax toxin on the immune system is to cause immunosuppression [2,6], allowing very high concentrations of Bacillus anthracis to accumulate in the host, thereby increasing the likelihood of future rounds of infection. Although this immunosuppression facilitates infection, it does not explain the deleterious effects of LT that occur independent of infection.

One central feature of intoxication is the compromise of host barrier integrity. Although the lethal effects of fractionated LT were first described by Stanley and Smith in early 1961 [7], subsequent studies performed at Fort Detrick during the 1960’s provided further insights into the underlying pathogenic mechanisms [8,9]. These studies revealed that LT has a deleterious effect on barrier function in rats, leading to lethal pulmonary edema and hemoconcentration. A comprehensive examination of the mechanisms underlying in vivo lethality with recombinant LT was not undertaken until 2003 by Moayeri et al. [3]. This murine study revealed that anthrax LT administration results in multiple signs of host barrier dysfunction, including splenic hemorrhage, fecal blood, and pleural and peritoneal edema [3]. More recent investigations have documented the effects of LT on the integrity of the endothelial cell barriers that line blood vessels and epithelial barriers that protect the gastrointestinal and respiratory tracts [10,11,12,13]. The effects of LT on barrier function not only contribute to the spread of infection, but also disrupt physiological homeostasis. This article will provide an overview of the effects of anthrax LT on a variety of host tissue barriers, as well as our current understanding of the pathogenic mechanisms underlying these effects.

Host barriers are essential for maintaining physiological homeostasis, by establishing functional boundaries between the host and its environment or between different tissue types or body cavities [14]. These boundaries facilitate the development of molecular gradients between tissue compartments, as well as provide protection from potentially harmful organisms and molecules located in the adjacent environment. Epithelial and endothelial tissues have several features that contribute to their barrier function. Luminal secretions such as mucus commonly coat the outermost surface of host barriers, forming a physical and chemical barrier to pathogens and large molecules. The mucus layer overlies the central structure of the barrier, a single layer of specialized endothelial or epithelial cells, whose lipid plasma membranes and highly specific membrane transport systems regulate transepithelial and/or transendothelial passage of most molecules. Adhesive junctional complexes between epithelial or endothelial cells are required to maintain an effective barrier, and have been a topic of detailed reviews [15,16]. Tight junction (TJ) and adherens junction (AJ) complexes consist of transmembrane proteins that link adjacent cells to the actin cytoskeleton through cytoplasmic scaffolding proteins. TJs are located most apically and are responsible for sealing the intercellular space, regulating selective paracellular ionic solute transport, and maintaining cell polarity. AJs are formed by cadherin-catenin interactions and are important in the mechanical linkage of adjacent cells. TJs and AJs are also important in the regulation of cellular proliferation, polarization, and differentiation. Desmosomes, which exist between epithelial cells (but not endothelial cells), provide additional mechanical linkages between neighboring epithelial cells. At their basal surfaces, epithelial or endothelial cells are anchored to the underlying basal lamina (basement membrane) through cell-matrix adhesions [17]. Epithelial layers are avascular and are nourished by substances diffusing from the blood vessels into the underlying tissue. The basement membrane acts as a selectively permeable membrane, determining which substances enter the epithelium through this direction. Subepithelial connective, nervous, and muscular tissues minimally contribute to the permeability of epithelial and endothelial barriers [18,19].

The natural cycle of Bacillus anthracis infection has been reviewed extensively [20,21]. The infection cycle generally involves herbivores, which are infected following exposure to spores present in the soil during feeding. Upon death of the animal, large numbers of bacteria are released into the soil where they sporulate, forming the basis for the next round of infection. Humans become infected with Bacillus anthracis when exposed to infected animal products, through either handling or consumption. Anthrax infection can also occur following exposure to aerosolized spores during manufacturing with animal products (e.g., leather processing) or by the malevolent actions of their own species, as highlighted by the U.S. bioterrorism attacks of 2001 [22].

Anthrax infection can take on three distinct clinical forms: cutaneous, inhalation, and gastrointestinal. The manifestation of a specific form is dependent upon the route of infection. Despite differences in their clinical presentations, these clinical syndromes share one important feature, each requires the penetration of protective host barriers by Bacillus anthracis.

Cutaneous anthrax is the most common form of anthrax infection, comprising nearly 95% of all human anthrax infections [21,23]. This form of anthrax occurs when Bacillus anthracis spores penetrate the dermal layers of the skin, usually through a cut or abrasion. Local edema becomes apparent 24–48 h post infection as a result of spore germination and toxin production. Approximately two days post infection, patients typically develop a large round ulcer, which is followed shortly by the formation of a characteristically painless, depressed black eschar. In some cases, the infection breaches the epithelial layers of the skin, entering into the lymphatic system. Once this breach occurs, patients may develop lymphangitis and lymphadenopathy with subsequent systemic complications. In these rare cases, the infection can lead to septicemia, shock, hemolytic anemia, coagulopathy, hyponatremia, renal failure, and death. Anthrax infection has also been acquired trans-cutaneously in injection drug abusers. In these cases, the ensuing soft tissue infection is not necessarily accompanied by eschar formation, rendering the diagnosis more difficult [24,25].

Gastrointestinal anthrax occurs when a large number of vegetative bacilli are ingested, usually through the consumption of infected, but insufficiently cooked meat [21]. Differential manifestations of the infection are observed depending on whether the upper or lower gastrointestinal tracts are involved. If infection occurs in the upper gastrointestinal tract or oropharyngeal tract, patients develop oral or esophageal ulcers. These lesions can, in turn, progress to regional lymphadenopathy, edema, and sepsis, in a similar fashion as cutaneous anthrax. Infection in the lower gastrointestinal tract often leads to lesions in the terminal ileum or cecum [23]. Patients subsequently develop nausea, vomiting, bloody diarrhea, severe gastric lesions, and, in some cases, ascites and sepsis [26]. However, cases that involve intestinal eschar formation with surrounding tissue edema are nearly always fatal, highlighting the functional importance of the intestinal barrier during infection [27].

Inhalation anthrax is considered to be the most serious form of anthrax infection, resulting in high levels of morbidity and mortality. This clinical form of anthrax ensues when Bacillus anthracis spores are inhaled and dispersed throughout the alveolar space. Extrapolation from animal studies suggests that the inhalation of 2500 to 55,000 Bacillus anthracis spores is sufficient to produce an LD₅₀ in humans [28]. However, an analysis from 2002 suggested that one to three spores would be sufficient to cause infection (LD₁), based on an LD₅₀ of 4100–8000 spores [29]. Once germination of the spores ensues, inhalation anthrax has been reported to have a mortality rate as high as 89%, although the survival in the 2001 bioterrorism cases was somewhat higher (six out of the eleven manifested inhalational cases), suggesting that mortality rates can be improved with proper antibiotic treatment and supportive care [23,30]. Moreover, the relative ease with which spores can be aerosolized makes inhalation anthrax a serious concern from a bioterrorism perspective.

In a typical inhalation infection, spores first encounter the pulmonary epithelium and are subsequently endocytosed by pulmonary macrophages and/or dendritic cells. Pulmonary phagocytes then translocate to the regional lymph nodes in the mediastinum, where germination occurs. One of the classic signs of inhalation anthrax is widening of the mediastinum, likely due to the production of anthrax toxin by bacteria germinating at this site [23,31,32]. Presumably, dissemination occurs mainly via the invasion of the bacteria into blood vessels surrounding the mediastinal lymph nodes. Although it has recently been observed in some animal model systems that germination can also occur in the intra-alveolar space, whether this can lead directly to dissemination is unclear [33,34].

Systemic dissemination is possible for each of the distinct forms of clinical anthrax infection. In this regard, Bacillus anthracis demonstrates an intrinsic ability to compromise a variety of epithelial and endothelial barriers that are present in many mammalian organ systems. The initial breach is facilitated either by existing barrier imperfections or by the host phagocytes serving as “Trojan horses” [35]. The regional lymph nodes form another line of defense that is overcome, leading to invasion of the blood vessels and dissemination. It is widely held that the anthrax toxin released by replicating Bacillus anthracis contributes greatly to systemic infection by disrupting the integrity and functionality of various barrier tissues (Figure 1).

Anthrax meningitis/meningoencephalitis is the main neurological complication of anthrax infection. It is exhibited in up to half of the cases in outbreaks of inhalational anthrax [36,73]. In experimental inhalational anthrax infection models in monkeys, meningitis has occurred in 50–77% of cases examined [38,74]. The high occurrence of meningitis in anthrax infections indicates that vegetative Bacillus anthracis bacteria are capable of penetrating the BBB. The BBB is formed by a single layer of specialized endothelial cells, known as brain microvascular endothelial cells (BMEC). These cells are closely sealed by tight junctions. Bacillus anthracis is able to invade human brain microvascular endothelial cells and to penetrate the BBB in vitro and in vivo, but this activity is toxin-coding plasmid (pXO1)-dependent, as a pXO1-deficient strain exhibits significantly reduced adherent and invasive properties (Figure 1) [75]. The ability of anthrax toxin to facilitate the penetration of bacteria into the brain correlates with its action to suppress the innate defenses of the BBB [75]. Whether LT directly disrupts the BBB structure in vivo remains to be explored. Early studies involving the direct application of LT into the cerebrospinal fluid of monkeys via the cisterna magna resulted in the death of non-human primates by an unknown mechanism [76]. However, these models are not ideal for evaluating the effect on BBB due to the rapid death that follows injection (within 10 min). Limited in vitro data derived from experiments with cultured human brain microvascular endothelial cells (hBMECs) suggest a role for LT-dependent cadherin deregulation in disrupting cellular junctions in the BBB [43].

It is also important to note that anthrax LT also has strain- and species-specific effects on the production of cytokines, depending on whether the animal encodes a resistant vs. susceptible allele of the Nalp1b gene [5]. For example, LT treatment leads to activation of caspase-1 and the release of activated IL-1β and IL-18 in BALB/c mice and macrophages derived from this line [3,4,5]. Cytokines such as IL-1β enhance endothelial permeability in the BBB [77]. However, LT-induced cytokine release is largely absent in strains and species with resistant alleles of Nalp1b, indicating that LT-induced disruption of endothelial barriers occurs through mechanisms independent of cytokine production as well [5]. LT might synergize with other pathogenic factors, such as Bacillus anthracis S-layer protein A (BslA). This newly identified pXO1-encoded protein is likely to play a critical role in breakdown of the BBB by Bacillus anthracis, as mice challenged with BslA-deficient Bacillus anthracis exhibit a significant decrease in the frequency of brain infections compared to mice injected with the parental Sterne strain [78,79].

It has been well documented that LT disrupts the anatomic and functional integrity of critical host barriers. These effects not only allow Bacillus anthracis to establish infection, but also they are leading contributors to morbidity and mortality. A central theme of recent studies is that LT causes dysregulation of junctional complexes by altering the localization of key junction associated proteins, including the cadherins and ZO1 [12,52]. Intriguing new data suggest that the blockade in cadherin trafficking, in turn, results from the activity of LT to directly target Rab11/Sec15 exocysts and thereby disrupt endocytic recycling [43].

Nevertheless, important questions remain to be addressed, including whether other pathogen- and host-associated factors work in concert with LT. For example, administration of lethal doses of edema toxin (ET) into mice leads to extensive pathological lesions in a broad range of organ/tissues including barrier tissues [54]. In addition, ET and LT have unique effects on cardiovascular and renal function in a canine model of intoxication [80]. These effects act synergistically. Other uncharacterized Bacillus anthracis-derived factors may also contribute to the pathology in barrier tissues (e.g., BslA). Investigation of the interactions of these bacterial factors with LT should be undertaken to assess the overall effect of LT in vivo. It will also be important to delineate the MKK pathways targeted by LT that underlie toxic effects on host barriers. In this regard, the MKK1/2 pathways appear to be promising candidates.

Although important strides have been made toward understanding the mechanisms underlying LT-induced barrier dysfunction, we have much to learn regarding the exact molecular and cellular interactions between LT and toxin-targeted host barrier tissues. It will be critical to determine whether the effects of anthrax LT on junctional complexes represent critical and universal mechanisms that would amenable to therapeutic targeting. With regard to the potential to develop anthrax therapies, treatment strategies for anthrax infection have focused thus far on antibiotics and, more recently, upstream anti-toxin therapeutics (e.g., neutralizing polyclonal and monoclonal anti-toxin antibodies, receptor binding inhibitors, and LF enzyme inhibitors) [81,82]. A consideration of downstream therapeutic targets such as those associated with toxin-induced barrier dysfunction might provide new possibilities for therapies that are effective, even if delayed in their administration following infection. Moreover, it is important to note that Bacillus anthracis is just one of several enteric pathogens that disrupt MAPK signaling as a virulence mechanism. Virulence factors of Shigella, Yersinia and Salmonella also target and inactivate MAPK signaling pathways [83,84,85,86]. For this reason, it is reasonable to speculate that an improved understanding of the role of the three major MAPK signaling pathways in maintaining barrier function will not only have therapeutic implications for anthrax infection, but for a variety of other infectious diseases and inflammatory bowel conditions as well.