What makes anthrax so virulent

The findings were published in Thursday's issue of the journal Nature. More than three months ago, letters tainted with a powerful strain of anthrax were mailed to politicians and media figures in New York and Washington, as well as a supermarket tabloid in Florida. Since then, the Centers for Disease Control and Prevention in Atlanta have confirmed 18 cases of anthrax infection nationwide cases of inhalation anthrax and seven through the skin.

Five people have died, all from inhaled anthrax. Tang said researchers also have found that the three anthrax proteins work as a team in the attack.

If enough anthrax spores are inhaled into the lungs, they can cause inhalation anthrax, the deadliest form of the disease. But it's also the hardest form of anthrax to get. That's because it takes a large number of spores--from eight to ten thousand of them--to cause the illness.

And those individual spores all have to be inhaled very deeply into the lungs to cause serious problems. When the individual dormant anthrax spores lodge in the tiny air sacs in the lungs known as the alveoli, the immune system recognizes and attacks them and can kill many of them.

But some spores may also travel to the lymph nodes near the lungs. That's where they germinate into live anthrax bacteria, which release toxins that destroy the body's cells. Without treatment to kill the anthrax bacteria, those toxins build up and cause a variety of problems that quickly lead to death within a few days. Inhalational and gastrointestinal anthrax are difficult to diagnose and are associated with high mortality [ 1 ].

The first case of anthrax infection by injection was described in , with a more severe outbreak in [ 3 , 4 ]. It constitutes a new form of anthrax infection that mainly affects soft tissue, and patients require antibiotic treatment as well as surgery to remove the necrotic tissues [ 5 , 6 ]. Even if the decline of anthrax infection in animals and humans has been significant during the last century, the multiple outbreaks among drug-injection users and the terrorist attacks clearly show that B.

The B. Sporulation of B. The spore is formed of several layers, the core containing the genetic material decorated by protective small acid-soluble proteins [ 9 ] surrounded by an inner cortex that will be the precursor of the cell wall of the vegetative state.

A thick proteinaceous coat surrounds the cortex and protects the inner core, preventing dehydration. Finally, spores are the infectious agents leading to anthrax infection.

The vegetative form of B. These virulence factors are mainly encoded by two virulence plasmids called pXO1, responsible for the bacterium exotoxins, and pXO2 that encodes for the biosynthetic machinery responsible for the production of the capsule. Here we will review recent findings that highlight how anthrax toxin hijacks different cellular mechanisms to increase its intoxication efficiency and describe new structural data that explain the molecular mechanisms responsible for the almost unmatched efficiency of this toxin to disrupt cellular functions and kill its host.

Once the spores have germinated in the body of the infected host, the bacteria will produce anthrax toxin. This tripartite AB toxin is composed of the receptor-binding subunit, protective antigen PA , and the two enzymatic subunits, lethal and edema factor LF and EF. To affect the cells of the host, the toxins needs to gain access to the cell cytoplasm where the two enzymatic subunits of the toxin act.

To do so, anthrax toxin has hijacked cellular pathways, a common strategy of pathogens. The physiological role of these two highly homologous receptors is poorly understood. Most of the results obtained to date point to an involvement of the receptors in the homeostasis of the extracellular matrix [ 16 ].

Given their similarity to integrins, they seem to bind to proteins of the extracellular matrix ECM , such as collagens and fibronectin [ 17 , 18 , 19 ] and might regulate the accumulation of these in vivo [ 20 , 21 , 22 ]. Given that mice are completely resistant to anthrax toxin challenge when CMG2 is knocked out [ 26 ], the proteins described above might be only accessory, rather modulating than mediating entry. This could partly explain why CMG2 is the main toxin receptor in mice [ 27 ].

After binding to the receptor at the cell surface, the 83 kDa form of PA PA 83 is cleaved by furin-like proteases and produces a shorter form, PA 63 [ 29 , 30 ].

Only this cleaved form of PA is able to assemble into oligomers, either heptamers or octamers [ 31 , 32 ]. The receptor-toxin oligomers cluster in lipid rafts, which are subdomains of the plasma membrane [ 33 ]. The localization of the oligomers to these subdomains is important, as disruption of lipid rafts leads to a defect in subsequent internalization of the toxin [ 33 ].

Partitioning into lipid rafts is controlled by palmitoylation of the tail of TEM8. Palmitoylation keeps the receptor out of lipid rafts and palmitoylation-deficient TEM8 will rapidly internalize, thereby perturbing the finely tuned timing of this process [ 34 ]. This timing and correct functioning of these processes is also mediated by other posttranslational modifications of the receptor tail. Both receptors are phosphorylated on tyrosine residues by Src or Fyn in response to PA binding.

This is not crucial for formation of the oligomers but impairs toxin entry [ 35 ]. Another important modification is ubiquitination. This leads to the recruitment of an E3 ubiquitin enzyme and the toxin-induced ubiquitination of the two receptors [ 37 ].

Schematic overview of cellular entry of anthrax toxin and progression through the endocytic pathway A B. After oligomerization in lipid rafts dark grey , the receptor-toxin complex is internalized by endocytosis.

This endocytosis seems to largely depend on clathrin, dynamin, AP-1 and actin. B After trafficking to the early endosome, PA can undergo a conformational change, leading to pore formation and translocation of the enzymatic subunits across the membrane. LF and EF get to the cytoplasm by either direct translocation or by backfusion of intraluminal vesicles ILVs of late endosomes.

LF cleaves MEKs and thereby leads to necrosis and hypoxia. EF leads to the elevation of intracellular cAMP and causes edema. LF and EF will only bind to PA oligomers, with a stoichiometry of enzymatic subunit to heptamer and to octamer. The hetero-oligomeric toxin-receptor complex is subsequently internalized, mainly by clathrin-mediated endocytosis, although alternate, context-dependent pathways may exist [ 37 , 38 ].

Interestingly, the clathrin-dependent endocytosis of anthrax toxin seems to depend on the unconventional adaptor AP1 rather than on the more common AP2 [ 37 ]. Another important player in enabling entry of the toxin into cells is the actin cytoskeleton.

Endocytosis of both receptors after toxin binding depends on actin and TEM8 appears to be pre-organized by actin at the cell surface Figure 1 A [ 37 ]. It has also been proposed that toxin entry depends on the reorganization of the actin cytoskeleton by calpain [ 39 ]. Finally, a genome-wide screen for anthrax susceptibility genes led to the identification of ARAP3, a protein involved in Rho-mediated actin remodeling [ 40 ], further linking actin to toxin entry.

Once internalized from the cell surface, PA undergoes a conformational change triggered by the low pH in endosomes and forms a pore that acts as a translocation channel for the enzymatic subunits. The precise site along the endocytic pathway where pore formation occurs may depend on whether entry is mediated by TEM8 or CMG2.

If PA is bound to TEM8, dissociation and pore formation occurs at earlier steps in the endocytic pathway, presumably in early endosomes, whereas for CMG2, the pH threshold is lower and seems to require trafficking to late endosomes [ 41 ]. To get to the cytoplasm, EF and LF translocate through the PA pore, either directly to the cytoplasm [ 42 ] or first by translocation into intraluminal vesicles ILV and then backfusion of these with the limiting membrane of endosomes [ 43 ].

The latter, more complex pathway, offers two major advantages. First, the enzymatic subunits can remain in the lumen of ILV protected from lysosomal enzymes, allowing long-term action of the toxin, as observed in vivo.

Also, by residing in ILVs, the toxin can be released to the extracellular environment as exosomes and taken up into cells independently of the receptor and invisible to the immune system [ 44 ]. This entire mechanism allows both long-term storage and long-range transmission of the toxin.

The long-term and very potent activity of the anthrax toxin might explain why patients, even without detectable bacterial load after successful antibiotic treatment, still succumb to the disease. Apart from entry via the plasma membrane, the enzymatic subunits LF and EF might also reach the cytoplasm via a different pathway. In a scenario described by Banks et al. Retrotranslocation is achieved by formation of the PA pore directly in the limiting membrane and depends on the presence of the anthrax toxin receptor [ 45 ].

The poly- d -glutamic acid capsule is an excellent defense of the bacteria against phagocytosis and is an important determinant of establishing successful infection in vitro and in vivo [ 12 , 46 ]. More factors have been postulated but remain to be tested for their exact role in anthrax.

The anthrax toxin, together with the capsule, however, seem to remain the two most important virulence factors. Lethal factor LF is a zinc-dependent metalloprotease, which cleaves members of the MAP kinase kinase family MEK , leading to their inactivation [ 47 ].

Edema factor EF is a highly efficient calmodulin-dependent adenylyl cyclase. It can convert up to molecules of ATP to cAMP per second, significantly increasing the concentration of this second messenger [ 48 ]. Both rely exclusively on PA to get into cells. Anthrax toxin acts both at early and late stages of infection and both EF and LF seem to have preferential target sites and complementary roles to ensure successful progression of the disease.

After entering the host and germination, the first mission for anthrax toxin is to incapacitate the immune response. It seems to first target myeloid cells, such as macrophages and neutrophils [ 49 ], paving the way for the bacterial replication.

Mice that were specifically deleted for CMG2 in their myeloid lineage were resistant to anthrax infection, underlining the importance of these immune cells in the establishment of a successful infection [ 49 ]. The primary effector here seems to be LF, which inactivates MEKs, thereby promoting apoptosis of the cells [ 50 , 51 , 52 ]. Some mice strains and rats harbor a polymorphism in NLRP1, a component of the NLRP1 inflammasome, which makes it susceptible to cleavage by LF, thereby leading to a rapid, caspasedependent apoptosis [ 53 ].

Interestingly, having susceptible macrophages seems to protect these mice more from infection than having resistant macrophages [ 54 , 55 ]. This seemingly counterintuitive result can be explained by an elevated secretion of inflammatory cytokines by susceptible macrophages, thereby leading to an activation of the immune response, which then efficiently controls the spread of the bacteria.

Therefore, LF-induced apoptosis has to be a rather slow process, which is also thought to help bacteria reach lymph nodes, a key environment for replication and dissemination [ 56 , 57 , 58 ]. After B. To achieve this, EF adenylate cyclase activity is believed to delay the LF-mediated macrophage apoptosis. This counteracts the pro-apoptotic cleavage of MEK by LF, resulting in a balance between survival and programmed cell death.

This balance is finely tuned to ensure maximum infection efficiency and dissemination of the pathogen to the lymph nodes [ 62 ]. In addition to this, EF can upregulate the expression of CMG2 via the transcriptional regulator CREB, increasing susceptibility of the cells to the toxin [ 63 , 64 ] and can delay general protein clearance in serum in vivo by a still unknown mechanism, thereby prolonging toxin effects in circulation [ 65 ].

Other effects of EF have been described but are less clear. Some studies show EF as an effector in increasing motility of macrophages, helping in dissemination of the bacteria [ 66 , 67 ], whereas others claim it blocks motility and migration of immune cells, impairing their arrival at inflammatory sites [ 68 , 69 ].

In some cases, infection depends largely on the presence of the toxins, whereas in others, pathogenicity can be more toxin independent [ 70 , 71 ]. Also, the relative contribution of LF and EF to disease progression can vary. LF has been repeatedly described as more important to sustain infection in different animal models [ 72 , 73 , 74 ], yet other reports also highlight the importance of EF at different stages of infection [ 66 , 75 , 76 ].

However, the consensus view is that both anthrax toxins contribute to pathogenicity and fatality of anthrax infections by disabling the immune system and by damaging vital functions of the host. More specifically, both LF and EF target and block the immune response in an early stage of systemic infection, allowing the infection to progress to later stages. The first stage of infection can be asymptomatic, followed by an acute stage, with rather non-specific symptoms, such as fever, sore throat, vomiting and diarrhea for inhalational and gastrointestinal anthrax [ 77 ].

The cause of death is a combination of bacterial sepsis, as bacteria rapidly multiply, and also toxemia, caused by a high level of anthrax toxin in circulation, affecting multiple organs [ 78 ].

That toxins are important for these late stages becomes clear with the fact that even patients with no detectable bacterial load after antibiotic treatment can die from a systemic infection. This is probably due to a prolonged action of the toxins in circulation [ 44 ]. As CMG2 is a rather ubiquitiously expressed receptor, toxins could potentially affect a large variety of tissues in the host.

However, both LF and EF seem to have a preference for certain tissues: LF targets cardiomyocytes and smooth muscle cells, thereby affecting the cardiovascular system, whereas EF primarily targets hepatocytes, damaging the liver [ 79 ].

This indicates that LF targets primarily the heart, but EF has other tissue targets apart from the liver [ 79 ]. EF, as the name implies, causes edema in skin and liver, though the exact mechanism remains unclear.

Protective antigen is the non-catalytic subunit of the anthrax toxin that binds to CMG2 and TEM8 and triggers toxin uptake [ 33 ]. PA is composed of four domains [ 80 ].

Domain 1 residues 1 to is cleaved by furin [ 30 ] after a RKKR motif localized on an solvent-exposed loop Figure 2 A.

This cleavage is a prerequisite for PA oligomerization. Domain 3 residues to resembles domain A of toxic-shock-syndrome toxin 1 [ 82 ]. This domain is strongly involved in receptor binding Figure 2 B.

Protective Antigen structure in monomeric and oligomeric form. US Coast Guard hazardous material workers in protective suits and breathing systems stand together before entering the sealed US Senate hart building as contamination tests continue searching for anthrax spores inside the US Capitol building in Washington,DC credit: Paul J. Recommended Anti-Islamic armed bikers confronted by protesters One World Trade Centre observatory opens to the public Isis fighter trained in counterintelligence by state department.

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