Anthrax Lethal Factor

Introduction

Lethal Factor (LF) is one of the enzymatic components belonging to the Anthrax Toxin. Anthrax toxin is a three protein exotoxin secreted by the bacterium Bacillus anthracis made up of a binding protein known as the protective antigen (PA) and two enzyme components known as edema factor (EF) and lethal factor (LF). ^[1]

Anthrax Toxin, encoded by plasmid pXO2, is considered an AB toxin, with two A domains (EF and LF) and one B domain (PA). ^{[1] [2]} On their own, these three domains are nontoxic, but any combination involving PA with EF and/or LF is what causes the physiological effects. ^[3] Initially PA is a 83kDa protein which binds to the host Anthrax toxin Receptor (ATR). Upon binding, PA is cleaved into two fragments by a furin proteases to become a 63 kDa protein bound to the ATR. Cleavage of PA allows self associate to occur which forms a ring shaped heptamer know as the pore precursor (prepore). The prepore is now able to bind up to three molecules of EF and/or LF, leading to endocytosis of the complex. In the endosome, the prepore converts to a transmembrane pore, allowing translocation of EF and LF to the cytosol through a mechanism that is not will understood. EF and LF are now able to carry out their enzymatic activity on the host cell. ^[1]

EF is a calmodulin and calcium dependent adenylate cyclase which causes the edema associated with the disease. ^{[1] [4]}

LF is a Zinc dependent protease that cleaves certain MAP kinase kinases (MAPKK)leading to the disruption of many cellular signalling pathways, which eventually leads to cell death. ^{[1] [5]}

This is a list of the possible combinations that can occur with Anthrax Toxin: ^[6]

PA+LF Leads to lethal activity

EF+PA Leads to edema

EF+LF Non-toxic

PA+LF+EF Leads to lethal activity and edema

Human Interaction

Anthrax is primarily a disease of domesticated and wild animals. Herbivores such as cattle, sheep, horses, mules and goats are primarily affected because these animals maybe grazing on soils contaminated with Bacillus Anthracis endospores. ^{[1] [7]} The blood of an animal that dies of anthrax can contain upward of 10^9 vegetative bacteria per milliliter and as the carcass decays, the bacteria form highly infectious endospores, which contaminate the local environment and can remain viable for long time periods. ^[1] The endosopres produced by Bacillus Anthracis remains viable for lengthy periods do to the poly-D-glutamic acid capsule, which itself is nontoxic. This capsule functions to protect the endospore from complement and other bactericidal components found in serum. This capsule plays an important role during the infection of anthrax, but is not important during the disease phase, which is caused by PA, EF, LF. Genes encoding this plasmid are located on plasmid pXO2. ^{[1] [8]} Anthrax is not a common disease among humans because it can not be transmittable from human to human. Humans become infected by being exposed to farm animals or contaminated animal products such as wool, hides, flesh and blood. There are three ways in which Anthrax can be transmitted to humans: ^{[1] [9]}

Cutaneous Anthrax is the most common form of the disease. This usually occurs when endospores enter the body through injured skin and germinates. In most cases the bacteria remain contain at the site of infection and present as a lesion. In rare case, the infection could spread to the blood stream and cause septicemia. Treatment is often not needed. ^[10]

Gastrointestinal Anthrax occurs through the ingestion of spore contaminated meat. The spores then invade the mucosa through a preexisting lesion. After germination, spores spread from the mucosal lesion into the lymphatic system. This form of Anthrax is associated with a high mortality rate but is considered the rarest of the three types of infection. ^[11]

Inhalation Anthrax is the most fatal of the three infections. Also know as woolsorters' disease, this form involves the inhalation of spore usually contained in animal hair and hides. The spores colonize the alveolar macrophages and its believe the macrophages serve both as the sites of germination and as vehicles for transporting the bacteria. At this point the bacteria can rapidly spread throughout the body. If left untreated death is certain. Even with antibiotics mortality rates are high. ^[12]

Treatments

Antibiotics are used to treat Cutaneous and Inhalation Anthrax infections. The primary antibiotics used are Ciprofloxacin and Doxycycline. Antibiotics should to be administered before symptoms arise, which will likely decrease the fatality rate. In the case of inhalation, treat should be received with 24 hours. If administered after symptoms arise, there is a high rate of fatality. The duration of treatment is 60 days of antibiotics to ensure all spores have germinated. In some cases more than two antibiotics are administered. ^[13].

Structure of Lethal Factor

Anthrax Lethal Factor is composed of four domains:

Domain I functions in the binding of Lethal Factor to Protective Antigen 63 (PA63), which is the membrane translocation component of Anthrax Toxin.^[1] The actual location where interacts with PA is unknown. This domain, made up of amino acids residues 1-263, is perched above the other three domains and is connected to the rest of the domains through an abrupt turn at the end of the last helix.^[1] Domain I consist of 12-helix bundle, packs against one face of a mixed four-stranded beta-sheet.^{[1] [14]}

Domain II consist of residues 263-297 and 385-550; also it has a similar structure with that of B. Cereus toxin catalytic domain VIP2, which contains a NAD binding pocket. Lethal Factor domain II lacks ADP-ribosylating activity due to the lack of conserved residues.^{[1] [15]}

Domain III residues 303-383, Sequence analysis had revealed the presence of a 101-residue segment comprising five tandem repeats residues 282-382, and suggested that repeats 2-5 arose from a duplication of repeat 1. The crystal structure reveals that repeat 1 actually forms the second helix-turn element of domain II, whereas repeats 2-5 form the four helix-turn elements of the helical bundle. Required for LF activity, shares same hydrophobic surface as domain IV and its location restricts access to the active site. Also, it contributes to substrate specificity by making interactions with the substrate.^[1][16]

Domain IV residues 552-776, consists of a nine-helix bundle packed against a four-stranded beta-sheet and contains a HExxH motif. The first six helices and the beta-sheet can be superposed with those of the metalloprotease. A zinc ion (Zn2+) is coordinated tetrahedrally by a water molecule and three protein side chains (Fig. 3), in an arrangement typical of the thermolysin family. Two coordinating residues are the histidines from the HExxH motif (His 686 and His 690) and Glu 735. ^[1][17]

Domains II, III, and IV for the binding pocket for the substrate.

Function of Lethal Factor

^[18]

^[19]

Biowarfare

1J7N

References