Anthrax Toxin Mechanism of Action

One of the key causes of anthrax virulence is the production of three specific factors by
the gram-positive spore forming bacteria Bacill

us anthracis. Even with successful antibiotic treatment, anthrax toxins can remain in the
circulation and cause lethality. The toxins produced by anthrax bacteria are derived from
three genes: lethal factor (LF), protective antigen (PA) and edema factor (EF). The entry
of toxin into cells begins with the recognition of a recently identified cellular receptor in
the plasma membrane by PA. Proteolytic cleavage of cell-bound PA creates a smaller
fragment that then multimerizes into a pore-like structure in the plasma membrane. The
LF and EF proteins bind to the PA pre-pore, followed by internalization of the entire
structure through receptor-mediated endocytosis. In the endosomal compartment, the
acidic pH causes a conformational change that inserts PA fragments and releases LF and
EF into the cytoplasm. In the cytoplasm, LF acts as a protease that cleaves MAP kinase
kinase (MAPKK 1 and MAPKK 2), inhibiting pathways that rely on this kinase family and

causing cell death. Edema factor is an adenylate cyclase that inhibits the immune
response, including phagocytosis by macrophages. Several potential mechanisms could
be used to block anthrax toxin action, one of which was demonstrated by the design of a
multivalent protein inhibitor of toxin interaction with PA.

De novo synthesis of pyrimidine nucleotides

The pyrimidine ribonucleotides, those found in RNA, are uracil and cytosine, each
containing the sugar ribose and a nitrogenous base. Pyrimidine nucleotides contain a
single ring in the nitrogenous base. In contrast to the synthesis of purines, the
pyrimidine ring is synthesized first and then joined to ribose. This pathway is conserved
from bacteria to man, occurring in essentially the same manner for all species. The
pathway begins with aspartate and carbamoyl phosphate as the first components to
build the pyrimidine base. Carbamoyl phosphate is synthesized from bicarbonate ions
and a nitrogen from glutamine. The condensation of aspartate and carbamoyl phosphate
creates carbamoyl aspartate, which goes through ring closure and oxidation to create the
precursor pyrimidine orotate. Orotate is joined to ribose phosphate and UMP is finally
formed through decarboxylation. CTP is synthesized from UTP through amination using
glutamine as the donor in mammals and ammonia in E.coli. This pathway is closely
regulated to provide pyrimidines only when they are needed. The pathway is activated
by ATP and inhibited by the end product, pyrimidines, providing an example of feedback
inhibition. The deoxyribonucleotides found in DNA are synthesized through reduction of

the ribose ring in ribonucleotides.

Although the bacterial and human pathways are similar, the enzymes that catalyze the
pathway reactions are quite distinct. In bacteria, the first three steps in the pathway are
catalyzed by three separate polypeptides, but in humans a single polypeptide has all
three enzymatic activities. Multifunctional enzyme complexes of this nature occur in
other pathways as well, and increase the overall efficiency of a pathway by directing
pathway intermediates directly from one step to the next rather than relying on diffusion
between enzymes in the pathway.

Biosynthesis of Arginine in Bacteria

Ceramide Signaling Pathway

Over 1,000 papers and reviews have been written about the role of ceramide in the
production of programmed cell death or apoptosis. Ceramide is a sphingosine-based
lipid-signaling molecule involved in the regulation of cellular differentiation, proliferation,
and apoptosis. This diagram represents some of the current understanding of the
cascades that couple ceramide to specific signaling pathways. These cascades illustrate
that ceramide can be a growth stimulus or proapototic signal. The ultimate ceramide
action is determined within the context of other stimuli and by the subcellular topology
of its production and is cell-type specific.
There are 2 forms of sphingomyelinase, acid (acid-sphingomyelinase:A-SMase) and
neutral (neutral-sphingomyelinase N-SMase), that can produce ceramide. TNF-alpha can
stimulate either form of sphingomyelinase as can other death receptors. Different
domanis of TNF-alpha stimulate the different Smases. N-SMase stimulation is enhanced
by the receptor for activated-C kinase 1 (RACK1). The activity of each form is dependent
on the local intracellular pH. In the illustration the forms are seperated to reduce
confusion however ceramide produced by either method can stimulate either cascade
depending on the presence of specific co-factors and activators.
A-SMase has been recognized as one of the required molecules to mediate proapoptotic
signalling in cell death induced by a diverse array of stresses such as H2O2, Heat, UV
exposure and Radiation. ROS generation in mitochondria activates caspase-3 via
cooperation of cytochrome c, Aif and caspase-9 and stimulates or increases ceramide
generation through A-SMase in a proaptotic activation cycle. Caspase-3 further increases

its own activation by proteolytically cleaving ceramide inhibited catalase which is an

inhibitor of ROS generation.
Ceramide-activated protein kinase(CARK) also known as Kinase Supressor of RAS (KSR)
activity is in some cases the switch point in the balance between proapoptotic and
antiapoptotic signals and is also cell-type specific. In endothelial cells for example the
activation of KSR is required for apoptosis. In contrast in epithelial cells activation of KSR
is required for cell proliferation. An additional switch point is the availability of Bad in the
cell. Activation of KSR leads to further mitocondrial stimulation or association with RAS
and activation of the Raf1 cascade leading to proliferation or differentiation.