Bacillus anthracis (B. anthracis) is a major bioterrorism threat in the United States (US) today. In the 2001 bioterrorism attack, the mortality rate in all patients who required vasopressor support was 100%. This contrasts with more commonly encountered types of bacterial sepsis in which mortality rates in patients requiring similar treatment is typically close to 60%. Although production of both lethal and edema toxins (LeTx and ETx respectively) contribute to the shock associated with B. anthracis, administration of these toxins alone do not result in the full spectrum of tissue injury noted with live bacterial infection. This suggests that other virulence factors contribute to shock with this organism. The cell wall of B. anthracis is one such candidate. ? ? The cell wall of gram positive bacteria is composed of several structural macromolecules, including peptoglycans (PG) and teichoic acids (TA). The peptidoglycan layer is a crystal lattice structure formed from linear chains of two alternating amino sugars, N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc). This layer is substantially thicker in Gram-positive bacteria (20 to 80 nm) than in Gram-negative bacteria (7 to 8 nm) and comprises around 90% of the dry weight of the former bacteria but only 10% of the latter. Teichoic acids are polymers of glycerol or ribitol linked via phosphodiester bonds. They are not found in gram-negative bacteria. They can be either covalently bonded to N-acetylmuramic acid of the peptidoglycan layer or linked to the plasma membrane lipids found in the cytoplasmic membrane forming lipoteichoic acids (LTA). These components serve a structural role in the bacterial cell wall, giving the wall shape and structural strength, as well as counteracting the osmotic pressure of the cytoplasm. In addition to their structural functions however, these molecules, just like lipopolysaccharide (LPS) from gram-negative bacteria, can also serve as powerful stimulants of the innate immune system. Interaction of peptidoglycan with PG recognition proteins (PGRPs), toll-like receptor 2 (TLR-2), nucleotide-binding oligomerization domain (NOD) proteins (NOD1, NOD2 or cryopyrin) on the surface of some gram-positive bacteria results in the activation of Caspase-1, NF-kB and MAP kinases and the subsequent expression and release of a variety of pro-inflammatory cytokines and chemokines. Lipotechoic acid from some bacteria can also interact with CD14, CD36, TLR-2 and possibly LBP to induce cytokine and chemokine release. Activation of the inflammatory response by these cell wall components can in turn produce the hemodynamic instability and tissue injury associated with sepsis and septic shock. ? ? The specific structure and immunostimulatory effects of cell wall peptidoglycan and lipotechoic acid varies among gram-positive bacteria. The cell wall of some, such as highly virulent S. aureus, is a strong inflammatory stimulant and is highly toxic when administered in vivo. The cell wall from other bacteria types however, like nonvirulent B. subtilis, induces little inflammatory response. Data now available from in vitro studies indicates that B. anthracis cell wall has considerable pro-inflammatory effects. In a series of experiments, Popov et al. has shown that B. anthracis cell wall strongly stimulates peripheral blood monocyte production of the inflammatory cytokines TNF??, IL-1?O, and IL-6 (17). It remains to be tested however, whether these stimulatory effects result in observable injury in vivo. It is also important to note that lethal infection with B. anthracis both in animals and man is associated with bacterial loads that are much greater than is typically observed with virulent bacteria. Thus, the amount of cell wall that may contribute to injury with B. anthracis, may be much greater than with other bacteria types.? ? In a series of experiments and studies, we have defined the effects of highly purified forms of LeTx and ETx, both alone and together on cardiopulmonary function, inflammatory cytokine and chemokine production, nitric oxide release and histological changes in rats. In the model we developed, toxin is infused over 24 h to better simulate the pattern that occurs during live bacterial infection. ? ? The present study is employing the rat model to study the effects of B. anthracis cell wall. In experiments to date we have noted that although infusion of cell wall is associated with dose dependent reductions in circulating white blood cells and platelet, consistent with an inflammatory response, blood pressure is not decreased and lethality, despite using fairly high cell wall doses, is minimal. Administration of cell wall as a bolus creates similar effects on circulating cells and platelets, but does produce some hemodynamic instability and more lethality. Overall however these studies support the possibility that B. anthracis cell wall must be present in very amounts to contribute to shock and lethality during B. anthracis infections. Abstracts are being prepared describing early work in this protocol and other studies are ongoing.
