The Effects of B. anthracis Cell Wall in a Small Animal Model

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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. <br><br>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 Q, IL-1 R, 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.<br><br>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. <br><br>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 infusion of cell wall is associated with dose dependent reductions in circulating white blood cells and platelet and increases in cytokine and nitric oxide levels, consistent with an inflammatory response. These changes are associated with abnormalities of oxygenation as reflected by increased alveolar to arterial oxygen gradients (AaO2). These increases in AaO2 are greater with lethal compared to nonlethal doses of anthrax cell wall and are very similar to ones produced by cell wall from a pathogenic strain of S. aureus. Based on these findings we hypothesized that anthrax cell wall would worsen outcome with LeTx infusion. However, in a highly unexpected pattern, administration of nonlethal doses of anthrax cell wall actually protected animals from lethal doses of LeTx. Further studies are underway to undertstand the basis for this effect.