SummaryPolymorphonuclear leucocytes (PMNs) play a protective role during Bacillus anthracis infection. However, B. anthracis is able to subvert the PMN response effectively as evidenced by the high mortality rates of anthrax. One major virulence factor produced by B. anthracis, lethal toxin (LT), is necessary for dissemination in the BSL2 model of mouse infection. While human and mouse PMNs kill vegetative B. anthracis, short in vitro half-lives of PMNs have made it difficult to determine how or if LT alters their bactericidal function. Additionally, the role of LT intoxication on PMN's ability to migrate to inflammatory signals remains controversial. LF concentrations in both serum and major organs were determined from mice infected with B. anthracis Sterne strain at defined stages of infection to guide subsequent administration of purified toxin. Bactericidal activity of PMNs assessed using ex vivo cell culture assays showed significant defects in killing B. anthracis. In vivo PMN recruitment to inflammatory stimuli was significantly impaired at 24 h as assessed by realtime analysis of light-producing PMNs within the mouse. The observations described above suggest that LT serves dual functions; it both attenuates accumulation of PMNs at sites of inflammation and impairs PMNs bactericidal activity against vegetative B. anthracis.

Bacillus anthracis can cause inhalational anthrax. Murine inhalational B. anthracis infections have two portals of entry, the nasal mucosa-associated lymphoid tissue (NALT) and the lumen of the lungs. Analysis of the dissemination from these sites is hindered because infections are asynchronous and asymptomatic until the hosts near death. To further understand and compare how B. anthracis disseminates from these two different environments, clonal analysis was employed using a library of equally virulent DNA-tagged clones of a luminescent Sterne strain. Luminescence was used to determine the origin of the infection and monitor the dissemination in vivo. The number of clones and their proportions in the portals of entry, lymph nodes draining the portals, and kidneys were analyzed. Clonal analysis indicated a bottleneck for both portals of entry, yet the extent and location of the reduction in represented clones differed between the routes. In NALT-based infections, all clones were found to germinate in the NALT, but they underwent a bottleneck as the infection spread to the cervical lymph node. However, lung-based infections underwent a bottleneck in a focal region of growth within the lung lumen and did not need to spread through the mediastinal lymph nodes to cause a systemic infection. Further, the average number of clones found in the kidney and the rate at which genetic drift was affecting the disseminated populations were significantly higher in lung-based infections. Collectively, the data suggested that differences in the host environment alter dissemination of B. anthracis depending on the site of initial colonization and growth.