The Bacillus anthracis chromosome encodes four unique, putative superoxide dismutase (sod) genes. During exponential growth and sporulation, sodA1, sodA2, and sodC are transcribed constitutively throughout the growth cycle as individual genes. In contrast, the transcription of sod15 occurs mainly during late exponential and sporulation phases as part of a four-gene operon that may be involved in spore formation. Vegetative cell and spore lysates of wild-type Sterne and superoxide dismutase deletion (⌬sod) mutants show detectable SOD activity for SODA1 and SODA2, and protein analysis suggests that these two proteins form active homodimers and heterodimers. A comparison of the growth of parental versus ⌬sod mutants under various chemical oxidative stresses indicates that ⌬sodA1 mutants are particularly sensitive to endogenously produced superoxide, whereas ⌬sodA2, ⌬sod15, and ⌬sodC mutants remain as resistant to this stress as the parental strain. In addition, in mouse survival assays, ⌬sod15 and ⌬sodA1 were responsible for less end-point death, but the level of decreased virulence does not fall within a statistically significant range. Collectively, these data show that sodA1 acts as a major protectant from intracellular superoxide stress, that sod15 is transcribed as part of an operon that may play a role in cell morphology, and that sodA2 and sodC may have minor roles that are not apparent in the conditions tested here.

In the environment, the gram-positive bacterium Bacillus anthracis persists as a metabolically dormant endospore. Upon inoculation into the host the endospores germinate and outgrow into vegetative bacilli able to cause disease. The dramatic morphogenic changes to the bacterium during germination and outgrowth are numerous and include major rearrangement of and modifications to the bacterial surface. Such modifications occur during a time in the B. anthracis infectious cycle when the bacterium must guard against a multitude of innate immune mediators. The dltABCD locus of B. anthracis encodes a cell wall D-alanine esterification system that is initiated by transcriptional activation during endospore outgrowth. The level of transcription from the dltABCD operon determined B. anthracis resistance to cationic antibacterial peptides during vegetative growth and cationic peptide, enzymatic, and cellular mediators of innate immunity during outgrowth. Mutation of dltABCD was also attenuating in a mouse model of infection. We propose that the dltABCD locus is important for protection of endosporeforming bacteria from environmental assault during outgrowth and that such protection may be critical during the establishment phase of anthrax.

Although traditionally considered to be an extracellular pathogen, Bacillus anthracis has a brief intracellular step to initiate anthrax. At the onset of infection, B. anthracis must withstand the bactericidal activities of the macrophage. Recently, three phospholipases C (PLCs) were shown to contribute to macrophage-associated growth of B. anthracis by presumably aiding in the escape of the bacterium from phagocytic vacuoles following phagocytosis. However, in the absence of all three PLCs, vegetative bacilli were still observed growing in association with the macrophage, albeit to a lesser extent, implicating that additional factors are involved in this process. In this study, the contributions of the previously identified cholesterol-dependent cytolysin anthrolysin O (ALO) to B. anthracis pathogenesis were investigated following challenges of bone marrow-derived macrophages and intratracheal inoculations of mice. Disruption of ALO alone yielded no differences in virulence in mice. However, combinatorial deletions of ALO with the three PLCs resulted in attenuation in both tissue culture and murine challenges, suggesting that these toxins may have overlapping roles in anthrax pathogenesis.

Several models of anthrax pathogenesis suggest that early in the infectious process Bacillus anthracis endospores germinate and outgrow into vegetative bacilli within phagocytes before being released into the blood. Here, we define the respective contributions of three phospholipases C (PLCs) to the pathogenesis of B. anthracis. Genetic deletions of the PLCs were made in the Sterne 7702 background, resulting in the respective loss of their activities. The PLCs were redundant both in tissue culture and in murine models of anthrax. Deletion of all three PLC genes was required for attenuation of virulence in mice after intratracheal inoculation. This attenuation may be attributed to the inability of the PLC-null strain to grow in association with the macrophage. Complementation of these defects in both models of anthrax was achieved by expression of the PLC genes in trans. The functional redundancy between PLCs in the virulence of B. anthracis implies that their activities are important for anthrax pathogenesis.

C57BL/6 mice develop an allergic bronchopulmonary mycosis following intratracheal inoculation ofCryptococcus neoformans 24067. We determined that only low levels of tumor necrosis factor alpha (TNF-␣) are produced in the lungs following infection. Thus, the objective of the present studies was to determine whether treatment with a TNF-␣-expressing adenoviral vector (adenoviral vector with the murine TNF-␣ transgene under the control of the human cytomegalovirus promoter [AdTNF␣]) could switch the type 2 (T2) T-cell response/T1 T-cell response balance toward the T1 T-cell response. AdTNF␣ induced an increase in TNF-␣ expression at days 3 and 7. At days 7 to 14, the number of cryptococcal lung CFU continued to increase in both untreated and control adenoviral vector (empty adenovirus type 5 backbone)-treated mice, but the number was ultimately 100-fold lower following AdTNF␣ treatment. AdTNF␣ markedly increased neutrophil and macrophage numbers, and pulmonary eosinophilia did not develop. CXCL1, CXCL2, and gamma interferon were also up-regulated, while eotaxin, interleukin-4 (IL-4), and IL-5 were down-regulated. AdTNF␣ treatment also increased the number of CD80؉ and CD40 ؉ cells and decreased the number of CD86 ؉ cells (CD11b ؉ and CD11c ؉ ) in the lungs. Major histocompatibility complex class II levels on CD11b ؉ cells were increased. Whole-lung expression of inducible nitric oxide synthase was increased, while YM2 expression and acidic mammalian chitinase expression were decreased. None of these effects were observed with the control (empty) adenoviral vector. Overall, these results support the hypothesis that early TNF-␣ expression promotes a shift in T-cell and macrophage polarization from T2/alternatively activated macrophages toward T1/classically activated macrophages, resulting in control of the fungal infection and prevention of the allergic response.