The effects of antioxidants on the interaction of Bacillus anthracis with phagocytes

Mariana Bernui

doi:10.17918/00000476

Bacillus anthracis (BA) is a gram-positive bacterium that causes the disease Anthrax. Pulmonary anthrax in humans is initiated by introduction of B. anthracis (BA) spores into the body through the respiratory epithelium. Once inhaled, anthrax spores reach the bronchioles and alveoli of the lung, and most are then rapidly and efficiently phagocytosed by alveolar macrophages. Infected macrophages then transport germinating spores to the regional lymph nodes from where they will finally invade the blood stream eventually reaching >10⁸/ml and killing the host. BA achieves this remarkable lethality by producing several potent exotoxins, the most recently discovered being Anthrolysin O (rALO). Therefore, the germination of spores and the emergence of bacilli, in an environment allowing rapid out-growth in the body, are essential to during the early stages of disease. Once the bacilli become extracellular and a systemic infection has been successfully established, the secreted exotoxins will lead to death of the host. Finding a therapeutic agent that: (i) alters the spores' ability to germinate, (ii) optimizes phagocytes to kill BA, and (iii) hinders the effect of BA's virulence factors upon the host's immunological machinery, could potentially prevent anthrax. Our studies focus on N-acetylcysteine (NAC), an antioxidant which our results suggest possesses these three properties. Specific amino acids can activate or inhibit BA germination in vitro. Therefore, we hypothesize that specific amino acid and nucleoside derivatives can control germination both in vitro and in vivo . Several combination of amino acids act as potent germinants in vitro. The amino acid derivative we have focused on is NAC. NAC is a thiol and a precursor of intracellular L-cysteine and reduced glutathione in mammalian cells. It is a source of sulfhydryl groups in cells and scavenger of free radicals as it interacts with ROS such as OH· and H₂O₂. Herein we show that NAC, as well as other thiol-based reducing agents, completely but reversibly inhibit BA spore germination without compromising spore viability. This suggests a reversible redox event on the spore surface. Oxidized NAC does not inhibit BA spore germination, confirming that NAC reduces components of the BA germination pathway. Use of different amino acid germinant combinations showed that germination receptors gerK and gerL are specifically targeted. Thus, in the absence of macrophages, 5 mM NAC reduced germination of BA spores >95% in a 90 min study. Importantly, NAC was not toxic to spores or vegetative bacilli at the times or concentrations used in our studies. In vitro, there is contradicting results about how successful macrophages are at clearing a BA infection. Most agree spore uptake is very inefficient and that there is extensive adherence of spores and vegetative cells to the outside of the macrophages. Four to 6 hours post-infection, there is extensive germination and vegetative cell outgrowth with long chains of bacteria extending across several macrophages. The degree of BA clearance varies depending on the type of macrophage cell line and the methodology used in each study. In our model we deliver antioxidants in liposomal form in addition to antioxidants in solution. The patented product we utilize is called STIMAL. STIMAL is the only product known to prevent death by mustard gas analogs in various animal models. In studies presented here, we use (i) human monocyte-derived macrophages (hMDMs), (ii) the RAW 264.7 murine macrophage cell line, or (iii) the differentiated human THP-1 cell line, infected with BA Sterne strain 7702 spores, to study the effect of NAC and STIMAL on the interaction between BA and macrophages. In our first set of studies, we incubated various macrophages or macrophage cell lines with or without 5 mM NAC, washed NAC out, and subsequently infected them with BA spores. At 2 hrs post-infection, untreated and treated macrophages have similar numbers of cell-associated spores. However, starting at 4 hrs, spores germinate 2 hrs earlier in NAC treated hMDMs, compared to untreated cells, and the germinated BA are rapidly killed. In addition, and logically following the intracellular killing of germinated spores, there is a >3-fold decrease in the number of extracellular vegetative bacteria 6 hrs post-infection in NAC treated hMDMs, compared to untreated controls. A similar phenomenon occurs with RAW 264.7 or differentiated THP-1 macrophages. Via in vitro viability assays and light microscopy, a significantly greater percentage of NAC pretreated macrophages are viable at the end of assays (8 hours) compared to untreated macrophages. Thus, macrophages pre-treated with 5 mM NAC kill germinating BA spores more effectively than do untreated macrophages, strictly limit total BA growth, and remain viable. (Abstract shortened by UMI.).

The effects of antioxidants on the interaction of Bacillus anthracis with phagocytes

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The effects of antioxidants on the interaction of Bacillus anthracis with phagocytes

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