Phagocytes generate nitric oxide (NO) and other reactive oxygen and nitrogen species in large quantities to combat infecting bacteria. Here, we report the surprising observation that in vivo survival of a notorious pathogen-Bacillus anthracis-critically depends on its own NO-synthase (bNOS) activity. Anthrax spores (Sterne strain) deficient in bNOS lose their virulence in an A/J mouse model of systemic infection and exhibit severely compromised survival when germinating within macrophages. The mechanism underlying bNOS-dependent resistance to macrophage killing relies on NO-mediated activation of bacterial catalase and suppression of the damaging Fenton reaction. Our results demonstrate that pathogenic bacteria use their own NO as a key defense against the immune oxidative burst, thereby establishing bNOS as an essential virulence factor. Thus, bNOS represents an attractive antimicrobial target for treatment of anthrax and other infectious diseases.anthrax ͉ bacterial NO-synthase ͉ oxidative stress T he spore-producing Gram-positive soil organism, Bacillus anthracis, is the causative agent of anthrax, an acute lifethreatening infection in humans and domestic animals. Inhalation of B. anthracis spores results in a high rate of mortality, because effective treatment must be provided within a very short time after exposure (1). Deliberate dispersal of anthrax spores through the United States Postal Service in 2001 emphasized the importance of developing effective treatments to combat this potential biological scourge.Although the innate immune response is the first line of defense against B. anthracis, its spores survive, germinate, and proliferate in macrophages, eventually bursting them to produce a lethal titer of infectious particles. The mechanism by which B. anthracis evades immune attack is not fully understood. Most studies have been focused on major virulence factors found on two plasmids (pXO1 and pXO2) that are responsible for exotoxins, capsule formation, and spore germination (2). These plasmid-borne virulence factors have been the prime candidates for anti-anthrax drug design. However, the ability of pathogens such as B. anthracis to survive in phagocytes also depends critically on the state of their oxidative stress defense system. Reactive oxygen species (ROS) play essential roles in innate immunity against many types of microorganisms (3-5). The antibacterial effects of ROS have been largely attributed to DNA and protein damage mediated by the Fenton reaction (6). This process generates hydroxyl radicals that react with DNA bases, sugar moieties, and amino acid side chains, causing various types of lesions (7). We showed that nonpathogenic B. subtilis utilizes its own nitric oxide (NO) to gain rapid protection against sudden oxidative damage (8). The mechanism of protection does not rely on transcriptional gene induction but rather on rapid suppression of DNA damage by preventing the Fenton reaction and direct activation of catalase (8). Here, we describe the key role of NO-synthase (bNOS)-derived ...

Bacterial nitric-oxide (NO) synthases (bNOSs) are smaller than their mammalian counterparts. They lack an essential reductase domain that supplies electrons during NO biosynthesis. This and other structural peculiarities have raised doubts about whether bNOSs were capable of producing NO in vivo. Here we demonstrate that bNOS enzymes from Bacillus subtilis and Bacillus anthracis do indeed produce NO in living cells and accomplish this task by hijacking available cellular redox partners that are not normally committed to NO production. These "promiscuous" bacterial reductases also support NO synthesis by the oxygenase domain of mammalian NOS expressed in Escherichia coli. Our results suggest that bNOS is an early precursor of eukaryotic NOS and that it acquired its dedicated reductase domain later in evolution. This work also suggests that alternatively spliced forms of mammalian NOSs lacking their reductase domains could still be functional in vivo. On a practical side, bNOS-containing probiotic bacteria offer a unique advantage over conventional chemical NO donors in generating continuous, readily controllable physiological levels of NO, suggesting a possibility of utilizing such live NO donors for research and clinical needs.

Sequential gene silencing in zebrafish embryos has been achieved using caged morpholino oligonucleotides with spectrally distinct triggers. Using these optochemical tools, the genetic interactions that dynamically regulate mesoderm patterning have been examined.

We report the visualization of NO production using fluorescence in tissue slices of the mouse main olfactory bulb. This discovery was possible through the use of a novel, cell-trappable probe for intracellular nitric oxide detection based on a symmetric scaffold with two NO-reactive sites. Ester moieties installed onto the fluorescent probe are cleaved by intracellular esterases to yield the corresponding negatively charged, cell-impermeable acids. The trappable probe Cu 2 ðFL2EÞ and the membrane-impermeable acid derivative Cu 2 ðFL2AÞ respond rapidly and selectively to NO in buffers that simulate biological conditions, and application of Cu 2 ðFL2EÞ leads to detection of endogenously produced NO in cell cultures and olfactory bulb brain slices.fluorescent sensing | NO | olfaction | trappable probe | fluorescence microscopy N itric oxide (NO) is important for biological signaling. It activates soluble guanylyl cyclase, initiating a signaling cascade that promotes vascular smooth muscle dilation (1-3). Nitric oxide produced in the nervous system has been implicated in neurotransmission (4), and the immune system generates NO as a defense against pathogens (5). Unregulated nitric oxide production has been associated with pathological conditions such as cancer, ischemia, septic shock, inflammation, and neurodegeneration (6).Because of its various biological consequences, investigating the generation, translocation, and utilization of NO continues to be an active area of research. A major limitation to advances in the field, however, has been the dearth of selective tools for visualization of biological NO, for example by fluorescence microscopy. Detection of nitric oxide offers many challenges. NO reacts rapidly in vivo with dioxygen, oxygen-generated radicals such as superoxide, amines, thiols, and metal centers (7,8). It also diffuses readily from its point of origin (9), making rapid detection desirable for uncovering both its production site and function. Moreover, NO is produced at concentrations as low as ∼100 picomolar, so it is essential to have an NO probe with a low detection limit (10). Fluorescent sensors can be designed to accommodate the properties of NO under physiological conditions, making this technique particularly valuable for in vivo nitric oxide imaging.Transition metal complexes have been investigated as platforms for NO detection (11). The strategy is to incorporate a fluorophore into a ligand that is quenched either by intracellular photoinduced electron transfer and/or by coordination to a paramagnetic or heavy metal ion. Fluorescence is restored by reduction of the metal and/or displacement of the ligand upon reaction of the probe with NO. CuFL1 (Fig. 1) is an excellent example of such a metal-based cellular NO imaging agent (12, 13). CuFL1 satisfies many of the requirements of a good sensor. It is nontoxic, cell membrane permeable, has low energy excitation and emission wavelengths, responds directly and selectively to NO, and exhibits dramatic fluorescence enhancement upon reaction with...

Forebrain dopamine plays a critical role in motivated behavior. According to the classic view, mesolimbic dopamine selectively guides behavior motivated by positive reinforcers. However, this has been challenged in favor of a wider role encompassing aversively motivated behavior. This controversy is particularly striking in the case of nicotine, with opposing claims that either the rewarding or the aversive effect of nicotine is critically dependent on mesolimbic dopamine transmission. In the present study, the effects of 6-hydroxydopamine lesions of nucleus accumbens core vs. medial shell on intravenous nicotine conditioned place preference and conditioned taste aversion were examined in male adult rats. Dopaminergic denervation in accumbens medial shell was associated with decreased nicotine conditioned place preference. Conversely, denervation in accumbens core was associated with an increase in conditioned place preference. In addition, dopaminergic denervation of accumbens core but not medial shell abolished conditioned taste aversion for nicotine. We conclude that nucleus accumbens core and medial shell dopaminergic innervation exert segregated effects on rewarding and aversive effects of nicotine. More generally, our findings indicate that dopaminergic transmission may mediate or enable opposing motivational processes within functionally distinct domains of the accumbens.