Lindsey E. McQuade

Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages

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.

Bacterial Nitric-oxide Synthases Operate without a Dedicated Redox Partner

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.

Fluorescent probes to investigate nitric oxide and other reactive nitrogen species in biology (truncated form: fluorescent probes of reactive nitrogen species)

Reactive nitrogen species (RNS), such as nitric oxide and its higher oxides, play important roles in cell signaling during many physiological and pathological events. Elucidation of the exact functions of these important biomolecules has been hampered by the inability to detect RNS reliably under biological conditions. A surge of research into RNS chemistry has resulted in the design of a new generation of fluorescent probes that are specific and sensitive for their respective RNS analytes. Progress in the field of nitric oxide, peroxynitrite, and nitroxyl sensing promises to advance our knowledge of important signaling events involving these species and lead to a better understanding of oxidative biochemistry crucial to health and disease.

Visualization of nitric oxide production in the mouse main olfactory bulb by a cell-trappable copper(II) fluorescent probe.

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 Cu2(FL2E) and the membrane-impermeable acid derivative Cu2(FL2A) respond rapidly and selectively to NO in buffers that simulate biological conditions, and application of Cu2(FL2E) leads to detection of endogenously produced NO in cell cultures and olfactory bulb brain slices.

Detecting and Understanding the Roles of Nitric Oxide in Biology

We are pursuing a dual strategy for investigating the chemistry of nitric oxide as a biological signaling agent. In one approach, metal-based fluorescent sensors for the detection of NO in living cells are evaluated, and a sensor based on a copper fluorescein complex has proved to be a valuable lead compound. Sensors of this class permit identification of NO from both inducible and constitutive forms of nitric oxide synthase and facilitate investigation of different NO functions in response to external stimuli. In the other approach, we employ synthetic model complexes of iron-sulfur clusters to probe their reactivity toward nitric oxide as biomimics of the active sites of iron-sulfur proteins. Our studies reveal that NO disassembles the Fe-S clusters to form dinitrosyl iron complexes.

Fluorescence-Based Nitric Oxide Sensing by Cu(II) Complexes That Can Be Trapped in Living Cells

A series of symmetrical, fluorescein-derived ligands appended with two derivatized 2-methyl-8-aminoquinolines were prepared and spectroscopically characterized. The ligands FL2, FL2E, and FL2A were designed to improve the dynamic range of previously described asymmetric systems, and the copper complex Cu(2)(FL2E) was constructed as a trappable NO probe that is hydrolyzed intracellularly to form Cu(2)(FL2A). The ligands themselves are only weakly emissive, and the completely quenched Cu(II) complexes, generated in situ by combining each ligand with 2 equiv of CuCl(2), were investigated as fluorescent probes for nitric oxide. Upon introduction of excess NO under anaerobic conditions to buffered solutions of Cu(2)(FL2), Cu(2)(FL2E), and Cu(2)(FL2A), the fluorescence increased by factors of 23 +/- 3, 17 +/- 2, and 27 +/- 3, respectively. The corresponding rate constants for fluorescence turn-on were determined to be 0.4 +/- 0.2, 0.35 +/- 0.05, and 0.6 +/- 0.1 min(-1). The probes are highly specific for NO over other biologically relevant reactive oxygen and nitrogen species, as well as Zn(II), the metal ion for which similar probes were designed to detect.

Seminaphthofluorescein-Based Fluorescent Probes for Imaging Nitric Oxide in Live Cells

Fluorescent turn-on probes for nitric oxide based on seminaphthofluorescein scaffolds were prepared and spectroscopically characterized. The Cu(II) complexes of these fluorescent probes react with NO under anaerobic conditions to yield a 20-45-fold increase in integrated emission. The seminaphthofluorescein-based probes emit at longer wavelengths than the parent FL1 and FL2 fluorescein-based generations of NO probes, maintaining emission maxima between 550 and 625 nm. The emission profiles depend on the excitation wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for NO over other biologically relevant reactive nitrogen and oxygen species including NO(3)(-), NO(2)(-), HNO, ONOO(-), NO(2), OCl(-), and H(2)O(2). The seminaphthofluorescein-based probes can be used to visualize endogenously produced NO in live cells, as demonstrated using Raw 264.7 macrophages.

Mechanism of nitric oxide reactivity and fluorescence enhancement of the NO-specific probe CuFL1

The mechanism of the reaction of CuFL1 (FL1 = 2-{2-chloro-6-hydroxy-5-[(2-methylquinolin-8-ylamino)methyl]-3-oxo-3H-xanthen-9-yl}benzoic acid) with nitric oxide (NO) to form the N-nitrosated product FL1-NO in buffered aqueous solutions was investigated. The reaction is first-order in [CuFL1], [NO], and [OH(-)]. The observed rate saturation at high base concentrations is consistent with a mechanism in which the protonation state of the secondary amine of the ligand is important for reactivity. This information provides a rationale for designing faster-reacting probes by lowering the pK(a) of the secondary amine. Activation parameters for the reaction of CuFL1 with NO indicate an associative mechanism (DeltaS(double dagger) = -120 +/- 10 J/mol.K) with a modest thermal barrier (DeltaH(double dagger) = 41 +/- 2 kJ/mol; E(a) = 43 +/- 2 kJ/mol). Variable-pH electron paramagnetic resonance experiments reveal that, as the secondary amine of CuFL1 is deprotonated, electron density shifts to yield a new spin-active species having electron density localized on the deprotonated amine nitrogen atom. This result suggests that FL1-NO formation occurs when NO attacks the deprotonated secondary amine of the coordinated ligand, followed by inner-sphere electron transfer to Cu(II) to form Cu(I) and release of FL1-NO from the metal.

Specific Visualization of Nitric Oxide in the Vasculature with Two-Photon Microscopy Using a Copper Based Fluorescent Probe

To study the role and (sub) cellular nitric oxide (NO) constitution in various disease processes, its direct and specific detection in living cells and tissues is a major requirement. Several methods are available to measure the oxidation products of NO, but the detection of NO itself has proved challenging. We visualized NO production using a NO-sensitive copper-based fluorescent probe (Cu 2FL2E) and two-photon laser scanning microscopy (TPLSM). Cu 2FL2E demonstrated high sensitivity and specificity for NO synthesis, combined with low cytotoxicity. Furthermore, Cu 2FL2E showed superior sensitivity over the conventionally used Griess assay. NO specificity of Cu 2FL2E was confirmed in vitro in human coronary arterial endothelial cells and porcine aortic endothelial cells using various triggers for NO production. Using TPLSM on ex vivo mounted murine carotid artery and aorta, the applicability of the probe to image NO production in both endothelial cells and smooth muscle cells was shown. NO-production and time course was detected for multiple stimuli such as flow, acetylcholine and hydrogen peroxide and its correlation with vasodilation was demonstrated. NO-specific fluorescence and vasodilation was abrogated in the presence of NO-synthesis blocker L-NAME. Finally, the influence of carotid precontraction and vasorelaxation validated the functional properties of vessels. Specific visualization of NO production in vessels with Cu 2FL2E-TPLSM provides a valid method for studying spatial-temporal synthesis of NO in vascular biology at an unprecedented level. This approach enables investigation of the pathways involved in the complex interplay between NO and vascular (dys) function.