Reductive activation and thiol reactivity of benzazolo[3,2-a]quinolinium salts

Derivatives of benzazolo[3,2-a]quinolium salts (QSDs) are reductively activated by the enzymatic reducing agents hypoxanthine (or xanthine)/xanthine oxidase and NADH dehydrogenase as evidenced by the increase in rates of ferricytochrome c (Cyt(III)c) reduction and oxygen consumption, respectively. No correlation between Michaelis-Menten parameters and QSDs redox potentials was found regarding anaerobic or aerobic Cyt(III)c reduction, although maximum rates were observed for nitro-containing QSDs. However, oxygen consumption rates correlate with QSDs redox potentials when NADH dehydrogenase is used as reducing agent. QSDs bind covalently to bovine serum albumin (BSA) under anaerobic conditions, in the presence, and less in the absence, of HX/XO and only if the nitro group is present at the QSD. QSDs react with glutathione (GSH) in the presence of HX/XO but not in its absence, under anaerobic conditions. The amount of reacted GSH increases, and the relative amount of GSSG formed decreases, with an increase in the QSD reduction potential, thus indicating that GSH reacts with reduced nitro-containing QSDs mainly in a manner which does not involve the production of GSSG, presumably, through the formation of the nitroso-QSD-GSH conjugate. QSDs are, thus, novel nitro-containing heterocyclic compounds which could be bioreductively activated to react with oxygen and thiols.     

Nitric oxide and redox mechanisms in the immune response

The role of redox molecules, such as NO and ROS, as key mediators of immunity has recently garnered renewed interest and appreciation. To regulate immune responses, these species trigger the eradication of pathogens on the one hand and modulate immunosuppression during tissue-restoration and wound-healing processes on the other. In the acidic environment of the phagosome, a variety of RNS and ROS is produced, thereby providing a cauldron of redox chemistry, which is the first line in fighting infection. Interestingly, fluctuations in the levels of these same reactive intermediates orchestrate other phases of the immune response. NO activates specific signal transduction pathways in tumor cells, endothelial cells, and monocytes in a concentration-dependent manner. As ROS can react directly with NO-forming RNS, NO bioavailability and therefore, NO response(s) are changed. The NO/ROS balance is also important during Th1 to Th2 transition. In this review, we discuss the chemistry of NO and ROS in the context of antipathogen activity and immune regulation and also discuss similarities and differences between murine and human production of these intermediates.     

Type IV pili interactions promote intercellular association and moderate swarming of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous bacterium that survives in many environments, including as an acute and chronic pathogen in humans. Substantial evidence shows that P. aeruginosa behavior is affected by its motility, and appendages known as flagella and type IV pili (TFP) are known to confer such motility. The role these appendages play when not facilitating motility or attachment, however, is unclear. Here we discern a passive intercellular role of TFP during flagellar-mediated swarming of P. aeruginosa that does not require TFP extension or retraction. We studied swarming at the cellular level using a combination of laboratory experiments and computational simulations to explain the resultant patterns of cells imaged from in vitro swarms. Namely, we used a computational model to simulate swarming and to probe for individual cell behavior that cannot currently be otherwise measured. Our simulations showed that TFP of swarming P. aeruginosa should be distributed all over the cell and that TFP−TFP interactions between cells should be a dominant mechanism that promotes cell−cell interaction, limits lone cell movement, and slows swarm expansion. This predicted physical mechanism involving TFP was confirmed in vitro using pairwise mixtures of strains with and without TFP where cells without TFP separate from cells with TFP. While TFP slow swarm expansion, we show in vitro that TFP help alter collective motion to avoid toxic compounds such as the antibiotic carbenicillin. Thus, TFP physically affect P. aeruginosa swarming by actively promoting cell−cell association and directional collective motion within motile groups to aid their survival.The bacterium Pseudomonas aeruginosa is a ubiquitous organism that is a known opportunistic pathogen, causing both chronic and acute infections in susceptible populations, including individuals with cystic fibrosis or burn wounds, or Intensive Care Unit patients (1). Among questions that remain unanswered for nonobligate pathogens like P. aeruginosa is how these bacteria initiate infections after entering the host from the environment. Given that P. aeruginosa is among many bacteria that grow as a biofilm during infection, there is a need to understand how individual cells coordinate in space with each other to colonize new surfaces and subsequently transition to stationary biofilms.Many organisms coordinate their movement as a population, emerging as self-organized swarming groups. Even the untrained eye would note the coordinated swarming behavior of fish, birds, and insects. Many bacteria also exhibit collective motion by swarming over surfaces in a coordinated manner to move unimpeded at the same time (2–4). Our knowledge of the specific actions used by individual cells during collective motion is limited; the behavior of single cells within a dense population is difficult to discern experimentally. Previous attempts to study bacterial collective behavior have used computational models to test mechanisms hypothesized to influence collective motion, including directional reversals (5), slime deposition and chemoregulation (6), quorum sensing and surfactant production (7), and escape-and-pursuit response (8). Cell-to-cell alignment is an included feature of many of these computational models and an experimental measurement frequently used to characterize ordering of cells within populations (9, 10). For example, assumption of higher alignment among cells to improve collective motion in model simulations was crucial to recreation of the density wave propagating with the velocity of the experimentally observed traveling wave in P. aeruginosa swarms (7). However, it is not yet clear if groups of bacteria truly coordinate (e.g., align) over longer distances and time scales to swarm. Such investigation has been limited to Escherichia coli, for which patterns of coordination have only been shown over short distances (11).Swarming is often considered to be a transition step before formation of stationary biofilm communities. For P. aeruginosa, it has been demonstrated that biofilm formation and swarming are inversely regulated by intracellular concentrations of cis(3′–5′)-cyclic-diguanylate-monophosphate (c-di-GMP). Low levels of c-di-GMP promote surface swarming, whereas elevated levels of c-di-GMP cue production of P. aeruginosa matrix polysaccharides and the initiation of a sessile biofilm (12, 13). The diguanylate cyclase WspR, for example, up-regulates Pel polysaccharide synthesis in a contact-dependent manner (14). However, the specific physical interaction(s) between P. aeruginosa and surfaces (e.g., swarming substrates, surfaces of attachment, or other P. aeruginosa cells) have yet to be elucidated in specific detail.Most motile bacteria use either flagella or type IV pili (TFP), but P. aeruginosa is one of few bacteria that possess both of these motile appendage types. P. aeruginosa TFP or flagella confer multiple motility modes in addition to swarming, including swimming, twitching, crawling, and walking (15–17); P. aeruginosa requires a functional flagellum to swarm (18, 19). Although the fastest swarming bacteria (i.e., species of Vibrio or Proteus) transition to a hyperflagellated state or can evolve as hyperflagellated mutants (20–23), for this study, we have investigated monoflagellated bacteria characteristic of P. aeruginosa swarming. Both TFP and flagella are important to P. aeruginosa biofilm formation (24) and mediate attachment to different surfaces, including eukaryotic epithelial cells (25). Previous research suggests that TFP do not lead to faster swarming. For example, P. aeruginosa mutants of TFP pilin genes pilA, pilW, pilX, or pilY1 (rendering them TFP deficient) exhibit an increased swarming phenotype (19, 26, 27), and retraction-impaired mutations, such as pilH, exhibit decreased swarming (28). Thus, in this paper, we addressed the question, “What role do TFP play in swarming?” More specifically, we were interested in studying how TFP contribute to collective motion during (flagellar-mediated) swarming of P. aeruginosa. Although separate studies suggest a broader regulatory role for some TFP-associated genes (26, 27), we judged the increased swarming exhibited by select TFP mutants as allowing for the possibility of a physical role of TFP during swarming.In this paper, we present evidence that P. aeruginosa promotes physical cell−cell interactions during swarming via their TFP to control their collective motion and limit lone cell movement in swarms. Because of the difficulty of specifically identifying the influence and dynamics of TFP upon swarming cells using a traditional experimental approach, we used a series of coordinated laboratory and computational experiments to study the physical influence of TFP among groups of P. aeruginosa cells. Using simulations, we showed that prior reports of improved swarming by P. aeruginosa TFP-deficient mutants can be caused by TFP-deficient cells displaying increased displacement compared with wild-type cells. We confirmed this prediction in vitro by showing that a TFP-deficient mutant could outcompete P. aeruginosa wild type in coculture experiments to reach the swarm edge first. We also infer from our experiments that TFP interact strongly with other TFP during swarming and conclude that P. aeruginosa initiates TFP-based community building within motile swarms. We show the benefit of TFP-mediated collective motion control by showing that wild-type, but not TFP-deficient, P. aeruginosa alters its swarming to avoid high concentrations of the antibiotic carbenicillin.     

The specificity of nitroxyl chemistry is unique among nitrogen oxides in biological systems

The importance of nitric oxide in mammalian physiology has been known for nearly 30 years. Similar attention for other nitrogen oxides such as nitroxyl (HNO) has been more recent. While there has been speculation as to the biosynthesis of HNO, its pharmacological benefits have been demonstrated in several pathophysiological settings such as cardiovascular disorders, cancer, and alcoholism. The chemical biology of HNO has been identified as related to, but unique from, that of its redox congener nitric oxide. A summary of these findings as well as a discussion of possible endogenous sources of HNO is presented in this review.     

Comparing the chemical biology of NO and HNO

For the past couple of decades nitric oxide (NO) and nitroxyl (HNO) have been extensively studied due to the important role they play in many physiological and/or pharmacological processes. Many researchers have reported important signaling pathways as well as mechanisms of action of these species, showing direct and indirect effects depending on the environment. Both NO and HNO can react with, among others, metals, proteins, thiols and heme proteins via unique and distinct chemistry leading to improvement of some clinical conditions. Understanding the basic chemistry of NO and HNO and distinguishing their mechanisms of action as well as methods of detection are crucial for understanding the current and potential clinical applications. In this review, we summarize some of the most important findings regarding NO and HNO chemistry, revealing some of the possible mechanisms of their beneficial actions.