Nitric Oxide and Hydrogen Sulfide interplay and tolerance in Pseudomonas aeruginosa: role of sulfide catabolism and aerobic respiration

Hydrogen sulfide (H₂S), recognized as the third gasotransmitter alongside nitric oxide (NO) and carbon monoxide (CO), plays a significant role in various biological systems. A key feature of gasotransmitters is their ability to exhibit functional redundancy and cross-regulate each other's bioavailability by reciprocally modulating their synthesis and breakdown. While the functions of H₂S are well-established in mammalian systems, particularly regarding the regulation of several physiological and pathophysiological processes, its role is still debated in bacteria. There H₂S is reportedly implicated in crucial processes such as virulence, antibiotic resistance, and biofilm formation; in contrast, elevated levels of H₂S are toxic to bacteria, as they impair crucial biological functions, such as oxidative phosphorylation and signaling via post-translational protein modifications, and cause accumulation of reactive oxygen and sulfur species (ROS and RSS), leading to cell death. To survive in H₂S-rich environments, bacteria have evolved protective mechanisms, such as the expression of sulfide-insensitive bd-type respiratory oxidases or evolutionarily conserved enzymes affording H₂S detoxification. These mechanisms are especially critical during host infection, when bacteria encounter not only H₂S but also NO produced by the host immune system, classifying both bd-type oxidases and H2S-detoxifying enzymes as possible drug targets for the development of novel antibiotics and adjuvants. In this PhD thesis work, H₂S and NO tolerance and interplay were investigated in Pseudomonas aeruginosa, a multidrug-resistant pathogen responsible of a wide range of nosocomial infections. Namely, focusing on this pathogen, this thesis has aimed to gain insights on: (i) the effects of H₂S and NO on aerobic respiration; (ii) the biochemical properties of enzymes involved in H₂S breakdown; and (iii) novel mechanisms through which H₂S and NO mutually regulate their bioavailability. Firstly, by performing in vivo studies and high resolution respirometric measurements on membranes of P. aeruginosa mutant strains, the bd-type Cyanide Insensitive Oxidase (CIO) was shown to be i) insensitive to sulfide inhibition, differently from the other respiratory oxidases, and ii) upregulated and facilitating bacterial growth in the presence of high levels of this compound. These results highlight the critical role of CIO in enabling P. aeruginosa grow in sulfide-rich environments. Also, NO-inhibited CIO was shown to rapidly and completely restore its control activity after NO removal from solution, proving to be less prone to irreversible nitrosative damage compared to other P. aeruginosa respiratory oxidases. Concerning sulfide catabolism, the P. aeruginosa H2S-detoxifying enzymes Persulfide Dioxygenase (PDO) and Sulfide:Quinone Oxidoreductase (SQR) were characterized both in vivo and in vitro as recombinant proteins isolated from Escherichia coli. Both enzymes were found to be significantly contribute to H2S detoxification in vivo, as deletion of the PDO or SQR gene in the P. aeruginosa PAO1 strain resulted in ca. 4-fold higher sulfide levels than the wild type. The crystallographic structure of PDO was successfully solved, showing distinct features with respect to the human homolog, and the catalytic properties of both PDO and SQR were thoroughly characterized by high resolution respirometry and time-resolved absorption spectroscopy. Lastly, as a novel finding, both P. aeruginosa PDO and its human homolog were found to be reversibly inhibited by NO, suggesting that this gas can modulate H2S bioavailability according to an unprecedented mechanism evolutionarily conserved from bacteria to humans. In conclusion, these studies shed light on key players of both aerobic respiration and H2S-detoxification in P. aeruginosa, recognized as potential pharmacological targets in this harmful pathogen.