Regulation of ribonucleotide reduction in facultative anaerobic pathogens and its influence in bacterial fitness, virulence and biofilm formation

Abstract

The perpetuation of life depends on the ability to reproduce. All known forms of life use DNA as genetic information storage. Ribonucleotide reduction is the process by which ribonucleotides (NTPs) are transformed into deoxyribonucleotides (dNTPs), thereby forming the building blocks for DNA synthesis and repair. This reaction is catalyzed by a family of sophisticated enzymes, the ribonucleotide reductases (RNRs). All RNRs are metalloproteins that use a common, free-radical-based catalytic mechanism. However, depending on the specific mechanisms they use for radical generation, the type of cofactor they require, and differences in the structure of their protein complexes, RNR are divided into three different classes, namely class I, class II and class III. RNR classes also present different relationships with oxygen: class I RNRs are oxygen-dependent, class II RNRs are oxygen-independent, and class III RNRs are oxygen-sensitive. While eukaryotic organisms use only class I RNR, bacteria can encode all RNR classes in any possible combinations, which provides them with a valuable tool for to adapt to different environmental conditions. Facultative anaerobic bacteria can thrive in the presence or absence of oxygen. Numerous species significant for their clinical relevance are facultative anaerobes, as many environments inside host bodies feature hypoxic or anoxic conditions; microaerobic or anaerobic environments are also found in biofilms in chronic infections. However, a facultative anaerobic lifestyle also implies an increased cost in regulation complexity. This effect extends to the ribonucleotide reductases network, which in facultative anaerobic pathogens must be thoroughly regulated to react to the different challenges imposed by different oxygenation conditions, changing growth speeds, host defense mechanisms, etc. This study is focused on facultative anaerobic pathogens and the strategies they use to modulate ribonucleotide reduction and balance it under different environmental stimuli, variable oxygenation conditions, and during infection or biofilm formation. We used two very well-studied species: Pseudomonas aeruginosa and Escherichia coli. First, in P. aeruginosa, an opportunistic pathogen well-known for its chronic pulmonary infections, we explore the effects of the AlgZR two-components system, one of the main regulatory elements responsible for biofilm formation and chronification, in the control of the RNR network. We then conducted a comprehensive characterization of the master regulator of ribonucleotide reductases NrdR, from its general role to its specific mechanism of action, in both E. coli and P. aeruginosa. We also explored the differential roles of RNR classes during biofilm formation and the function that the master regulators of anaerobic metabolism play in their control. Finally, we used a technique developed in this work, a continuous-culture method named AnaeroTrans, to characterize in detail the gradual adaptation E. coli and P. aeruginosa withstand during the aerobic-anaerobic transition and, isolating this effect from all other consequences of biofilm formation, explore the dynamic actions of RNR classes and anaerobic regulators in the microaerobic range. Overall, this works provides a comprehensive description of the different roles ribonucleotide reductases play in anaerobic facultative pathogens and the regulatory mechanisms that control them.

La reducción de ribonucleótidos es el proceso por el que éstos se convierten en desoxirribonucleótidos (dNTPs), formando así los precursores para la síntesis y la reparación del ADN. Esta reacción es catalizada por las ribonucleótido reductasas (RNR). Aunque todas las RNR son metaloproteínas que comparten un mismo mecanismo basado en radicales libres, éstas pueden separarse en tres clases (clase I, clase II, clase III) dependiendo del mecanismo que utilizan para la síntesis del radical, el tipo de cofactores que requieren, o las diferencias estructurales que presentan. Los organismos eucarióticos utilizan exclusivamente RNR de clase I, pero las bacterias pueden codificar todas las clases en cualquier combinación posible. Las bacterias anaeróbicas facultativas pueden crecer en presencia o ausencia de oxígeno. Numerosas especies significativas por su importancia clínica son anaeróbicas facultativas. No obstante, también han de asumir un coste superior en cuando a la complejidad de su regulación genética. Este efecto se extiende a las ribonucleótido reductasas, que, en patógenos anaeróbicos facultativos, deben poder responder a distintas concentraciones de oxígeno, cambios en la velocidad de crecimiento, mecanismos de defensa del anfitrión, etc. Este trabajo se centra en los patógenos anaeróbicos facultativos y las estrategias que usan para regular y equilibrar la síntesis de ribonucleótidos bajo diferentes estímulos ambientales y condiciones variables de oxigenación, así como durante la infección y la formación de biofilm. En primer lugar, exploramos el efecto sobre la red de las RNR del sistema de dos componentes AlgZR de P. aeruginosa, un sistema regulatorio relacionado con la síntesis de alginato, la formación de biofilm y la cronificación. Posteriormente, realizamos una caracterización completa de NrdR, el regulador principal de las ribonucleótido reductasas, analizando tanto su papel general sobre la red de las RNR como su mecanismo molecular, tanto en P. aeruginosa como en E. coli. También analizamos el papel que desempeñan las distintas clases de RNR durante la formación de biofilm y la función de los reguladores anaeróbicos generales en su control. Finalmente, usamos una técnica de cultivo continuo desarrollada para este trabajo que denominamos AnaeroTrans para el estudio detallado de la transición aeróbica-anaeróbica en P. aeruginosa y E. coli.