Abstract
Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of nosocomial and chronic infections contributing to morbidity and mortality in cystic fibrosis patients. One of the reasons for its success as a pathogen is its ability to adapt to a broad range of circumstances. Here, we show the involvement of the general nitrogen regulator NtrBC, which is structurally conserved but functionally diverse across species, in pathogenic and adaptive states of P. aeruginosa. The role of NtrB and NtrC was examined in progressive or chronic infections, which revealed that mutants (ΔntrB, ΔntrC, and ΔntrBC) were reduced in their ability to invade and cause damage in a high-density abscess model in vivo. Progressive infections were established with mutants in the highly virulent PA14 genetic background, whereas chronic infections were established with mutants in the less virulent clinical isolate LESB58 genetic background. Characterization of adaptive lifestyles in vitro confirmed that the double ΔntrBC mutant demonstrated >40% inhibition of biofilm formation, a nearly complete inhibition of swarming motility, and a modest decrease and altered surfing motility colony appearance; with the exception of swarming, single mutants generally had more subtle or no changes. Transcriptional profiles of deletion mutants under swarming conditions were defined using RNA-Seq and unveiled dysregulated expression of hundreds of genes implicated in virulence in PA14 and LESB58 chronic lung infections, as well as carbon and nitrogen metabolism. Thus, transcriptional profiles were validated by testing responsiveness of mutants to several key intermediates of central metabolic pathways. These results indicate that NtrBC is a global regulatory system involved in both pathological and physiological processes relevant to the success of Pseudomonas in high-density infection.
Highlights
Nitrogen is an essential element of life that is critical for the normal synthesis of proteins and nucleic acids constituting 3% of the human body by mass (Rohmer et al, 2011)
Forming high-density subcutaneous abscesses with mutants in the PA14 rather than LESB58 genetic background allowed us to examine this feature since this strain is capable of dissemination to distal organs, which is associated with significant mortality of mice within 36 h (Pletzer et al, 2017)
Due to the deficiency in swarming motility in a strain PA14 ntrC mutant (Yeung et al, 2009), it was interesting to examine if this impacted on dissemination from a subcutaneous localized abscess to distal organs, including the heart, lungs, liver, spleen and kidneys of mice (Table 2 and Supplementary Figure S1)
Summary
Nitrogen is an essential element of life that is critical for the normal synthesis of proteins and nucleic acids constituting 3% of the human body by mass (Rohmer et al, 2011). Pools of inorganic and organic nitrogen are found in the soil, water and atmosphere (Johnson et al, 2010). Despite their abundance, environmental forms of nitrogen are largely inaccessible to plants and animals and must be transformed for biological use. Transformation of nitrogen into its many oxidation states is dependent on microbial, especially bacterial, activity and can occur through nitrification, denitrification or nitrogen fixation among other processes (Vicente and Dean, 2017). P. aeruginosa is known for its ability to adapt to many environmental circumstances, which is reflected by numerous regulatory networks essential for sensing and responding appropriately to stimuli (Galan-Vasquez et al, 2011). The rapid response to environmental changes is often mediated by signaling through two-component systems, which are often encoded as a sensor kinase and cognate response regulator under the control of a single promoter, and these systems can be activated by the binding of a particular stimulant to the sensor kinase (Rodrigue et al, 2000; Francis et al, 2018)
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