Abstract
Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome coxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e. ‘orphan’) operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen Pseudomonas aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets.
Bacteria often form communities called biofilms to make them stronger and more ‘invincible’. However, when these communities become too crowded, oxygen levels can drop, which makes it harder for them to survive. Some types of bacteria, such as Pseudomonas aeruginosa, have found different ways to cope with lower levels of oxygen. For example, they produce enzymes that use oxygen more efficiently or are better at scavenging low concentrations of oxygen.
When organisms – including bacteria – produce energy, they break down nutrients into small molecules to extract electrons. These electrons are then transported along their membrane until they reach their final destination – an oxygen molecule. Studies of P. aeruginosa grown in the laboratory have shown that it uses several types of enzymes called terminal oxidases to complete this last electron transfer. The bacterium can also make chemicals that help to shuttle electrons to remote oxygen sources. For example, they can produce compounds called phenazines that can transport electrons and also compensate for low oxygen levels.
However, the conditions in biofilms can be very different to those in a laboratory environment, and until now it was not known what role the different oxidases play in biofilm communities, or how phenazines can compensate for low oxygen levels.
To investigate this further, Jo et al. studied P. aeruginosa in an artificial biofilm environment and in a nematode worm host. The results showed that a specific part of the terminal oxidases – a protein called CcoN4 – was necessary for P. aeruginosa to grow optimally in both instances. Mutant bacteria that lacked CcoN4 struggled to survive. Moreover, bacteria containing CcoN4 were able to deliver the electrons to phenazines. This suggests that CcoN4 is also needed for phenazines to work properly.
This study shows that blocking terminal oxidases that contain CcoN4 can weaken P. aeruginosa and consequently its ability to cause infections. Furthermore, these types of terminal oxidases are only found in bacteria, which makes them attractive targets for potential drugs that would have minimal side effects on the host’s metabolism. P. aeruginosa infections are a leading cause of death for people suffering from cystic fibrosis, a genetic condition that affects the lungs and the digestive system. A better understanding of what makes P. aeruginosa so infectious will help to find new treatments for these patients.