Catecholamine-mediated resistance to iron limitation in Neisseria gonorrhoeae

About This Grant

PROJECT SUMMARY/ABSTRACT Neisseria gonorrhoeae (Gc) causes the sexually transmitted infection gonorrhea. Rates of gonorrhea are increasing, along with resistance to multiple antibiotics, and there is no licensed vaccine. New approaches are needed to combat this urgent public health threat. One approach is to target the mechanisms used by Gc to successfully establish infection, including its ability to obtain essential metals like iron from its obligate human host. Gc produces outer membrane transporters that bind and strip iron from human transferrin and lactoferrin, bind hemoglobin and import its heme moiety, and steal the iron-binding siderophores made by other bacteria. The metal is then imported into the cytoplasm for incorporation into biomolecules. However, how Gc overcomes iron limitation in the context of human infection remains enigmatic. Symptomatic infection is characterized by influx of neutrophils and a resulting purulent exudate that is unable to clear Gc. We discovered that neutrophil- conditioned, Chelex-treated (metal-stripped) defined rich medium supports Gc growth in an iron-dependent manner. The growth-supportive activity was in the flow-through of a < 3 kDa filtration unit, yet this fraction did not have significantly more iron. By untargeted metabolomics, the < 3kDa fraction contained catecholamines, in particular norepinephrine (NE). Addition of NE to the Chelex-treated medium was sufficient to significantly increase Gc growth. Catecholamines have been implicated in inter-kingdom signaling and induction of virulence gene expression in pathogens; they have also been shown to act as pseudo-siderophores for iron acquisition. However, potential homologs of the catecholamine receptors in other pathogens were dispensable for Gc growth with NE. By RNAseq, Gc grown with NE had increased expression of iron- and Fur-regulated genes. A fur mutant with reduced repressive activity grew significantly better in Chelex-treated medium, and its growth was not further enhanced by adding NE. These findings lead to the overarching hypothesis that Gc responds to NE in neutrophil- conditioned medium by derepressing the fur regulon to enhance growth in low-iron conditions. We will test this hypothesis in two Specific Aims. In Aim 1, we will determine how NE affects iron levels and iron uptake in Gc. We will also measure NE and other catecholamines in neutrophil-conditioned supernatants and determine their necessity for Gc growth. In Aim 2, we will examine how NE treatment affects Fur levels and Fur binding to defined gene targets. We will evaluate changes in expression of ferredoxins and bacterioferritin in NE-treated Gc, and we will test how an uncharacterized ferredoxin that is significantly upregulated with NE treatment contributes to NE-stimulated growth of Gc in low iron conditions. Results arising from these studies will uncover the interplay between Gc and catecholamines in the context of iron limitation. These findings can reveal targets for novel interventions to combat drug-resistant gonorrhea.