Characterization of the Neisseria gonorrhoeae Iron and Fur Regulatory Network

Abstract

The Neisseria gonorrhoeae ferric uptake regulator (Fur) protein controls expression of iron homeostasis genes in response to intracellular iron levels. In this study, using transcriptome sequencing (RNA-seq) analysis of an N. gonorrhoeae fur strain, we defined the gonococcal Fur and iron regulons and characterized Fur-controlled expression of an ArsR-like DNA binding protein.We observed that 158 genes (8% of the genome) showed differential expression in response to iron in an N. gonorrhoeae wildtype or fur strain, while 54 genes exhibited differential expression in response to Fur. The Fur regulon was extended to additional regulators, including NrrF and 13 other small RNAs (sRNAs), and two transcriptional factors. One transcriptional factor, coding for an ArsR-like regulator (ArsR), exhibited increased expression under iron-replete conditions in the wild-type strain but showed decreased expression across iron conditions in the fur strain, an effect that was reversed in a fur-complemented strain. Fur was shown to bind to the promoter region of the arsR gene downstream of a predicted 70 promoter region. Electrophoretic mobility shift assay (EMSA) analysis confirmed binding of the ArsR protein to the norB promoter region, and sequence analysis identified two additional putative targets, NGO1411 and NGO1646. A gonococcal arsR strain demonstrated decreased survival in human endocervical epithelial cells compared to that of the wild-type and arsR-complemented strains, suggesting that the ArsR regulon includes genes required for survival in host cells. Collectively, these results demonstrate that the N. gonorrhoeae Fur functions as a global regulatory protein to repress or activate expression of a large repertoire of genes, including additional transcriptional regulatory proteins. IMPORTANCEGene regulation in bacteria in response to environmental stimuli, including iron, is of paramount importance to both bacterial replication and, in the case of pathogenic bacteria, successful infection. Bacterial DNA binding proteins are a common mechanism utilized by pathogens to control gene expression under various environmental conditions. Here, we show that the DNA binding protein Fur, expressed by the human pathogen Neisseria gonorrhoeae, controls the expression of a large repertoire of genes and extends this regulon by controlling expression of additional DNA binding proteins. One of these proteins, an ArsRlike regulator, was required for N. gonorrhoeae survival within host cells. These results show that the Fur regulon extends to additional regulatory proteins, which together contribute to gonococcal mechanisms of pathogenesis.

Global Network Analysis of Neisseria gonorrhoeae Identifies Coordination between Pathways, Processes, and Regulators Expressed during Human Infection

“…5B) but, to date, have not yet been shown to be targets of Fur. Several of these genes were also regulated via iron based on a previous analysis (17), including NGO0639, NGO2071, NGO2092, and NGO2111. In addition, several of these same genes were also regulated as a function of the absence of Fur, including NGO0639, NGO2092, and NGO2111 (17).…”

“…The reasoning for this is unclear but is likely due to the fact that an environmental condition will induce changes in a greater number of genes than a genetic perturbation. As an example, studies of iron and Fur regulation in N. gonorrhoeae have found that while only 54 genes respond to a lack of the Fur protein (a genetic perturbation), 158 genes responded to changes in iron in the growth media (an environmental perturbation) (17). As transcriptomic data for N. gonorrhoeae becomes more available in the coming years, it may be beneficial to infer networks made primarily, if not completely, from transcriptomes resulting from environmental changes rather than genetic perturbations.…”

“…Like other human pathogens, gene expression in N. gonorrhoeae is under tight control and achieved via a number of mechanisms, including classical DNA binding regulatory proteins and small regulatory RNAs (12)(13)(14). A central regulatory protein targeting several genes is the ferric uptake regulator (Fur), which controls genes involved in iron homeostasis (15)(16)(17). The gonococcal Fur protein also controls expression of the gene encoding the regulator MisR (CpxR), which confers resistance to antimicrobial peptides (18,19), and the gene encoding the regulator OxyR, which confers resistance to oxidative stress (20,21).…”

“…Several recent studies have utilized transcriptomic analysis to define the N. gonorrhoeae global response to conditions encountered during human infection. These include exposure to hydrogen peroxide (22), iron limitation (17), lack of oxygen (23), and incubation with human endothelial cells (14). Transcriptome analysis has also been performed using deletion mutants of many of the known gonococcal DNA binding regulatory proteins to better define potential regulatory pathways (17,18,24).…”

“…These include exposure to hydrogen peroxide (22), iron limitation (17), lack of oxygen (23), and incubation with human endothelial cells (14). Transcriptome analysis has also been performed using deletion mutants of many of the known gonococcal DNA binding regulatory proteins to better define potential regulatory pathways (17,18,24). Our group has also recently defined the gonococcal transcriptional response during natural mucosal infection in men and women (25,26).…”