Genome-wide Phenotypic Profiling Identifies and Categorizes Genes Required for Mycobacterial Low Iron Fitness

Marte S Dragset,Eric J Rubin,Zekarias Ginbot,Magnus Steigedal,James C Sacchettini,Mali Mærk,Yanjia J Zhang,Thomas R Ioerger,Trude H Flo

doi:10.1038/s41598-019-47905-y

Abstract

Iron is vital for nearly all living organisms, but during infection, not readily available to pathogens. Infectious bacteria therefore depend on specialized mechanisms to survive when iron is limited. These mechanisms make attractive targets for new drugs. Here, by genome-wide phenotypic profiling, we identify and categorize mycobacterial genes required for low iron fitness. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can scavenge host-sequestered iron by high-affinity iron chelators called siderophores. We take advantage of siderophore redundancy within the non-pathogenic mycobacterial model organism M. smegmatis (Msmeg), to identify genes required for siderophore dependent and independent fitness when iron is low. In addition to genes with a potential function in recognition, transport or utilization of mycobacterial siderophores, we identify novel putative low iron survival strategies that are separate from siderophore systems. We also identify the Msmeg in vitro essential gene set, and find that 96% of all growth-required Msmeg genes have a mutual ortholog in Mtb. Of these again, nearly 90% are defined as required for growth in Mtb as well. Finally, we show that a novel, putative ferric iron ABC transporter contributes to low iron fitness in Msmeg, in a siderophore independent manner.

Highlights

Www.nature.com/scientificreports as membrane microvesicle production14), and the activity of diverse iron-containing enzymes[7,15]
We take advantage of siderophore redundancy within M. smegmatis (Msmeg) and define and categorize genes required for mycobactin, exochelin or siderophore independent modes of low iron growth by transposon insertion sequencing (Tnseq)[21]
We found that most (96%) of the Msmeg required (ES and growth defect (GD)) genes had a mutual ortholog in Mycobacterium tuberculosis (Mtb). 90% of these genes were required for optimal Mtb in vitro growth (Fig. 1c), indicating that, under the given conditions, Msmeg and Mtb largely depend on the same mechanisms for growth

Summary

Introduction

Www.nature.com/scientificreports as membrane microvesicle production14), and the activity of diverse iron-containing enzymes[7,15]. We demonstrated the need of ESX-3, a type VII secretion system, for mycobactin-mediated iron uptake using Msmeg[17,18], a finding later confirmed in Mtb[19,20]. We take advantage of siderophore redundancy within Msmeg and define and categorize genes required for mycobactin, exochelin or siderophore independent modes of low iron growth by transposon insertion sequencing (Tnseq). (the power of Msmeg siderophore redundancy to identify genetic interaction partners to siderophore pathways was previously demonstrated by Judd el al, in a proof-of-principle synthetic genetic array[22]). We found that the vast majority (90%) of Msmeg essential genes had a growth-required mutual ortholog in Mtb. our low iron screens identified candidate genes for recognition, transport or utilization of siderophores, as well as new siderophore-independent low iron fitness mechanisms. We constructed a directed knockout of one of our hits, msmeg_3635 (a homologue of a ferric iron ABC permease), and found that this gene is important during Msmeg iron starvation and appears to function independently of siderophores

Methods

Results

Conclusion