Discovery of a Siderophore Export System Essential for Virulence of Mycobacterium tuberculosis

Abstract

Iron is an essential nutrient for most bacterial pathogens, but is restricted by the host immune system. Mycobacterium tuberculosis (Mtb) utilizes two classes of small molecules, mycobactins and carboxymycobactins, to capture iron from the human host. Here, we show that an Mtb mutant lacking the mmpS4 and mmpS5 genes did not grow under low iron conditions. A cytoplasmic iron reporter indicated that the double mutant experienced iron starvation even under high-iron conditions. Loss of mmpS4 and mmpS5 did not change uptake of carboxymycobactin by Mtb. Thin layer chromatography showed that the ΔmmpS4/S5 mutant was strongly impaired in biosynthesis and secretion of siderophores. Pull-down experiments with purified proteins demonstrated that MmpS4 binds to a periplasmic loop of the associated transporter protein MmpL4. This interaction was corroborated by genetic experiments. While MmpS5 interacted only with MmpL5, MmpS4 interacted with both MmpL4 and MmpL5. These results identified MmpS4/MmpL4 and MmpS5/MmpL5 as siderophore export systems in Mtb and revealed that the MmpL proteins transport small molecules other than lipids. MmpS4 and MmpS5 resemble periplasmic adapter proteins of tripartite efflux pumps of Gram-negative bacteria, however, they are not only required for export but also for efficient siderophore synthesis. Membrane association of MbtG suggests a link between siderophore synthesis and transport. The structure of the soluble domain of MmpS4 (residues 52–140) was solved by NMR and indicates that mycobacterial MmpS proteins constitute a novel class of transport accessory proteins. The bacterial burden of the mmpS4/S5 deletion mutant in mouse lungs was lower by 10,000-fold and none of the infected mice died within 180 days compared to wild-type Mtb. This is the strongest attenuation observed so far for Mtb mutants lacking genes involved in iron utilization. In conclusion, this study identified the first components of novel siderophore export systems which are essential for virulence of Mtb.

Self-poisoning of Mycobacterium tuberculosis by interrupting siderophore recycling

“…We recently showed that the Mtb ΔmmpS4/S5 mutant displays a lowiron [c(Fe 3+ ) = 0.5 μM] growth defect and produces less siderophores but is fully capable of siderophore uptake and sensing an iron-deficient state (14). Interestingly, the ΔmmpS4/S5 Significance Many bacteria acquire iron from the environment by secreting small iron-binding molecules called siderophores.…”

“…S3B). We next sought to determine whether the iron from cMBT was metabolized by the ΔmmpS4/S5 mutant and therefore added cMBT to midlogarithmically growing cells harboring an iron-dependent regulator (IdeR)-dependent GFP reporter (14,20). The cytoplasmic iron reporter indicates iron starvation by fluorescence.…”

“…MBT was one of the first siderophores characterized (12,13), yet little is known about siderophore transport in this organism. We recently demonstrated that MBT and cMBT are exported across the inner membrane by a process involving the membrane proteins MmpS4 and MmpS5 interacting with the resistance-nodulation-cell division (RND)-type transporters MmpL4 and MmpL5 (14). The siderophore export mutant, ΔmmpS4/S5, showed one of the strongest virulence defects known for Mtb mutants in mice establishing siderophore secretion as a valid drug target (14).…”

“…We recently demonstrated that MBT and cMBT are exported across the inner membrane by a process involving the membrane proteins MmpS4 and MmpS5 interacting with the resistance-nodulation-cell division (RND)-type transporters MmpL4 and MmpL5 (14). The siderophore export mutant, ΔmmpS4/S5, showed one of the strongest virulence defects known for Mtb mutants in mice establishing siderophore secretion as a valid drug target (14). In this study, we demonstrate that the MmpS4/MmpL4 and MmpS5/MmpL5 export systems are also required for siderophore recycling.…”

Potenciais alvos moleculares para o desenvolvimento de novos fármacos antituberculose

“…101 MmpL3, MmpL4 e MmpL5 colaboram com o transporte de ferro. 102 MmpL8 tem um papel importante na biogênese de sulfatídeos e também contribui para a virulência do MTB. 103 A proteína MmpL3 tem um papel crucial durante o crescimento do MTB e está envolvida no transporte de ferro, ácidos micólicos e monomicolato trealose.…”