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.
Highlights
Iron is an essential micronutrient for most forms of life on earth because of its vital role as a redox cofactor of proteins required for critical cellular processes
Since no lowiron growth defect was observed with the single DmmpS4 and DmmpS5 mutants (Figs. 1, S1–2), and expression of both mmpS4 and mmpS5 is induced under iron-limited conditions [14] we suspected that they might have redundant functions
Growth of the double mutant was partially rescued when glycerol-alanine salts (GAS) agar plates were supplemented with 5 mM hemoglobin that was previously shown to function as an iron source for Mycobacterium tuberculosis (Mtb) (Fig. 1C) [15]
Summary
Iron is an essential micronutrient for most forms of life on earth because of its vital role as a redox cofactor of proteins required for critical cellular processes. Pathogenic bacteria have evolved an array of intricate mechanisms to scavenge limited iron from the host [1]. Mtb meets its iron demands by stripping host iron stores employing two hydroxyphenyloxazoline siderophores, mycobactin (MBT) and carboxymycobactin (cMBT). To counteract these bacterial iron acquisition processes, the alveolar macrophage in which Mtb thrives, keeps phagosomal iron levels extremely low by the natural resistance-associated macrophage protein Nramp in particular after activation by interferon-c [2,3]. MBT and cMBT increase the biologically available iron within the phagosomal compartment almost by 20-fold indicating that the Mtb siderophores can overcome these host defense mechanisms [4]. Studies using siderophore biosynthesis and uptake mutants underpin the importance of siderophore-mediated iron acquisition to the virulence of Mtb [5,6,7]
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