UC Berkeley

Bacterial pathogens acquire the iron they need for survival and growth in a host by using siderophores. The structures of siderophores are specialized for binding ferric ion with high affinity. Siderophore structures are also specialized to specifically interact with the proteins that mediate siderophore function. These proteins include the bacterial proteins involved in siderophore uptake and utilization, as well as host proteins that inhibit bacterial iron acquisition by intercepting siderophores.

The interactions between siderophores and iron underlie biological function. The fundamental coordination chemistry of catecholate and hydroxamate siderophores affects protein interactions during siderophore uptake and host defense. Chapter 1 reviews previous studies on siderophore coordination chemistry and the effects it has on protein interactions and biology with emphasis on research from the Raymond laboratory.

The human protein siderocalin defends against siderophore-mediated iron acquisition. Human siderocalin recognizes the metal center of catecholate siderophores, including enterobactin, with high affinity. Several pathogens modify the catecholate metal binding units to make stealth siderophores that are not recognized by human siderocalin. As presented in Chapter 2, the pathogens Vibrio fluvialis and Vibrio cholerae use the siderophores fluvibactin and vibriobactin, respectively, which have catechol-oxazoline metal-binding units. The catechol-oxazoline had been proposed to be a stealth mechanism, but the results herein presented clearly demonstrate that it is not a stealth mechanism. Catechol-oxazoline coordinates iron in either a catecholate mode or a phenolate-oxazoline mode. The phenolate-oxazoline mode is not recognized by siderocalin while the catecholate mode is. Siderocalin stabilizes the catecholate mode sufficiently to cause a shift from the phenolate-oxazoline mode at physiological pH. The high affinity recognition of the ferric triscatecholate metal center allows siderocalin to defend against iron acquisition by a large number of bacterial siderophores.

Siderocalin defense has been identified in hosts other than humans. Ex-FABP is a protein found in chickens that has high structural homology to human siderocalin. Chapter 3 reports that Ex-FABP binds many of the same siderophores that are bound by human siderocalin. Unlike human siderocalin, the binding pocket of Ex-FABP is expanded to allow it to bind glucosylated enterobactin. Many of the pathogens specific to chickens use glucosylated enterobactin siderophores known as salmochelins. Salmochelins are stealth siderophores in humans. The siderophore recognition of Ex-FABP demonstrates that siderophore binding proteins may be a general host defense mechanism, and that siderocalins have adapted to the pathogens most frequently encountered by the host. Siderocalin defense and stealth siderophores are at the edge of the arms race for iron.

Siderophores carry iron into a bacterial cell through specific transport systems. Once inside the cell, the iron must be removed from the siderophore. Bacteria that use ferric enterobactin remove the iron by hydrolyzing the backbone with an esterase followed by reduction of the ferric ion. Hydrolysis is necessary because the high stability of intact ferric enterobactin prevents biological reduction and iron release. V. cholerae had been reported to use ferric enterobactin, but it does not have an esterase to hydrolyze the backbone. Chapter 4 reports that V. cholerae does not use intact ferric enterobactin, but that it most likely uses ferric complexes of enterobactin hydrolysis products.

Uptake of ferric siderophores relies on specific cell membrane receptors. Many siderophore receptors recognize the apo-siderophores as well as the ferric complexes. Binding apo-siderophores does not directly deliver iron to the bacteria, but it plays a role in the uptake mechanism. Chapter 5 describes YxeB, the ferrichrome/ferrioxamine receptor of Bacillus cereus. YxeB transports the siderophores using a Gram-positive siderophore-shuttle in which metal exchange between a ferric siderophore and the bound apo-siderophore is facilitated by the receptor. Metal exchange is not required for uptake, but the siderophore-shuttle is faster than transport without metal exchange.

Metal exchange, iron release, and the sterics and electronics of the metal center are coordination chemistry principles that influence the interactions between siderophores and proteins. Proteins usher siderophores through the biological functions of removing iron from the host, passing through the bacterial membrane, and releasing iron to the cell. Therefore, siderophores act as the intermediaries between ferric ion and the biology of bacterial iron uptake.