Amonabactin-mediated iron acquisition from transferrin and lactoferrin by Aeromonas hydrophila: direct measurement of individual microscopic rate constants

The effectiveness and mechanism of iron acquisition from transferrin or lac toferrin by Aeromonas hydrophila has been analyzed with regard to the patho genesis of this microbe. The ability of A. hydrophila's siderophore, amonab actin, to remove iron from transferrin was evaluated with in vitro competit ion experiments. The kinetics of iron removal from the three molecular form s of ferric transferrin (diferric, N- and C-terminal monoferric) were inves tigated by separating each form by urea gel electrophoresis. The first dire ct determination of individual microscopic rates of iron removal from difer ric transferrin is a result. A. hydrophila 495A2 was cultured in an iron-st arved defined medium and the growth monitored. Addition of transferrin or l actoferrin promoted bacterial growth. Growth promotion was independent of t he level of transferrin or lactoferrin iron saturation (between 30 and 100% ), even when the protein was sequestered inside dialysis tubing. Siderophor e production was also increased when transferrin or lactoferrin was enclose d in a dialysis tube. Cell yield and growth rate were identical in experime nts where transferrin was present inside or outside the dialysis tube, indi cating that binding of transferrin was not essential and that the sideropho re plays a major role in iron uptake from transferrin. The rate of iron rem oval from diferric transferrin shows a hyperbolic dependence on amonabactin concentration. Surprisingly, amonabactin cannot remove iron from the more weakly binding N-terminal site of monoferric transferrin, while it is able to remove iron from the more strongly binding C-terminal site of monoferric transferrin. Iron from both sites is removed from diferric transferrin and it is the N-terminal site (which does not release iron in the monoferric p rotein) that releases iron more rapidly! It is apparent that there is a sig nificant interaction of the two lobes of the protein with regard to the che lator access. Taken together, these results support an amonabactin-dependen t mechanism for iron removal by A. hydrophila from transferrin and lactofer rin. The implications of these findings for an amonabactin-dependent mechan ism for iron removal by A. hydrophila from transferrin and lactoferrin are discussed.