TheMajorIron-Containing Protein ofLegionella pneumophila Isan Aconitase Homologous withtheHumanIron- Responsive Element-Binding Protein

Legionela pneumophila hashighironrequirements, anditsintracellular growth inhumanmonocytes is dependent ontheavailability ofintracellular iron. Tolearn more about iron metabolism inL.pneumophila, we haveundertaken an analysis oftheironproteins ofthebacterium. We first developed an assaytoidentify proteins by"9Fe labelling andnondenaturing polyacrylamide gelelectrophoresis. Theassayrevealed seven iron proteins (IPs) withapparent molecular weights of500, 450, 250,210,150,130,and85.IUP150 comigrates withsuperoxide dismutase activity andisprobably theFe-superoxide dismutase ofL.pneunophla. IP210 isthemajoriron-containing protein (MICP). Toidentify andcharacterize MICP,we purified theprotein and cloned andsequenced its gene.MICPisa monomeric protein containing 891aminoacids, andithasa calculated molecular mass of98,147 Da.Analysis ofthesequencerevealed thatMICPhastwointeresting homologies. First, MICPishighly homologous withthehumaniron-responsive element-binding protein, consistent withthehypothesis that this critical iron-regulatory molecule ofhumanshasaprokaryotic ancestor. Second, MICPishigly homologous withtheEwherichia coliaconitase andtoalesser extent withporcine heart mitochondrial aconitase. Consistent withthis, we found that MICPexhibits aconitase activity. Incontrast to other aconitases, MICPhasa single aminoacidchange ofapotentiaLly deleterious tpe atasite thought tobe critical forsubstrate binding andenzymatic activity. However, thespecificactivity ofMICPisroughly comparable tothat ofother aconitases, suggesting that themutation hasatmostamildeffect ontheaconitase activity ofMICP.Theabundance ofMICPinL.pneumophika suggests either that L.pneumophila requires highaconitase andperhaps tricarboxylic acidcycle activity orthatthebacterium requires large amountsof this protein toservean additional roleinbacterial physiology. A needforlarge amountsofMICP,which contains fourFeatomspermolecule whenfully loaded, could atleast partly explain L.pneumophiIa's high metabolic requirement foriron. Ironplays acritical roleintheimmunobiology ofthe facultative intracellular parasite Legionella pneumophila, theagent ofLegionnaires' disease. L.pneumophila hasa particularly highmetabolic requirement foriron. Whereas pathogenic bacteria, including gram-negative andfacultative intracellular bacteria, commonly require 0.3to1.6,uMiron foroptimal growth, L.pneumophila requires 3to13,uMiron forminimal growth and>20,uMiron foroptimal growth (14, 26a, 32,60). Inhumanmonocytes, L.pneumophila's growth isstrictly dependent ontheavailability ofiron, whichit obtains viahostiron transport andstorage systems, includ- ingthetransferrin-transferrin receptor system, thelactofer- rin-lactoferrin receptor system, andferritin (4-7). Conse- quently, agents whichreduce intracellular ironavailability inhibit L.pneumophila intracellular multiplication. These agents include (i) ironchelators, suchasthenonphysiologic ironchelator deferoxamine andthephysiologic iron chelator apolactoferrin; (ii) theweakbaseschloroquine andammo- niumchloride, whichraise endocytic andlysosomal pHand consequently inhibit thepH-dependent release ofiron from endocytized iron transferrin andthepH-dependent proteol- ysis offerritin andiron-lactoferrin andrelease ofiron from these molecules; and(iii) gammainterferon, whichcoordi- nately downregulates transferrin receptor expression and