RELATIVE IMPORTANCE OF 3 IRON-REGULATED OUTER-MEMBRANE PROTEINS FOR IN-VIVO GROWTH OF VIBRIO-CHOLERAE

Iron is an essential nutrient to support the growth of most bacterial species, However, iron is not easily available to microorganisms infec ting mammalian hosts, because it is largely sequestered by iron-bindin g proteins, such as transferrin or lactoferrin, or complexed to heme, In response to environmental iron stress, Vibrio cholerae produces the siderophore vibriobactin as well as a number of iron-induced outer me mbrane proteins, Previous data on the role of iron acquisition systems for the intraintestinal growth Of mucosal pathogens such as V. choler ae are conflicting, In this report, we isolated mutants of V. cholerae with TnphoA fusions in each of viuA, hutA, and irgA, as well as strai ns mutant in each pair of these genes and all three simultaneously, to analyze the role of these iron-induced outer membrane protein recepto rs for in vivo growth of V. cholerae. The fusion between hutA and Tnph oA in a single copy on the chromosome allowed the study of in vitro re gulation of hutA in response to iron, fur, and irgB; transcription of hutA was tightly iron regulated (70-fold) and dependent on a functiona l Fur but did not require IrgB, To investigate the effects of mutation s in these iron-induced outer membrane proteins on in vivo growth, we inoculated ileal loops in a rabbit model of infection, This avoids exp osure of organisms to the potential killing effects of gastric acid, a llows several logarithmic increases in growth in the in vivo environme nt, and facilitates direct comparison of multiple strains in the same animal to avoid any differences between animals, We grew each mutant t o be tested in competition with the wild-type strain in the same loop, to provide an internal control, We confirmed that the inocula for the se experiments were grown under conditions of iron stress prior to in vivo inoculation, by measuring the alkaline phosphatase activity of th e iron-regulated fusion in each strain, The results confirmed that mut ation of irgA produced a much more substantial in vivo growth defect t han mutation of either hutA or viuA alone, Double mutants of irgA with either viuA or hutA, or the strain mutant in all three genes, showed an in vivo growth defect comparable to the strain mutant in irgA only, suggesting that mutation of irgA was the most relevant for in vivo gr owth, The strain mutant in both hutA and viuA was also markedly impair ed for in vivo growth, suggesting that mutation of both of these iron uptake systems simultaneously can also produce a substantial in vivo g rowth defect.