Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection.

In order to determine the role of ferrous iron transport in Legionella pathogenesis, we identified and mutated the feoB gene in virulent Legionella pneumophila strain 130b. As it is in Escherichia coli, the L. pneumophila feoB gene was contained within a putative feoAB operon. L. pneumophila feoB insertion mutants exhibited decreased ferrous but not ferric iron uptake compared to the wild type. Growth on standard buffered charcoal yeast extract agar or buffered yeast extract broth was unaffected by the loss of L. pneumophila FeoB. However, the L. pneumophila feoB mutant had a reduced ability to grow on buffered charcoal yeast extract agar with a reduced amount of its usual iron supplementation, a phenotype that could be complemented by the addition of feoB in trans. In unsupplemented buffered yeast extract broth, the feoB mutant also had a growth defect, which was further exacerbated by the addition of the ferrous iron chelator, 2,2'-dipyridyl. The feoB mutant was also 2.5 logs more resistant to streptonigrin than wild-type 130b, confirming its decreased ability to acquire iron during extracellular growth. Decreased replication of the feoB mutant was noted within iron-depleted Hartmannella vermiformis amoebae and human U937 cell macrophages. The reduced intracellular infectivity of the feoB mutant was complemented by the introduction of a plasmid containing feoAB. The L. pneumophila feoB gene conferred a modest growth advantage for the wild type over the mutant in a competition assay within the lungs of A/J mice. Taken together, these results indicate that L. pneumophila FeoB is a ferrous iron transporter that is important for extracellular and intracellular growth, especially in iron-limited environments. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.

A new chromosomal locus associated with gut-modulated phenotypes in Salmonella enterica serotype typhimurium.

A cosmid DNA library had been constructed previously from 40-kb fragments of genomic DNA from a virulent invasive strain of Salmonella enterica serotype Typhimurium (TML) in an avirulent hypo-invasive Typhimurium strain (LT7). Selection of invasive clones from the library was attempted by iterative passage through a rabbit ileal organ culture. After the fourth passage, a clone, designated LT7(pHC20-2), was isolated. Exposure to both gut tissue and Caco-2 cells enhanced the growth, invasiveness for gut and Caco-2 cells, and flagellin expression of LT7(pHC20-2) although its invasiveness was less than that of strain TML. Expression of appendages (surface structures c. 60-70 nm diameter) was shown to play a role in but not to confer invasiveness, and was demonstrated in the absence of direct contact with eukaryotic cells. Exposure to gut tissue also affected the expression of several outer-membrane proteins (OMPs) in all four Salmonella strains--TML, LT7, LT7(pHC79), LT7(pHC20 2)--used in this work. As the genes involved in flagella, invasin and porin expression are distributed around the salmonella chromosome, it is possible that pHC20-2 encodes a pleiotropic regulator of genes involved in gastro-enteritic virulence and adaptation to the in-vivo gut environment. pHC20-2 mapped at c. centisome 25 on the salmonella chromosome close to, but distinct from, SPI-5.