Major type IV pilin of Legionella pneumophila enhances bacterial iron acquisition/assimilation, independently of its role in piliation

ABSTRACT We identified a locus within the genome of Legionella pneumophila strain 130b that encodes two related type IV pilins, PilA1 and PilA2. Based on in silico analyses, both proteins were predicted to be major pilins in all L. pneumophila strains and nearly all Legionella species. Utilizing immunoblot analysis of sheared-cell supernatants, immunofluorescence microscopy, and whole-cell ELISA, we confirmed that PilA2 (but not PilA1) behaves as a major pilin, and mutant analysis documented that PilA2-containing type IV pili (T4P) are required for DNA uptake, and subject to retraction by PilT. Unexpectedly, a pilA2 mutant, but not its complement, had impaired growth on solid and liquid media depleted of iron, indicating that PilA2 can promote growth in low-iron conditions. However, mutants lacking PilT, PilD, or PilS/PilR did not exhibit this phenotype, implying that PilA2’s newfound role does not involve inclusion into T4P, cleavage/maturation from prepilin to pilin, or the signal transduction pathway that often modulates the expression of pilin, along with other genes. Mutants lacking the FeoB Fe 2+ transporter, LbtC siderophore transporter, or Ccm cytochrome maturation showed even greater defects on low-iron agar media when pilA2 mutations were introduced into them, suggesting that PilA2 functions within a novel iron-uptake pathway. Compatible with this, the pilA2 mutant, but not its complement, exhibited increased resistance to streptonigrin and heightened expression of iron-regulated genes. Finally, although unimpaired for growth within amoebae, the pilA2 mutant displayed increased biofilm formation. Overall, this study reveals a novel role for a type IV pilin in bacterial growth in iron acquisition/assimilation. IMPORTANCE Legionella pneumophila is the main cause of Legionnaires’ disease, an increasingly prevalent pneumonia. Prior studies indicated that L. pneumophila expresses type IV pili, a common form of pili that is mainly known for mediating DNA uptake, twitching motility, and attachment to biotic and abiotic surfaces. By utilizing a variety of in silico and experimental approaches, wehave helped redefine the major structural component (pilin) of the L. pneumophila pili. More significantly, we uncovered a heretofore unknown and seemingly novel role for the pilin in L. pneumophila growth in low-iron conditions and iron acquisition/assimilation. These findings have broad implications for understanding other bacteria and infectious diseases, as well as offering fresh insight into the roles of pilins in bacterial physiology and the mechanisms of iron assimilation.