A novel siderophore-independent strategy of iron uptake in the genus Burkholderia.

Like many other bacteria, Burkholderia sp. take up iron in its ferric form via siderophore-dependent transporters. We observed that mutant strains of B. cenocepacia H111 unable to synthesize siderophores did not exhibit any growth defect under iron limited conditions. This finding suggested that this opportunistic pathogen can adopt an alternative iron uptake strategy to compensate for the loss of siderophores. We identified a putative iron uptake locus, ftrBcc ABCD, in the genome of B. cenocepacia H111, which is also conserved in other members of the genus Burkholderia. Mutants deficient in both siderophore-dependent and FtrBcc ABCD systems failed to grow under iron-limited conditions and radiolabelled iron transport assays showed that these mutants were impaired in iron uptake. In addition, expression of ftrBcc ABCD can restore growth of an E. coli strain lacking all known high-affinity iron transport systems under iron-limited conditions. We show that all four proteins encoded by ftrBcc ABCD are essential for iron uptake. Furthermore, our results indicate that the expression of ftrBcc ABCD is regulated at the transcriptional level by iron concentration. This study provides evidence of an alternative, siderophore-independent, iron uptake system in Burkholderia species.

A novel siderophore system is essential for the growth of Pseudomonas aeruginosa in airway mucus.

Pseudomonas aeruginosa establishes airway infections in Cystic Fibrosis patients. Here, we investigate the molecular interactions between P. aeruginosa and airway mucus secretions (AMS) derived from the primary cultures of normal human tracheal epithelial (NHTE) cells. PAO1, a prototype strain of P. aeruginosa, was capable of proliferating during incubation with AMS, while all other tested bacterial species perished. A PAO1 mutant lacking PA4834 gene became susceptible to AMS treatment. The ΔPA4834 mutant was grown in AMS supplemented with 100 µM ferric iron, suggesting that the PA4834 gene product is involved in iron metabolism. Consistently, intracellular iron content was decreased in the mutant, but not in PAO1 after the AMS treatment. Importantly, a PAO1 mutant unable to produce both pyoverdine and pyochelin remained viable, suggesting that these two major siderophore molecules are dispensable for maintaining viability during incubation with AMS. The ΔPA4834 mutant was regrown in AMS amended with 100 µM nicotianamine, a phytosiderophore whose production is predicted to be mediated by the PA4836 gene. Infectivity of the ΔPA4834 mutant was also significantly compromised in vivo. Together, our results identify a genetic element encoding a novel iron acquisition system that plays a previously undiscovered role in P. aeruginosa airway infection.

Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system.

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.

Biochemical characterization of a Haemophilus influenzae periplasmic iron transport operon.

Bacterial iron transport is critical for growth of pathogens in the host environment, where iron is limited as a form of nonspecific immunity. For Gram-negative bacteria such as Haemophilus influenzae, iron first must be transported across the outer membrane and into the periplasmic space, then from the periplasm to the cytosol. H. influenzae express a periplasmic iron-binding protein encoded by the hitA gene. This gene is organized as the first of a three-gene operon purported to encode a classic high affinity iron acquisition system that includes hitA, a cytoplasmic permease (hitB), and a nucleotide binding protein (hitC). In this study we describe the cloning, overexpression, and purification of the H. influenzae hitA gene product. The function of this protein is unambiguously assigned by demonstrating its ability to compete for iron bound to the chemical iron chelator 2,2'-dipyridyl, both in vitro and within the periplasmic space of a siderophore-deficient strain of Escherichia coli. Finally, the importance of a functional hitABC operon for iron acquisition is demonstrated by complementation of this siderophore-deficient E. coli to growth on dipyridyl-containing medium. These studies represent a detailed genetic, biochemical, and physiologic description of an active transport system that has evolved to efficiently transport iron and consequently is widely distributed among Gram-negative pathogenic bacteria.