Iron acquisition from the host is an important step in the pathogenic process. While Yersinia pestis has multiple iron transporters, the yersiniabactin (Ybt) siderophore-dependent system plays a major role in iron acquisition in vitro and in vivo. In this study, we determined that the Ybt system is required for the use of iron bound by transferrin and lactoferrin and examined the importance of the Ybt system for virulence in mouse models of bubonic and pneumonic plague. Y. pestis mutants unable to either transport Ybt or synthesize the siderophore were both essentially avirulent via subcutaneous injection (bubonic plague model). Surprisingly, via intranasal instillation (pneumonic plague model), we saw a difference in the virulence of Ybt biosynthetic and transport mutants. Ybt biosynthetic mutants displayed an ϳ24-fold-higher 50% lethal dose (LD 50 ) than transport mutants. In contrast, under iron-restricted conditions in vitro, a Ybt transport mutant had a more severe growth defect than the Ybt biosynthetic mutant. Finally, a ⌬pgm mutant had a greater loss of virulence than the Ybt biosynthetic mutant, indicating that the 102-kb pgm locus encodes a virulence factor, in addition to Ybt, that plays a role in the pathogenesis of pneumonic plague.Nearly all organisms require trace amounts of iron. Pathogens must overcome host iron-and heme-binding proteins to cause an infection and disease. The importance of iron acquisition mechanisms has been demonstrated in a number of bacterial pathogens (14,15,27,32,85). Yersinia pestis, the causative agent of plague, has a number of proven and putative iron and heme transport systems. Of these systems, the yersiniabactin (Ybt) siderophore-dependent iron transport system plays a major role in the virulence of bubonic plague in mice (7,8,38,76,79).All identified genes required for the regulation, synthesis, and transport of Ybt, except for ybtD, are carried within a high-pathogenicity island (HPI) that has been spread among enteric pathogens but is essentially identical in the pathogenic yersiniae (13,59,79). In Y. pestis, the ϳ36-kb HPI is located within the 102-kb pgm locus; the entire pgm locus undergoes spontaneous deletion in vitro at a frequency of about 10 Ϫ5 (25, 39, 62). The Ybt system produces a siderophore composed of one salicylate, one thiazoline, and two thiazolidine rings via a nonribosomal peptide/polyketide synthesis mechanism involving high-molecular-weight protein 1 (HMWP1), HMWP2, YbtD, YbtT, YbtE, YbtU, and YbtS (76, 79, 94). The formation constant of this siderophore with ferric iron is 4 ϫ 10 36 , and the crystal structure of the ferric complex has been solved (68, 78).Iron from the Ybt-Fe complex is transported into the cell via the TonB-dependent outer membrane (OM) receptor Psn (which is also required for sensitivity to the bacteriocin pesticin) and an ABC transporter consisting of two inner membrane (IM), fused-function permease/ATP-binding proteins, YbtP and YbtQ. A mutation in any of these three genes prevents Ybt-dependent uptake of iron but does ...

Little is known about Zn homeostasis inZinc (Zn) is an essential trace metal required to preserve the biological function and/or structural integrity of numerous enzymes and proteins in all eukaryotic and prokaryotic cells (3,49,99). Procuring sufficient Zn to sustain growth during mammalian infection is a considerable challenge for bacterial pathogens (56). Serum levels of Zn are in the micromolar range, and the metal's bioavailability is restricted further because it is tightly bound to proteins and not freely exchangeable (35,85,88,103). In addition, as with iron (Fe), mammals sequester Zn systemically and locally in an attempt to deprive invading pathogens of this critical micronutrient (35,85,88,103). Bacteria, therefore, must depend upon the expression of high-affinity Zn uptake systems to compete successfully with the mammalian host for this metal. Although Zn is essential, high concentrations are toxic because of its proclivity to occupy ligand sites intended for other transition metals, such as Mn and Fe (39); consequently, bacteria must strictly control intracellular Zn levels to avoid disruption of physiological processes (49). Two major mechanisms by which Zn homeostasis is achieved are metal effluxers and regulation of Zn uptake systems.The discovery of the cluster 9 (C9) family of transition metal ATP-binding cassette (ABC) transporters significantly advanced our understanding of bacterial Zn metabolism (19). The function of the C9 family was revealed primarily through genetic studies in which the growth defects of mutants under metal-limiting conditions were reversed by supplementation with Zn (27,77) or Mn (8,27,59). Bioinformatic analyses of the C9 solute-binding protein (SBP) components, which capture metals within the periplasmic space and ferry them to the cytoplasmic membrane-bound permease complex, revealed a bimodal clustering pattern appearing to correlate with experimentally proven metal specificities (19). One subcluster con-* Corresponding author. Mailing address:

The gene designated BAB1_1460 in the Brucella abortus 2308 genome sequence is predicted to encode the manganese transporter MntH. Phenotypic analysis of an isogenic mntH mutant indicates that MntH is the sole high-affinity manganese transporter in this bacterium but that MntH does not play a detectable role in the transport of Fe 2؉ , Zn 2؉ , Co 2؉ , or Ni 2؉ . Consistent with the apparent selectivity of the corresponding gene product, the expression of the mntH gene in B. abortus 2308 is repressed by Mn 2؉ , but not Fe 2؉ , and this Mn-responsive expression is mediated by a Mur-like repressor. The B. abortus mntH mutant MWV15 exhibits increased susceptibility to oxidative killing in vitro compared to strain 2308, and a comparative analysis of the superoxide dismutase activities present in these two strains indicates that the parental strain requires MntH in order to make wild-type levels of its manganese superoxide dismutase SodA. The B. abortus mntH mutant also exhibits extreme attenuation in both cultured murine macrophages and experimentally infected C57BL/6 mice. These experimental findings indicate that Mn 2؉ transport mediated by MntH plays an important role in the physiology of B. abortus 2308, particularly during its intracellular survival and replication in the host.Brucella abortus is a gram-negative bacterium that is responsible for the zoonotic disease brucellosis. Brucellosis causes spontaneous abortion and sterility in ruminants (27) and a debilitating febrile illness in humans known as undulant fever (17). The ability of brucellae to cause disease is directly related to their capacity to establish and maintain intracellular infection in host macrophages (63). Within the phagosomal compartment in these host cells, brucellae must cope with oxidative stress, low pH, and nutrient deprivation. The availability of metal ions is restricted within this environment due in part to the activity of the host natural resistance-associated macrophage protein (NRAMP-1), which transports divalent cations out of the phagosome (40). Mn 2ϩ serves as an important cofactor for a variety of bacterial enzymes, including those involved in carbon metabolism, induction of the stringent response, and detoxification of reactive oxygen species (ROS) (55). Consequently, the inability of brucellae to acquire sufficient levels of this divalent cation may compromise their ability to successfully adapt to the environmental conditions encountered during residence in their intracellular niche.Manganese uptake by bacteria is typically accomplished through the activity of either ABC-type transporters such as the SitABC complex (4, 42, 59, 65) or H ϩ -dependent manganese transporters such as MntH (37, 41, 52, 60). Many bacteria possess both types of Mn 2ϩ transporters (55), but a survey of the publicly available Brucella genome sequences (14,20,36,57) suggests that these bacteria do not produce a SitABC-type transporter and rely solely on an MntH homolog for the highaffinity transport of Mn 2ϩ . Escherichia coli MntH was originally des...

Although Yersinia pestis epidemic biovars and Yersinia pseudotuberculosis are recently diverged, highly related species, they cause different diseases via disparate transmission routes. Since iron transport systems are important for iron acquisition from hosts and for survival in the environment, we have analyzed potential iron transport systems encoded by epidemic and non-epidemic or endemic strains of Y. pestis as well as two virulent Y. pseudotuberculosis strains. Computational biology analysis of these genomes showed a high degree of identity/similarity among 16 proven or possible iron/heme transporters identified. Of these, 7 systems were essentially the same in all seven genomes analyzed. The remaining 9 loci had 2-6 genetic variations among these genomes. Two untested, potential siderophore-dependent systems appear intact in Y. pseudotuberculosis but are disrupted or absent in all the endemic Y. pestis strains as well as the epidemic strains from the antiqua and mediaevalis biovars. Only one of these two loci are obviously disrupted in Y. pestis CO92 (epidemic orientalis biovar). Experimental studies failed to identify a role for hemin uptake systems in the virulence of pneumonic plague and suggest that Y. pestis CO92 does not make a siderophore other than Ybt.

The gene annotated BAB2_1150 in the Brucella abortus 2308 genome sequence is predicted to encode a homolog of the well-characterized heme transporter ShuA of Shigella dysenteriae and accordingly has been given the designation bhuA (Brucella heme utilization). Phenotypic analysis of an isogenic bhuA mutant derived from B. abortus 2308 verified that there is a link between BhuA and the ability of the parent strain to use heme as an iron source in in vitro assays. Maximum expression of bhuA in B. abortus 2308 is observed during stationary phase when this strain in cultivated in low-iron minimal medium, and a comparison of the growth characteristics of the B. abortus bhuA mutant and 2308 in this medium suggested that heme serves as an important iron source for the parent strain during stationary phase. The B. abortus bhuA mutant HR1703 exhibits significant attenuation in cultured murine macrophages compared to strain 2308, and unlike its parent strain, the B. abortus bhuA mutant is unable to maintain a chronic spleen infection in experimentally infected BALB/c mice. These experimental findings suggest that heme and/or heme-containing proteins represent important iron sources for B. abortus 2308 during its residence in the mammalian host and that BhuA is required for efficient utilization of these iron sources.The capacity of a bacterium to acquire sufficient amounts of iron is almost always linked to its ability to survive in its biological niche. Accomplishing this in the iron-restricted environment of a mammalian host represents a major virulence determinant in the case of bacterial pathogens (41). Indeed, pathogens often employ specialized transport machinery to counteract the iron restriction associated with the host innate immune defense.Brucella abortus is a gram-negative zoonotic pathogen that causes abortion and infertility in ruminants and a chronic debilitating disease known as undulant fever in humans (1). Survival and replication of the brucellae within host macrophages are critical for virulence (38). These bacteria produce the monocatechol 2,3-dihydroxybenzoic acid (2,3-DHBA) (22) and a more complex 2,3-DHBA-based compound known as brucebactin (18) in response to iron limitation in vitro. Experimental evidence indicates that both of these compounds function as siderophores. Biochemical studies (23) and genome analysis (39) have also suggested that these are the only siderophores produced by the brucellae. Neither 2,3-DHBA nor brucebactin is required for the survival and replication of B. abortus 2308 in cultured murine macrophages or for wild-type virulence of this strain in experimentally infected BALB/c or C57BL6 mice (4, 18, 31). Consequently, the question of what iron source(s) the brucellae use during their prolonged residence in host macrophages during the chronic stage of infection remains unanswered (39).Brucella melitensis 16M and B. abortus 2308 have been shown to utilize heme as an iron source in vitro (5, 11). Due to the central role of macrophages in heme recycling in mammals (10, 45) heme...