In the ancient anaerobic environment, ferrous iron (Fe2+) was one of the first metal cofactors. Oxygenation of the ancient world challenged bacteria to acquire the insoluble ferric iron (Fe3+) and later to defend against reactive oxygen species (ROS) generated by the Fenton chemistry. To acquire Fe3+, bacteria produce low-molecular weight compounds, known as siderophores, which have extremely high affinity for Fe3+. However, during infection the host restricts iron from pathogens by producing iron- and siderophore-chelating proteins, by exporting iron from intracellular pathogen-containing compartments, and by limiting absorption of dietary iron. Ferric Uptake Regulator (Fur) is a transcription factor which utilizes Fe2+ as a corepressor and represses siderophore synthesis in pathogens. Fur, directly or indirectly, controls expression of enzymes that protect against ROS damage. Thus, the challenges of iron homeostasis and defense against ROS are addressed via Fur. Although the role of Fur as a repressor is well documented, emerging evidence demonstrates that Fur can function as an activator. Fur activation can occur through three distinct mechanisms 1) indirectly via small RNAs, 2) binding at cis regulatory elements that enhance recruitment of the RNA polymerase holoenzyme (RNAP), and 3) functioning as an antirepressor by removing or blocking DNA binding of a repressor of transcription. In addition, Fur homologs control defense against peroxide stress (PerR) and control uptake of other metals such as zinc (Zur) and manganese (Mur) in pathogenic bacteria. Fur family members are important for virulence within bacterial pathogens since mutants of fur, perR, or zur exhibit reduced virulence within numerous animal and plant models of infection. This review focuses on the breadth of Fur regulation in pathogenic bacteria.

Salmonella entericaserovar Typhimurium must successfully transition the broad fluctuations in oxygen concentrations encountered in the host. InEscherichia coli, FNR is one of the main regulatory proteins involved in O₂sensing. To assess the role of FNR in serovar Typhimurium, we constructed an isogenicfnrmutant in the virulent wild-type strain (ATCC 14028s) and compared their transcriptional profiles and pathogenicities in mice. Here, we report that, under anaerobic conditions, 311 genes (6.80% of the genome) are regulated directly or indirectly by FNR; of these, 87 genes (28%) are poorly characterized. Regulation by FNR in serovar Typhimurium is similar to, but distinct from, that inE. coli.Thus, genes/operons involved in aerobic metabolism, NO· detoxification, flagellar biosynthesis, motility, chemotaxis, and anaerobic carbon utilization are regulated by FNR in a fashion similar to that inE. coli. However, genes/operons existing inE. colibut regulated by FNR only in serovar Typhimurium include those coding for ethanolamine utilization, a universal stress protein, a ferritin-like protein, and a phosphotransacetylase. Interestingly,Salmonella-specific genes/operons regulated by FNR include numerous virulence genes withinSalmonellapathogenicity island 1 (SPI-1), newly identified flagellar genes (mcpAC,cheV), and the virulence operon (srfABC). Furthermore, the role of FNR as a positive regulator of motility, flagellar biosynthesis, and pathogenesis was confirmed by showing that the mutant is nonmotile, lacks flagella, is attenuated in mice, and does not survive inside macrophages. The inability of the mutant to survive inside macrophages is likely due to its sensitivity to the reactive oxygen species generated by NADPH phagocyte oxidase.

Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogen's life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogen's survival in its vector host. The Lyme disease pathogen Borrelia burgdorferi has two sets of two-component systems, Hk1-Rrp1 and Hk2-Rrp2. The Hk2-Rrp2 signaling system has been shown to modulate differential expression of numerous surface lipoprotein genes and to play an essential role in spirochete transformation from a tick colonizer to a mammalian host-adapted state. In this study, we show that Rrp1, the only diguanylate cyclase in B. burgdorferi, is not required for mammalian infection but is essential for spirochete survival in the tick vector. We identify over 39 genes whose expression is influenced by this c-di-GMP signaling system. We further demonstrate that one set of the Rrp1-dependent genes, the glp operon for glycerol transport and metabolism, plays an important role in the spirochete adaptation to tick environment and partially accounts for the essentiality of c-di-GMP for B. burgdorferi survival in ticks.

Cyclic di-AMP (c-di-AMP) is a recently discovered second messenger in bacteria. Most of work on c-di-AMP signaling has been done in Gram-positive bacteria, firmicutes, and actinobacteria, where c-di-AMP signaling pathways affect potassium transport, cell wall structure, and antibiotic resistance. Little is known about c-di-AMP signaling in other bacteria.Borrelia burgdorferi, the causative agent of Lyme disease, is a spirochete that has a Gram-negative dual membrane. In this study, we demonstrated thatB. burgdorferiBB0619, aDHH-DHHA1 domainprotein (herein designated DhhP), functions as c-di-AMP phosphodiesterase. Recombinant DhhP hydrolyzed c-di-AMP to pApA in a Mn²⁺- or Mg²⁺-dependent manner. In contrast to c-di-AMP phosphodiesterases reported thus far, DhhP appears to be essential forB. burgdorferigrowth bothin vitroand in the mammalian host. Inactivation of the chromosomaldhhPgene could be achieved only in the presence of a plasmid-encoded inducibledhhPgene. The conditionaldhhPmutant had a dramatic increase in intracellular c-di-AMP level in comparison to the isogenic wild-type strain. Unlike what has been observed in Gram-positive bacteria, elevated cellular c-di-AMP inB. burgdorferidid not result in an increased resistance to β-lactamase antibiotics, suggesting that c-di-AMP's functions in spirochetes differ from those in Gram-positive bacteria. In addition, thedhhPmutant was defective in induction of the σ^Sfactor, RpoS, and the RpoS-dependent outer membrane virulence factor OspC, which uncovers an important role of c-di-AMP inB. burgdorferivirulence.

Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella , the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA , as well as of other HilA-dependent genes, invF and sipC . The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA . Instead, the expression of the gene coding for the histone-like protein, hns , was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns . Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo , to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA . In conclusion, Fur activates hilA by repressing the expression of hns .

Outer surface protein C (OspC) is one of the major lipoproteins expressed on the surface ofBorrelia burgdorferiduring tick feeding and the early phase of mammalian infection. OspC is required forB. burgdorferito establish infection in both immunocompetent and SCID mice and has been proposed to facilitate evasion of innate immune defenses. However, the exact biological function of OspC remains elusive. In this study, we showed that theospC-deficient spirochete could not establish infection in NOD-scid IL2rγ^nullmice that lack B cells, T cells, NK cells, and lytic complement. TheospCmutant also could not establish infection in anti-Ly6G-treated SCID and C3H/HeN mice (depletion of neutrophils). However, depletion of mononuclear phagocytes at the skin site of inoculation in SCID and C3H/HeN mice allowed theospCmutant to establish infectionin vivo. In phagocyte-depleted mice, theospCmutant was able to colonize the joints and triggered neutrophilia during dissemination. Furthermore, we found that phagocytosis of green fluorescent protein (GFP)-expressingospCmutant spirochetes by murine peritoneal macrophages and human THP-1 macrophage-like cells, but not in PMN-HL60, was significantly higher than parental wild-typeB. burgdorferistrains, suggesting that OspC has an antiphagocytic property. In addition, overproduction of OspC in spirochetes also decreased the uptake of spirochetes by murine peritoneal macrophages. Together, our findings provide evidence that mononuclear phagocytes play a key role in clearance of theospCmutant and that OspC promotes spirochetes' evasion of macrophages during early Lyme borreliosis.