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Riemerella anatipestifer is an important bacterial pathogen in ducks and causes heavy economic losses in the duck industry. However, the pathogensis of this bacterium is poorly understood. In this study, a putative outer membrane hemin receptor gene B739&#95;1208 in R. anatipestifer CH-1 was deleted to determine the relationship between iron uptake and virulence. The R. anatipestifer CH-1ΔB739&#95;1208 mutants grew significantly more slowly than the wild-type bacteria in TSB liquid medium. Further characterization revealed that the R. anatipestifer CH-1ΔB739&#95;1208 mutants were deficient in iron uptake. Animal experiments indicated that the median lethal dose of the wild-type RA-CH-1 in ducklings was 3.89×10 8 , whereas the median lethal dose of the R. anatipestifer CH-1ΔB739&#95;1208 mutant in ducklings was 5.68×10 9 . The median lethal dose of the complementation strain in ducklings was 9.84×10 8 . Additional analysis indicated that bacterial loads in the blood, liver, and brain tissues in the R. anatipestifer CH-1ΔB739&#95;1208-infected ducklings were significantly decreased compared to those in the wild-type R. anatipestifer CH-1 infected ducklings. In a duck co-infection model with R. anatipestifer CH-1 and R. anatipestifer CH-1ΔB739&#95;1208, the R. anatipestifer CH-1B739&#95;1208 mutant was outcompeted by the wild-type R. anatipestifer CH-1 in the blood (P<0.002), livers (P<0.001) and brains (P<0.001) of infected ducks, indicating that B739&#95;1208 gene expression provided a competitive advantage in these organs. Our results demonstrate that the B739&#95;1208 gene is a virulence factor in R. anatipestifer CH-1., (Copyright © 2017 Elsevier B.V. All rights reserved.)

One of the important elements for most bacterial growth is iron, the bioavailability of which is limited in hosts. Riemerella anatipestifer ( R. anatipestifer , RA), an important duck pathogen, requires iron to live. However, the genes involved in iron metabolism and the mechanisms of iron transport are largely unknown. Here, we investigated the transcriptomic effects of iron limitation condition on R. anatipestifer CH-1 using the RNA-Seq and RNA-Seq-based analysis. Data analysis revealed genes encoding functions related to iron homeostasis, including a number of putative TonB-dependent receptor systems, a HmuY-like protein-dependent hemin (an iron-containing porphyrin) uptake system, a Feo system, a gene cluster related to starch utilization, and genes encoding hypothetical proteins that were significantly upregulated in response to iron limitation. Compared to the number of upregulated genes, more genes were significantly downregulated in response to iron limitation. The downregulated genes mainly encoded a number of outer membrane receptors, DNA-binding proteins, phage-related proteins, and many hypothetical proteins. This information suggested that RNA-Seq-based analysis in iron-limited medium is an effective and fast method for identifying genes involved in iron uptake in R. anatipestifer CH-1.

Riemerella anatipestifer (R. anatipestifer) is one of the most important pathogens in ducks. The bacteria causes acute or chronic septicemia characterized by fibrinous pericarditis and meningitis. The R. anatipestifer genome encodes multiple iron/hemin-uptake systems that facilitate adaptation to iron-limited host environments. These systems include several TonB-dependent transporters and three TonB proteins responsible for energy transduction. These three tonB genes are present in all the R. anatipestifer genomes sequenced so far. Two of these genes are contained within the exbB-exbD-tonB1 and exbB-exbD-exbD-tonB2 operons. The third, tonB3, forms a monocistronic transcription unit. The inability to recover derivatives deleted for this gene suggests its product is essential for R. anatipestifer growth. Here, we show that deletion of tonB1 had no effect on hemin uptake of R. anatipestifer, though disruption of tonB2 strongly decreases hemin uptake, and disruption of both tonB1 and tonB2 abolishes the transport of exogenously added hemin. The ability of R. anatipestifer to grow on iron-depleted medium is decreased by tonB2 but not tonB1 disruption. When expressed in an E. coli model strain, the TonB1 complex, TonB2 complex, and TonB3 protein from R. anatipestifer cannot energize heterologous hemin transporters. Further, only the TonB1 complex can energize a R. anatipestifer hemin transporter when co-expressed in an E. coli model strain.

The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival.

Bartonella are hemotropic bacteria responsible for emerging zoonoses. Most Bartonella species appear to share a natural cycle that involves an arthropod transmission, followed by exploitation of a mammalian host in which they cause long-lasting intra-erythrocytic bacteremia. Persistence in erythrocytes is considered an adaptation to transmission by bloodsucking arthropod vectors and a strategy to obtain heme required for Bartonella growth. Bartonella genomes do not encode for siderophore biosynthesis or a complete iron Fe(3+) transport system. Only genes, sharing strong homology with all components of a Fe(2+) transport system, are present in Bartonella genomes. Also, Bartonella genomes encode for a complete heme transport system. Bartonella must face various environments in their hosts and vectors. In mammals, free heme and iron are rare and oxygen concentration is low. In arthropod vectors, toxic heme levels are found in the gut where oxygen concentration is high. Bartonella genomes encode for 3-5 heme-binding proteins. In Bartonella henselae heme-binding proteins were shown to be involved in heme uptake process, oxidative stress response, and survival inside endothelial cells and in the flea. In this report, we discuss the use of the heme uptake and storage system of B. henselae during its infection cycle. Also, we establish a comparison with the iron and heme uptake systems of Yersinia pestis used during its infection cycle.

Serratia marcescens, like several other Gram-negative bacteria, possesses two functional haem uptake systems. The first, referred to as the Hem system, can transport haem present at a concentration equal to or above 10(-6) M. It requires an active outer-membrane receptor which uses proton-motive force energy transmitted by the inner-membrane TonB protein. The other system, Has, takes up haem at lower concentrations and utilizes a small secreted haem-binding protein (haemophore) and its cognate TonB-dependent outer-membrane receptor HasR. Various combinations of mutations were used to examine haem uptake activity by the two systems in S. marcescens. The Hem uptake system enables S. marcescens to take up haem at a concentration of 10(-6) M in the presence of various levels of iron depletion. The Has system, which enables such uptake even in the presence of lower haem concentrations, requires higher iron depletion conditions for function. Has haem uptake requires the presence of HasB, a TonB paralogue encoded by the has operon. These two systems enable S. marcescens to take up haem under various conditions from different sources, reflecting its capacity to confront conditions encountered in natural biotopes.

Exogenous inorganic pyrophosphate increases the biomass yield of Escherichia coli. In this report, we show that the effect of pyrophosphate is related to iron uptake. We have found that addition of pyrophosphate, ammonium iron (III) citrate or iron (III) chloride, in M63 minimal medium containing 1.7 microM of iron, causes an increase in growth yield. In contrast to iron chloride or ammonium iron (III) citrate, exogenous pyrophosphate is deleterious to strains unable to synthesize enterobactin. Thus the positive effect of pyrophosphate is related to the enterobactin uptake system expressed in a low iron content medium. Pyrophosphate in minimal medium has a repressing effect on the expression of Fur-regulated genes. In iron rich medium where enterobactin synthesis is strongly decreased, addition of pyrophosphate increases expression of Fur-regulated genes. Furthermore, this latter regulatory effect of pyrophosphate in iron-rich medium is enhanced in the absence of enterobactin synthesis. It has also been shown that addition of pyrophosphate protects the cell against the oxidative stress caused by the presence of hydrogen peroxide in an iron-rich containing medium. These results indicate that pyrophosphate acts as an iron-chelating agent, could trigger the enterobactin-dependent iron uptake system and could promote an increased binding of iron to enterobactin.