Actinobacillus pleuropneumoniae iron transport: a set of exbBD genes is transcriptionally linked to the tbpB gene and required for utilization of transferrin-bound iron.
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ABSTRACT:
SUBMITTER: Tonpitak W
PROVIDER: S-EPMC97262 | biostudies-literature | 2000 Mar
REPOSITORIES: biostudies-literature
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Project description:The transferrin-binding proteins TbpA and TbpB enable Neisseria gonorrhoeae to obtain iron from human transferrin. The lipoprotein TbpB facilitates, but is not strictly required for, TbpA-mediated iron acquisition. The goal of the current study was to determine the contribution of two conserved regions within TbpB to the function of this protein. Using site-directed mutagenesis, the first mutation we constructed replaced the lipobox (LSAC) of TbpB with a signal I peptidase cleavage site (LAAA), while the second mutation deleted a conserved stretch of glycine residues immediately downstream of the lipobox. We then evaluated the resulting mutants for effects on TbpB expression, surface exposure, and transferrin iron utilization. Western blot analysis and palmitate labeling indicated that the lipobox, but not the glycine-rich motif, is required for lipidation of TbpB and tethering to the outer membrane. TbpB was released into the supernatant by the mutant that produces TbpB LSAC. Neither mutation disrupted the transport of TbpB across the bacterial cell envelope. When these mutant TbpB proteins were produced in a strain expressing a form of TbpA that requires TbpB for iron acquisition, growth on transferrin was either abrogated or dramatically diminished. We conclude that surface tethering of TbpB is required for optimal performance of the transferrin iron acquisition system, while the presence of the polyglycine stretch near the amino terminus of TbpB contributes significantly to transferrin iron transport function. Overall, these results provide important insights into the functional roles of two conserved motifs of TbpB, enhancing our understanding of this critical iron uptake system.
| S-EPMC3754215 | biostudies-literature
Project description:Urea transporters (UTs) effect rapid flux of urea across biological membranes. In the mammalian kidney, UT activity is essential for effective urine concentration. In bacteria, UT-mediated urea uptake permits intracellular urease to degrade urea to ammonia and CO(2), a process that either buffers acid loads or provides nutrient nitrogen. We have characterized the urea transport channel protein ApUT from Actinobacillus pleuropneumoniae. Kinetic analysis of bacterial inside-out membranes enriched in ApUT showed approximately 28-fold increase in urea permeability (3.3 +/- 0.4 x 10(-4) cm/s) compared with control vesicles (0.11 +/- 0.02 x 10(-4) cm/s). In addition to urea, ApUT also conducts water. Urea and water transport across the channel was phloretin and mercury inhibitable, and the site of inhibition may be located on the cytoplasmic side of the protein. Glycerol and urea analogs, such as methylamine, dimethylurea, formamide, acetamide, methylurea, propanamide, and ethylamine did not permeate across ApUT.
| S-EPMC2698602 | biostudies-literature
Project description:Catecholamines are host stress hormones that can induce the growth of many bacteria by facilitating iron utilization and/or regulate the expression of virulence genes through specific hormone receptors. Whether these two responsive pathways are interconnected is unknown. In our previous study, it was found that catecholamines can regulate the expression of a great number of genes of Actinobacillus pleuropneumoniae, an important swine respiratory pathogen. However, bacterial growth was not affected by catecholamines in rich medium. In this study, it was discovered that catecholamines affected A. pleuropneumoniae growth in chemically defined medium (CDM). We found that serum inhibited A. pleuropneumoniae growth in CDM, while epinephrine, norepinephrine and dopamine promoted A. pleuropneumoniae growth in the CDM containing serum. The known bacterial hormone receptor QseC didn't play roles in this process. Ion-supplementation and transcriptome analysis indicated that serum addition resulted in iron-restricted conditions which were alleviated by the addition of catecholamines. Transferrin, one of the components in serum, inhibited the growth of A. pleuropneumoniae in CDM, an effect reversed by addition of catecholamines in a TonB2-dependent manner. Our data demonstrate that catecholamines promote A. pleuropneumoniae growth by regulating iron-acquisition and metabolism, which is independent of the adrenergic receptor QseC.
| S-EPMC4388731 | biostudies-literature
Project description:BACKGROUND: To better understand effects of iron restriction on Actinobacillus pleuropneumoniae and to identify new potential vaccine targets, we conducted transcript profiling studies using a DNA microarray containing all 2025 ORFs of the genome of A. pleuropneumoniae serotype 5b strain L20. This is the first study involving the use of microarray technology to monitor the transcriptome of A. pleuropneumoniae grown under iron restriction. RESULTS: Upon comparing growth of this pathogen in iron-sufficient versus iron-depleted medium, 210 genes were identified as being differentially expressed. Some genes (92) were identified as being up-regulated; many have confirmed or putative roles in iron acquisition, such as the genes coding for two TonB energy-transducing proteins and the hemoglobin receptor HgbA. Transcript profiling also led to identification of some new iron acquisition systems of A. pleuropneumoniae. Genes coding for a possible Yfe system (yfeABCD), implicated in the acquisition of chelated iron, were detected, as well as genes coding for a putative enterobactin-type siderophore receptor system. ORFs for homologs of the HmbR system of Neisseria meningitidis involved in iron acquisition from hemoglobin were significantly up-regulated. Down-regulated genes included many that encode proteins containing Fe-S clusters or that use heme as a cofactor. Supplementation of the culture medium with exogenous iron re-established the expression level of these genes. CONCLUSION: We have used transcriptional profiling to generate a list of genes showing differential expression during iron restriction. This strategy enabled us to gain a better understanding of the metabolic changes occurring in response to this stress. Many new potential iron acquisition systems were identified, and further studies will have to be conducted to establish their role during iron restriction.
| S-EPMC1832192 | biostudies-literature
Project description:Neisseria gonorrhoeae has the capacity to acquire iron from its human host by removing this essential nutrient from serum transferrin. The transferrin binding proteins, TbpA and TbpB constitute the outer membrane receptor complex responsible for binding transferrin, extracting the tightly bound iron from the host-derived molecule, and transporting iron into the periplasmic space of this Gram-negative bacterium. Once iron is transported across the outer membrane, ferric binding protein A (FbpA) moves the iron across the periplasmic space and initiates the process of transport into the bacterial cytosol. The results of the studies reported here define the multiple steps in the iron transport process in which TbpA and TbpB participate. Using the SUPREX technique for assessing the thermodynamic stability of protein-ligand complexes, we report herein the first direct measurement of periplasmic FbpA binding to the outer membrane protein TbpA. We also show that TbpA discriminates between apo- and holo-FbpA; i.e. the TbpA interaction with apo-FbpA is higher affinity than the TbpA interaction with holo-FbpA. Further, we demonstrate that both TbpA and TbpB individually can deferrate transferrin and ferrate FbpA without energy supplied from TonB resulting in sequestration by apo-FbpA.
| S-EPMC2749328 | biostudies-literature
Project description:Iron acquisition in vivo by Actinobacillus pleuropneumoniae depends upon a functional TonB system. Tonpitak et al. (W. Tonpitak, S. Thiede, W. Oswald, N. Baltes, and G.-F. Gerlach, Infect. Immun. 68:1164-1170, 2000) have described one such system, associated with tbpBA encoding the transferrin receptor, and here we report a second, termed tonB2. This gene cluster (exbB2-exbD2-tonB2) is highly homologous to those in other Pasteurellaceae, unlike the earlier system described (now termed tonB1), suggesting that it is the indigenous system for this organism. Both tonB2 and tonB1 are upregulated upon iron restriction. TonB2, but not TonB1, was found to be essential for growth in vitro when the sole source of iron was hemin, porcine hemoglobin, or ferrichrome. In the case of iron provided as iron-loaded porcine transferrin, neither tonB mutant was viable. The tonB1 phenotype could be explained by a polar effect of the mutation on transcription of downstream tbp genes. We propose that TonB2 is crucial for the acquisition of iron provided in this form, interacting with accessory proteins of the TonB1 system that have been demonstrated to be necessary by Tonpitak et al. TonB2 appears to play a much more important role in A. pleuropneumoniae virulence than TonB1. In an acute porcine infection model, the tonB2 mutant was found to be highly attenuated, while the tonB1 mutant was not. We hypothesize that acquisition of the tonB1-tbp gene cluster confers a biological advantage through its capacity to utilize transferrin-iron but that TonB1 itself plays little or no part in this process.
| S-EPMC321588 | biostudies-literature
Project description:In healthy individuals, virtually all blood plasma iron is bound by transferrin. However, in several diseases and clinical conditions, hazardous non-transferrin-bound iron (NTBI) species occur. NTBI represents a potentially toxic iron form, being a direct cause of oxidative stress in the circulating compartment and tissue iron loading. The accumulation of these species can cause cellular damage in several organs, namely, the liver, spleen, and heart. Despite its pathophysiological relevance, the chemical nature of NTBI remains elusive. This has precluded its use as a clinical biochemical marker and the development of targeted therapies. Herein, we make a critical assessment of the current knowledge of NTBI speciation. The currently accepted hypotheses suggest that NTBI is mostly iron bound to citric acid and iron bound to serum albumin, but the chemistry of this system remains fuzzy. We explore the complex chemistry of iron complexation by citric acid and its implications towards NTBI reactivity. Further, the ability of albumin to bind iron is revised and the role of protein post-translational modifications on iron binding is discussed. The characterization of the NTBI species structure may be the starting point for the development of a standardized analytical assay, the better understanding of these species' reactivity or the identification of NTBI uptake mechanisms by different cell types, and finally, to the development of new therapies.
| S-EPMC8951307 | biostudies-literature
Project description:The swine pathogen Actinobacillus pleuropneumoniae possesses a 75-kDa outer membrane protein (OMP), FhuA, the receptor for ferrichrome, a hydroxamate-type siderophore. Polyclonal serum to FhuA reacted with OMP preparations from 12 serotypes of A. pleuropneumoniae under conditions of iron repletion and restriction. Reverse transcription-PCR confirmed that A. pleuropneumoniae fhuA expression is not upregulated in response to low iron levels. An A. pleuropneumoniae fhuA deletion mutant was generated and showed abolishment of ferrichrome uptake.
| S-EPMC153255 | biostudies-literature
Project description:The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumoniae, a lethal respiratory infectious disease causing great economic losses in the swine industry worldwide. In order to better interpret the genetic background of serotypic diversity, nine genomes of A. pleuropneumoniae reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12, and 13 were sequenced by using rapid high-throughput approach. Based on 12 genomes of corresponding serovar reference strains including three publicly available complete genomes (serovars 3, 5b, and 7) of this bacterium, we performed a comprehensive analysis of comparative genomics and first reported a global genomic characterization for this pathogen. Clustering of 26,012 predicted protein-coding genes showed that the pan genome of A. pleuropneumoniae consists of 3,303 gene clusters, which contain 1,709 core genome genes, 822 distributed genes, and 772 strain-specific genes. The genome components involved in the biogenesis of capsular polysaccharide and lipopolysaccharide O antigen relative to serovar diversity were compared, and their genetic diversity was depicted. Our findings shed more light on genomic features associated with serovar diversity of A. pleuropneumoniae and provide broader insight into both pathogenesis research and clinical/epidemiological application against the severe disease caused by this swine pathogen.
| S-EPMC2953695 | biostudies-literature
Project description:Actinobacillus pleuropneumoniae has been considered nonmotile and nonflagellate. In this work, it is demonstrated that A. pleuropneumoniae produces flagella composed of a 65-kDa protein with an N-terminal amino acid sequence that shows 100% identity with those of Escherichia coli, Salmonella, and Shigella flagellins. The DNA sequence obtained through PCR of the fliC gene in A. pleuropneumoniae showed considerable identity (93%) in its 5' and 3' ends with the DNA sequences of corresponding genes in E. coli, Salmonella enterica, and Shigella spp. The motility of A. pleuropneumoniae was observed in tryptic soy or brain heart infusion soft agar media, and it is influenced by temperature. Flagella and motility may be involved in the survival and pathogenesis of A. pleuropneumoniae in pigs.
| S-EPMC145319 | biostudies-literature