scholarly journals Gonococcal Genes Encoding Transferrin-Binding Proteins A and B Are Arranged in a Bicistronic Operon but Are Subject to Differential Expression

2001 ◽  
Vol 69 (10) ◽  
pp. 6336-6347 ◽  
Author(s):  
Chalinee Ronpirin ◽  
Ann E. Jerse ◽  
Cynthia Nau Cornelissen
Keyword(s):  
Binding Proteins ◽  
Iron Acquisition ◽  
Sole Source ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Upstream Promoter ◽  
Genes Encoding ◽  
Transferrin Binding Proteins ◽  
Iron Binding Proteins ◽  
Fusion Analysis

ABSTRACT Neisseria gonorrhoeae is capable of utilizing host iron-binding proteins, such as transferrin, lactoferrin, and hemoglobin, as the sole source of iron. The receptor involved in transferrin iron acquisition is composed of two distinct transferrin-binding proteins, TbpA and TbpB. The genes that encode these proteins are linked on the chromosome in the ordertbpB-tbpA but are separated by an inverted repeat of unknown function. In this study, we sought to understand the transcriptional organization and regulation of thetbp genes, using a combination of lacZtranscriptional fusion analysis and reverse transcriptase PCR (RT-PCR). First, we demonstrated that tbpB and tbpAare cotranscribed and coregulated from the common upstream promoter that precedes tbpB. Using β-galactosidase activity as a surrogate for tbp-specific transcription, we found that tbpB-specific transcripts were more prevalent thantbpA-specific transcripts after 2 h of growth under iron stress conditions. We confirmed the results obtained by fusion analysis by using RT-PCR applied to native RNA isolated from wild-type gonococci. Three different varieties of RT-PCR were employed: relative, competitive, and real time quantitative. The results of all analyses indicated that tbpB-specific transcripts were approximately twofold more prevalent than tbpA-specific transcripts at steady state. In iron-stressed cultures, the ratio oftbpB- to tbpA-specific message was approximately 2; however, in iron-replete cultures, this ratio dropped to 1. Using these techniques, we also quantitated the effects of iron, external pH, and presence of ligand on tbp mRNA levels.

Transferrin-mediated iron acquisition by pathogenic Neisseria

2002 ◽  
Vol 30 (4) ◽  
pp. 705-707 ◽  
Author(s):  
R. W. Evans ◽  
J. S. Oakhill
Keyword(s):  
Bacterial Cell ◽  
Iron Uptake ◽  
Binding Proteins ◽  
Current Knowledge ◽  
Iron Acquisition ◽  
Direct Interaction ◽  
Acquisition System ◽  
Uptake System ◽  
Short Account ◽  
Transferrin Binding Proteins

The pathogenic Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and transferrin. In the meningococcus this uptake system is dependent on two surface-exposed transferrin-binding proteins. This short account will review our current knowledge of the transferrin-mediated iron-acquisition system of pathogenic Neisseria.

scholarly journals Identification of TbpA Residues Required for Transferrin-Iron Utilization by Neisseria gonorrhoeae

2008 ◽  
Vol 76 (5) ◽  
pp. 1960-1969 ◽  
Author(s):  
Jennifer M. Noto ◽  
Cynthia Nau Cornelissen
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Iron Uptake ◽  
Binding Proteins ◽  
Iron Acquisition ◽  
Uptake System ◽  
Wild Type ◽  
Conserved Motif ◽  
Alanine Substitution ◽  
Transferrin Binding Proteins ◽  
Iron Utilization

ABSTRACT Neisseria gonorrhoeae requires iron for survival in the human host and therefore expresses high-affinity receptors for iron acquisition from host iron-binding proteins. The gonococcal transferrin-iron uptake system is composed of two transferrin binding proteins, TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter critical for iron acquisition, while TbpB is a surface-exposed lipoprotein that increases the efficiency of iron uptake. The precise mechanism by which TbpA mediates iron acquisition has not been elucidated; however, the process is distinct from those of characterized siderophore transporters. Similar to these TonB-dependent transporters, TbpA is proposed to have two distinct domains, a β-barrel and a plug domain. We hypothesize that the TbpA plug coordinates iron and therefore potentially functions in multiple steps of transferrin-mediated iron acquisition. To test this hypothesis, we targeted a conserved motif within the TbpA plug domain and generated single, double, and triple alanine substitution mutants. Mutagenized TbpAs were expressed on the gonococcal cell surface and maintained wild-type transferrin binding affinity. Single alanine substitution mutants internalized iron at wild-type levels, while the double and triple mutants showed a significant decrease in iron uptake. Moreover, the triple alanine substitution mutant was unable to grow on transferrin as a sole iron source; however, expression of TbpB compensated for this defect. These data indicate that the conserved motif between residues 120 and 122 of the TbpA plug domain is critical for transferrin-iron utilization, suggesting that this region plays a role in iron acquisition that is shared by both TbpA and TbpB.

scholarly journals Antigenic and Sequence Diversity in Gonococcal Transferrin-Binding Protein A

2000 ◽  
Vol 68 (8) ◽  
pp. 4725-4735 ◽  
Author(s):  
Cynthia Nau Cornelissen ◽  
James E. Anderson ◽  
Ian C. Boulton ◽  
P. Frederick Sparling
Keyword(s):  
Synthetic Peptides ◽  
Pathogenic Bacteria ◽  
Binding Proteins ◽  
Specific Binding ◽  
Protein A ◽  
Sequence Diversity ◽  
Western Blots ◽  
Gram Negative ◽  
Genes Encoding ◽  
Transferrin Binding Proteins

ABSTRACT Neisseria gonorrhoeae is a gram-negative pathogen that is capable of satisfying its iron requirement with human iron-binding proteins such as transferrin and lactoferrin. Transferrin-iron utilization involves specific binding of human transferrin at the cell surface to what is believed to be a complex of two iron-regulated, transferrin-binding proteins, TbpA and TbpB. The genes encoding these proteins have been cloned and sequenced from a number of pathogenic, gram-negative bacteria. In the current study, we sequenced four additional tbpA genes from other N. gonorrhoeaestrains to begin to assess the sequence diversity among gonococci. We compared these sequences to those from other pathogenic bacteria to identify conserved regions that might be important for the structure and function of these receptors. We generated polyclonal mouse sera against synthetic peptides deduced from the TbpA sequence from gonococcal strain FA19. Most of these synthetic peptides were predicted to correspond to surface-exposed regions of TbpA. We found that, while most reacted with denatured TbpA in Western blots, only one antipeptide serum reacted with native TbpA in the context of intact gonococci, consistent with surface exposure of the peptide to which this serum was raised. In addition, we evaluated a panel of gonococcal strains for antigenic diversity using these antipeptide sera.

Cloning and characterization of Neisseria meningitidis genes encoding the transferrin-binding proteins Tbp1 and Tbp2

Gene ◽  
1993 ◽  
Vol 130 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Michèle Legrain ◽  
Véronique Mazarin ◽  
Scan W. Irwin ◽  
Bernadette Bouchon ◽  
Marie-José Quentin-Millet ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Binding Proteins ◽  
Genes Encoding ◽  
Transferrin Binding Proteins

Negative Correlation Between Serum Levels of Homocysteine and Apolipoprotein M

2019 ◽  
Vol 19 (2) ◽  
pp. 120-126
Author(s):  
J. Wei ◽  
Y. Yu ◽  
Y. Feng ◽  
J. Zhang ◽  
Q. Jiang ◽  
...  
Keyword(s):  
Negative Correlation ◽  
Hepg2 Cells ◽  
Serum Levels ◽  
Significant Negative Correlation ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Apolipoprotein M ◽  
Protein Mass ◽  
Protein Levels ◽  
Phosphoinositide 3 Kinase

Background: Homocysteine (Hcy) has been suggested as an independent risk factor for atherosclerosis. Apolipoprotein M (apoM) is a constituent of the HDL particles. The goal of this study was to examine the serum levels of homocysteine and apoM and to determine whether homocysteine influences apoM synthesis. Methods: Serum levels of apoM and Hcy in 17 hyperhomocysteinemia (HHcy) patients and 19 controls were measured and their correlations were analyzed. Different concentrations of homocysteine (Hcy) and LY294002, a specific phosphoinositide 3- kinase (PI3K) inhibitor, were used to treat HepG2 cells. The mRNA levels were determined by RT-PCR and the apoM protein mass was measured by western blot. Results: We found that decreased serum apoM levels corresponded with serum HDL levels in HHcy patients, while the serum apoM levels showed a statistically significant negative correlation with the serum Hcy levels. Moreover, apoM mRNA and protein levels were significantly decreased after the administration of Hcy in HepG2 cells, and this effect could be abolished by addition of LY294002. Conclusions: resent study demonstrates that Hcy downregulates the expression of apoM by mechanisms involving the PI3K signal pathway.

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