Mutational Analysis of Hemoglobin Binding and Heme Utilization by a Bacterial Hemoglobin Receptor

Fusco, William G.; Choudhary, Neelima; Collins, Edward J.; Leduc, Isabelle

doi:10.1128/jb.00199-13

Cited by 10 publications

(7 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6, step 2). Consistent with this idea, mutagenesis studies of the Hb receptor A (HgbA) from Gram-negative Haemophilus ducreyi indicate that there is likewise a physical separation of the Hb-binding and heme uptake interfaces (66,67). This similarity suggests that an architecture involving multiple interaction interfaces might have a functional advantage for heme extraction from Hb in a range of systems.…”

Section: Discussionmentioning

confidence: 87%

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Dickson

Kumar

Jacques

et al. 2014

Journal of Biological Chemistry

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 87%

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Dickson

Kumar

Jacques

et al. 2014

Journal of Biological Chemistry

View full text Add to dashboard Cite

“…Site-directed mutagenesis studies of TBDT HgbA from Haemophilus ducreyi demonstrate that motifs in the extracellular loops are required to bind hemoglobin, a prerequisite to heme transfer 19,32 . Thus, removal of an essential extra-cellular loop region of ShuA was expected to disrupt the ShuA-hemoglobin interaction.…”

Section: Resultsmentioning

confidence: 99%

Dissecting binding of a β-barrel membrane protein by phage display

et al. 2017

View full text Add to dashboard Cite

Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal. Here, we apply a new approach for the expedited protein characterization of the TBDT ShuA from Shigella dysenteriae, and elucidate the protein’s initial steps during heme-uptake. ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable MP variants for rapid characterization of binding to various ligands. Site-directed mutagenesis studies targeting each extracellular loop region of ShuA demonstrate no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. The results identify a loop susceptible to antibody binding, and also a small molecule motif capable of disrupting ShuA from S. dysenteriae. The approach is generalizable to the dissection of other phage-displayed TBDTs and MPs.

show abstract

“…Iron acquisition is an important aspect of pathogenesis for many pathogens, and bacterial strategies for the acquisition of iron have been described for many decades [ 24 ]. It has been shown that the inactivation of genes involved in iron acquisition in Shigella [ 25 ], Y .…”

Section: Discussionmentioning

confidence: 99%

Roles of B739_1343 in iron acquisition and pathogenesis in Riemerella anatipestifer CH-1 and evaluation of the RA-CH-1ΔB739_1343 mutant as an attenuated vaccine

Liu

Shui

et al. 2018

PLoS ONE

View full text Add to dashboard Cite

Iron is one of the most important elements for bacterial survival and pathogenicity. The iron uptake mechanism of Riemerella anatipestifer (R. anatipestifer, RA), a major pathogen that causes septicemia and polyserositis in ducks, is largely unknown. Here, the functions of the putative TonB-dependent iron transporter of RA-CH-1, B739_1343, in iron utilization and pathogenicity were investigated. Under iron-starved conditions, the mutant strain RA-CH-1ΔB739_1343 exhibited more seriously impaired growth than the wild-type strain RA-CH-1, and the expression of B739_1343 in the mutant strain restored growth. qRT-PCR results showed that the transcription of B739_1343 was not regulated by iron conditions. In an animal model, the median lethal dose (LD50) of the mutant strain RA-CH-1ΔB739_1343 increased more than 104-fold (1.6×1012 CFU) compared to that of the wild-type strain RA-CH-1 (1.43×108 CFU). In a duck co-infection model, the mutant strain RA-CH-1ΔB739_1343 was outcompeted by the wild-type RA-CH-1 in the blood, liver and brain of infected ducks, indicating that B739_1343 is a virulence factor of RA-CH-1. Finally, immunization with live bacteria of the mutant strain RA-CH-1ΔB739_1343 protected 83.33% of ducks against a high-dose (100-fold LD50) challenge with the wild-type strain RA-CH-1, suggesting that the mutant strain RA-CH-1ΔB739_1343 could be further developed as a potential live attenuated vaccine candidate for the duck industry.

show abstract

Mutational Analysis of Hemoglobin Binding and Heme Utilization by a Bacterial Hemoglobin Receptor

Cited by 10 publications

References 26 publications

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Dissecting binding of a β-barrel membrane protein by phage display

Roles of B739_1343 in iron acquisition and pathogenesis in Riemerella anatipestifer CH-1 and evaluation of the RA-CH-1ΔB739_1343 mutant as an attenuated vaccine

Contact Info

Product

Resources

About