Hydrogen-deuterium exchange coupled to mass spectrometry to investigate ligand-receptor interactions.

A method for exploring protein-protein interactions using hydrogen/deuterium exchange coupled to mass spectrometry is described. The method monitors the exchange of backbone (amide) hydrogens in solutions of deuterated water that primarily occur on portions of the protein exposed to solvent. In the presence of a protein binding partner, regions that experience reduced exchange are either part of the protein-protein interaction interface or undergo conformational changes to reduce accessibility to solvent. This method has the advantage of being used under physiological conditions with unmodified proteins. In this chapter, we describe an approach suitable for probing interactions among relatively large proteins using conventional mass spectrometry systems. The interaction between human transferrin and the Neisseria meningitidis receptor protein, transferrin binding protein B, provides a challenging system as an example.

Delineating the regions of human transferrin involved in interactions with transferrin binding protein B from Neisseria meningitidis.

Pathogenic bacteria in the Neisseriaceae possess a surface receptor mediating iron acquisition from human transferrin (hTf) that consists of a transmembrane iron transporter (TbpA) and a surface-exposed lipoprotein (TbpB). In this study, we used hydrogen/deuterium exchange coupled to mass spectrometry (H/DX-MS) to elucidate the effects on hTf by interaction with TbpB or derivatives of TbpB. An overall conserved interaction was observed between hTf and full-length or N-lobe TbpB from Neisseria meningitidis strains B16B6 or M982 that represent two distinct subtypes of TbpB. Changes were observed exclusively in the C-lobe of hTf and were caused by the interaction with the N-lobe of TbpB. Regions localized to the 'lip' of the C1 and C2 domains that flank the interdomain cleft represent sites of direct contact with TbpB whereas the peptides within the interdomain cleft that encompass iron binding ligands are inaccessible in the closed (holo) conformation. Although substantial domain separation upon binding TbpB cannot be excluded by the H/DX-MS data, the preferred model of interaction involves binding hTf C-lobe in the closed conformation. Alternate explanations are provided for the substantial protection from deuteration of the peptides encompassing iron binding ligands within the interdomain cleft but cannot be differentiated by the H/DX-MS data.

The mviN homolog in Burkholderia pseudomallei is essential for viability and virulence.

The virulence factors of Burkholderia pseudomallei, the causative agent of melioidosis, are not fully understood. We have identified a gene with homology to the Salmonella typhimurium mouse virulence gene, mviN, a member of the mouse virulence factor family. Expression studies with an insertional mutant containing a lux operon demonstrated that the expression of the gene is influenced by free-iron availability in the media and by growth phase. The mutant displayed an increased LD50 value in the hamster infection model and a loss of the ability to invade human lung epithelial cells. The mutant has a slower growth rate than that of the wild type. Both defects were restored to various degrees when complemented in trans with the mviN gene. The mutant contains an insertion at 1229 bp of the 1548 bp gene, resulting in a truncated protein that is presumably responsible for the defects. Deletion mutants of the entire B. pseudomallei mviN gene were obtained only in the presence of the complement vector. This result and the inability of the complemented deletion mutant to lose the plasmid in the absence of antibiotic selection suggest that the gene is essential to B. pseudomallei.

Perspectives on interactions between lactoferrin and bacteria.

Lactoferrin has long been recognized for its antimicrobial properties, initially attributed primarily to iron sequestration. It has since become apparent that interaction between the host and bacteria is modulated by a complex series of interactions between lactoferrin and bacteria, lactoferrin and bacterial products, and lactoferrin and host cells. The primary focus of this review is the interaction between lactoferrin and bacteria, but interactions with the lactoferrin-derived cationic peptide lactoferricin will also be discussed. We will summarize what is currently known about the interaction between lactoferrin (or lactoferricin) and surface or secreted bacterial components, comment on the potential physiological relevance of the findings, and identify key questions that remain unanswered.

Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarrays.

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation.