The Negatively Charged Regions of Lactoferrin Binding Protein B, an Adaptation against Anti-Microbial Peptides

Ari Morgenthau,Amanda Beddek,Anthony B. Schryvers,Paul J. Planet

doi:10.1371/journal.pone.0086243

Abstract

Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein’s C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides.

Highlights

Lactoferrin, an 80 Kda bi-lobed host iron-binding glycoprotein, is found at varying levels throughout the body, the highest being at mucosal surfaces and sites of inflammation [1,2]
In a previous study it was shown that lactoferrin binding protein B (LbpB) and not lactoferrin binding protein A (LbpA) conferred protection against the cationic antimicrobial peptide, lactoferricin, and was postulated that this might be due to the relatively large clusters of negatively charged amino acids present in C-terminal lobe of the LbpB [10]
The removal of the negatively charged regions from the N. meningitidis MC58 LbpB did not impact the stability of the protein (Figure 1), or its export to the cell surface (Figure 4B), enabling us to determine whether or not these regions contributed to protection against the killing activity of lactoferricin on meningococcal cells

Summary

Introduction

Lactoferrin, an 80 Kda bi-lobed host iron-binding glycoprotein, is found at varying levels throughout the body, the highest being at mucosal surfaces and sites of inflammation [1,2]. A primary role of lactoferrin is to sequester available iron in the extracellular milieu, it has many other important functions that combat pathogens within the host (Reviewed in [3]). Lactoferrin is a major component of the secondary granules of neutrophils and is released in its iron free form at sites of inflammation resulting in high local concentrations. LbpB differs from TbpB in multiple important aspects, in the presence of negatively charged regions localized to the LbpB CLobe. The presence of negatively charged regions have previously been used to distinguish LbpB from TbpB in bioinformatics analyses [10]

Methods

Results

Conclusion