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

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.

A comparative, cross-species investigation of the properties and roles of transferrin- and lactoferrin-binding protein B from pathogenic bacteria

“…The presence of large clusters of negatively charged residues in the LbpB C-lobe from from N. meningitidis and M. catarrhalis led us to propose that these proteins would be involved in protection against lactoferricin H (an 11 residue peptide representing lactoferrin peptides released from human Lf) which was experimentally demonstrated (Morgenthau et al 2012). Subsequent experiments demonstrated that the protection with the meningococcal LbpB was associated with the clusters of negatively charged residues and that this protection extended to other cationic antimicrobial peptides (CAPs), including mouse cathelicidin related antimicrobial peptide (mCRAMP) (Morgenthau et al 2014a;Morgenthau et al 2014b). In this study we demonstrate that protection is extended to extracts from both human and mouse neutrophils (Figures III and IV) that would contain CAPs such as CRAMP and CAPs released by proteolysis D r a f t of Lf.…”

“…For the alternate model that has been proposed (Noinaj et al 2013), which involves the Lf C-lobe, other bacterial components would have to be invoked in order to explain the positive selection analysis results that primarily impact the Lf N-lobe. These could potentially include the PspA protein from Streptococcus (Shaper et al 2004) and the negatively charged regions of the LbpB C-lobe (Morgenthau et al 2014a), although it is difficult to envision how the latter could confer species specificity.…”

“…LbpA but not LbpB is required for growth in vitro with Lf as an exogenous iron source (Bonnah and Schryvers 1998;Pettersson et al 1998), comparable to what was observed with the Tf receptor proteins. A notable difference between LbpBs and TbpBs from Neisseria meningitidis and Moraxella catarrhalis, is the presence of clusters of negatively charged residues in the C-terminal lobe of LbpB (Bonnah and Schryvers 1998;Pettersson et al 1998) (Yu and Schryvers 2002) that have been shown to function in the protection against cationic antimicrobial peptides (Morgenthau et al 2014a). This feature is not present in the LbpB from M. bovis (Yu and Schryvers 2002).…”

“…The presence of large clusters of negatively charged amino acids in the C-lobe of LbpB from N. meningitidis and M. catarrhalis prompted us to explore whether these could be involved in protection against lactoferricin, a cationic antimicrobial peptide (CAP) released by pepsin digestion of human lactoferrin, a process that occurs when mother's milk is ingested by infants (Morgenthau et al 2012;Morgenthau et al 2014a). Subsequent studies demonstrated that the protective effect extended to other CAPs (Morgenthau et al 2014b).…”

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“…It affects hemostasis by reducing the time of clot formation, and prevents platelet aggregation (Brock, 2002). Its anti-inflammatory properties are also linked to the ability to bind components of bacterial cell walls (LPS) as well as the specific receptors (Morgenthau, Beddek, Schryvers, 2014). Lipopolysaccharides, potent mediators of the inflammatory response, cause the activation of leukocytes which results in the hyperproduction of free radicals.…”