Abstract
The increased interest in predatory bacteria due to their ability to kill antibiotic resistant bacteria has also highlighted their inherent plethora of hydrolytic enzymes, and their potential as natural sources of novel therapeutic agents and biotechnological tools. Here, we have identified and characterized a novel protease from the predatory bacterium Bdellovibrio bacteriovorus: BspE (Bdellovibrio elastase-like serine protease). Mapping preferential sites of proteolytic activity showed a single proteolytic cleavage site of native plasma IgA (pIgA) in the Fc-tail; as well as in the secretory component (SC) of secretory IgA (SIgA). Proteolysis of other native immunoglobulins and plasma proteins was either absent (IgG1 and 2, IgM, albumin and orosomucoid) or unspecific with multiple cleavage sites (IgG3 and 4, IgE, IgD). BspE displayed a broad activity against most amino acid bonds in shorter peptides and denatured proteins, with a slight preference for hydrolysis C-terminal of Y, V, F, S, L, R, P, E, and K. BspE autoproteolysis results in numerous cleavage products sustaining activity for more than 6 h. The enzymatic activity remained stable at pH 5.0–9.0 but was drastically reduced in the presence of MnCl2 and completely inhibited by ZnCl2. The hydrolysis of pIgA was subsequently utilized for the specific glycan characterization of the released pIgA Fc-tail (Asn459). Besides contributing to the basic knowledge of Bdellovibrio biology and proteases, we propose that BspE could be used as a potential tool to investigate the importance, and biological function of the pIgA Fc-tail.IMPORTANCEAntibodies are well-established as key components of the immune system, and the importance of antibody glycosylation is steadily gaining recognition. Modifications of antibodies by glycosylation creates a vast repertoire of antibody glycovariants with distinctive and diverse functions in the immune system. Most of the available information regarding antibody glycosylation is based on studies with IgG, which have contributed greatly to the advance of therapeutic antibody treatments. However, much is still unknown regarding the importance of glycosylation and the Fc-structure for the remaining antibody classes. Such research has proven to be technically challenging and demonstrates a need for novel tools to facilitate such investigations. Here we have identified and characterized a novel protease from B. bacteriovorus, facilitating the study of plasma IgA by cleaving the Fc-tail, including the Asn459 N-glycan. This further highlights the potential of B. bacteriovorus as a source to identify potential novel biotechnological tools.
Highlights
With the advent of an increasing number of multi-resistant bacteria, researchers have started looking into alternatives to antibiotics
We have identified and characterized the enzyme behind this phenomenon: BspE, a broad-spectrum serine protease, which despite its broad activity displays a specificity toward cleaving the Fc-tail on native plasma or serum IgA (pIgA)
As Bdellovibrio produces a vast array of proteases, the broad activity of BspE could possibly be involved in the degradation of other proteases, acting protectively against excessive proteolytic damage to Bdellovibrio self or the prey host
Summary
With the advent of an increasing number of multi-resistant bacteria, researchers have started looking into alternatives to antibiotics. Bdellovibrio has a broad host range, restricted to specific Gram negative bacteria (Koval and Hynes, 1991; Hobley et al, 2006; Sockett, 2009) It is capable of killing many antibiotic-resistant, clinical pathogens including Acinetobacter baumannii and Klebsiella pneumoniae in vitro, and reduces the general bacterial burden in vivo (Negus et al, 2017). This bacterium has been studied by a limited number of scientists since the 1960s but has, with the rise of antimicrobial resistance (AMR), gained increasing attention. Previous studies have shown positive correlations between the presence of B. bacteriovorus and health (Iebba et al, 2013), suggesting it may act as an environmental balancer, aiding in sustaining a beneficial microflora and contributing to good health (Pérez et al, 2016)
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