Analyzing the molecular mechanism of lipoprotein localization in Brucella

Abstract

Bacterial lipoproteins possess diverse structure and functionality, ranging from bacterial physiology to pathogenic processes. As such many lipoproteins, originating from Brucella are exploited as potential vaccines to countermeasure brucellosis infection in the host. These membrane proteins are translocated from the cytoplasm to the cell membrane where they are anchored peripherally by a multifaceted targeting mechanism. Although much research has focused on the identification and classification of Brucella lipoproteins and their potential use as vaccine candidates for the treatment of Brucellosis, the underlying route for the translocation of these lipoproteins to the outer surface of the Brucella (and other pathogens) outer membrane (OM) remains mostly unknown. This is partly due to the complexity of the organism and evasive tactics used to escape the host immune system, the variation in biological structure and activity of lipoproteins, combined with the complex nature of the translocation machinery. The biosynthetic pathway of Brucella lipoproteins involves a distinct secretion system aiding translocation from the cytoplasm, where they are modified by lipidation, sorted by the lipoprotein localization machinery pathway and thereafter equipped for export to the OM. Surface localized lipoproteins in Brucella may employ a lipoprotein flippase or the β-barrel assembly complex for translocation. This review provides an overview of the characterized Brucella OM proteins that form part of the OM, including a handful of other characterized bacterial lipoproteins and their mechanisms of translocation. Lipoprotein localization pathways in gram negative bacteria will be used as a model to identify gaps in Brucella lipoprotein localization and infer a potential pathway. Of particular interest are the dual topology lipoproteins identified in Escherichia coli and Haemophilus influenza. The localization and topology of these lipoproteins from other gram negative bacteria are well characterized and may be useful to infer a solution to better understand the translocation process in Brucella.

Keywords: Brucella lipoprotein; Brucella vaccine target; Lol pathway; lipoprotein localization; lipoprotein secretion; outer membrane protein; pathogen-associated molecular patterns; toll-like receptors.

FIGURE 1

The biosynthesis and sorting of bacterial lipoproteins. Prelipoproteins synthesized in the cytoplasm are translocated to the outer surface of the IM by the general secretory (Sec), two-arginine (Tat) pathways. Prelipoproteins possess a consensus motif termed the lipobox, which is a component of the N-terminal signal peptide cleavage site. It is the +1 cysteine residue of the lipobox that undergoes lipid modification in a sequential process catalyzed by three periplasmic enzymes to yield the mature lipoproteins. The first enzyme, preprolipoprotein diacylglyceryl transferase (Lgt) generates a thioether linkage by transferring a diacylglyceryl molecule to the sulfhydryl group of the +1 cysteine. The second enzyme, lipoprotein signal peptidase (Lsp) catalyzes the cleavage of the N-terminal signal peptide at +1 S-diacylglyceryl cysteine. Lastly, lipoprotein N’N-acyl transferase (Lnt) catalyzes aminoacylation of the α-amino group of the S-diacylglyceryl cysteine, producing a mature triacylated lipoprotein. Depending upon the lipoprotein sorting signal the lipoprotein is then targeted for the inner membrane (IM) or the outer membrane (OM). Abbreviations used: inner membrane (IM), general secretory (Sec), two-arginine (Tat), preprolipoprotein diacylglyceryl transferase (Lgt), lipoprotein signal peptidase (Lsp), lipoprotein N’N-acyl transferase (Lnt), outer membrane (OM).

FIGURE 2

The proposed translocation pathway for the dual topology E. coli Braun’s lipoprotein (Lpp). After generating the mature Lpp (green structure), the lipoprotein in the inner membrane (IM) interacts with the lipoprotein localization machinery (Lol) located in the periplasm. Adenosine triphosphate (ATP) hydrolysis, an ABC transporter (LolCDE), a carrier protein (LolA) and outer membrane (OM) lipoprotein receptor (LolB) mediates the “mouth-to-mouth” the transfer of Lpp from the IM to the OM. Lpp may reside in the periplasm anchored to the inner leaflet of the OM (bound form) or spans the OM resulting in the surface exposure of its C terminus (free form). Abbreviations used: Braun’s lipoprotein (Lpp), inner membrane (IM), lipoprotein localization machinery (Lol), adenosine triphosphate (ATP), outer membrane (OM), ATP binding cassette (ABC).

FIGURE 3

The translocation pathways for the surface exposure of lipoproteins in Brucella. Prelipoproteins synthesized in the cytoplasm undergo lipid modification generating a mature, triacylated lipoprotein. Brucella lipoproteins (orange, green, and blue structures) may be released from the inner membrane (IM) to the outer membrane (OM) using the lipoprotein localization machinery (Lol) pathway, which is propelled by Adenosine triphosphate (ATP hydrolysis), an ATP binding cassette (ABC) transporter (LolCDE) and a carrier protein (LolA). LolB is absent in α-Proteobacteria, as such it is proposed lipoprotein flippase located in the OM transfers lipoproteins to the OM. Another proposed translocation pathway involves the export of the Brucella lipoproteins through the β- barrel domain of β- barrel assembly complex (Bam) complex embedded in the OM, assisted by the periplasmic chaperones (SurA) to the surface of the OM. The Brucella Bam complex lacks the BamB and BamC components found in other gram negative bacteria, however, a BamF component with a conserved sequence motif related to the BamC component may perform a similar role. Abbreviations used: inner membrane (IM), outer membrane (OM), lipoprotein localization machinery (Lol), adenosine triphosphate (ATP), ATP binding cassette (ABC), β- barrel assembly complex (Bam).