Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria Yogesh Hooda 1† , Christine Chieh-Lin Lai 1† , Andrew Judd 1 , Carolyn M. Buckwalter 2 , Hyejin Esther Shin 1 , Scott D. Gray-Owen 2 and Trevor F. Moraes 1 * Lipoproteins decorate the surface of many Gram-negative bacterial pathogens, playing essential roles in immune evasion and nutrient acquisition. In Neisseria spp., the causative agents of gonorrhoea and meningococcal meningitis, surface lipoproteins (SLPs) are required for virulence and have been extensively studied as prime candidates for vaccine development. However, the machinery and mechanism that allow for the surface display of SLPs are not known. Here, we describe a transposon (Tn5)-based search for the proteins required to deliver SLPs to the surface of Neisseria meningitidis, revealing a family of proteins that we have named the surface lipoprotein assembly modulator (Slam). N. meningitidis contains two Slam proteins, each exhibiting distinct substrate preferences. The Slam proteins are sufficient to reconstitute SLP transport in laboratory strains of Escherichia coli, which are otherwise unable to efficiently display these lipoproteins on their cell surface. Immunoprecipitation and domain probing experiments suggest that the SLP, TbpB, interacts with Slam during the transit process; furthermore, the membrane domain of Slam is sufficient for selectivity and proper surface display of SLPs. Rather than being a Neisseria-specific factor, our bioinformatic analysis shows that Slam can be found throughout proteobacterial genomes, indicating a conserved but until now unrecognized virulence mechanism. G ram-negative bacteria are encapsulated by a double mem- brane that is separated by a thin peptidoglycan layer and peri- plasmic space 1 . The outer membrane confers protection against toxic compounds and host defence molecules. However, this benefit creates unique challenges for the bacteria with respect to the delivery of effector proteins to the cell surface. The molecular processes required to insert proteins into the outer membrane are well described 2 , but many Gram-negative organisms also display peripherally associated nutrient scavenging and immune defence proteins on their surface via a covalently associated lipid anchor inserted into the outer membrane, collectively termed bacterial surface lipoproteins (SLPs) 3–5 . The human pathogen Neisseria meningitidis uses SLPs to acquire nutrients and evade the host immune defences to achieve a sus- tained infection 6 . For example, the neisserial factor H-binding protein (fHbp) is an SLP that binds the human serum protein factor H to avoid the complement-mediated bactericidal activity of the blood 7 . SLPs involved in iron acquisition have also been studied extensively, with the uptake process being initiated by a bipartite receptor composed of a surface-exposed lipoprotein (trans- ferrin or lactoferrin binding protein B, TbpB or LbpB, or haemo- globin–haptoglobin using protein A, HpuA) and an integral outer membrane protein 8,9 . In accordance with their roles in these pro- cesses and the fact that their functions depend on direct, specific interactions with host proteins on the bacterial cell surface 10 , these SLPs elicit bactericidal antibodies 11–13 and have been successfully used as vaccine antigens to prevent meningococcal disease 14–16 . To properly traffic to the cell surface, the neisserial SLPs require signalling motifs to translocate across the bacterial cell envelope (Supplementary Fig. 1a–d). SLPs contain the canonical signal peptide and consensus lipobox motif that are necessary for translocation through the Sec secretion machinery and lipidation of their mature amino-terminal cysteine 17 . This was confirmed for neisserial SLPs by the detection of unprocessed full-length TbpB build-up in N. meningitidis and Escherichia coli with globomycin treatment 18 (Supplementary Fig. 1b). Lipoproteins can traverse the periplasm through the Lol system. The E. coli LolA R43L mutation inhibits the transfer of lipoproteins to LolB and effectively traps LolA–lipo- protein complexes 19 . Expression of TbpB together with LolA R43L stalls lipoprotein translocation and the complex of TbpB bound to LolA R43L can be detected in pulldowns (Supplementary Fig. 1c), confirming the use of the Lol system by neisserial SLPs. Although TbpB expressed in laboratory strains of E. coli is able to reach the outer membrane (Supplementary Fig. 1d), it is not displayed on the surface (Supplementary Fig. 1e). This suggests that neisserial SLPs undergo an additional translocation step that ‘flips’ them across the outer membrane to the surface of the cell. Until now, it remained unknown how this latter step occurrred. Results Identification of surface lipoprotein assembly modulator (Slam) in N. meningitidis. To identify the component(s) required for SLP translocation to the N. meningitidis cell surface, we constructed a genome-wide random Tn5-based transposon mutant library within the N. meningitidis strain B16B6. Using a whole cell solid-phase binding assay, 4,000 mutants from this library were analysed for the ability to display TbpB on the cell surface (Supplementary Fig. 2). One of the defective mutants had incorporated a Tn5 into a genetic region that aligns with the ORF nmb0313 of the published N. meningitidis MC58 genome sequence 20 . This nmb0313::Tn5 mutant (nmb0313/tn5) has effectively lost TbpB from its cell surface when compared with the wild-type 1 Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada. 2 Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. † These authors contributed equally to this work. *e-mail: trevor.moraes@utoronto.ca ARTICLES PUBLISHED: 29 FEBRUARY 2016 | ARTICLE NUMBER: 16009 | DOI: 10.1038/NMICROBIOL.2016.9 NATURE MICROBIOLOGY | VOL 1 | APRIL 2016 | www.nature.com/naturemicrobiology 1 © 2016 Macmillan Publishers Limited. All rights reserved