Identification of a Large Family of Slam-Dependent Surface Lipoproteins in Gram-Negative Bacteria

Abstract

The surfaces of many Gram-negative bacteria are decorated with soluble proteins anchored to the outer membrane via an acylated N-terminus; these proteins are referred to as surface lipoproteins or SLPs. In Neisseria meningitidis, SLPs such as transferrin-binding protein B (TbpB) and factor-H binding protein (fHbp) are essential for host colonization and infection because of their essential roles in iron acquisition and immune evasion, respectively. Recently, we identified a family of outer membrane proteins called Slam (Surface lipoprotein assembly modulator) that are essential for surface display of neisserial SLPs. In the present study, we performed a bioinformatics analysis to identify 832 Slam related sequences in 638 Gram-negative bacterial species. The list included several known human pathogens, many of which were not previously reported to possess SLPs. Hypothesizing that genes encoding SLP substrates of Slams may be present in the same gene cluster as the Slam genes, we manually curated neighboring genes for 353 putative Slam homologs. From our analysis, we found that 185 (~52%) of the 353 putative Slam homologs are located adjacent to genes that encode a protein with an N-terminal lipobox motif. This list included genes encoding previously reported SLPs in Haemophilus influenzae and Moraxella catarrhalis, for which we were able to show that the neighboring Slams are necessary and sufficient to display these lipoproteins on the surface of Escherichia coli. To further verify the authenticity of the list of predicted SLPs, we tested the surface display of one such Slam-adjacent protein from Pasteurella multocida, a zoonotic pathogen. A robust Slam-dependent display of the P. multocida protein was observed in the E. coli translocation assay indicating that the protein is a Slam-dependent SLP. Based on multiple sequence alignments and domain annotations, we found that an eight-stranded beta-barrel domain is common to all the predicted Slam-dependent SLPs. These findings suggest that SLPs with a TbpB-like fold are found widely in Proteobacteria where they exist with their interaction partner Slam. In the future, SLPs found in pathogenic bacteria can be investigated for their role in virulence and may also serve as candidates for vaccine development.

Lipoprotein Transport: Greasing the Machines of Outer Membrane Biogenesis

“…Recent discovery of the Slam protein family in Neisseria demonstrates that some OMPs could be dedicated translocators of lipoproteins . The Slams contain conserved C‐terminal β‐barrel domains and are found scattered throughout proteobacteria . An important outstanding question remains: do Slams catalyze successive rounds of flipping?…”

Description of a widespread bacterial secretion system with chemically diverse substrates

“…Recently, a mechanism of lipoprotein tethering to the outer leaflet of the outer membrane was identified in Neisseria meningitidis and termed the Slam (Surface lipoprotein assembly modulator) machinery [13,14]. In this system, prolipoproteins are initially transported across the inner membrane by the Sec or Tat translocases, and subsequently localized and tethered to either the inner or outer membrane by the Lol (localization of lipoprotein) system [15].…”

“…In N. meningitidis, Slam proteins containing a beta-barrel domain of the protein family DUF560 are then required in a third step for surface presentation of certain lipoproteins, especially those that capture host-metal chaperones and allow proliferation within a host exhibiting nutritional immunity [13]. In addition, two different Slam proteins from the same strain exhibit differential specificity for lipoprotein targets [13,14]. DUF560 family proteins are present throughout Proteobacteria, and only a few members have been characterized [13,14,16,17].…”

“…In addition, two different Slam proteins from the same strain exhibit differential specificity for lipoprotein targets [13,14]. DUF560 family proteins are present throughout Proteobacteria, and only a few members have been characterized [13,14,16,17]. One characterized DUF560 homolog, NilB, was identified as a host-association and species-specificity factor in the entomopathogenic nematode symbiont Xenorhabdus nematophila, a proteobacterium in the family Morganellaceae [16][17][18][19][20].…”

“…Slam activity has been demonstrated for DUF560 representatives from Neisseria meningitidis, Pasteurella multocida, Moraxella catarrhalis, and Haemophilus influenzae [13,14] but it remains unclear whether lipoprotein translocation is a universal attribute of DUF560 proteins. Some characterized Slams are encoded adjacent to genes encoding their experimentally-verified lipoprotein targets [13,14].…”