Conserved Regions of Gonococcal TbpB Are Critical for Surface Exposure and Transferrin Iron Utilization

Abstract

The transferrin-binding proteins TbpA and TbpB enable Neisseria gonorrhoeae to obtain iron from human transferrin. The lipoprotein TbpB facilitates, but is not strictly required for, TbpA-mediated iron acquisition. The goal of the current study was to determine the contribution of two conserved regions within TbpB to the function of this protein. Using site-directed mutagenesis, the first mutation we constructed replaced the lipobox (LSAC) of TbpB with a signal I peptidase cleavage site (LAAA), while the second mutation deleted a conserved stretch of glycine residues immediately downstream of the lipobox. We then evaluated the resulting mutants for effects on TbpB expression, surface exposure, and transferrin iron utilization. Western blot analysis and palmitate labeling indicated that the lipobox, but not the glycine-rich motif, is required for lipidation of TbpB and tethering to the outer membrane. TbpB was released into the supernatant by the mutant that produces TbpB LSAC. Neither mutation disrupted the transport of TbpB across the bacterial cell envelope. When these mutant TbpB proteins were produced in a strain expressing a form of TbpA that requires TbpB for iron acquisition, growth on transferrin was either abrogated or dramatically diminished. We conclude that surface tethering of TbpB is required for optimal performance of the transferrin iron acquisition system, while the presence of the polyglycine stretch near the amino terminus of TbpB contributes significantly to transferrin iron transport function. Overall, these results provide important insights into the functional roles of two conserved motifs of TbpB, enhancing our understanding of this critical iron uptake system.

Comprehensive Spatial Analysis of the Borrelia burgdorferi Lipoproteome Reveals a Compartmentalization Bias toward the Bacterial Surface

“…Some Gram-negative bacteria express surface-exposed lipoproteins (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) but, with the exception of recently discovered surface lipoproteins in the phylum Bacteroidetes (43, 45), they remain relatively rare. In the Gram-negative model organism E. coli, only 7 of the about 90 expressed lipoproteins (46, 47) were detected on the bacterial surface but remained restricted to a proportion of total protein and certain protein domains, indicating rather complex and dynamic OM topologies (39,42,(48)(49)(50)(51)(52).…”

Description of a widespread bacterial secretion system with chemically diverse substrates

“…Further, TpbB is a substrate of DUF560 Slam1 in N. meningitidis [13,14], strengthening the notion that despite the absence of a lipid anchor, TbpB_B_Dsol homologs may be substrates for DUF560 homologs for extracellular exposure. As further support for potential TbpB_B_Dsol export, N. gonorrhoeae FA19 TbpB lacking its lipidation site is found in the supernatant in contrast to WT TbpB which is surface exposed but cell anchored, indicating that even without lipid anchoring, TbpB can be exported and can interact with TbpA [50].…”

Universal fluorescent sensors of high-affinity iron transport, applied to ESKAPE pathogens

“…The FD sensor approach may fail for certain transport processes. The Tbpmediated iron uptake systems of pathogenic Neisseria, which do not deplete their external transferrin ligand (40), illustrate this point. Although a neisserial TbpB-FM sensor that discriminates between holo-and apotransferrin (e.g.)…”