The structure of HiSiaQM defines the architecture of tripartite ATP-independent periplasmic (TRAP) transporters

SummaryTripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria and archaea and provide important uptake routes for many metabolites 1–3. They consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains) that form a functional unit 4. While the structures of the P-domains are well-known, an experimental structure of any QM-domain has been elusive. HiSiaPQM is a TRAP transporter for the monocarboxylate sialic acid, which plays a key role in the virulence of pathogenic bacteria 5. Here, we present the first cryo-electron microscopy structure of the membrane domains of HiSiaPQM reconstituted in lipid nanodiscs. The reconstruction reveals that TRAP transporters consist of 15 transmembrane helices and are structurally related to elevator-type transporters, such as GltPh and VcINDY 6, 7. Whereas the latter proteins function as multimers, the idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. Structural and mutational analyses together with an AlphaFold 8 model of the tripartite (PQM) complex reveal the structural and conformational coupling of the substrate-binding protein to the transporter domains. Furthermore, we characterize high-affinity VHHs that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake in vivo. Thereby, they also confirm the orientation of the protein in the membrane. Our study provides the first structure of any binding-protein dependent secondary transporter and provides starting points for the development of specific inhibitors.

Site 2 of the Yersinia pestis substrate-binding protein YfeA is a dynamic surface metal-binding site

The substrate-binding protein YfeA (also known as YPO2439 or y1897) is a polyspecific metal-binding protein that is crucial for nutrient acquisition and virulence in Yersinia pestis, the causative microbe of plague. YfeA folds into a monomeric c-clamp like other substrate-binding proteins and has two metal-binding sites (sites 1 and 2). Site 2 is a bidentate surface site capable of binding Zn and Mn atoms and is a unique feature of YfeA. Occasionally, the site 2 residues of two YfeA molecules will cooperate with the histidine tag of a third YfeA molecule in coordinating the same metal and lead to metal-dependent crystallographic packing. Here, three crystal structures of YfeA are presented at 1.85, 2.05 and 2.25 Å resolution. A comparison of the structures reveals that the metal can be displaced at five different locations ranging from ∼4 to ∼16 Å away from the canonical site 2. These observations reveal different configurations of site 2 that enable cooperative metal binding and demonstrate how site 2 is dynamic and freely available for inter-protein metal coordination.

Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that lipidated MetQ is required for interaction

NmMetQ is a substrate-binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To elucidate the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the presence and absence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function and localization, playing a role in NmMetNI-mediated transport at the inner membrane and moonlighting on the bacterial surface.

Characterization and protective activity of monoclonal antibodies directed against Fe (3+) ABC transporter substrate-binding protein of Glaesserella parasuis

Glässer’s disease is caused by the agent Glaesserella parasuis and is difficult to prevent and control. Candidate screening for subunit vaccines contributes to the prevention of this disease. Therefore, in this study, the inactivated G. parasuis reference serovar 5 strain (G. parasuis-5) was used to generate specific monoclonal antibodies (mAbs) to screen subunit vaccine candidates. Six mAbs (1A12, 3E3, 4C6, 2D1, 3E6, and 4B2) were screened, and they all reacted with the G. parasuis serovar 5 strain according to laser confocal microscopy and flow cytometry (FCM). Indirect enzyme-linked immunosorbent assay (ELISA) showed that one mAb 2D1, can react with all 15 reference serovars of G. parasuis. Protein mass spectrometry and Western blot analysis demonstrated that mAb 2D1 specifically reacts with Fe (3+) ABC transporter substrate-binding protein. A complement killing assay found that the colony numbers of bacteria were significantly reduced in the G. parasuis-5 group incubated with mAb 2D1 (p < 0.01) in comparison with the control group. Opsonophagocytic assays demonstrated that mAb 2D1 significantly enhanced the phagocytosis of 3D4/21 cells by G. parasuis (p < 0.05). RAW264.7 cells with stronger phagocytic ability were also used for the opsonophagocytic assay, and the difference was highly significant (p < 0.01). Passive immunization of mice revealed that mAb 2D1 can eliminate the bacteria in the blood and provide protection against G. parasuis-5. Our study found one mAb that can be used to prevent and control G. parasuis infection in vivo and in vitro, which may suggest that Fe (3+) ABC transporter substrate-binding protein is an immunodominant antigen and a promising candidate for subunit vaccine development.

Selective Nutrient Transport in Bacteria: Multicomponent Transporter Systems Reign Supreme

Multicomponent transporters are used by bacteria to transport a wide range of nutrients. These systems use a substrate-binding protein to bind the nutrient with high affinity and then deliver it to a membrane-bound transporter for uptake. Nutrient uptake pathways are linked to the colonisation potential and pathogenicity of bacteria in humans and may be candidates for antimicrobial targeting. Here we review current research into bacterial multicomponent transport systems, with an emphasis on the interaction at the membrane, as well as new perspectives on the role of lipids and higher oligomers in these complex systems.

The Pseudomonas aeruginosa substrate-binding protein Ttg2D functions as a general glycerophospholipid transporter across the periplasm

In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. Here we use the crystallographic structure of Ttg2D at 2.5 Å resolution to reveal that this protein can accommodate four acyl chains. Analysis of the available structures of Ttg2D orthologs shows that they conform a new substrate-binding-protein structural cluster. Native and denaturing mass spectrometry experiments confirm that Ttg2D, produced both heterologously and homologously and isolated from the periplasm, can carry two diacyl glycerophospholipids as well as one cardiolipin. Binding is notably promiscuous, allowing the transport of various molecular species. In vitro binding assays coupled to native mass spectrometry show that binding of cardiolipin is spontaneous. Gene knockout experiments in P. aeruginosa multidrug-resistant strains reveal that the Ttg2 system is involved in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes.

The Role of a Dipeptide Transporter in the Virulence of Human Pathogen, Helicobacter pylori

Helicobacter pylori harbors a dipeptide (Dpp) transporter consisting of a substrate-binding protein (DppA), two permeases (DppB and C), and two ATPases (DppD and F). The Dpp transporter is responsible for the transportation of dipeptides and short peptides. We found that its expression is important for the growth of H. pylori. To understand the role of the Dpp transporter in the pathogenesis of H. pylori, the expression of virulence factors and H. pylori-induced IL-8 production were investigated in H. pylori wild-type and isogenic H. pylori Dpp transporter mutants. We found that expression of CagA was downregulated, while expression of type 4 secretion system (T4SS) components was upregulated in Dpp transporter mutants. The DppA mutant strain expressed higher levels of outer membrane proteins (OMPs), including BabA, HopZ, OipA, and SabA, and showed a higher adhesion level to gastric epithelial AGS cells compared with the H. pylori 26695 wild-type strain. After infection of AGS cells, H. pylori ΔdppA induced a higher level of NF-κB activation and IL-8 production compared with wild-type. These results suggested that in addition to supporting the growth of H. pylori, the Dpp transporter causes bacteria to alter the expression of virulence factors and reduces H. pylori-induced NF-κB activation and IL-8 production in gastric epithelial cells.

A Systematic Review of Campylobacter jejuni Vaccine Candidates for Chickens

Campylobacter jejuni infection linked to the consumption of contaminated poultry products is one of the leading causes of human enteric illness worldwide. Vaccination of chickens is one of the potential strategies that could be used to control C. jejuni colonization. To date, various C. jejuni vaccines using potential antigens have been evaluated, but a challenge in identifying the most effective formulation is the wide variability in vaccine efficacies reported. A systematic review was undertaken to compare C. jejuni vaccine studies. Based upon specific selection criteria eligible papers were identified and included in the analysis. Vaccine efficacy reported from different C. jejuni antigens, vaccine types, and vaccination regimens reported in these papers were reviewed. Our analysis shows that total outer membrane proteins and cysteine ABC transporter substrate-binding protein were among the most efficacious vaccine antigen candidates reported. This review also highlights the importance of the need for increased consistency in the way C. jejuni vaccine studies in poultry are designed and reported in order to be able to undertake a robust comparison of C. jejuni vaccine candidates.