Structure of a SLC26 Anion Transporter STAS Domain in Complex with Acyl Carrier Protein: Implications for E. coli YchM in Fatty Acid Metabolism

SummaryEscherichia coli YchM is a member of the SLC26 (SulP) family of anion transporters with an N-terminal membrane domain and a C-terminal cytoplasmic STAS domain. Mutations in human members of the SLC26 family, including their STAS domain, are linked to a number of inherited diseases. Herein, we describe the high-resolution crystal structure of the STAS domain from E. coli YchM isolated in complex with acyl-carrier protein (ACP), an essential component of the fatty acid biosynthesis (FAB) pathway. A genome-wide genetic interaction screen showed that a ychM null mutation is synthetically lethal with mutant alleles of genes (fabBDHGAI) involved in FAB. Endogenous YchM also copurified with proteins involved in fatty acid metabolism. Furthermore, a deletion strain lacking ychM showed altered cellular bicarbonate incorporation in the presence of NaCl and impaired growth at alkaline pH. Thus, identification of the STAS-ACP complex suggests that YchM sequesters ACP to the bacterial membrane linking bicarbonate transport with fatty acid metabolism

Utility of Hybrid Transferrin Binding Protein Antigens for Protection Against Pathogenic Neisseria Species

The surface transferrin receptor proteins from Neisseria gonorrhoeae have been recognized as ideal vaccine targets due to their critical role in survival in the human male genitourinary tract. Recombinant forms of the surface lipoprotein component of the receptor, transferrin binding protein B (TbpB), can be readily produced at high levels in the Escherichia coli cytoplasm and is suitable for commercial vaccine production. In contrast, the integral outer membrane protein, transferrin binding protein A (TbpA), is produced at relatively low levels in the outer membrane and requires detergents for solubilization and stabilization, processes not favorable for commercial applications. Capitalizing on the core β-barrel structural feature common to the lipoprotein and integral outer membrane protein we engineered the lipoprotein as a scaffold for displaying conserved surface epitopes from TbpA. A stable version of the C-terminal domain of TbpB was prepared by replacing four larger exposed variable loops with short linking peptide regions. Four surface regions from the plug and barrel domains of Neisseria TbpA were transplanted onto this TbpB C-lobe scaffold, generating stable hybrid antigens. Antisera generated in mice and rabbits against the hybrid antigens recognized TbpA at the surface of Neisseria meningitidis and inhibited transferrin-dependent growth at levels comparable or better than antisera directed against the native TbpA protein. Two of the engineered hybrid antigens each elicited a TbpA-specific bactericidal antibody response comparable to that induced by TbpA. A hybrid antigen generated using a foreign scaffold (TbpB from the pig pathogen Haemophilus parasuis) displaying neisserial TbpA loop 10 was evaluated in a model of lower genital tract colonization by N. gonorrhoeae and a model of invasive infection by N. meningitidis. The loop 10 hybrid antigen was as effective as full length TbpA in eliminating N. gonorrhoeae from the lower genital tract of female mice and was protective against the low dose invasive infection by N. meningitidis. These results demonstrate that TbpB or its derivatives can serve as an effective scaffold for displaying surface epitopes of integral outer membrane antigens and these antigens can elicit protection against bacterial challenge

Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326)

IntroductionSyphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp’s repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages.MethodsThree overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed “Epivolve” (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold (PfTrxBamA/ECL4). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse assay. Sera from Tp-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using PfTrxBamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays.ResultsEach of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of PfTrxBamA/ECL4. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope.DiscussionEpivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection

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

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

Reconstitution of surface lipoprotein translocation through the Slam translocon.

Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient acquisition and immune evasion in the host. While the factors that are involved in the synthesis and delivery of SLPs in the inner membrane are well characterized, the molecular machinery required for the movement of SLPs to the surface are still not fully elucidated. In this study, we investigated the translocation of a SLP TbpB through a Slam1-dependent pathway. Using purified components, we developed an in vitro translocation assay where unfolded TbpB is transported through Slam1-containing proteoliposomes, confirming Slam1 as an outer membrane translocon. While looking to identify factors to increase translocation efficiency, we discovered the periplasmic chaperone Skp interacted with TbpB in the periplasm of Escherichia coli. The presence of Skp was found to increase the translocation efficiency of TbpB in the reconstituted translocation assays. A knockout of Skp in Neisseria meningitidis revealed that Skp is essential for functional translocation of TbpB to the bacterial surface. Taken together, we propose a pathway for surface destined lipoproteins, where Skp acts as a holdase for Slam-mediated TbpB translocation across the outer membrane

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor

PilN Binding Modulates the Structure and Binding Partners of thePseudomonas aeruginosaType IVa Pilus Protein PilM

General view of Art Deco hote