Structural characterization of closely related O-antigen lipopolysaccharide (LPS) chain length regulators

The surface O-antigen polymers of Gram-negative bacteria exhibit a modal length distribution that depends on dedicated chain length regulator periplasmic proteins (polysaccharide co-polymerases, PCPs) anchored in the inner membrane by two transmembrane helices. In an attempt to determine whether structural changes underlie the O-antigen modal length specification, we have determined the crystal structures of several closely related PCPs, namely two chimeric PCP-1 family members solved at 1.6 and 2.8 Å and a wild-type PCP-1 from Shigella flexneri solved at 2.8 Å. The chimeric proteins form circular octamers, whereas the wild-type WzzB from S. flexneri was found to be an open trimer. We also present the structure of a Wzz^FepE mutant, which exhibits severe attenuation in its ability to produce very long O-antigen polymers. Our findings suggest that the differences in the modal length distribution depend primarily on the surface-exposed amino acids in specific regions rather than on the differences in the oligomeric state of the PCP protomers.     

Domain Interactions Control Complex Formation and Polymerase Specificity in the Biosynthesis of the Escherichia coli O9a Antigen

The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for bacterial glycan synthesis and export by an ATP-binding cassette transporter-dependent pathway. The O9a O-PS possesses a tetrasaccharide repeat unit comprising two α-(1→2)- and two α-(1→3)-linked mannose residues and is extended on a polyisoprenoid lipid carrier by the action of a polymerase (WbdA) containing two glycosyltransferase active sites. The N-terminal domain of WbdA possesses α-(1→2)-mannosyltransferase activity, and we demonstrate in this study that the C-terminal domain is an α-(1→3)-mannosyltransferase. Previous studies established that the size of the O9a polysaccharide is determined by the chain-terminating dual kinase/methyltransferase (WbdD) that is tethered to the membrane and recruits WbdA into an active enzyme complex by protein-protein interactions. Here, we used bacterial two-hybrid analysis to identify a surface-exposed α-helix in the C-terminal mannosyltransferase domain of WbdA as the site of interaction with WbdD. However, the C-terminal domain was unable to interact with WbdD in the absence of its N-terminal partner. Through deletion analysis, we demonstrated that the α-(1→2)-mannosyltransferase activity of the N-terminal domain is regulated by the activity of the C-terminal α-(1→3)-mannosyltransferase. In mutants where the C-terminal catalytic site was deleted but the WbdD-interaction site remained, the N-terminal mannosyltransferase became an unrestricted polymerase, creating a novel polymer comprising only α-(1→2)-linked mannose residues. The WbdD protein therefore orchestrates critical localization and coordination of activities involved in chain extension and termination. Complex domain interactions are needed to position the polymerase components appropriately for assembly into a functional complex located at the cytoplasmic membrane.     

Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis

The O-antigen polymerase of Gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly¹⁷⁶, Asp¹⁷⁷, Gly³²³, and Tyr³²⁴) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface.     

Identification of the Flagellin Glycosylation System in Burkholderia cenocepacia and the Contribution of Glycosylated Flagellin to Evasion of Human Innate Immune Responses

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-d-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.     

The Origin of 8-Amino-3,8-dideoxy-d-manno-octulosonic Acid (Kdo8N) in the Lipopolysaccharide of Shewanella oneidensis

Lipopolysaccharide (LPS; endotoxin) is an essential component of the outer monolayer of nearly all Gram-negative bacteria. LPS is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. In nearly all species, the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-d-manno-octulosonic acid (Kdo), and thus it is critically important for LPS biosynthesis. Modifications to lipid A have been shown to be important for resistance to antimicrobial peptides as well as modulating recognition by the mammalian innate immune system. Therefore, lipid A derivatives have been used for development of vaccine strains and vaccine adjuvants. One derivative that has yet to be studied is 8-amino-3,8-dideoxy-d-manno-octulosonic acid (Kdo8N), which is found exclusively in marine bacteria of the genus Shewanella. Using bioinformatics, a candidate gene cluster for Kdo8N biosynthesis was identified in Shewanella oneidensis. Expression of these genes recombinantly in Escherichia coli resulted in lipid A containing Kdo8N, and in vitro assays confirmed their proposed enzymatic function. Both the in vivo and in vitro data were consistent with direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of LPS by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase. To our knowledge, this oxidase is the first enzyme shown to oxidize an alcohol using a metal and molecular oxygen, not NAD(P)⁺. Creation of an S. oneidensis in-frame deletion strain showed increased sensitivity to the cationic antimicrobial peptide polymyxin as well as bile salts, suggesting a role in outer membrane integrity.     

Structure of the Bacterial Deacetylase LpxC Bound to the Nucleotide Reaction Product Reveals Mechanisms of Oxyanion Stabilization and Proton Transfer

The emergence of antibiotic-resistant strains of pathogenic bacteria is an increasing threat to global health that underscores an urgent need for an expanded antibacterial armamentarium. Gram-negative bacteria, such as Escherichia coli, have become increasingly important clinical pathogens with limited treatment options. This is due in part to their lipopolysaccharide (LPS) outer membrane components, which dually serve as endotoxins while also protecting Gram-negative bacteria from antibiotic entry. The LpxC enzyme catalyzes the committed step of LPS biosynthesis, making LpxC a promising target for new antibacterials. Here, we present the first structure of an LpxC enzyme in complex with the deacetylation reaction product, UDP-(3-O-(R-3-hydroxymyristoyl))-glucosamine. These studies provide valuable insight into recognition of substrates and products by LpxC and a platform for structure-guided drug discovery of broad spectrum Gram-negative antibiotics.     

Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major nosocomial pathogen that infects cystic fibrosis and immunocompromised patients. The impermeability of the P. aeruginosa outer membrane contributes substantially to the notorious antibiotic resistance of this human pathogen. This impermeability is partially imparted by the outer membrane protein H (OprH). Here we have solved the structure of OprH in a lipid environment by solution NMR. The structure reveals an eight-stranded β-barrel protein with four extracellular loops of unequal size. Fast time-scale dynamics measurements show that the extracellular loops are disordered and unstructured. It was previously suggested that the function of OprH is to provide increased stability to the outer membranes of P. aeruginosa by directly interacting with lipopolysaccharide (LPS) molecules. Using in vivo and in vitro biochemical assays, we show that OprH indeed interacts with LPS in P. aeruginosa outer membranes. Based upon NMR chemical shift perturbations observed upon the addition of LPS to OprH in lipid micelles, we conclude that the interaction is predominantly electrostatic and localized to charged regions near both rims of the barrel, but also through two conspicuous tyrosines in the middle of the bilayer. These results provide the first molecular structure of OprH and offer evidence for multiple interactions between OprH and LPS that likely contribute to the antibiotic resistance of P. aeruginosa.     

Characterization of the Six Glycosyltransferases Involved in the Biosynthesis of Yersinia enterocolitica Serotype O:3 Lipopolysaccharide Outer Core

Yersinia enterocolitica (Ye) is a Gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage φR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage φR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.     

A Membrane-located Glycosyltransferase Complex Required for Biosynthesis of the d-Galactan I Lipopolysaccharide O Antigen in Klebsiella pneumoniae

d-Galactan I is a polysaccharide with the disaccharide repeat unit structure [→3-β-d-Galf-(1→3)-α-d-Galp-(1→]. This glycan represents the lipopolysaccharide O antigen found in many Gram-negative bacteria, including several Klebsiella pneumoniae O serotypes. The polysaccharide is synthesized in the cytoplasm prior to its export via an ATP-binding cassette transporter. Sequence analysis predicts three galactosyltransferases in the d-galactan I genetic locus. They are WbbO (belonging to glycosyltransferase (GT) family 4), WbbM (GT-family 8), and WbbN (GT-family 2). The WbbO and WbbM proteins are each predicted to contain two domains, with the GT modules located toward their C termini. The N-terminal domains of WbbO and WbbM exhibit no similarity to proteins with known function. In vivo complementation assays suggest that all three glycosyltransferases are required for d-galactan I biosynthesis. Using a bacterial two-hybrid system and confirmatory co-purification strategies, evidence is provided for protein-protein interactions among the glycosyltransferases, creating a membrane-located enzyme complex dedicated to d-galactan I biosynthesis.     

Selective sorting of cargo proteins into bacterial membrane vesicles

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.     

Outer Membrane Protein I of Pseudomonas aeruginosa Is a Target of Cationic Antimicrobial Peptide/Protein

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and α-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and α-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric α-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of α-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.     

Biosynthesis of the Polymannose Lipopolysaccharide O-antigens from Escherichia coli Serotypes O8 and O9a Requires a Unique Combination of Single- and Multiple-active Site Mannosyltransferases

The Escherichia coli O9a and O8 O-antigen serotypes represent model systems for the ABC transporter-dependent synthesis of bacterial polysaccharides. The O9a and O8 antigens are linear mannose homopolymers containing conserved reducing termini (the primer-adaptor), a serotype-specific repeat unit domain, and a terminator. Synthesis of these glycans occurs on the polyisoprenoid lipid-linked primer, undecaprenol pyrophosphoryl-GlcpNAc, by two conserved mannosyltransferases, WbdC and WbdB, and a serotype-specific mannosyltransferase, WbdA. The glycan structure and pattern of conservation in the O9a and O8 mannosyltransferases are not consistent with the existing model of O9a biosynthesis. Here we establish a revised pathway using a combination of in vivo (mutant complementation) experiments and in vitro strategies with purified enzymes and synthetic acceptors. WbdC and WbdB synthesize the adaptor region, where they transfer one and two α-(1→3)-linked mannose residues, respectively. The WbdA enzymes are solely responsible for forming the repeat unit domains of these O-antigens. WbdA^O9a has two predicted active sites and polymerizes a tetrasaccharide repeat unit containing two α-(1→3)- and two α-(1→2)-linked mannopyranose residues. In contrast, WbdA^O8 polymerizes trisaccharide repeat units containing single α-(1→3)-, α-(1→2)-, and β-(1→2)-mannopyranoses. These studies illustrate assembly systems exploiting several mannosyltransferases with flexible active sites, arranged in single- and multiple-domain formats.     

Elongation Factor G Is a Critical Target during Oxidative Damage to the Translation System of Escherichia coli

Elongation factor G (EF-G), a key protein in translational elongation, is known to be particularly susceptible to oxidation in Escherichia coli. However, neither the mechanism of the oxidation of EF-G nor the influence of its oxidation on translation is fully understood. In the present study, we investigated the effects of oxidants on the chemical properties and function of EF-G using a translation system in vitro derived from E. coli. Treatment of EF-G with 0.5 mm H(2)O(2) resulted in the complete loss of translational activity. The inactivation of EF-G by H(2)O(2) was attributable to the oxidation of two specific cysteine residues, namely, Cys(114) and Cys(266), and subsequent formation of an intramolecular disulfide bond. Replacement of Cys(114) by serine rendered EF-G insensitive to oxidation and inactivation by H(2)O(2). Furthermore, generation of the translation system in vitro with the mutated EF-G protected the entire translation system from oxidation, suggesting that EF-G might be a primary target of oxidation within the translation system. Oxidized EF-G was reactivated via reduction of the disulfide bond by thioredoxin, a ubiquitous protein that mediates dithiol-disulfide exchange. Our observations indicate that the translational machinery in E. coli is regulated, in part, by the redox state of EF-G, which might depend on the balance between the supply of reducing power and the degree of oxidative stress.