Transition transferases prime bacterial capsule polymerization.

Capsules are long-chain carbohydrate polymers that envelop the surfaces of many bacteria, protecting them from host immune responses. Capsule biosynthesis enzymes are potential drug targets and valuable biotechnological tools for generating vaccine antigens. Despite their importance, it remains unknown how structurally variable capsule polymers of Gram-negative pathogens are linked to the conserved glycolipid anchoring these virulence factors to the bacterial membrane. Using Actinobacillus pleuropneumoniae as an example, we demonstrate that CpsA and CpsC generate a poly(glycerol-3-phosphate) linker to connect the glycolipid with capsules containing poly(galactosylglycerol-phosphate) backbones. We reconstruct the entire capsule biosynthesis pathway in A. pleuropneumoniae serotypes 3 and 7, solve the X-ray crystal structure of the capsule polymerase CpsD, identify its tetratricopeptide repeat domain as essential for elongating poly(glycerol-3-phosphate) and show that CpsA and CpsC stimulate CpsD to produce longer polymers. We identify the CpsA and CpsC product as a wall teichoic acid homolog, demonstrating similarity between the biosynthesis of Gram-positive wall teichoic acid and Gram-negative capsules.

Identification of a second glycoform of the clinically prevalent O1 antigen from Klebsiella pneumoniae.

Carbapenemase and extended ß-lactamase-producing Klebsiella pneumoniae isolates represent a major health threat, stimulating increasing interest in immunotherapeutic approaches for combating Klebsiella infections. Lipopolysaccharide O antigen polysaccharides offer viable targets for immunotherapeutic development, and several studies have described protection with O-specific antibodies in animal models of infection. O1 antigen is produced by almost half of clinical Klebsiella isolates. The O1 polysaccharide backbone structure is known, but monoclonal antibodies raised against the O1 antigen showed varying reactivity against different isolates that could not be explained by the known structure. Reinvestigation of the structure by NMR spectroscopy revealed the presence of the reported polysaccharide backbone (glycoform O1a), as well as a previously unknown O1b glycoform composed of the O1a backbone modified with a terminal pyruvate group. The activity of the responsible pyruvyltransferase (WbbZ) was confirmed by western immunoblotting and in vitro chemoenzymatic synthesis of the O1b terminus. Bioinformatic data indicate that almost all O1 isolates possess genes required to produce both glycoforms. We describe the presence of O1ab-biosynthesis genes in other bacterial species and report a functional O1 locus on a bacteriophage genome. Homologs of wbbZ are widespread in genetic loci for the assembly of unrelated glycostructures in bacteria and yeast. In K. pneumoniae, simultaneous production of both O1 glycoforms is enabled by the lack of specificity of the ABC transporter that exports the nascent glycan, and the data reported here provide mechanistic understanding of the capacity for evolution of antigenic diversity within an important class of biomolecules produced by many bacteria.

Mechanism and linkage specificities of the dual retaining ß-Kdo glycosyltransferase modules of KpsC from bacterial capsule biosynthesis.

KpsC is a dual-module glycosyltransferase (GT) essential for "group 2" capsular polysaccharide biosynthesis in Escherichia coli and other Gram-negative pathogens. Capsules are vital virulence determinants in high-profile pathogens, making KpsC a viable target for intervention with small-molecule therapeutic inhibitors. Inhibitor development can be facilitated by understanding the mechanism of the target enzyme. Two separate GT modules in KpsC transfer 3-deoxy-ß-d-manno-oct-2-ulosonic acid (ß-Kdo) from cytidine-5'-monophospho-ß-Kdo donor to a glycolipid acceptor. The N-terminal and C-terminal modules add alternating Kdo residues with ß-(2→4) and ß-(2→7) linkages, respectively, generating a conserved oligosaccharide core that is further glycosylated to produce diverse capsule structures. KpsC is a retaining GT, which retains the donor anomeric carbon stereochemistry. Retaining GTs typically use an SNi (substitution nucleophilic internal return) mechanism, but recent studies with WbbB, a retaining ß-Kdo GT distantly related to KpsC, strongly suggest that this enzyme uses an alternative double-displacement mechanism. Based on the formation of covalent adducts with Kdo identified here by mass spectrometry and X-ray crystallography, we determined that catalytically important active site residues are conserved in WbbB and KpsC, suggesting a shared double-displacement mechanism. Additional crystal structures and biochemical experiments revealed the acceptor binding mode of the ß-(2→4)-Kdo transferase module and demonstrated that acceptor recognition (and therefore linkage specificity) is conferred solely by the N-terminal α/ß domain of each GT module. Finally, an Alphafold model provided insight into organization of the modules and a C-terminal membrane-anchoring region. Altogether, we identified key structural and mechanistic elements providing a foundation for targeting KpsC.

Investigation of core machinery for biosynthesis of Vi antigen capsular polysaccharides in Gram-negative bacteria.

Salmonella enterica serovar Typhi causes typhoid fever. It possesses a Vi antigen capsular polysaccharide coat that is important for virulence and is the basis of a current glycoconjugate vaccine. Vi antigen is also produced by environmental Bordetella isolates, while mammal-adapted Bordetella species (such as Bordetella bronchiseptica) produce a capsule of undetermined structure that cross-reacts with antibodies recognizing Vi antigen. The Vi antigen backbone is composed of poly-α-(1→4)-linked N-acetylgalactosaminuronic acid, modified with O-acetyl residues that are necessary for vaccine efficacy. Despite its biological and biotechnological importance, some central aspects of Vi antigen production are poorly understood. Here we demonstrate that TviE and TviD, two proteins encoded in the viaB (Vi antigen production) locus, interact and are the Vi antigen polymerase and O-acetyltransferase, respectively. Structural modeling and site-directed mutagenesis reveal that TviE is a GT4-family glycosyltransferase. While TviD has no identifiable homologs beyond Vi antigen systems in other bacteria, structural modeling suggests that it belongs to the large SGNH hydrolase family, which contains other O-acetyltransferases. Although TviD possesses an atypical catalytic triad, its O-acetyltransferase function was verified by antibody reactivity and 13C NMR data for tviD-mutant polysaccharide. The B. bronchiseptica genetic locus predicts a mode of synthesis distinct from classical S. enterica Vi antigen production, but which still involves TviD and TviE homologs that are both active in a reconstituted S. Typhi system. These findings provide new insight into Vi antigen production and foundational information for the glycoengineering of Vi antigen production in heterologous bacteria.

A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family.

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-ß-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.

Biosynthesis of a conserved glycolipid anchor for Gram-negative bacterial capsules.

Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved 3-deoxy-ß-D-manno-oct-2-ulosonic acid (ß-Kdo) oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining the structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two cytidine 5'-monophosphate (CMP)-Kdo-dependent glycosyltransferase catalytic centers with different linkage specificities. The structure of the N-terminal ß-(2→4) Kdo transferase from KpsC reveals two α/ß domains, supplemented by several helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared with canonical GT-B folds in glycosyltransferases. The similar structure of the C-terminal ß-(2→7) Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 ß-Kdo transferases. This enzyme provides the prototype for the GT107 family.

Substrate recognition by a carbohydrate-binding module in the prototypical ABC transporter for lipopolysaccharide O-antigen from Escherichia coli O9a.

Escherichia coli serotype O9a provides a model for export of lipopolysaccharide (LPS) O-antigen polysaccharide (O-PS) via ABC transporters. In O9a biosynthesis, a chain-terminator enzyme, WbdD, caps the nonreducing end of the glycan with a methylphosphate moiety and thereby establishes chain-length distribution. A carbohydrate-binding module (CBM) in the ABC transporter recognizes terminated glycans, ensuring that only mature O-PS is exported and incorporated into LPS. Here, we addressed two questions arising from this model. Are both residues in the binary terminator necessary for termination and export? And is a terminal methylphosphate moiety sufficient for export of heterologous glycans? To answer the first question, we uncoupled WbdD kinase and methyltransferase activities. WbdD mutants revealed that although the kinase activity is solely responsible for chain-length regulation, both activities are essential for CBM recognition and export. Consistent with this observation, a saturation transfer difference NMR experiment revealed a direct interaction between the CBM and the terminal methyl group. To determine whether methylphosphate is the sole determinant of substrate recognition by the CBM, we exploited Klebsiella pneumoniae O7, whose O-PS repeat-unit structure differs from O9a, but, as shown here, offers the second confirmed example of a terminal methylphosphate serving in substrate recognition. In vitro and in vivo experiments indicated that each CBM can bind the O-PS only with the native repeat unit, revealing that methylphosphate is essential but not sufficient for substrate recognition and export. Our findings provide important new insight into the structural determinants in a prototypical quality control system for glycan assembly and export.

Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification.

A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-d-Galp-(1→3)-ß-d-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-d-Galp-(1→3)-ß-d-Galp-(1→] or O2c [→3)-ß-d-GlcpNAc-(1→5)-ß-d-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp-dependent GT-A galactosyltransferase adding ß-(1→3)-linked d-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)-linked d-Galp These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Galf-dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf. Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria.

Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control.

Lipopolysaccharides (LPS) are essential outer membrane glycolipids in most gram-negative bacteria. Biosynthesis of the O-antigenic polysaccharide (OPS) component of LPS follows one of three widely distributed strategies, and similar processes are used to assemble other bacterial surface glycoconjugates. This study focuses on the ATP-binding cassette (ABC) transporter-dependent pathway, where glycans are completed on undecaprenyl diphosphate carriers at the cytosol:membrane interface, before export by the ABC transporter. We describe Raoultella terrigena WbbB, a prototype for a family of proteins that, remarkably, integrates several key activities in polysaccharide biosynthesis into a single polypeptide. WbbB contains three glycosyltransferase (GT) modules. Each of the GT102 and GT103 modules characterized here represents a previously unrecognized GT family. They form a polymerase, generating a polysaccharide of [4)-α-Rhap-(1→3)-ß-GlcpNAc-(1→] repeat units. The polymer chain is terminated by a ß-linked Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) residue added by a third GT module belonging to the recently discovered GT99 family. The polymerase GT modules are separated from the GT99 chain terminator by a coiled-coil structure that forms a molecular ruler to determine product length. Different GT modules in the polymerase domains of other family members produce diversified OPS structures. These findings offer insight into glycan assembly mechanisms and the generation of antigenic diversity as well as potential tools for glycoengineering.

Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae.

Klebsiella pneumoniae is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing Klebsiella infections. Lipopolysaccharide O antigens are attractive candidates because of the relatively small range of known O-antigen polysaccharide structures, but immunotherapeutic applications require a complete understanding of the structures found in clinical settings. Currently, the precise number of Klebsiella O antigens is unknown because available serological tests have limited resolution, and their association with defined chemical structures is sometimes uncertain. Molecular serotyping methods can evaluate clinical prevalence of O serotypes but require a full understanding of the genetic determinants for each O-antigen structure. This is problematic with Klebsiella pneumoniae because genes outside the main rfb (O-antigen biosynthesis) locus can have profound effects on the final structure. Here, we report two new loci encoding enzymes that modify a conserved polysaccharide backbone comprising disaccharide repeat units [→3)-α-d-Galp-(1→3)-ß-d-Galf-(1→] (O2a antigen). We identified in serotype O2aeh a three-component system that modifies completed O2a glycan in the periplasm by adding 1,2-linked α-Galp side-group residues. In serotype O2ac, a polysaccharide comprising disaccharide repeat units [→5)-ß-d-Galf-(1→3)-ß-d-GlcpNAc-(1→] (O2c antigen) is attached to the non-reducing termini of O2a-antigen chains. O2c-polysaccharide synthesis is dependent on a locus encoding three glycosyltransferase enzymes. The authentic O2aeh and O2c antigens were recapitulated in recombinant Escherichia coli hosts to establish the essential gene set for their synthesis. These findings now provide a complete understanding of the molecular genetic basis for the known variations in Klebsiella O-antigen carbohydrate structures based on the O2a backbone.

High-Throughput "FP-Tag" Assay for the Identification of Glycosyltransferase Inhibitors.

Bacterial capsular polysaccharides are important virulence factors. Capsular polysaccharides from several important Gram-negative pathogens share a conserved glycolipid terminus containing 3-deoxy-ß-d- manno-oct-2-ulosonic acid (ß-Kdo). The ß-Kdo glycosyltransferases responsible for synthesis of this conserved glycolipid belong to a new family of glycosyltransferases that shares little homology with other such enzymes, thereby representing an attractive antivirulence target. Here, we report the development of a fluorescence polarization-based, high-throughput screening assay (FP-tag) for ß-Kdo glycosyltransferases, and use it to identify a class of marine natural products as lead inhibitors. This "FP-tag" assay should be readily adaptable to high-throughput screens of other glycosyltransferases.

Unique lipid anchor attaches Vi antigen capsule to the surface of Salmonella enterica serovar Typhi.

Polysaccharide capsules are surface structures that are critical for the virulence of many Gram-negative pathogenic bacteria. Salmonella enterica serovar Typhi is the etiological agent of typhoid fever. It produces a capsular polysaccharide known as "Vi antigen," which is composed of nonstoichiometrically O-acetylated α-1,4-linked N-acetylgalactosaminuronic acid residues. This glycan is a component of currently available vaccines. The genetic locus for Vi antigen production is also present in soil bacteria belonging to the genus Achromobacter Vi antigen assembly follows a widespread general strategy with a characteristic glycan export step involving an ATP-binding cassette transporter. However, Vi antigen producers lack the enzymes that build the conserved terminal glycolipid characterizing other capsules using this method. Achromobacter species possess a Vi antigen-specific depolymerase enzyme missing in S enterica Typhi, and we exploited this enzyme to isolate acylated Vi antigen termini. Mass spectrometry analysis revealed a reducing terminal N-acetylhexosamine residue modified with two ß-hydroxyl acyl chains. This terminal structure resembles one half of lipid A, the hydrophobic portion of bacterial lipopolysaccharides. The VexE protein encoded in the Vi antigen biosynthesis locus shares similarity with LpxL, an acyltransferase from lipid A biosynthesis. In the absence of VexE, Vi antigen is produced, but its physical properties are altered, its export is impaired, and a Vi capsule structure is not assembled on the cell surface. The structure of the lipidated terminus dictates a unique assembly mechanism and has potential implications in pathogenesis and vaccine production.

Bacterial ß-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99).

Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) is an eight-carbon sugar mostly confined to Gram-negative bacteria. It is often involved in attaching surface polysaccharides to their lipid anchors. α-Kdo provides a bridge between lipid A and the core oligosaccharide in all bacterial LPSs, whereas an oligosaccharide of ß-Kdo residues links "group 2" capsular polysaccharides to (lyso)phosphatidylglycerol. ß-Kdo is also found in a small number of other bacterial polysaccharides. The structure and function of the prototypical cytidine monophosphate-Kdo-dependent α-Kdo glycosyltransferase from LPS assembly is well characterized. In contrast, the ß-Kdo counterparts were not identified as glycosyltransferase enzymes by bioinformatics tools and were not represented among the 98 currently recognized glycosyltransferase families in the Carbohydrate-Active Enzymes database. We report the crystallographic structure and function of a prototype ß-Kdo GT from WbbB, a modular protein participating in LPS O-antigen synthesis in Raoultella terrigena The ß-Kdo GT has dual Rossmann-fold motifs typical of GT-B enzymes, but extensive deletions, insertions, and rearrangements result in a unique architecture that makes it a prototype for a new GT family (GT99). The cytidine monophosphate-binding site in the C-terminal α/ß domain closely resembles the corresponding site in bacterial sialyltransferases, suggesting an evolutionary connection that is not immediately evident from the overall fold or sequence similarities.

Biochemical Characterization of Bifunctional 3-Deoxy-ß-d-manno-oct-2-ulosonic Acid (ß-Kdo) Transferase KpsC from Escherichia coli Involved in Capsule Biosynthesis.

3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an essential component of bacterial lipopolysaccharides, where it provides the linkage between lipid and carbohydrate moieties. In all known LPS structures, Kdo residues possess α-anomeric configurations, and the corresponding inverting α-Kdo transferases are well characterized. Recently, it has been shown that a large group of capsular polysaccharides from Gram-negative bacteria, produced by ATP-binding cassette transporter-dependent pathways, are also attached to a lipid anchor through a conserved Kdo oligosaccharide. In the study reported here, the structure of this Kdo linker was determined by NMR spectroscopy, revealing alternating ß-(2→4)- and ß-(2→7)-linked Kdo residues. KpsC contains two retaining ß-Kdo glycosyltransferase domains belonging to family GT99 that are responsible for polymerizing the ß-Kdo linker on its glycolipid acceptor. Full-length Escherichia coli KpsC was expressed and purified, together with the isolated N-terminal domain and a mutant protein (KpsC D160A) containing a catalytically inactivated N-terminal domain. The Kdo transferase activities of these proteins were determined in vitro using synthetic acceptors, and the reaction products were characterized using TLC, mass spectrometry, and NMR spectroscopy. The N- and C-terminal domains were found to catalyze formation of ß-(2→4) and ß-(2→7) linkages, respectively. Based on phylogenetic analyses, we propose the linkage specificities of the glycosyltransferase domains are conserved in KpsC homologs from other bacterial species.

Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism.

Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O-antigen glycans, altering the structure of the bacteriophage receptor and resulting in serotype conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O-antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation potential of a model O-antigen produced in an ATP-binding cassette (ABC) transporter-dependent system. Although O-antigen production is cryptic in Escherichia coli K-12, because of a mutation in the synthesis genes, it possesses a prophage glucosylation cluster, which modifies the GlcNAc residue in an α-l-Rha-(1→3)-d-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O-antigen possessing the same disaccharide motif, but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O-antigen displaying reduced reactivity toward antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways.

Variations in O-antigen biosynthesis and O-acetylation associated with altered phage sensitivity in Escherichia coli 4s.

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmental Escherichia coli isolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen of E. coli O22, differing only in side-chain α-D-glucosylation in the former, mediated by a gtr locus on the chromosome. Spontaneous mutations of E. coli 4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions of E. coli 4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.

Hypermucoviscosity Regulator RmpD Interacts with Wzc and Controls Capsular Polysaccharide Chain Length.

Klebsiella pneumoniae is a leading cause of nosocomial infections, including pneumonia, bacteremia, and urinary tract infections. Treatment options are increasingly restricted by the high prevalence of resistance to frontline antibiotics, including carbapenems, and the recently identified plasmid-conferred colistin resistance. The classical pathotype (cKp) is responsible for most of the nosocomial infections observed globally, and these isolates are often multidrug resistant. The hypervirulent pathotype (hvKp) is a primary pathogen capable of causing community-acquired infections in immunocompetent hosts. The hypermucoviscosity (HMV) phenotype is strongly associated with the increased virulence of hvKp isolates. Recent studies demonstrated that HMV requires capsule (CPS) synthesis and the small protein RmpD but is not dependent on the increased amount of capsule associated with hvKp. Here, we identified the structure of the capsular and extracellular polysaccharide isolated from hvKp strain KPPR1S (serotype K2) with and without RmpD. We found that the polymer repeat unit structure is the same in both strains and that it is identical to the K2 capsule. However, the chain length of CPS produced by strains expressing rmpD demonstrates more uniform length. This property was reconstituted in CPS from Escherichia coli isolates that possess the same CPS biosynthesis pathway as K. pneumoniae but naturally lack rmpD. Furthermore, we demonstrate that RmpD binds Wzc, a conserved capsule biosynthesis protein required for CPS polymerization and export. Based on these observations, we present a model for how the interaction of RmpD with Wzc could impact CPS chain length and HMV. IMPORTANCE Infections caused by Klebsiella pneumoniae continue to be a global public health threat; the treatment of these infections is complicated by the high frequency of multidrug resistance. K. pneumoniae produces a polysaccharide capsule required for virulence. Hypervirulent isolates also have a hypermucoviscous (HMV) phenotype that increases virulence, and we recently demonstrated that a horizontally acquired gene, rmpD, is required for HMV and hypervirulence but that the identity of the polymeric product(s) in HMV isolates is uncertain. Here, we demonstrate that RmpD regulates capsule chain length and interacts with Wzc, a part of the capsule polymerization and export machinery shared by many pathogens. We further show that RmpD confers HMV and regulates capsule chain length in a heterologous host (E. coli). As Wzc is a conserved protein found in many pathogens, it is possible that RmpD-mediated HMV and increased virulence may not be restricted to K. pneumoniae.

Inhibiting fatty acid synthesis overcomes colistin resistance.

Treating multidrug-resistant infections has increasingly relied on last-resort antibiotics, including polymyxins, for example colistin. As polymyxins are given routinely, the prevalence of their resistance is on the rise and increases mortality rates of sepsis patients. The global dissemination of plasmid-borne colistin resistance, driven by the emergence of mcr-1, threatens to diminish the therapeutic utility of polymyxins from an already shrinking antibiotic arsenal. Restoring sensitivity to polymyxins using combination therapy with sensitizing drugs is a promising approach to reviving its clinical utility. Here we describe the ability of the biotin biosynthesis inhibitor, MAC13772, to synergize with colistin exclusively against colistin-resistant bacteria. MAC13772 indirectly disrupts fatty acid synthesis (FAS) and restores sensitivity to the last-resort antibiotic, colistin. Accordingly, we found that combinations of colistin and other FAS inhibitors, cerulenin, triclosan and Debio1452-NH3, had broad potential against both chromosomal and plasmid-mediated colistin resistance in chequerboard and lysis assays. Furthermore, combination therapy with colistin and the clinically relevant FabI inhibitor, Debio1452-NH3, showed efficacy against mcr-1 positive Klebsiella pneumoniae and colistin-resistant Escherichia coli systemic infections in mice. Using chemical genomics, lipidomics and transcriptomics, we explored the mechanism of the interaction. We propose that inhibiting FAS restores colistin sensitivity by depleting lipid synthesis, leading to changes in phospholipid composition. In all, this work reveals a surprising link between FAS and colistin resistance.

Genetic analysis of the O-antigen of Providencia alcalifaciens O30 and biochemical characterization of a formyltransferase involved in the synthesis of a Qui4N derivative.

O-Antigen is a component of the outer membrane of Gram-negative bacteria and one of the most variable cell surface constituents, giving rise to major antigenic variability. The diversity of O-antigen is almost entirely attributed to genetic variations in O-antigen gene clusters. Bacteria of the genus Providencia are facultative pathogens, which can cause urinary tract infections, wound infections and enteric diseases. Recently, the O-antigen gene cluster of Providencia was localized between the cpxA and yibK genes in the genome. However, few genes involved in the synthesis of Providencia O-antigens have been functionally identified. In this study, the putative O-antigen gene cluster of Providencia alcalifaciens O30 was sequenced and analyzed. Almost all putative genes for the O-antigen synthesis were found, including a novel formyltransferase gene vioF that was proposed to be responsible for the conversion of dTDP-4-amino-4,6- dideoxy-D-glucose (dTDP-D-Qui4N) to dTDP-4,6-dideoxy-4-formamido-D-glucose (dTDP-D-Qui4NFo). vioF was cloned, and the enzyme product was expressed as a His-tagged fusion protein, purified and assayed for its activity. High-performance liquid chromatography was used to monitor the enzyme-substrate reaction, and the structure of the product dTDP-D-Qui4NFo was established by electrospray ionization tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Kinetic parameters of VioF were determined, and effects of temperature and cations on its activity were also examined. Together, the functional analyses support the identification of the O-antigen gene cluster of P. alcalifaciens O30.

Localization and molecular characterization of putative O antigen gene clusters of Providencia species.

Enterobacteria of the genus Providencia are opportunistic human pathogens associated with urinary tract and wound infections, as well as enteric diseases. The lipopolysaccharide (LPS) O antigen confers major antigenic variability upon the cell surface and is used for serotyping of Gram-negative bacteria. Recently, Providencia O antigen structures have been extensively studied, but no data on the location and organization of the O antigen gene cluster have been reported. In this study, the four Providencia genome sequences available were analysed, and the putative O antigen gene cluster was identified in the polymorphic locus between the cpxA and yibK genes. This finding provided the necessary information for designing primers, and cloning and sequencing the O antigen gene clusters from five more Providencia alcalifaciens strains. The gene functions predicted in silico were in agreement with the known O antigen structures; furthermore, annotation of the genes involved in the three-step synthesis of GDP-colitose (gmd, colD and colC) was supported by cloning and biochemical characterization of the corresponding enzymes. In one strain (P. alcalifaciens O39), no polysaccharide product of the gene cluster in the cpxA-yibK locus was found, and hence genes for synthesis of the existing O antigen are located elsewhere in the genome. In addition to the putative O antigen synthesis genes, homologues of wza, wzb, wzc and (in three strains) wzi, required for the surface expression of capsular polysaccharides, were found upstream of yibK in all species except Providencia rustigianii, suggesting that the LPS of these species may be attributed to the so-called K LPS (K(LPS)). The data obtained open a way for development of a PCR-based typing method for identification of Providencia isolates.