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.

Trapped translocation intermediates establish the route for export of capsular polysaccharides across Escherichia coli outer membranes.

The outer membrane (OM) of Gram-negative bacteria is designed to exclude potentially harmful molecules. This property presents a challenge for bacteria that must secrete proteins and large glycoconjugates to grow, divide, and persist. Proteins involved in trafficking such molecules have been identified, but their precise roles are often unresolved due to the difficulty in capturing "snapshots" during the export pathway. Wza is the prototype for the large family of OM polysaccharide export proteins. In Escherichia coli, Wza is essential for the assembly of a capsule, a protective surface coat composed of long-chain polysaccharides. Wza creates an octameric α-helical channel spanning the OM, but the bulk of the protein exists as a large periplasmic structure enclosing an extensive lumen. Residues within the lumen of Wza were targeted for site-specific incorporation of the UV photo-cross-linkable unnatural amino acid p-benzoyl-L-phenylalanine. Using this in vivo photo-cross-linking strategy, we were able to trap polysaccharide translocation intermediates within the lumen of Wza, providing the first unequivocal evidence to our knowledge that nascent capsular polysaccharide chains exit the cell through the Wza portal.

Genetic, Biochemical, and Structural Analyses of Bacterial Surface Polysaccharides.

Surface polysaccharides are an often essential component of the outer surface of bacteria. They may serve to protect organisms from harsh environmental conditions and to increase virulence. The focus of this review will be to introduce polysaccharide biosynthesis and export from the cell, and the associated techniques used to determine these glycostructures. Protein interactions and proteomics will then be discussed while introducing systems biology approaches used to determine protein-protein and protein-polysaccharide interactions. The final section will address related screening methods used to study gene regulation in bacteria relating to polysaccharide gene clusters and their associated regulators. The goal of this review will be to highlight key studies that have increased our knowledge of glycobiology and discuss novel methods that examine this field at the cellular level using systems biology.

The UDP-glucose dehydrogenase of Escherichia coli K-12 displays substrate inhibition by NAD that is relieved by nucleotide triphosphates.

UDP-glucose dehydrogenase (Ugd) generates UDP-glucuronic acid, an important precursor for the production of many hexuronic acid-containing bacterial surface glycostructures. In Escherichia coli K-12, Ugd is important for biosynthesis of the environmentally regulated exopolysaccharide known as colanic acid, whereas in other E. coli isolates, the same enzyme is required for production of the constitutive group 1 capsular polysaccharides, which act as virulence determinants. Recent studies have implicated tyrosine phosphorylation in the activation of Ugd from E. coli K-12, although it is not known if this is a feature shared by bacterial Ugd proteins. The activities of Ugd from E. coli K-12 and from the group 1 capsule prototype (serotype K30) were compared. Surprisingly, for both enzymes, site-directed Tyr → Phe mutants affecting the previously proposed phosphorylation site retained similar kinetic properties to the wild-type protein. Purified Ugd from E. coli K-12 had significant levels of NAD substrate inhibition, which could be alleviated by the addition of ATP and several other nucleotide triphosphates. Mutations in a previously identified UDP-glucuronic acid allosteric binding site decreased the binding affinity of the nucleotide triphosphate. Ugd from E. coli serotype K30 was not inhibited by NAD, but its activity still increased in the presence of ATP.

The Platelet Fraction Is a Novel Reservoir to Detect Lyme Borrelia in Blood.

Serological diagnosis of Lyme disease suffers from considerable limitations. Yet, the technique cannot currently be replaced by direct detection methods, such as bacterial culture or molecular analysis, due to their inadequate sensitivity. The low bacterial burden in vasculature and lack of consensus around blood-based isolation of the causative pathogen, Borrelia burgdorferi, are central to this challenge. We therefore addressed methodological optimization of Borrelia recovery from blood, first by analyzing existing protocols, and then by using experimentally infected human blood to identify the processing conditions and fractions that increase Borrelia yield. In this proof-of-concept study, we now report two opportunities to improve recovery and detection of Borrelia from clinical samples. To enhance pathogen viability and cultivability during whole blood collection, citrate anticoagulant is superior to more commonly used EDTA. Despite the widespread reliance on serum and plasma as analytes, we found that the platelet fraction of blood concentrates Borrelia, providing an enriched resource for direct pathogen detection by microscopy, laboratory culture, Western blot, and PCR. The potential for platelets to serve as a reservoir for Borrelia and its diagnostic targets may transform direct clinical detection of this pathogen.

Immunogenicity of the Lyme disease antigen OspA, particleized by cobalt porphyrin-phospholipid liposomes.

Outer surface protein A (OspA) is a Borrelia lipoprotein and an established Lyme disease vaccine target. Admixing non-lipidated, recombinant B. burgdorferi OspA with liposomes containing cobalt porphyrin-phospholipid (CoPoP) resulted in rapid, particulate surface display of the conformationally intact antigen. Particleization was serum-stable and led to enhanced antigen uptake in murine macrophages in vitro. Mouse immunization using CoPoP liposomes that also contained a synthetic monophosphoryl lipid A (PHAD) elicited a Th1-biased OspA antibody response with higher IgG production compared to other vaccine adjuvants. Antibodies were reactive with intact B. burgdorferi spirochetes and Borrelia lysates, and induced complement-mediated borreliacidal activity in vitro. One year after initial immunization, mice maintained high levels of circulating borreliacidal antibodies capable of blocking B. burgdorferi transmission from infected ticks to human blood in a feeding chamber.

The structure of Escherichia coli signal recognition particle revealed by scanning transmission electron microscopy.

Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances that were consistent with our model of SRP. Docking our model onto the bacterial ribosome suggests a mechanism for signal recognition involving interdomain movement of Ffh into and out of the nascent chain exit site and suggests how SRP could interact and/or compete with the ribosome-bound chaperone, trigger factor, for a nascent chain during translation.

Lyme Disease Frontiers: Reconciling Borrelia Biology and Clinical Conundrums.

Lyme disease is a complex tick-borne zoonosis that poses an escalating public health threat in several parts of the world, despite sophisticated healthcare infrastructure and decades of effort to address the problem. Concepts like the true burden of the illness, from incidence rates to longstanding consequences of infection, and optimal case management, also remain shrouded in controversy. At the heart of this multidisciplinary issue are the causative spirochetal pathogens belonging to the Borrelia Lyme complex. Their unusual physiology and versatile lifestyle have challenged microbiologists, and may also hold the key to unlocking mysteries of the disease. The goal of this review is therefore to integrate established and emerging concepts of Borrelia biology and pathogenesis, and position them in the broader context of biomedical research and clinical practice. We begin by considering the conventions around diagnosing and characterizing Lyme disease that have served as a conceptual framework for the discipline. We then explore virulence from the perspective of both host (genetic and environmental predispositions) and pathogen (serotypes, dissemination, and immune modulation), as well as considering antimicrobial strategies (lab methodology, resistance, persistence, and clinical application), and borrelial adaptations of hypothesized medical significance (phenotypic plasticity or pleomorphy).

Wzi is an outer membrane lectin that underpins group 1 capsule assembly in Escherichia coli.

Many pathogenic bacteria encase themselves in a polysaccharide capsule that provides a barrier to the physical and immunological challenges of the host. The mechanism by which the capsule assembles around the bacterial cell is unknown. Wzi, an integral outer-membrane protein from Escherichia coli, has been implicated in the formation of group 1 capsules. The 2.6 Å resolution structure of Wzi reveals an 18-stranded ß-barrel fold with a novel arrangement of long extracellular loops that blocks the extracellular entrance and a helical bundle that plugs the periplasmic end. Mutagenesis shows that specific extracellular loops are required for in vivo capsule assembly. The data show that Wzi binds the K30 carbohydrate polymer and, crucially, that mutants functionally deficient in vivo show no binding to K30 polymer in vitro. We conclude that Wzi is a novel outer-membrane lectin that assists in the formation of the bacterial capsule via direct interaction with capsular polysaccharides.

TPR motifs: hallmarks of a new polysaccharide export scaffold.

Bacteria produce a remarkable range of surface and secreted polysaccharides. Two pathways have been defined for the biosynthesis and export of capsular polysaccharides and exopolysaccharides in Gram-negative bacteria. A structure of AlgK described in this issue provides structural insight into a third previously unrecognized pathway associated with important biopolymers (Keiski et al., 2010).

Pivotal roles of the outer membrane polysaccharide export and polysaccharide copolymerase protein families in export of extracellular polysaccharides in gram-negative bacteria.

Many bacteria export extracellular polysaccharides (EPS) and capsular polysaccharides (CPS). These polymers exhibit remarkably diverse structures and play important roles in the biology of free-living, commensal, and pathogenic bacteria. EPS and CPS production represents a major challenge because these high-molecular-weight hydrophilic polymers must be assembled and exported in a process spanning the envelope, without compromising the essential barrier properties of the envelope. Emerging evidence points to the existence of molecular scaffolds that perform these critical polymer-trafficking functions. Two major pathways with different polymer biosynthesis strategies are involved in the assembly of most EPS/CPS: the Wzy-dependent and ATP-binding cassette (ABC) transporter-dependent pathways. They converge in an outer membrane export step mediated by a member of the outer membrane auxiliary (OMA) protein family. OMA proteins form outer membrane efflux channels for the polymers, and here we propose the revised name outer membrane polysaccharide export (OPX) proteins. Proteins in the polysaccharide copolymerase (PCP) family have been implicated in several aspects of polymer biogenesis, but there is unequivocal evidence for some systems that PCP and OPX proteins interact to form a trans-envelope scaffold for polymer export. Understanding of the precise functions of the OPX and PCP proteins has been advanced by recent findings from biochemistry and structural biology approaches and by parallel studies of other macromolecular trafficking events. Phylogenetic analyses reported here also contribute important new insight into the distribution, structural relationships, and function of the OPX and PCP proteins. This review is intended as an update on progress in this important area of microbial cell biology.

Crystal structures of Wzb of Escherichia coli and CpsB of Streptococcus pneumoniae, representatives of two families of tyrosine phosphatases that regulate capsule assembly.

Many Gram-positive and Gram-negative bacteria utilize polysaccharide surface layers called capsules to evade the immune system; consequently, the synthesis and export of the capsule are a potential therapeutic target. In Escherichia coli K-30, the integral membrane tyrosine autokinase Wzc and the cognate phosphatase Wzb have been shown to be key for both synthesis and assembly of capsular polysaccharides. In the Gram-positive bacterium Streptococcus pneumoniae, the CpsCD complex is analogous to Wzc and the phosphatase CpsB is the corresponding cognate phosphatase. The phosphatases are known to dephosphorylate their corresponding autokinases, yet despite their functional equivalence, they share no sequence homology. We present the structure of Wzb in complex with phosphate and high-resolution structures of apo-CpsB and a phosphate-complexed CpsB. We show that both proteins are active toward Wzc and thereby demonstrate that CpsB is not specific for CpsCD. CpsB is a novel enzyme and represents the first solved structure of a tyrosine phosphatase from a Gram-positive bacterium. Wzb and CpsB have completely different structures, suggesting that they must operate by very different mechanisms. Although the mechanism of Wzb can be inferred from previous studies, CpsB appears to have a tyrosine phosphatase mechanism not observed before. We propose a chemical mechanism for CpsB based on site-directed mutagenesis and structural data.