Three-component systems represent a common pathway for extracytoplasmic addition of pentofuranose sugars into bacterial glycans.

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.

Cytosolic galectin-4 enchains bacteria, restricts their motility, and promotes inflammasome activation in intestinal epithelial cells.

Galectin-4, a member of the galectin family of animal glycan-binding proteins (GBPs), is specifically expressed in gastrointestinal epithelial cells and is known to be able to bind microbes. However, its function in host-gut microbe interactions remains unknown. Here, we show that intracellular galectin-4 in intestinal epithelial cells (IECs) coats cytosolic Salmonella enterica serovar Worthington and induces the formation of bacterial chains and aggregates. Galectin-4 enchains bacteria during their growth by binding to the O-antigen of lipopolysaccharides. Furthermore, the binding of galectin-4 to bacterial surfaces restricts intracellular bacterial motility. Galectin-4 enhances caspase-1 activation and mature IL-18 production in infected IECs especially when autophagy is inhibited. Finally, orally administered S. enterica serovar Worthington, which is recognized by human galectin-4 but not mouse galectin-4, translocated from the intestines to mesenteric lymph nodes less effectively in human galectin-4-transgenic mice than in littermate controls. Our results suggest that galectin-4 plays an important role in host-gut microbe interactions and prevents the dissemination of pathogens. The results of the study revealed a novel mechanism of host-microbe interactions that involves the direct binding of cytosolic lectins to glycans on intracellular microbes.

Helicobacter pylori employs a general protein glycosylation system for the modification of outer membrane adhesins.

Helicobacter pylori infection is associated with the development of several gastric diseases including gastric cancer. To reach a long-term colonization in the host stomach, H. pylori employs multiple outer membrane adhesins for binding to the gastric mucosa. However, due to the redundancy of adhesins that complement the adhesive function of bacteria, targeting each individual adhesin alone usually achieves nonideal outcomes for preventing bacterial adhesion. Here, we report that key adhesins AlpA/B and BabA/B in H. pylori are modified by glycans and display a two-step molecular weight upshift pattern from the cytoplasm to the inner membrane and from the inner membrane to the outer membrane. Nevertheless, this upshift pattern is missing when the expression of some enzymes related to lipopolysaccharide (LPS) biosynthesis, including the LPS O-antigen assembly and ligation enzymes WecA, Wzk, and WaaL, is disrupted, indicating that the underlying mechanisms and the involved enzymes for the adhesin glycosylation are partially shared with the LPS biosynthesis. Loss of the adhesin glycosylation not only reduces the protease resistance and the stability of the tested adhesins but also changes the adhesin-binding ability. In addition, mutations in the LPS biosynthesis cause a significant reduction in bacterial adhesion in the in vitro cell-line model. The current findings reveal that H. pylori employs a general protein glycosylation system related to LPS biosynthesis for adhesin modification and its biological significance. The enzymes required for adhesin glycosylation rather than the adhesins themselves are potentially better drug targets for preventing or treating H. pylori infection.

Attenuated Salmonella Typhimurium with truncated LPS and outer membrane-displayed RGD peptide for cancer therapy.

Gram-negative, facultatively anaerobic bacteria Salmonella Typhimurium is a candidate agent or delivery vector for cancer therapy. Effective targeted therapies in addition to radiotherapy, chemotherapy and surgery have been urgently needed as an alternative or supplement. This study expected to further improve the tumor-targeting ability of Salmonella bacteria through genetic modifications. Based on an auxotrophic Salmonella bacterial strain (D2), we constructed Salmonella mutants with altered LPS length to facilitate displaying the RGD4C targeting peptide on the outer membrane surface of Salmonella. The expression of RGD4C peptide in fusion with OmpA was identified by outer membrane protein extraction and WB detection in different mutant strains. However, flow cytometry analysis following immunofluorescence staining demonstrated that the extracellular length of Salmonella LPS did affect the surface display of RGD4C peptide. The strain D2-RGD4C that synthesized intact LPS including lipid A, core oligosaccharides and O antigen polysaccharides could hardly display RGD4C peptide, showing the same fluorescence signal intensity as the strains not expressing RGD4C peptide. Among different strains, D2 ∆rfaJ-RGD4C that synthesized truncated LPS including lipid A and partial core oligosaccharides was capable of displaying RGD4C peptide most efficiently and showed the highest ability to target HUVECs expressing αV integrin and tumor tissue with abundant neovascularization. Animal experiments also demonstrated that this tumor-targeting attenuated Salmonella strain to simultaneously deliver endostatin and TRAIL, two agents with different anti-tumor activities, could significantly inhibit tumor growth and prolong mouse survival. Thus, our studies revealed that Salmonella could be genetically engineered to improve its tumor targeting via the truncation of LPS and surface display of targeting peptides, thereby eliciting superior anti-tumor effects through targeted delivery of drug molecules.

The biosynthetic origin of ribofuranose in bacterial polysaccharides.

Bacterial surface polysaccharides are assembled by glycosyltransferase enzymes that typically use sugar nucleotide or polyprenyl-monophosphosugar activated donors. Characterized representatives exist for many monosaccharides but neither the donor nor the corresponding glycosyltransferases have been definitively identified for ribofuranose residues found in some polysaccharides. Klebsiella pneumoniae O-antigen polysaccharides provided prototypes to identify dual-domain ribofuranosyltransferase proteins catalyzing a two-step reaction sequence. Phosphoribosyl-5-phospho-D-ribosyl-α-1-diphosphate serves as the donor for a glycan acceptor-specific phosphoribosyl transferase (gPRT), and a more promiscuous phosphoribosyl-phosphatase (PRP) then removes the residual 5'-phosphate. The 2.5-Å resolution crystal structure of a dual-domain ribofuranosyltransferase ortholog from Thermobacillus composti revealed a PRP domain that conserves many features of the phosphatase members of the haloacid dehalogenase family, and a gPRT domain that diverges substantially from all previously characterized phosphoribosyl transferases. The gPRT represents a new glycosyltransferase fold conserved in the most abundant ribofuranosyltransferase family.

Detection of a disulphide bond and conformational changes in Shigella flexneri Wzy, and the role of cysteine residues in polymerase activity.

Shigella flexneri utilises the Wzy-dependent pathway for the production of a plethora of complex polysaccharides, including the lipopolysaccharide O-antigen (Oag) component. The inner membrane protein WzySF polymerises Oag repeat units, whilst two co-polymerase proteins, WzzSF and WzzpHS-2, together interact with WzySF to regulate production of short- (S-Oag) and very long- (VL-Oag) Oag modal lengths, respectively. The 2D arrangement of WzySF transmembrane and soluble regions has been previously deciphered, however, attaining information on the 3D structural and conformational arrangement of WzySF, or any homologue, has proven difficult. For the first time, the current study detected insights into the in situ WzySF arrangement. In vitro assays using thiol-reactive PEG-maleimide were used to probe WzySF conformation, which additionally detected novel, unique conformational changes in response to interaction with intrinsic factors, including WzzSF and WzzpHS-2, and extrinsic factors, such as temperature. Site-directed mutagenesis of WzySF cysteine residues revealed the presence of a putative intramolecular disulphide bond, between cysteine moieties 13 and 60. Subsequent analyses highlighted both the structural and functional importance of WzySF cysteines. Substitution of WzySF cysteine residues significantly decreased biosynthesis of the VL-Oag modal length, without disruption to S-Oag production. This phenotype was corroborated in the absence of co-polymerase competition for WzySF interaction. These data suggest WzySF cysteine substitutions directly impair the interaction between Wzy/WzzpHS-2, without altering the Wzy/WzzSF interplay, and in combination with structural data, we propose that the N- and C-termini of WzySF are arranged in close proximity, and together may form the unique WzzpHS-2 interaction site.

Attachment of Enterohemorrhagic Escherichia coli to Host Cells Reduces O Antigen Chain Length at the Infection Site That Promotes Infection.

Many enteropathogenic bacteria express a needle-like type III secretion system (T3SS) that translocates effectors into host cells promoting infection. O antigen (OAg) constitutes the outer layer of Gram-negative bacteria protecting bacteria from host immune responses. Shigella constitutively shortens the OAg molecule in its three-dimensional conformation by glucosylation, leading to enhanced T3SS function. However, whether and how other enteropathogenic bacteria shorten the OAg molecule that probably facilitates infection remain unknown. For the first time, we report a smart mechanism by which enterohemorrhagic Escherichia coli specifically reduces the size of the OAg molecule at the infection site upon sensing mechanical signals of intestinal epithelial cell attachment via the membrane protein YgjI. YgjI represses expression of the OAg chain length regulator gene fepE via the global regulator H-NS, leading to shortened OAg chains and injection of more T3SS effectors into host cells. However, bacteria express long-chain OAg in the intestinal lumen benefiting their survival. Animal experiments show that blocking this regulatory pathway significantly attenuates bacterial virulence. This finding enhances our understanding of interactions between the surfaces of bacterial and host cells and the way this interaction enhances bacterial pathogenesis. IMPORTANCE Little is known about the regulation of cell wall structure of enteropathogenic bacteria within the host. Here, we report that enterohemorrhagic Escherichia coli regulates its cell wall structure during the infection process, which balances its survival in the intestinal lumen and infection of intestinal epithelial cells. In the intestinal lumen, bacteria express long-chain OAg, which is located in the outer part of the cell wall, leading to enhanced resistance to antimicrobial peptides. However, upon epithelial cell attachment, bacteria sense this mechanical signal via a membrane protein and reduce the OAg chain length, resulting in enhanced injection into epithelial cells of T3SS effectors that mediate host cell infection. Similar regulation mechanisms of cell wall structure in response to host cell attachment may be widespread in pathogenic bacteria and closely related with bacterial pathogenesis.

The HLA Ligandome Comprises a Limited Repertoire of O-GlcNAcylated Antigens Preferentially Associated With HLA-B*07:02.

Mass-spectrometry based immunopeptidomics has provided unprecedented insights into antigen presentation, not only charting an enormous ligandome of self-antigens, but also cancer neoantigens and peptide antigens harbouring post-translational modifications. Here we concentrate on the latter, focusing on the small subset of HLA Class I peptides (less than 1%) that has been observed to be post-translationally modified (PTM) by a O-linked N-acetylglucosamine (GlcNAc). Just like neoantigens these modified antigens may have specific immunomodulatory functions. Here we compiled from literature, and a new dataset originating from the JY B cell lymphoblastoid cell line, a concise albeit comprehensive list of O-GlcNAcylated HLA class I peptides. This cumulative list of O-GlcNAcylated HLA peptides were derived from normal and cancerous origin, as well as tissue specimen. Remarkably, the overlap in detected O-GlcNAcylated HLA peptides as well as their source proteins is strikingly high. Most of the O-GlcNAcylated HLA peptides originate from nuclear proteins, notably transcription factors. From this list, we extract that O-GlcNAcylated HLA Class I peptides are preferentially presented by the HLA-B*07:02 allele. This allele loads peptides with a Proline residue anchor at position 2, and features a binding groove that can accommodate well the recently proposed consensus sequence for O-GlcNAcylation, P(V/A/T/S)g(S/T), essentially explaining why HLA-B*07:02 is a favoured binding allele. The observations drawn from the compiled list, may assist in the prediction of novel O-GlcNAcylated HLA antigens, which will be best presented by patients harbouring HLA-B*07:02 or related alleles that use Proline as anchoring residue.

O-Specific Antigen-Dependent Surface Hydrophobicity Mediates Aggregate Assembly Type in Pseudomonas aeruginosa.

Bacteria live in spatially organized aggregates during chronic infections, where they adapt to the host environment, evade immune responses, and resist therapeutic interventions. Although it is known that environmental factors such as polymers influence bacterial aggregation, it is not clear how bacterial adaptation during chronic infection impacts the formation and spatial organization of aggregates in the presence of polymers. Here, we show that in an in vitro model of cystic fibrosis (CF) containing the polymers extracellular DNA (eDNA) and mucin, O-specific antigen is a major factor determining the formation of two distinct aggregate assembly types of Pseudomonas aeruginosa due to alterations in cell surface hydrophobicity. Our findings suggest that during chronic infection, the interplay between cell surface properties and polymers in the environment may influence the formation and structure of bacterial aggregates, which would shed new light on the fitness costs and benefits of O-antigen production in environments such as CF lungs. IMPORTANCE During chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways; however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood. In this study, we found that changes in O-specific antigen determine the spatial organization of P. aeruginosa cells by altering the relative cell surface hydrophobicity. This finding suggests a role for O-antigen in regulating P. aeruginosa aggregate size and shape in cystic fibrosis airways.

Loss of the virulence plasmid by Shigella sonnei promotes its interactions with CD207 and CD209 receptors.

Introduction. Shigella sonnei, the cause of bacillary dysentery, belongs to Gram-negative enteropathogenic bacteria. S. sonnei contains a 210 kb virulence plasmid that encodes an O-antigen gene cluster of LPSs. However, this virulence plasmid is frequently lost during replication. It is well-documented that after losing the O-antigen and becoming rough strains, the Gram-negative bacteria may express an LPS core on its surface. Previous studies have suggested that by using the LPS core, Gram-negative bacteria can interact with several C-type lectin receptors that are expressed on antigen-presenting cells (APCs).Hypothesis/Gap Statement. S. sonnei by losing the virulence plasmid may hijack APCs via the interactions of LPS-CD209/CD207.Aim. This study aimed to investigate if the S. sonnei rough strain, by losing the virulence plasmid, interacted with APCs that express C-type lectins of human CD207, human CD209a and mouse CD209b.Methodology. SDS-PAGE silver staining was used to examine the O-antigen expression of S. sonnei WT and its rough strain. Invasion assays and inhibition assays were used to examine the ability of S. sonnei WT and its rough strain to invade APCs and investigate whether CD209 and CD207 are receptors for phagocytosis of rough S. sonnei. Animal assays were used to observe the dissemination of S. sonnei.Results. S. sonnei did not express O-antigens after losing the virulence plasmid. The S. sonnei rough strain invades with APCs, including human dendritic cells (DCs) and mouse macrophages. CD209 and CD207 are receptors for phagocytosis of rough S. sonnei. Expression of the O-antigen reduces the ability of the S. sonnei rough strain to be disseminated to mesenteric lymph nodes and spleens.Conclusion. This work demonstrated that S. sonnei rough strains - by losing the virulence plasmid - invaded APCs through interactions with CD209 and CD207 receptors.

Cryo-EM structure of the full-length WzmWzt ABC transporter required for lipid-linked O antigen transport.

O antigens are important cell surface polysaccharides in gram-negative bacteria where they extend core lipopolysaccharides in the extracellular leaflet of the outer membrane. O antigen structures are serotype specific and form extended cell surface barriers endowing many pathogens with survival benefits. In the ABC transporter-dependent biosynthesis pathway, O antigens are assembled on the cytosolic side of the inner membrane on a lipid anchor and reoriented to the periplasmic leaflet by the channel-forming WzmWzt ABC transporter for ligation to the core lipopolysaccharides. In many cases, this process depends on the chemical modification of the O antigen's nonreducing terminus, sensed by WzmWzt via a carbohydrate-binding domain (CBD) that extends its nucleotide-binding domain (NBD). Here, we provide the cryo-electron microscopy structure of the full-length WzmWzt transporter from Aquifex aeolicus bound to adenosine triphosphate (ATP) and in a lipid environment, revealing a highly asymmetric transporter organization. The CBDs dimerize and associate with only one NBD. Conserved loops at the CBD dimer interface straddle a conserved peripheral NBD helix. The CBD dimer is oriented perpendicularly to the NBDs and its putative ligand-binding sites face the transporter to likely modulate ATPase activity upon O antigen binding. Further, our structure reveals a closed WzmWzt conformation in which an aromatic belt near the periplasmic channel exit seals the transporter in a resting, ATP-bound state. The sealed transmembrane channel is asymmetric, with one open and one closed cytosolic and periplasmic portal. The structure provides important insights into O antigen recruitment to and translocation by WzmWzt and related ABC transporters.

Influence of Shigella flexneri 2a O Antigen Acetylation on Its Bacteriophage Sf6 Receptor Activity and Bacterial Interaction with Human Cells.

Shigella flexneri is a major causative agent of bacillary dysentery in developing countries, where serotype 2a2 is the prevalent strain. To date, approximately 30 serotypes have been identified for S. flexneri, and the major contribution to the emergence of new serotypes is chemical modifications of the lipopolysaccharide (LPS) component O antigen (Oag). Glucosylation, O-acetylation, and phosphoethanolamine (PEtN) modifications increase the Oag diversity, providing benefits to S. flexneri LPS Oag acts as a primary receptor for bacteriophage Sf6, which infects only a limited range of S. flexneri serotypes (Y and X). It uses its tailspike protein (Sf6TSP) to establish initial interaction with LPS Oags that it then hydrolyzes. Currently, there is a lack of comprehensive study on the parent and serotype variant strains from the same genetic background and an understanding of the importance of LPS Oag O-acetylations. Therefore, a set of isogenic strains (based on S. flexneri 2457T [2a2]) with deletions of different Oag modification genes (oacB, oacD, and gtrII) that resemble different naturally occurring serotype Y and 2a strains was created. The impacts of these Oag modifications on S. flexneri sensitivity to Sf6 and the pathogenesis-related properties were then compared. We found that Sf6TSP can hydrolyze serotype 2a LPS Oag, identified that 3/4-O-acetylation is essential for resistance of serotype 2a strains to Sf6, and showed that serotype 2a strains have better invasion ability. Lastly, we revealed two new serotype conversions for S. flexneri, thereby contributing to understanding the evolution of this important human pathogen.IMPORTANCE The emergence of antibiotic-resistant strains and lack of efficient vaccines have made Shigella a priority organism for the World Health Organization (1). Therefore, bacteriophage therapy has received increasing attention as an alternative therapeutic approach. LPS Oag is the most variable part of LPS due to chemical modifications and is the target of bacteriophage Sf6 (S. flexneri specific). We dissected the evolution of S. flexneri serotype Y to 2a2, which revealed a new role for a gene acquired during serotype conversion and furthermore identified new specific forms of LPS receptor for Sf6. Collectively, these results unfold the importance of the acquisition of those Oag modification genes and further our understanding of the relationship between Sf6 and S. flexneri.

A lipid gating mechanism for the channel-forming O antigen ABC transporter.

Extracellular glycan biosynthesis is a widespread microbial protection mechanism. In Gram-negative bacteria, the O antigen polysaccharide represents the variable region of outer membrane lipopolysaccharides. Fully assembled lipid-linked O antigens are translocated across the inner membrane by the WzmWzt ABC transporter for ligation to the lipopolysaccharide core, with the transporter forming a continuous transmembrane channel in a nucleotide-free state. Here, we report its structure in an ATP-bound conformation. Large structural changes within the nucleotide-binding and transmembrane regions push conserved hydrophobic residues at the substrate entry site towards the periplasm and provide a model for polysaccharide translocation. With ATP bound, the transporter forms a large transmembrane channel with openings toward the membrane and periplasm. The channel's periplasmic exit is sealed by detergent molecules that block solvent permeation. Molecular dynamics simulation data suggest that, in a biological membrane, lipid molecules occupy this periplasmic exit and prevent water flux in the transporter's resting state.

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus.

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight-stranded ß-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O-polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.

Sensing of Escherichia coli and LPS by mammary epithelial cells is modulated by O-antigen chain and CD14.

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14.

Escherichia coli and Pseudomonas aeruginosa lipopolysaccharide O-antigen ligases share similar membrane topology and biochemical properties.

WaaL is an inner membrane glycosyltransferase that catalyzes the transfer of O-antigen polysaccharide from its lipid-linked intermediate to a terminal sugar of the lipid A-core oligosaccharide, a conserved step in lipopolysaccharide biosynthesis. Ligation occurs at the periplasmic side of the bacterial cell membrane, suggesting the catalytic region of WaaL faces the periplasm. Establishing the membrane topology of the WaaL protein family will enable understanding its mechanism and exploit it as a potential antimicrobial target. Applying oxidative labeling of native methionine/cysteine residues, we previously validated a topological model for Escherichia coli WaaL, which differs substantially from the reported topology of the Pseudomonas aeruginosa WaaL, derived from the analysis of truncated protein reporter fusions. Here, we examined the topology of intact E. coli and P. aeruginosa WaaL proteins by labeling engineered cysteine residues with the membrane-impermeable sulfhydryl reagent polyethylene glycol maleimide (PEG-Mal). The accessibility of PEG-Mal to targeted engineered cysteine residues in both E. coli and P. aeruginosa WaaL proteins demonstrates that both ligases share similar membrane topology. Further, we also demonstrate that P. aeruginosa WaaL shares similar functional properties with E. coli WaaL and that E. coli WaaL may adopt a functional dimer conformation.

Novel Role of VisP and the Wzz System during O-Antigen Assembly in Salmonella enterica Serovar Typhimurium Pathogenesis.

Salmonella enterica serovars are associated with diarrhea and gastroenteritis and are a helpful model for understanding host-pathogen mechanisms. Salmonella enterica serovar Typhimurium regulates the distribution of O antigen (OAg) and presents a trimodal distribution based on Wzy polymerase and the WzzST (long-chain-length OAg [L-OAg]) and WzzfepE (very-long-chain-length OAg [VL-OAg]) copolymerases; however, several mechanisms regulating this process remain unclear. Here, we report that LPS modifications modulate the infectious process and that OAg chain length determination plays an essential role during infection. An increase in VL-OAg is dependent on Wzy polymerase, which is promoted by a growth condition resembling the environment of Salmonella-containing vacuoles (SCVs). The virulence- and stress-related periplasmic protein (VisP) participates in OAg synthesis, as a ΔvisP mutant presents a semirough OAg phenotype. The ΔvisP mutant has greatly decreased motility and J774 macrophage survival in a colitis model of infection. Interestingly, the phenotype is restored after mutation of the wzzST or wzzfepE gene in a ΔvisP background. Loss of both the visP and wzzST genes promotes an imbalance in flagellin secretion. L-OAg may function as a shield against host immune systems in the beginning of an infectious process, and VL-OAg protects bacteria during SCV maturation and facilitates intramacrophage replication. Taken together, these data highlight the roles of OAg length in generating phenotypes during S Typhimurium pathogenesis and show the periplasmic protein VisP as a novel protein in the OAg biosynthesis pathway.

Neutralization of Lipopolysaccharide by Heat Shock Protein in Pediococcus pentosaceus AK-23.

About 1000 species of bacteria are present in the human intestine. Some Gram-negative bacteria such as Escherichia coli or Salmonella spp. among intestinal bacteria have lipopolysaccharide (LPS), which might induce inflammation of human intestines. Actually, LPS, especially its lipid A constituent, is toxic. Small amounts of LPS in bacteria cause inflammation of mucosa and other tissues in humans. Such bacteria may be regulated by beneficial lactic acid bacteria to maintain human health. Many lactic acid bacteria show cancer prevention activity and anti-inflammatory activity in intestines. Recently, Pediococcus pentosaceus AK-23 was isolated from fermentative vegetable pickles for neutralization of LPS. For this study, a protein for LPS neutralization was purified partly from P. pentosaceus AK-23. For this study, a protein for LPS neutralization was purified partly from P. pentosaceus AK-23, by ultrafiltration using a 300 kDa membrane and a 100 kDa membrane after cell wall digestion by lysozyme. Gel running blue native electrophoresis revealed the existence of a 217 kDa protein. The band of the protein having the ability to bind LPS on the gel was analyzed for amino acid homology. As the result, it is revealed as part of a subunit of heat shock protein (HSP). Furthermore, it displayed LPS binding or hydrophobic motifs. The protein neutralized LPS to release fatty acid as myristic acid and glucose from polysaccharide. These findings suggest that HSP in P. pentosaceus AK-23 neutralizes LPS to decompose it compising fatty acid and polysaccharide.

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.

Characterization of Pseudomonas aeruginosa phage K5 genome and identification of its receptor related genes.

Phage genomic information and the nature of host-phage interactions are important for phage applications. In this study, Pseudomonas aeruginosa phage K5 is characterized as a linear double-stranded genomic DNA molecule of 93,754 bp with identical 1182-bp direct terminal repeats. Comparative genomic analysis reveals that phage K5 is highly homologous to the "PaP1-like" phages. Thirteen mutants resistant to phage K5 are screened in a transposon mutant library. The disrupted genetic loci are identified as gene Y880_RS05480 encoding a putative O-antigen polymerase Wzy and gene wapH encoding a glycosyltransferase. The mutants are confirmed by the complementation experiment. The production of biofilm and the profile of lipopolysaccharide (LPS) are further analyzed in the Y880_RS05480 mutant. Our data indicate that LPS is the receptor of phage K5.