Campylobacter jejuni CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to E. coli CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), C. jejuni CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of C. jejuni CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.

Identification of physiological modulators of nuclear hormone receptor (NHR) activity is paramount for understanding the link between metabolism and transcriptional networks that orchestrate development and cellular physiology. Using libraries of metabolic enzymes alongside their substrates and products, we identify 1-deoxysphingosines as modulators of the activity of NR2F1 and 2 (COUP-TFs), which are orphan NHRs that are critical for development of the nervous system, heart, veins, and lymphatic vessels. We show that these non-canonical alanine-based sphingolipids bind to the NR2F1/2 ligand-binding domains (LBDs) and modulate their transcriptional activity in cell-based assays at physiological concentrations. Furthermore, inhibition of sphingolipid biosynthesis phenocopies NR2F1/2 deficiency in endothelium and cardiomyocytes, and increases in 1-deoxysphingosine levels activate NR2F1/2-dependent differentiation programs. Our findings suggest that 1-deoxysphingosines are physiological regulators of NR2F1/2mediated transcription.

A hypervirulent A. hydrophila (vAh) pathotype has been identified as the etiologic agent responsible for disease outbreaks in farmed carp species and channel catfish (Ictalurus punctatus) in China and the Southeastern United States, respectively. The possible route of infection has previously been unknown; however, virulence is believed to be multifactorial, involving the production/secretion of several virulence factors, including a high molecular weight group 4 capsular polysaccharide. Here we present chemical structural evidence of a novel capsule- and LPS-associated O-antigen found present in vAh isolated during these disease outbreaks. In this study, the chemical structure of the vAh O-antigen was determined by chemical analysis, Smith degradation, mass spectrometry, and 2D proton and carbon nuclear magnetic resonance (NMR) spectroscopy and found to be unique among described bacterial O-antigens. The O-antigen consists of hexasaccharide repeating units featuring a 4)-α-l-Fucp-(1–3)-β-d-GlcpNAc-(1–4)-α-l-Fucp-(1–4)-β-d-Glcp-(1- backbone, substituted with single residue side chains of α-d-Glcp and α-d-Quip3NAc linked to O-3 of the two fucose residues. The polysaccharide is partially O-acetylated on O-6 of the 4-substituted β-Glcp residue.

The original version of this Article contained errors in Figures 1, 3 and 4. In panels b and d of Figure 1, the labels ‘Sdc4-/-’ were inadvertently replaced by ‘Sdc4+/+‘. In panels c and d of Figure 3, the labels ‘Sdc4-/-’ were replaced by ‘Sdc2-/-’. In panel f of Figure 3, the labels ‘FGF2’ were replaced by ‘VEGFA165’. In panel e of Figure 6, a ‘Sdc2-/-‘ label was inadvertently included. This has now been corrected in the PDF and HTML versions of the Article.

The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS-specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

The ceramide phosphoinositol glycan core (CPI-GC) of the lipophosphoglycan of Trichomonas vaginalis is a major virulent factor of this common genitourinary parasite. While its carbohydrate composition has been reported before, its structure has remained largely unknown. We isolated the glycan portions of CPI-GC by nitrous acid deamination and hydrofluoric acid treatment and investigated their structures by methylation analysis and 1- and 2-D NMR. We found that the α-anomer of galactose is a major constituent of CPI-GC. The β-anomer was found exclusively at the non-reducing end of CPI-GC side chains. Furthermore the data showed that the rhamnan backbone is more complex than previously thought and that the inositol residue at the reducing end is linked to a 4-linked α-glucuronic acid (GlcA) residue. This appears to be the most striking and novel feature of this GPI-anchor type molecule.

The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability.

Galactofuranose (Galf) is the five-membered ring form of galactose. Although it is absent from mammalian glycans, it occurs as a structural and antigenic component of important cell surface molecules in a variety of microbes, ranging from bacteria to parasites and fungi. One such organism is Cryptococcus neoformans, a pathogenic yeast that causes lethal meningoencephalitis in immunocompromised individuals, particularly AIDS patients. C. neoformans is unique among fungal pathogens in bearing a complex polysaccharide capsule, a critical virulence factor reported to include Galf. Notably, how Galf modification contributes to the structure and function of the cryptococcal capsule is not known. We have determined that Galf is beta 1,2-linked to an unusual tetrasubstituted galactopyranose of the glucuronoxylomannogalactan (GXMGal) capsule polysaccharide. This discovery fills a longstanding gap in our understanding of a major polymer of the cryptococcal capsule. We also engineered a C. neoformans strain that lacks UDP-galactopyranose mutase; this enzyme forms UDP-Galf, the nucleotide sugar donor required for Galf addition. Mutase activity was required for the incorporation of Galf into glucuronoxylomannogalactan but was dispensable for vegetative growth, cell integrity, and virulence in a mouse model.

The key challenge in the treatment of bacterial infection is rapid identification of bacteremia at an early stage of the diseases. Currently available imaging systems such as computed tomography (CT) and magnetic resonance imaging (MRI) can only detect bacterial infection after they have become systemic or have caused significant anatomical tissue damage, and at this stage infection are challenging to treat due to the high bacterial burden. To this day positron emission tomography (PET) imaging has showed the great potential for improving the diagnosis of bacterial infection because of the high sensitivity of PET radionuclides, capability of detecting molecular biology in details (even prior to anatomic change). Fluorodeoxyglucose (FDG) PET has been developed for bacterial imaging with incredible success. Whole body PET imaging with FDG for the diagnosis of bacterial infection and monitoring response to treatment has been well established. FDG-PET will not only help to accelerate the diagnosis of infection but improve the bacterial treatment. In this review, we focus on FDG-PET imaging for diagnosing bacterial infection in the clinic.

Burkholderia oklahomensis E0147 is a US isolated bacterium believed to express a similar O-antigen to type A structure of the highly pathogenic species, Burkholderia pseudomallei. Both species are genetically closely related. Lipopolysaccharide was collected from E0147 and structurally characterized to test this hypothesis. Glycosyl composition and linkage analyses in conjunction with 1D and 2D 1H and 13C NMR spectroscopy showed that the O-antigen was a repeating disaccharide with the following structure: [3)-β-d-Glcp-(1→3)-2OAc-α-l-6dTalp-(1→]n NMR spectroscopy also revealed the presence of a co-extracted exopolysaccharide previously described in B. pseudomallei, with the structure: [3)-2OAc-β-d-Galp-(1→4)-α-d-Galp-(1→3)-β-d-Galp-(1→5)-β-d-Kdop-(2→]n.

Capsular polysaccharides (CPSs) were isolated from O-polysaccharide deficient strains of Burkholderia mallei and Burkholderia pseudomallei using a modified hot phenol/water extraction procedure. Glycosyl composition, methylation, MALDI-TOF MS analyses as well as H-1 NMR spectroscopy including COSY, TOCSY, NOESY, HMBC and HSQC experiments identified the presence of two distinct CPS antigens in the samples exhibiting the following structures:-> 3)-2O-Ac-beta-6dHepp-(1 ->-> 3)-alpha-D-Manp-(1 ->This study confirms the ability of B. mallei to express a 6-deoxy-heptan CPS and represents the first report of a mannan CPS being expressed by these bacterial pathogens. (C) 2011 Elsevier Ltd. All rights reserved.

Permethylation is a valuable and widely used tool for the mass spectrometry of carbohydrates, improving sensitivity and fragmentation and increasing the amount of information that can be obtained from tandem mass spectrometric experiments. Permethylation of most glycans is easily performed with sodium hydroxide and iodomethane in dimethyl sulfoxide (DMSO). However, permethylation has not been widely used in the mass spectrometry of glycosaminoglycan (GAG) oligosaccharides, partly because it has required the use of the difficult Hakomori method employing the methylsulfinylmethanide ('dimsyl') base, which has to be made in a tedious process. Additionally, the Hakomori method is not as effective as the sodium hydroxide method in making fully methylated derivatives. A further problem in the permethylation of highly sulfated oligosaccharides is their limited solubility in DMSO. This paper describes the use of the triethylammonium counterion to overcome this problem, as well as the application of the sodium hydroxide method to make permethylated heparin disaccharides and their workup to yield fully methylated disaccharides for electrospray ionization mass spectrometry. The ease, speed, and effectiveness of the described methodology should open up permethylation of GAG oligosaccharides to a wider circle of mass spectrometrists and enable them to develop further derivatization schemes in the effort to rapidly elucidate the structure of these important molecules. Permethylation may also provide new ways of separating GAG oligosaccharides in LC/MS, their increased hydrophobicity making them amenable for reversed-phase chromatography without the need for ion pairing reagents.

The diagnosis of bacterial infections remains a major challenge in medicine. Although numerous contrast agents have been developed to image bacteria, their clinical impact has been minimal because they are unable to detect small numbers of bacteria in vivo, and cannot distinguish infections from other pathologies such as cancer and inflammation. Here, we present a family of contrast agents, termed maltodextrin-based imaging probes (MDPs), which can detect bacteria in vivo with a sensitivity two orders of magnitude higher than previously reported, and can detect bacteria using a bacteria-specific mechanism that is independent of host response and secondary pathologies. MDPs are composed of a fluorescent dye conjugated to maltohexaose, and are rapidly internalized through the bacteria-specific maltodextrin transport pathway, endowing the MDPs with a unique combination of high sensitivity and specificity for bacteria. Here, we show that MDPs selectively accumulate within bacteria at millimolar concentrations, and are a thousand-fold more specific for bacteria than mammalian cells. Furthermore, we demonstrate that MDPs can image as few as 10(5) colony-forming units in vivo and can discriminate between active bacteria and inflammation induced by either lipopolysaccharides or metabolically inactive bacteria.

Switchgrass (Panicum virgatum L.) has been identified for development into an efficient and environmentally friendly biomass energy crop. A recent 5 year study demonstrated that switchgrass grown for biofuel production produced 540% more energy than what is needed to grow, harvest, and process it into cellulosic ethanol. If switchgrass is grown on a scale useful for a bioenergy source, some of the material could be used by livestock as hay or pasture. Switchgrass has been reported to cause hepatogenous photosensitization in lambs (Ovis aries) and horses (Equus caballus). In this study, a simple extraction and rapid reversed phase high-performance liquid chromatography-electrospray ionization-mass spectrometry method was developed for quantifying the major saponins in switchgrass samples. Differences in the relative concentrations of different saponins were observed between switchgrass cultivars and plant parts.

The structure of galactoxylomannan, a capsular polysaccharide from the opportunistic yeast Cryptococcus neoformans, was re-examined by NMR spectroscopy and GC–MS. The residue that is 3-linked to the side chain galactose and was previously assigned as β-d-xylose [Vaishnav, V. V.; Bacon, B. E.; O’Neill, M.; Cherniak, R. Carbohydr. Res.1998, 306, 315–330] was determined to be β-d-glucuronic acid. A revised structure for this polymer is presented, along with a proposal that this compound be termed glucuronoxylomannogalactan (GXMGal).