Listeria monocytogenes wall teichoic acid decoration in virulence and cell‐to‐cell spread

Wall teichoic acid (WTA) comprises a class of glycopolymers covalently attached to the peptidoglycan of gram positive bacteria. In Listeria monocytogenes, mutations that prevent addition of certain WTA decorating sugars are attenuating. However, the steps required for decoration and the pathogenic process interrupted are not well described. We systematically examined the requirement for WTA galactosylation in a mouse oral‐virulent strain by first creating mutations in four genes whose products conferred resistance to a WTA‐binding bacteriophage. WTA biochemical and structural studies indicated that galactosylated WTA was directly required for bacteriophage adsorption and that mutant WTA lacked appreciable galactose in all except one mutant – which retained a level ca. 7% of the parent. All mutants were profoundly attenuated in orally infected mice and were impaired in cell‐to‐cell spread in vitro. Confocal microscopy of cytosolic mutants revealed that all expressed ActA on their cell surface and formed actin tails with a frequency similar to the parent. However, the mutant tails were significantly shorter – suggesting a defect in actin based motility. Roles for the gene products in WTA galactosylation are proposed. Identification and interruption of WTA decoration pathways may provide a general strategy to discover non‐antibiotic therapeutics for gram positive infections. © 2016 John Wiley & Sons Ltd     

Brushite cement additives inhibit attachment to cell culture beads

Brushite‐forming calcium phosphate cements are of great interest as bone replacement materials because they are resorbable in physiological conditions. Cell‐attached culture beads formed from this material could be of great use for cell therapy. Despite a significant amount of work on optimizing the physicochemical properties of these materials, there are very few studies that have evaluated the capacity of the materials to facilitate cell adhesion. In this study, we have formed resorbable calcium phosphate (brushite) culture beads and for the first time we showed that cell attachment to the surface of the brushite cement (BC) could be inhibited by the presence of an intermediate dicalcium phosphate–citrate complex, formed in the cement as a result of using citric acid, a retardant and viscosity modifier used in many cement formulations. The BC beads formed from the mixture of β‐TCP/orthophosphoric acid using citric acid did not allow cell attachment without further treatment. Ageing of BC beads in serum‐free Dulbecco's Modified Eagle's Medium (DMEM) solution at 37°C for 1 week greatly enhanced the cell adhesion capacity of the material. Scanning electron microscopy, X‐ray diffraction (XRD), and confocal Raman microspectrometry indicated the increased capacity for cell adhesion was due to the changes in phase composition of BC. XRD patterns collected before and after ageing in aqueous solution and a high initial mass loss, suggest the formation of a dicalcium phosphate–citrate complex within the matrix. Since compacts formed from brushite powder supported cell attachment, it was hypothesized that the dicalcium phosphate–citrate complex prevented attachment to the cement surface. Biotechnol. Bioeng. 2013; 110: 1487–1494. © 2012 Wiley Periodicals, Inc.     

Mucoid Helicobacter pylori isolates with fast growth under microaerobic and aerobic conditions

Background: Helicobacter pylori is microaerobic and turns into coccoid under aerobic conditions. In this study, two mucoid strains, A and D, were isolated from gastric biopsies which grew well on blood agar after 24‐hour incubation under aerobic as well as microaerobic conditions. The aim of this study was to identify these strains and compare their growth under aerobic and microaerobic conditions with that of control H. pylori. Materials and Methods: The two isolates A and D were identified as H. pylori according to microscopic morphology, urease, catalase and oxidase tests. Their growth under humidified aerobic and microaerobic conditions was compared with that of control H. pylori which grew only under microaerobic conditions. They were further identified by amplification of 16S rRNA, vacA alleles, cagA and ureAB genes by PCR. Their susceptibility to current antimicrobials was also examined. Results: The strains A and D produced mucoid colonies under aerobic and microaerobic conditions after 24‐hour, exhibiting the typical spiral morphology of H. pylori. The results of urease, catalase and oxidase tests were positive. Sequencing of amplified products showed 99–100% homology with those of the reference H. pylori strains in GenBank. Both strains exhibited resistance to the high concentrations of antimicrobials. Conclusions: This study reports the isolation of two mucoid strains of H. pylori with confluent growth under aerobic and microaerobic conditions. It appears that production of exopolysaccharide (EXP) could serve as a physical barrier to reduce oxygen diffusion into the bacterial cell and uptake of antibiotics. EXP protected the mucoid H. pylori isolates against stressful conditions, the result of which could be persistence of bacterial infection in the stomach.     

An engineered eukaryotic protein glycosylation pathway in Escherichia coli

We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins.     

Burkholderia mallei expresses a unique lipopolysaccharide mixture that is a potent activator of human Toll-like receptor 4 complexes

Burkholderia mallei, the aetiologic agent of glanders, causes a variety of illnesses in animals and humans ranging from occult infections to acute fulminating septicaemias. To better understand the role of lipopolysaccharide (LPS) in the pathogenesis of these diseases, studies were initiated to characterize the structural and biological properties of lipid A moieties expressed by this organism. Using a combination of chemical analyses and MALDI-TOF mass spectrometry, B. mallei was shown to express a heterogeneous mixture of tetra- and penta-acylated lipid A species that were non-stoichiometrically substituted with 4-amino-4-deoxy-arabinose residues. The major penta-acylated species consisted of bisphosphorylated d-glucosamine disaccharide backbones possessing two amide linked 3-hydroxyhexadecanoic acids, two ester linked 3-hydroxytetradecanoic acids [C14:0(3-OH)] and an acyloxyacyl linked tetradecanoic acid, whereas, the major tetra-acylated species possessed all but the 3'-linked C14:0(3-OH) residues. In addition, although devoid of hexa-acylated species, B. mallei LPS was shown to be a potent activator of human Toll-like receptor 4 complexes and stimulated human macrophage-like cells (THP-1 and U-937), monocyte-derived macrophages and dendritic cells to produce high levels of TNF-alpha, IL-6 and RANTES. Based upon these results, it appears that B. mallei LPS is likely to play a significant role in the pathogenesis of human disease.     

Structure of Compositionally Simple Lipopolysaccharide from Marine Synechococcus

Lipopolysaccharide (LPS) is the first defense against changing environmental factors for many bacteria. Here, we report the first structure of the LPS from cyanobacteria based on two strains of marine Synechococcus, WH8102 and CC9311. While enteric LPS contains some of the most complex carbohydrate residues in nature, the full-length versions of these cyanobacterial LPSs have neither heptose nor 3-deoxy-d-manno-octulosonic acid (Kdo) but instead 4-linked glucose as their main saccharide component, with low levels of glucosamine and galacturonic acid also present. Matrix-assisted laser desorption ionization mass spectrometry of the intact minimal core LPS reveals triacylated and tetraacylated structures having a heterogeneous mix of both hydroxylated and nonhydroxylated fatty acids connected to the diglucosamine backbone and a predominantly glucose outer core-like region for both strains. WH8102 incorporated rhamnose in this region as well, contributing to differences in sugar composition and possibly nutritional differences between the strains. In contrast to enteric lipid A, which can be liberated from LPS by mild acid hydrolysis, lipid A from these organisms could be produced by only two novel procedures: triethylamine-assisted periodate oxidation and acetolysis. The lipid A contains odd-chain hydroxylated fatty acids, lacks phosphate, and contains a single galacturonic acid. The LPS lacks any limulus amoebocyte lysate gelation activity. The highly simplified nature of LPSs from these organisms leads us to believe that they may represent either a primordial structure or an adaptation to the relatively higher salt and potentially growth-limiting phosphate levels in marine environments.Lipopolysaccharide (LPS) in the outer membrane layer is known to be the first line of defense against environmental factors in many gram-negative organisms, preventing lysis by complement, antimicrobial peptides and detergents (17, 21, 47). In proteobacteria, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and phosphate are key parts of the conserved inner core of the LPS which connects the less-well-conserved outer core and sometimes an attached polysaccharide to the lipid A anchor. Why heptose is so well conserved is a mystery, but the prevalence of Kdo and phosphate may be related to the charge which they impart to the outer membrane and to their ability to bind divalent cations. The Kdo-phosphate metal binding center is capable of binding calcium with a dissociation constant (K_d) of 12 to 13 μM (28). This high-affinity binding of divalent cations is known to be necessary for the low permeability of LPS bilayers to some antibiotics (32), and it has been hypothesized that divalent cation cross-bridges may link LPS molecules on the bacterial cell surfaces of enterobacteria into a giant complex with very low membrane permeability (16).Though the LPSs of many proteobacteria are well characterized, the LPSs from cyanobacteria are much less studied. The cell envelopes of cyanobacteria resemble those of gram-negative bacteria structurally, consisting of a cytoplasmic membrane, a peptidoglycan layer, an outer membrane containing LPS, and sometimes additional structures (9, 14). Previous chemical analyses have shown the LPS of some cyanobacteria to be devoid of phosphate, Kdo, and heptose (11, 12, 42, 43). Given the lack of Kdo in these organisms as well as the fact that the lability of the Kdo-glucosamine ketosidic linkage allows for the mild acid hydrolysis of LPS to lipid A, it is perhaps not surprising that many attempts at hydrolysis of cyanobacterial LPS to lipid A have failed (for an example, see reference 29).Within the cyanobacteria, the genus Synechococcus represents a polyphyletic group of unicellular morphotypes. Synechococcus cells are found in both freshwater and marine environments. Organisms from group A Synechococcus and its sister taxon Prochlorococcus are extremely important primary producers in marine environments, with multiple “clades” similar to “species” described for other bacteria, dominating in different environments (3, 22). Unlike enterobacteria, which must frequently contend with an onslaught of host factors, members of the Synechococcus face grazing by protists and bacteriophages as their primary survival challenges.The genome of Synechococcus sp. strain CC9311 has been shown to be devoid of the genes for Kdo biosynthesis, while strain WH8102 has several putative genes for Kdo biosynthesis (18, 20). This suggests that the LPS of cyanobacteria could be significantly different from that of enteric bacteria and could show species/strain variation as well. A comparison of the structures of LPS from cyanobacteria and enterobacteria would afford a unique opportunity to understand which elements of LPS structure are essential to bacterial survival and which are adaptations to the environment in which the bacteria live. To further this understanding, we present here an analysis of the LPS structure from two strains of marine Synechococcus: an open-ocean-dwelling strain having the putative genes for Kdo biosynthesis (strain WH8102; clade III) and a coastal strain lacking these genes (strain CC9311; clade I). We further present two novel methods for producing lipid A from bacteria lacking the labile Kdo ketosidic linkage.