Zbigniew Kaczyński

Application of Spectroscopic Methods for Structural Analysis of Chitin and Chitosan

Chitin, the second most important natural polymer in the world, and its N-deacetylated derivative chitosan, have been identified as versatile biopolymers for a broad range of applications in medicine, agriculture and the food industry. Two of the main reasons for this are firstly the unique chemical, physicochemical and biological properties of chitin and chitosan, and secondly the unlimited supply of raw materials for their production. These polymers exhibit widely differing physicochemical properties depending on the chitin source and the conditions of chitosan production. The presence of reactive functional groups as well as the polysaccharide nature of these biopolymers enables them to undergo diverse chemical modifications. A complete chemical and physicochemical characterization of chitin, chitosan and their derivatives is not possible without using spectroscopic techniques. This review focuses on the application of spectroscopic methods for the structural analysis of these compounds.

Alanine Esters of Enterococcal Lipoteichoic Acid Play a Role in Biofilm Formation and Resistance to Antimicrobial Peptides

ABSTRACT Enterococcus faecalis is among the predominant causes of nosocomial infections. Surface molecules like d-alanine lipoteichoic acid (LTA) perform several functions in gram-positive bacteria, such as maintenance of cationic homeostasis and modulation of autolytic activities. The aim of the present study was to evaluate the effect of d-alanine esters of teichoic acids on biofilm production and adhesion, autolysis, antimicrobial peptide sensitivity, and opsonic killing. A deletion mutant of the dltA gene was created in a clinical E. faecalis isolate. The absence of d-alanine in the LTA of the dltA deletion mutant was confirmed by nuclear magnetic resonance spectroscopy. The wild-type strain and the deletion mutant did not show any significant differences in growth curve, morphology, or autolysis. However, the mutant produced significantly less biofilm when grown in the presence of 1% glucose (51.1% compared to that of the wild type); adhesion to eukaryotic cells was diminished. The mutant absorbed 71.1% of the opsonic antibodies, while absorption with the wild type resulted in a 93.2% reduction in killing. Sensitivity to several cationic antimicrobial peptides (polymyxin B, colistin, and nisin) was considerably increased in the mutant strain, confirming similar results from other studies of gram-positive bacteria. Our data suggest that the absence of d-alanine in LTA plays a role in environmental interactions, probably by modulating the net negative charge of the bacterial cell surface, and therefore it may be involved in the pathogenesis of this organism.

Opsonic Antibodies to Enterococcus faecalis Strain 12030 Are Directed against Lipoteichoic Acid

ABSTRACT A teichoic acid (TA)-like polysaccharide in Enterococcus faecalis has previously been shown to induce opsonic antibodies that protect against bacteremia after active and passive immunization. Here we present new data providing a corrected structure of the antigen and the epitope against which the opsonic antibodies are directed. Capsular polysaccharide isolated from E. faecalis strain 12030 by enzymatic digestion of peptidoglycan and chromatography (enzyme-TA) was compared with lipoteichoic acid (LTA) extracted using butanol and purified by hydrophobic-interaction chromatography (BuOH-LTA). Structural determinations were carried out by chemical analysis and nuclear magnetic resonance spectroscopy. Antibody specificity was assessed by enzyme-linked immunosorbent assay and the opsonophagocytosis assay. After alanine ester hydrolysis, there was structural identity between enzyme-TA and BuOH-LTA of the TA-parts of the two molecules. The basic enterococcal LTA structure was confirmed: 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at position C-2 of the glycerol residues with d-Ala and kojibiose. We also detected a novel substituent at position C-2, [d-Ala→6]-α-d-Glcp-(1→2-[d-Ala→6]-α-d-Glcp-1→). Antiserum raised against enzyme-TA bound equally well to BuOH-LTA and dealanylated BuOH-LTA as to the originally described enzyme-TA antigen. BuOH-LTA was a potent inhibitor of opsonophagocytic killing by the antiserum to enzyme-TA. Immunization with antibiotic-killed whole bacterial cells did not induce a significant proportion of antibodies directed against alanylated epitopes on the TA, and opsonic activity was inhibited completely by both alanylated and dealanylated BuOH-LTA. In summary, the E. faecalis strain 12030 enzyme-TA is structurally and immunologically identical to dealanylated LTA. Opsonic antibodies to E. faecalis 12030 are directed predominantly to nonalanylated epitopes on the LTA molecule.

Modification of lipopolysaccharide with colanic acid (M-antigen) repeats in Escherichia coli

Colanic acid (CA) or M-antigen is an exopolysaccharide produced by many enterobacteria, including the majority of Escherichia coli strains. Unlike other capsular polysaccharides, which have a close association with the bacterial surface, CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. Herein we show that a highly mucoid strain of E. coli K-12 ligates CA repeats to a significant proportion of lipopolysaccharide (LPS) core acceptor molecules, forming the novel LPS glycoform we call MLPS.MLPS biosynthesis is dependent upon (i) CA induction, (ii) LPS core biosynthesis, and (iii) the O-antigen ligase WaaL. Compositional analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy of a purified MLPS sample confirmed the presence of a CA repeat unit identical in carbohydrate sequence, but differing at multiple positions in anomeric configuration and linkage, from published structures of extracellular CA. The attachment point was identified as O-7 of the l-glycero-d-manno-heptose of the outer LPS core, the same position used for O-antigen ligation. When O-antigen biosynthesis was restored in the K-12 background and grown under conditions meeting the above specifications, only MLPS was observed, suggesting E. coli can reversibly change its proximal covalently linked cell surface polysaccharide coat from O-antigen to CA in response to certain environmental stimuli. The identification of MLPS has implications for potential underlying mechanisms coordinating the synthesis of various surface polysaccharides.

Structure of the O-polysaccharide isolated from Cronobacter sakazakii 767

The Cronobacter spp., previously known as Enterobacter sakazakii, are Gram-negative enterobacterial pathogens that can cause necrotizing enterocolitis, meningitis, and septicemia with a high mortality rate in neonates. The O-specific polysaccharide (O-PS) was isolated from Cronobacter sakazakii strain 767 and structurally characterized using (1)H and (13)C NMR spectroscopy, including two-dimensional DQF-COSY, TOCSY, ROESY, HSQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods followed by GLC, and GLC-MS analysis. The repeating unit of O-PS isolated from C. sakazakii 767 was a branched heptasaccharide composed of l-Rha, d-Glc, d-GlcNAc, and d-GalA, and had the structure shown below. One of the Rha residues was partially O-acetylated at C-4. C. sakazakii 767 was originally isolated from a fatal neonatal meningitic case, and the structure of its O-PS significantly differs from the O-PS structures previously described for Cronobacter spp.

Serodiversity of Opsonic Antibodies against Enterococcus faecalis —Glycans of the Cell Wall Revisited

In a typing system based on opsonic antibodies against carbohydrate antigens of the cell envelope, 60% of Enterococcus faecalis strains can be assigned to one of four serotypes (CPS-A to CPS-D). The structural basis for enterococcal serotypes, however, is still incompletely understood. Here we demonstrate that antibodies raised against lipoteichoic acid (LTA) from a CPS-A strain are opsonic to both CPS-A and CPS-B strains. LTA-specific antibodies also bind to LTA of CPS-C and CPS-D strains, but fail to opsonize them. From CPS-C and CPS-D strains resistant to opsonization by anti-LTA, we purified a novel diheteroglycan with a repeating unit of →6)-β-Galf-(1→3)- β-D-Glcp-(1→ with O-acetylation in position 5 and lactic acid substitution at position 3 of the Galf residue. The purified diheteroglycan, but not LTA absorbed opsonic antibodies from whole cell antiserum against E. faecalis type 2 (a CPS-C strain) and type 5 (CPS-D). Rabbit antiserum raised against purified diheteroglycan opsonized CPS-C and CPS-D strains and passive protection with diheteroglycan-specific antiserum reduced bacterial counts by 1.4 – 3.4 logs in mice infected with E. faecalis strains of the CPS-C and CPS-D serotype. Diheteroglycan-specific opsonic antibodies were absorbed by whole bacterial cells of E. faecalis FA2-2 (CPS-C) but not by its isogenic acapsular cpsI-mutant and on native PAGE purified diheteroglycan co-migrated with the gene product of the cps-locus, suggesting that it is synthesized by this locus. In summary, two polysaccharide antigens, LTA and a novel diheteroglycan, are targets of opsonic antibodies against typeable E. faecalis strains. These cell-wall associated polymers are promising candidates for active and passive vaccination and add to our armamentarium to fight this important nosocomial pathogen.

Identification and Role of a 6-Deoxy-4-Keto-Hexosamine in the Lipopolysaccharide Outer Core of Yersinia enterocolitica Serotype O:3

The outer core (OC) region of Yersinia enterocolitica serotype O:3 lipopolysaccharide is a hexasaccharide essential for the integrity of the outer membrane. It is involved in resistance against cationic antimicrobial peptides and plays a role in virulence during early phases of infection. We show here that the proximal residue of the OC hexasaccharide is a rarely encountered 4-keto-hexosamine, 2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose (Sugp) and that WbcP is a UDP-GlcNAc-4,6-dehydratase enzyme responsible for the biosynthesis of the nucleotide-activated form of this rare sugar converting UDP-2-acetamido-2-deoxy-D-glucopyranose (UDP-D-GlcpNAc) to UDP-2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyranose (UDP- Sugp). In an aqueous environment, the 4-keto group of this sugar was present in the 4-dihydroxy form, due to hydration. Furthermore, evidence is provided that the axial 4-hydroxy group of this dihydroxy function was crucial for the biological role of the OC, that is, in the bacteriophage and enterocoliticin receptor structure and in the epitope of a monoclonal antibody.

Influence of the Chemical Structure and Physicochemical Properties of Chitin- and Chitosan-Based Materials on Their Biomedical Activity

Chitin and chitosan are an important family of linear polysaccharides consisting of varying amounts of ┚-(1→4)-linked 2-acetamido-2-deoxy-┚-D-glucopyranose (GlcNAc) and 2amino-2-deoxy-┚-D-glucopyranose (GlcN) units (Muzzarelli, 1973; Roberts, 1992). Chitin samples contain a high content of GlcNAc units; hence, they are insoluble in water and common organic solvents. On the other hand, they dissolve only in solvents such as N,Ndimethylacetamide, hexafluoroacetone or hexafluoro-2-propanol (Pillai et al., 2009; Austin, 1988; Kurita, 2001). When the degree of N-acetylation (defined as the average number of N-acetyl-D-glucosamine units per 100 monomers expressed as a percentage) is less than 50%, chitin becomes soluble in aqueous acidic solutions (pH < 6.0) and is called chitosan (Pillai et al., 2009). This means that the term “chitosan” represents a group of fully and partially deacetylated chitins, but a rigid nomenclature with respect to the degree of Ndeacetylation between chitin and chitosan has not been established (Ravi Kumar, 2000). Some authors consider that chitosan is a polysaccharide containing at least 60% GlcN residues (Aiba, 1992). According to the nomenclature proposed by the European Chitin Society (EUCHIS) (Roberts, 2007), chitin and chitosan should be classified on the basis of their insolubility and solubility in 0.1 M acetic acid; the insoluble material is called chitin, whereas the soluble one is chitosan. The structures of “ideal” chitin and “ideal” chitosan, and the “real“ structures of these compounds are presented in Figure 1. Chitin is the second most abundant polysaccharide (next to cellulose) synthesized by a great number of living organisms, serving in many functions where reinforcement and strength are required (Muzzarelli et al., 1986). In nature, chitin is found as structural components in the exoskeleton of arthropods or in the cell walls of fungi and yeast. It is also produced by a number of other living organisms in the lower plant and animal kingdoms. It has been

Definitive Structural Assessment of Enterococcal Diheteroglycan

Enterococcus faecalis is one of most important nosocomial and often multi-antibiotic resistant pathogens responsible for infections that are difficult to treat. Previously, a cell-wall polysaccharide termed diheteroglycan (DHG) was isolated and characterized as a promising vaccine candidate. However, the configuration of its lactic acid (LA) residue attached to the galactofuranoside unit was not assessed, although it influences conformation of DHG chain in terms of biological recognition and immune evasion. This study proves the R configuration of the LA residue by means of chemical analysis, investigation of intramolecular NMR nuclear Overhauser effects and molecular dynamics simulations of native DHG and corresponding R and S models, which were obtained by using pyranoside-into-furanoside rearrangement. As alternative treatment and prevention strategies for E. faecalis are desperately needed, this discovery may offer the prospect of a synthetic vaccine to actively immunize patients at risk.

Investigation of the chemical structure and biological activity of oligosaccharides isolated from rough-type Xanthomonas campestris pv. campestris B100 lipopolysaccharide

The rough-type lipopolysaccharide (LPS) of the phytopathogenic bacterium Xanthomonas campestris pv. campestris B 100 was isolated utilizing the hot phenol-water method and successively de-acylated by treatment with hydrazine and hot potassium hydroxide. Four compounds were separated by preparative high-performance anion-exchange chromatography and studied by sugar analysis and by 1D and 2D homonuclear and heteronuclear 1H-, 13C- and 31P-NMR spectroscopy as well as ESI FT-MS. The two main products were a heptasaccharide and a pentasaccharide of the structures α-D-Manp-(1→3)-α-D-Man p-(1→4)-β-D-Glcp-(1→4)-α-D-Manp-3P -(1→5)-α-Kdo-(2→6)-β-D-GlcpN-4P-(1→6)-α-D-Glc pN-1P (1) and β-D-Glcp-(1→4)-α-D-Man p-3P-(1→5)-α-Kdo-(2→6)-β-D-GlcpN-4 P-(1→6)-α-D-GlcpN-1P (2), respectively. The products in smaller amounts were a heptasaccharide and pentasaccharide possessing the above structures plus a phosphate group at C-4 of the Kdo residue (compounds 3 and 4). Both, heptasaccharide 1 and pentasaccharide 2 were able to induce an oxidative burst in cell cultures of the non-host plant tobacco.