Andrzej Ziółkowski

Polycations increase the permeability of Mycobacterium vaccae cell envelopes to hydrophobic compounds

Polycations [protamine, polymyxin B nonapeptide (PMBN) and polyethyleneimine (PEI)] have been shown to increase the cell wall permeability of Mycobacterium vaccae to highly hydrophobic compounds, as manifested in enhanced intracellular bioconversion of beta-sitosterol to 4-androsten-3,17-dione (AD) and 1,4-androstadien-3,17-dione (ADD), and cell sensitization to erythromycin and rifampicin. The quantity of AD(D) formed per biomass unit was twice as high in the presence of PMBN and PEI, and three times higher with protamine. The sensitization factor, i.e. the MIC(50) ratio of the control bacteria to those exposed to polycations, ranged from 4 to 16, depending on the polycation/antibiotic combination. Non-covalently bound free lipids were extracted from the control and polycation-treated cells and fractionated with the use of chloroform, acetone and methanol. Chloroform- and acetone-eluted fractions (mainly neutral lipids and glycolipids, respectively) showed significant polycation-induced alterations in their quantitative and qualitative composition. The fatty acid profile of neutral lipids was reduced in comparison to control, whereas acetone-derived lipids were characterized by a much higher level of octadecenoic acid (C(18:1)) and a considerably lower content of docosanoic acid (C(22:0)), the marker compound of mycolate-containing glycolipids. Methanol-eluted fractions remained unaltered. Cell-wall-linked mycolates obtained from delipidated cells were apparently unaffected by the action of polycations, as judged from the TLC pattern of mycolic acid subclasses, the mean weight of mycolate preparations and the C(22:0) acid content in the mycolates, determined by GC/MS and pyrolysis GC. The results suggest the involvement of the components of non-covalently bound lipids in the outer layer in the M. vaccae permeability barrier.

Structures of the O-antigens of Proteus bacilli belonging to OX group (serogroups O1–O3) used in Weil-Felix test

Structures of the O‐specific polysaccharide chains of lipopolysaccharides from Proteus group OX strains (serogroups O1–O3) used as antigens in Weil‐Felix test for diagnosis of rickettsiosis, were established. From them, the acid‐labile polysaccharide of Proteus vulgaris OX19 (O1) is built up of the following branched pentasaccharide repeating units connected via a phosphate group: where QuiNAc stands for 2‐acetamido‐2,6‐dideoxyglucose (N‐acetylquinovosamine). The basis of serospecificity of the Proteus group OX antigens and their cross‐reactivity with human anti‐rickettsial antibodies is discussed.

The effect of cell wall components on glycine-enhanced sterol side chain degradation to androstene derivatives by mycobacteria

Abstractβ-Sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683 results in the formation of androstene derivatives and is increased in the presence of glycine. As the sterol transformation is carried out inside the cell, higher product accumulation could indicate faster diffusion of highly hydrophobic substrate through the cell wall permeability barrier. Cell wall preparations were obtained to analyse the effect of glycine on peptidoglycan components. Peptidoglycan is known to be the target for glycine action. In glycine-treated preparations, the molar ratio of diaminopimelic acid:muramic acid, the marker compounds of tetrapeptides and glycan strands respectively, was about 60% lower than in the control. This indicates a possible reduction in cross-linking between peptide units and the destruction of peptidoglycan. Unexpectedly, glycine also caused changes in the relative proportion of mycolic acids to other lipids occurring in the strain used for this study. The enhancement of β-sitosterol side chain degradation is likely to result from disturbing the integrity of the cell wall components responsible for the permeability barrier in mycobacteria.

STRUCTURES OF NEW ACIDIC O-SPECIFIC POLYSACCHARIDES OF THE BACTERIUM PROTEUS MIRABILIS SEROGROUPS O26 AND O30

The polysaccharide chains of the lipopolysaccharides of the Proteus mirabilis serogroups O26 and O30 were studied using sugar and methylation analysis and 1H and 13C NMR spectroscopy, including two‐dimensional correlation spectroscopy and rotating‐frame NOE spectroscopy. The polysaccharides were found to be acidic due to the presence of d‐galacturonic acid and its amide with l‐lysine in serogroup O26 or d‐glucuronic acid in serogroup O30, and the structures of their tetrasaccharide repeating units were established. The O26‐specific polysaccharide is structurally and serologically related to the O‐specific polysaccharide of P. mirabilis O28, which includes amides of d‐GalA with l‐lysine and l‐serine [Radziejewska‐Lebrecht, J. et al. (1995) Eur. J. Biochem. 230, 705–712].

Structural and serological studies of the O-specific polysaccharide of the bacterium Proteus mirabilis O10 containing L-altruronic acid, a new component of O-antigens.

An acidic O‐specific polysaccharide from the lipopolysaccharide of Proteus mirabilis O10 contains 2‐acetamido‐2deoxy‐d‐glucose, 2‐acetamido‐2‐deoxy‐d‐galactose, d‐galacturonic acid, and l‐altruronic acid, the last‐named sugar having not been found hitherto in O‐antigens. Structure of a branched tetrasaccharide repeating unit of the polysaccharide was established by 1H and 13C NMR spectroscopy, including two‐dimensional COSY and rotating‐frame NOE spectroscopy. The lateral l‐altruronic acid residue plays the immunodominant role in manifestation of the O10 specificity of Proteus, whereas a disaccharide fragment of the main chain in common with the O‐specific polysaccharide of P. mirabilis O43 provides the one‐way serological cross‐reactivity between anti‐O10 serum and O43‐antigen.

Role of the complement‐lectin pathway in anaphylactoid reaction induced with lipopolysaccharide in mice

We show that Proteus vulgaris O25 (PO25) lipopolysaccharide (LPS) induced an anaphylactoid reaction not only in wild‐type and in lipid A non‐responding mice but also in recombinase‐activating gene‐2‐deficient (RAG‐2–/–) and in mast cell‐deficient (W/Wv) animals. Western blot analysis indicated that PO25 LPS bound to Ra‐reactive factor (RaRF), the complex of mannan‐binding lectins (MBL) and MBL‐associated serine proteases. Binding of RaRF to PO25 LPS led to the activation of C4 component without participation of either C1 or Ig, via the lectin pathway. Relative concentration of RaRF and hemolytic activity in mouse serum decreased rapidly during the process of anaphylactoid reaction. A significant drop of MBL‐A, but not MBL‐C level was observed. Administrationwith antiserum to RaRF prevented animals from death as a consequence of the inhibition of interaction of RaRF with the carbohydrate target and complement activation. These results indicate that complement‐lectin pathway activation is responsible for the anaphylactoid reaction induced with LPS in muramyldipeptide‐primed mice. RaRF also activated fibrinogen in vitro suggesting the involvement of the coagulation system in the process investigated.

Alterations in lipid composition of Mycobacterium vaccae cell wall outer layer enhance β-sitosterol degradation

The rate of transformation of β-sitosterol to 4-androsten-3,17-dione (AD) by Mycobacterium vaccae increased considerably in the presence of D,L-norleucine and m-fluorophenylalanine. These compounds inhibit the biosynthesis of the complex lipids in the cell wall outermost layer. Non-covalently linked (free) lipids were extracted from control and inhibitor-treated cells, and the fatty acid patterns were determined in the neutral lipid, glycolipid and phospholipid fractions. Profound differences were revealed in both the fatty acid composition and the quantities of the individual components in each analysed fraction, derived from the cells exposed to the action of the inhibitors. Partial disorganization of the outermost layer of M. vaccae cell is assumed to alter the cell wall permeability to β-sitosterol and to enhance its biotransformation.

Thermodynamical model of reversible R-phase transformation in TiNi shape memory alloy

Abstract An austenite pre-martensite (R-phase) transformation can be observed in a specially heat treated TiNi alloy. Recent tensile tests performed on Ti-53·3 Ni (wt%) wires exhibited an extremely small hysteresis loop. The uniaxial thermomechanical behaviour of shape memory alloys associated with R-phase transformations is experimentally investigated. An appropriate simple model of an ideal solid undergoing a reversible transformation is developed and applied to predict the uniaxial nonisothermal behaviour of TiNi alloys in the temperature range of R-phase transformations.

Structure of the O-specific polysaccharide of Providencia alcalifaciens O16 containing N-acetylmuramic acid

The O-specific polysaccharide of Providencia alcalifaciens O16 was obtained by mild-acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. It was found that the polysaccharide contains N-acetylmuramic acid, which was isolated by solvolysis with trifluoromethanesulfonic acid and identified by the specific optical rotation and NMR spectroscopy. The following structure of the trisaccharide repeating-unit of the polysaccharide was established:

Isolation using triflic acid solvolysis and identification of Nε-[(R)-1-carboxyethyl]-Nα-(d-galacturonoyl)-l-lysine as a component of the O-specific polysaccharide of Proteusmirabilis O13

Abstract An amino acid was released from the O-specific polysaccharide of Proteus mirabilis O13 by acid hydrolysis and identified as N e -[( R )-1-carboxyethyl]- l -lysine by comparison with the authentic sample. An amide of this amino acid with d -galacturonic acid was isolated from the polysaccharide by solvolysis with anhydrous trifluoromethanesulfonic (triflic) acid and characterised by 1 H and 13 C NMR spectroscopy. These and published data enabled determination of the full structure of the repeating unit of the polysaccharide.