Chain length heterogeneity of lipopolysaccharide released from Salmonella typhimurium by ethylenediaminetetraacetic acid or polycations

Abstract

Cells of two smooth Salmonella typhimurium strains (SL696 and SH4247) were treated with ethylenediaminetetraacetic acid (EDTA) and the polycations poly(L-lysine) and protamine to monitor both quantitatively and qualitatively the release of [ 14C] galactose-labelled lipopolysaccharide into the medium to find out whether these effector substances caused selective release of certain fractions from the initially heterogenous lipopolysaccharide population. Each one of the substances released considerable amounts of lipopolysaccharide into the medium. Analysis by sodium dodecyl sulphate/polyacrylamide gel electrophoresis followed by autoradiography showed that the total lipopolysaccharide (from isolated membranes) and the released materials produced coincident banding patterns, each with a high degree of 0 side-chain length heterogeneity. Densitometric scans of the autoradiograms were analyzed for possible differences in the distribution and relative abundance of lipopolysaccharide molecules with different 0 chain lengths. It was found that in SL696 the released materials were identical to the total lipopolysaccharide; in SH4247 subtle deviations from the total lipopolysaccharide were seen. We conclude from these results that lipopolysaccharide molecules with short and long 0 side chains are linked to and stabilized in the outer membrane by similar mechanisms equally susceptible to the effects of EDTA and polycations.In smooth gram-negative bacteria the outermost cell surface (the outer membrane; for a recent review see [I]) is covered by lipopolysaccharide (LPS). LPS consists of a lipophilic moiety termed lipid A (embedded in the outer membrane), an oligosaccharide core, and an 0-specific chain containing repeating oligosaccharide units (protruding outside from the cell surface). Recent studies employing sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) have revealed that the LPS of smooth bacteria are highly heterogenous with respect to the degree of polymerization of the 0-specific chain. For instance, Salmonella typhimurium LPS separated in SDS-PAGE and visualized either by silver staining or autoradiography typically appears to contain two predominant populations: one consisting of molecules having long 0 chains with even up to 50 repeating units, and the other having only a few or no repeating units (2 -61. The biological significance of these two populations is unclear; it is not known whether or not the different species of LPS molecules have different functions or separate location in the outer membrane.Leive and coworkers have shown that treatment of smooth bacteria with EDTA releases approximately half of their LPS into the surrounding medium [7-91. On this basis it was suggested [9] that LPS exists in the outer membrane in two distinct fractions, one being held there by divalent cations and releasable with EDTA, and the other organized by some other unspecified mechanism which is resistant to EDTA. On the other hand, it has been reported [lo] that EDTA does not release significant amoun...

Effect of EDTA on Salmonella enterica serovar Typhimurium involves a component not assignable to lipopolysaccharide release

“…Since the initial research by Leive (1965) it has been known that chelating agents such as EDTA destabilize the OM of Gram-negative bacteria by sequestering the stabilizing divalent cations. Such destabilization leads to the release of substantial proportions, up to 40 %, of LPS (Leive, 1965(Leive, , 1974Hukari et al, 1986;Alakomi et al, 2000), whereby EDTA-treated bacteria become susceptible to agents that normally do not penetrate the OM and, as a consequence, do not affect the bacteria, as summarized by Vaara (1992Vaara ( , 1999. This phenomenon is often referred to as permeabilization.…”

“…Smooth S. enterica Typhimurium E-95582 T cells were grown in LB at 37 uC with shaking (200 r.p.m.) to the desired growth phase and supplemented with [ 14 C]galactose (0?1 mCi ml 21 ) in order to label their LPS (Hukari et al, 1986). Labelling with [ 14 C]galactose was performed for 5 min at 37 uC with shaking (200 r.p.m.).…”

“…It is not known, for instance, why only a certain proportion of LPS is released. It was shown by Hukari et al (1986) that the macromolecular quality (LPS chain length distribution) of EDTA-released LPS vs cell-bound LPS was identical. It can, however, be postulated that the releasable fraction differs from the non-releasable one in some structural aspect that is related to the stabilizing effect of divalent cations, perhaps at the level of anionic groups in the core oligosaccaharide and lipid A.…”

Lactoferrin and transferrin damage of the Gram-negative outer membrane is modulated by Ca2+ and Mg2+

“…A wild-type 'smooth' Salmonella typhimurium strain, SL696, that also incorporates a large percentage of exogenous galactose into LPS was obtained from Dr I. M. Helander (National Public Health Institute, Helsinki, Finland) (Hukari et al, 1986). This strain was initially grown in Luria broth supplemented with 2 mM-CaC1, before testing for sensitivity to lactoferrin or transferrin-mediated LPS release (Hukari et al, 1986 LPS release assay.…”

“…This strain was initially grown in Luria broth supplemented with 2 mM-CaC1, before testing for sensitivity to lactoferrin or transferrin-mediated LPS release (Hukari et al, 1986 LPS release assay. Release of LPS from bacteria was determined using the assay described previously (Ellison et al, 1988).…”