Differences in Membrane Fluidity and Fatty Acid Composition between Phenotypic Variants of Streptococcus pneumoniae

Abstract

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P < 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P < 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P < 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition.Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease (27). The different appearance of bacterial colonies is assumed to result from the spontaneous and reversible phase variation of surface components, the identity of which is not yet clear. The frequency of switching is highly variable from isolate to isolate, ranging from 10 Ϫ3 to 10 Ϫ6 per generation. The significance of opacity variation in the biology of pneumococcal infection in vivo was examined by using animal models of nasopharyngeal colonization and bacteremia. Transparent variants persist in the nasopharynx in vivo and show greater adherence to human lung epithelial cells. However, experiments performed with an adult mouse model of sepsis showed a strong selection for organisms with the opaque morphology during invasive infections (28).Genetic experiments were used to isolate a single locus able to confer altered colony opacity at a higher frequency than the background rate (18). The opacity locus was found to be associated with two genes in the presumed glycerol operon, glpF and glpD, required for glycerol metabolism in other bacteria. This finding raises the possibility that phase variation in S. pneumoniae is linked to the mechani...

In Vitro Inhibition of Streptococcus mutans Biofilm Formation on Hydroxyapatite by Subinhibitory Concentrations of Anthraquinones

“…Although the average difference in anisotropy between DPH-labeled emodin-treated cells (0.1528) and untreated cells (0.1327) was low (15.13%) and not significant (P ϭ 0.512), these differences were observed consistently. In addition, small changes (approximately 10%) in fluorescence anisotropy may reflect marked changes (of about 25%) in membrane microviscosity (2,11). Together with the significant decrease in membrane lateral diffusion, the observed differences in fluorescence anisotropy strongly suggest an effect of emodin on membrane microviscosity.…”

“…Inhibition of autoinducer-2-based QS was studied using V. harveyi biosensor strains, as described previously (7). Lateral diffusion of fatty acids in the cell membrane was measured by the intermolecular excimerization of the fluorescent probe pyrene (2). To study the rotational diffusion of the fatty acid acyl chains in the interior of the membrane, fluorescence anisotropy was measured using 1,6-diphenyl 1,3,5-hexatriene (DPH) (2).…”

“…Lateral diffusion of fatty acids in the cell membrane was measured by the intermolecular excimerization of the fluorescent probe pyrene (2). To study the rotational diffusion of the fatty acid acyl chains in the interior of the membrane, fluorescence anisotropy was measured using 1,6-diphenyl 1,3,5-hexatriene (DPH) (2). Cellular fatty acid analysis was performed as described previously (21).…”

The Staphylococcus aureus Two-Component Regulatory System, GraRS, Senses and Confers Resistance to Selected Cationic Antimicrobial Peptides

“…In the current study, we documented changes in L-PG synthesis and outer membrane translocation that paralleled perturbations in the mprF expression profiles in graR or vraG mutant strains. It should be emphasized that, although the absolute quantitative changes in L-PG synthesis and flipping between parental and mutant strains was relatively small, such modest alterations in membrane lipids can have profound physiologic impacts (1,41). In addition, lowered expression of both mprF and dlt in the above mutants correlated well with both their reduced positive surface charge phenotype and their increased susceptibility to killing by two mammalian host defense CAPs of platelet and polymorphonuclear leukocyte origins.…”

Genetic Basis of Evolutionary Adaptation by Escherichia coli to Stressful Cycles of Freezing, Thawing and Growth

“…Laurdan is an amphipathic molecule that localizes near the lipid polar head groups in the cytoplasmic membrane and is therefore sensitive to changes at the water-lipid interface (Harris et al 2002;Vanounou et al 2002). DPH is useful for detecting changes in saturation of fatty acyl chains in the cytoplasmic membrane (Aricha et al 2004;Beney et al 2004). Either laurdan (5 3 10 À5 m added from a stock solution in methanol) or DPH (5 3 10 À5 m added from a stock solution in tetrahydrofuran) was incubated for 1 hr at room temperature in the dark with 0.2 ml of cultures sampled either at stationary phase or after a FT cycle.…”