Interactions between Mutant Bacterial Lipopolysaccharide (LPS-Ra) Surface Layers: Surface Vesicles, Membrane Fusion, and Effect of Ca²⁺and Temperature

Lipopolysaccharides (LPS) are a major component of the protective outer membrane of Gram-negative bacteria. Understanding how the solution conditions may affect LPS-containing membranes is important to optimizing the design of antibacterial agents (ABAs) which exploit electrostatic and hydrophobic interactions to disrupt the bacteria membrane. Here, interactions between surface layers of LPS (Ra mutants) in aqueous media have been studied using a surface force apparatus (SFA), exploring the effects of temperature and divalent Ca2+ cations. Complementary dynamic light scattering (DLS) characterization suggests that vesicle-like aggregates of diameter ∼28–80 nm are formed by LPS-Ra in aqueous media. SFA results show that LPS-Ra vesicles adsorb weakly onto mica in pure water at room temperature (RT) and the surface layers are readily squeezed out as the two surfaces approach each other. However, upon addition of calcium (Ca2+) cations at near physiological concentration (2.5 mM) at RT, LPS multilayers or deformed LPS liposomes on mica are observed, presumably due to bridging between LPS phosphate groups and between LPS phosphates and negatively charged mica mediated by Ca2+, with a hard wall repulsion at surface separation D0 ∼ 30–40 nm. At 40 °C, which is above the LPS-Ra β–α acyl chain melting temperature (Tm = 36 °C), fusion events between the surface layers under compression could be observed, evident from δD ∼ 8–10 nm steps in the force–distance profiles attributed to LPS-bilayers being squeezed out due to enhanced fluidity of the LPS acyl-chain, with a final hard wall surface separation D0 ∼ 8–10 nm corresponding to the thickness of a single bilayer confined between the surfaces. These unprecedented SFA results reveal intricate structural responses of LPS surface layers to temperature and Ca2+, with implications to our fundamental understanding of the structures and interactions of bacterial membranes

Inducible expression of DL1ICD variants in ES cells.

<p>(A) Schematic representations of the expression construct prior to and after Cre-mediated recombination, and of the pMP8 targeting vector. Black and white triangles indicate wild type loxP and mutant loxP2272 sites, respectively. (B) Western blot analysis of HA-tagged DΔECD expressed in CHO cells showing in addition to a γ-secretase-dependent cleavage product (arrow) a γ-secretase-independent proteolytic fragment (arrow head). (C) GFP expression in targeted ES cells indicating Cre-mediated activation of transgene expression. (D) Western blot analysis of cell lysates from wild type and DL1ICD-expressing E14tg2a cells using affinity purified polyclonal anti-DICD peptide antibodies.</p

Inefficient nuclear translocation and cleavage of DICD.

<p>(A) Detection of DICD stably (c, d) or transiently (e, f) expressed in CHO cells in the absence (c, e) or presence (d, f) of the proteasome inhibitor MG132. (B) Schematic representation of DICD-LexAVP16 fusion constructs. (C) Activation of lexA operator driven luciferase in CHO cells. (D) Western blot of cell lysates of CHO cells stably expressing DICD and fDICD.</p

Normal proliferation and neuronal differentiation of ES cells expressing DL1ICD variants.

<p>(A) Doubling times of targeted E14tg2a cells before and after Cre-mediated activation of DL1ICD expression. Doubling times were calculated from cell counts after non-linear regression using Prism software (GraphPad). Indicated are mean doubling times and upper and lower limit of 95% confidence intervals. (B) Western blot analysis of cell lysates of wild type and DL1ICD-expressing ES cells, CHO cells with or without transient expression of mouse p21, and HeLa nuclear extract. The arrow points to the position of p21, the asterisk marks a non-specific background band detected in ES cells. (C) Expression of the pan-neuronal marker Nefm in differentiated wild type and DL1ICD-expressing ES cells analyzed by qRT-PCR. Indicated are means and SEM of expression levels determined in differentiated wild type (n=16 pools of aggregates ) RA treated (n=14 pools of aggregates) and transgenic (DICD: n=13 pools of aggregates; fDICD: n=12 pools of aggregates; DΔECD: n=10 pools of aggregates) ES cells. ns: not significant (p>0.05). </p

Normal development of embryos expressing DL1ICD variants.

<p>(A) GFP expression in male, and hetero-and homozygous female transgenic embryos indicating Cre-mediated activation of transgene expression. (B) Western blot analysis of cell lysates from wild type and DL1ICD-expressing embryos. (C) Whole mount in situ hybridization of wild type (a, h) and DL1ICD-expressing (b-g, and i-n) embryos showing normal anterior-posterior somite patterning (a-g) and muscle differentiation (h-n). (D) Whole mount in situ hybridization of wild type (a) and DL1ICD-expressing (b-g) embryos showing normal neuronal differentiation. (E) qRT-PCR analysis of Nfem and Isl1 expression in wild type and DL1ICD-expressing embryos. Indicated are means and SEM of expression levels determined in individual wild type and transgenic embryos. ns: not significant (p>0.05).</p

Normal Notch target gene expression in embryos expressing DL1ICD variants.

<p>(A) Whole mount in situ hybridization of wild type and DL1ICD-expressing embryos showing normal Hey1 expression. (B) qRT-PCR analysis of Hes5 and Hey2 expression in wild type and DL1ICD-expressing embryos. Indicated are means and SEM of expression levels determined in individual wild type and transgenic embryos. ns: not significant (p>0.05).</p